Patent Grant
10045530
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: PRVI_010_01US_SeqList_ST25.txt, date created: Nov. 11, 2016, file size≈24 kilobytes).
Insects are estimated to cause global crop losses of approximately $250Bn—equivalent to 15% of global crop yield. Broad-spectrum insecticides, such as pyrethroid, organophosphate, and carbamate insecticide sprays, are currently used to combat such losses. However, these insecticides are harmful to both humans and the environment. In addition, the widespread use of insecticides has resulted in the evolution of resistant insects. For example, small-plot insecticide evaluations and scattered control failures in commercial sweet corn fields suggest that corn earworm populations in the Midwestern United States and southeastern Canada are gaining widespread resistance to pythrethroid-based insecticides. The rising frequency of resistant insects and the greater ease with which such insects migrate in a global economy have led to super-bugs that are causing multi-billion dollar losses. The cotton bollworm (Helicoverpa armigera) in Brazil and the corn rootworm (Diabrotica virgifera virgifera) in the United States are also contemporary illustrations of this trend. Furthermore, controlling infestations with broad-spectrum insecticides also reduces populations of beneficial insects, which leads to an outbreak of secondary pests, such as mites.
Thus, there exists a need for an insect management practice which prevents crop damage but does not have the harmful consequences of broad-spectrum insecticides.
In a first aspect, the disclosure provides for an insect pheromone composition for modifying the behavior of a target member of the order Lepidoptera. In some embodiments the pheromone composition comprises: (a) a first synthetically derived insect pheromone, having a chemical structure corresponding to that of a natural insect pheromone produced by a given target member of the order Lepidoptera; and (b) a positional isomer of said first synthetically derived insect pheromone, wherein said positional isomer is not naturally produced by the target member of the order Lepidoptera.
In some embodiments, the positional isomer is not produced by a member of the order Lepidoptera. In some embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the family Noctuidae or Plutellidae. In some embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the genus Helicoverpa, Plutella, Spodoptera, or Chrysodeixis. In some embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Helicoverpa zea. In other embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Helicoverpa armigera. In still other embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Plutella xylostella. In yet other embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Spodoptera frugiperda. In other embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Chrysodeixis includens.
In some embodiments, the first synthetically derived insect pheromone is present in the composition in a ratio of from about 99% to about 1%, relative to the positional isomer, which is present in the composition in a ratio of from about 1% to about 99%. In other embodiments, the first synthetically derived insect pheromone is present in the composition in an amount of from about 99% to about 1% w/w. In yet other embodiments, the positional isomer is present in the composition in an amount of from about 99% to about 1% w/w.
In some embodiments, the first synthetically derived insect pheromone is Z-11-hexadecenal. In some embodiments, the first synthetically derived insect pheromone is Z-11-hexadecenal and the positional isomer is Z-5-hexadecenal.
In another aspect, the insect pheromone composition further comprising: (c) a second synthetically derived insect pheromone, having a chemical structure corresponding to that of a natural insect pheromone produced by a given target member of the order Lepidoptera; and (d) optionally, a positional isomer of said second synthetically derived insect pheromone, wherein said positional isomer is not naturally produced by the target member of the order Lepidoptera. In some embodiments, the second synthetically derived insect pheromone is Z-9-hexadecenal. In some embodiments, the second synthetically derived insect pheromone is Z-9-hexadecenal and the positional isomer of the second synthetically derived insect pheromone is present and is Z-7-hexadecenal. In some embodiments, the first synthetically derived insect pheromone is Z-11-hexadecenal and the positional isomer of the first synthetically derived insect pheromone is Z-5-hexadecenal, and wherein the second synthetically derived insect pheromone is Z-9-hexadecenal and the positional isomer of the second synthetically derived insect pheromone is present and is Z-7-hexadecenal.
In some embodiments, the pheromone composition further comprises at least one additional synthetically derived insect pheromone. In some embodiments, the insect pheromone composition further comprises an agriculturally acceptable adjuvant or carrier.
In some embodiments, an insect pheromone composition for modifying the behavior of male Helicoverpa sp., is disclosed herein which comprises: (a) Z-11-hexadecenal and Z-5-hexadecenal; and (b) an agriculturally acceptable adjuvant or carrier. In some such embodiments, the Z-11-hexadecenal is present in the composition in a ratio of from about 99% to about 1%, relative to the Z-5-hexadecenal, which is present in the composition in a ratio of from about 1% to about 99%. In other embodiments, the Z-11-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w and the Z-5-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w. In further embodiments, the insect pheromone composition further comprises: Z-9-hexadecenal. In still further embodiments, the insect pheromone composition further comprises: Z-9-hexadecenal and Z-7-hexadecenal.
In some embodiments, the Z-11-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w, the Z-5-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w, the Z-9-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w, and the Z-7-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w.
In some embodiments, a method of attracting an adult male Helicoverpa sp. to a locus, comprises: presenting an effective amount of the insect pheromone composition of described herein to a locus. In some embodiments, a method of attracting and killing an adult male Helicoverpa sp., comprises: presenting an effective amount of the insect pheromone composition described herein to a locus, wherein said locus also comprises a mechanism to kill the Helicoverpa sp. In some embodiments, a method of suppressing a population of Helicoverpa sp. in a given area, comprises: applying an effective amount of the insect pheromone composition disclosed herein to a locus within said area. In some embodiments, a method of suppressing a population of Helicoverpa sp. in a given area, comprises: permeating the atmosphere within said area with an effective amount of the insect pheromone composition of disclosed herein. In some embodiments, the effective amount of the insect pheromone composition is sufficient to at least partially disrupt mating within the Helicoverpa sp. population.
In some embodiments, an insect pheromone composition for modifying the behavior of a target insect, comprises: (a) a first synthetically derived insect pheromone having a chemical structure corresponding to the chemical structure of a naturally occurring insect pheromone produced by the target insect, said structure comprising formula (1),
wherein R is located on a terminal carbon of an m-end of a carbon-carbon double bond in an unsaturated hydrocarbon substrate; and (b) positional isomer of said first synthetically derived insect pheromone, said positional isomer having a chemical structure of formula (2),
wherein the positional isomer has an R located on a terminal carbon of an n-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; wherein m and n are independently integers from 0 to 15, wherein a, c, and e are independently integers from 0 to 1, provided that at least one of a, c, or e is 1, wherein b and d are independently integers from 0 to 10, the m-end and the n-end are located on opposing sides of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; and each R is independently —OH, ═O, or —OAc.
In some embodiments, the insect pheromone composition further comprises an analog of said first synthetically derived insect pheromone, said analog having a chemical structure of formula (3)
wherein the analog has an R′ located on the n-end and the m-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; wherein m and n are independently integers from 0 to 15, wherein a, c, and e are independently integers from 0 to 1, provided that at least one of a, c, or e is 1, wherein b and d are independently integers from 0 to 10, the m-end and the n-end are located on opposing sides of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; and each R′ is independently H, —OH, ═O, —OAc, or —OOH.
In some embodiments, the insect pheromone composition further comprises:
a positional isomer of said first synthetically derived insect pheromone, said positional isomer having a chemical structure of formula (4),
wherein the positional isomer has an R located on a subterminal carbon on the m-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate thereby forming an i-end, wherein the i-end comprises a terminal carbon of the unsaturated hydrocarbon substrate; or a positional isomer of said first synthetically derived insect pheromone, said positional isomer having a chemical structure of formula 5,
wherein the positional isomer has an R located on a subterminal carbon on the n-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate, thereby forming an i-end, wherein the i-end comprises a terminal carbon of the unsaturated hydrocarbon substrate; wherein m, n, and i are independently integers from 0 to 15, wherein a, c, and e are independently integers from 0 to 1, provided that at least one of a, c, or e is 1, wherein b and d are independently integers from 0 to 10, the m-end and the n-end are located on opposing sides of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; and R is —OH, ═O, or —OAc.
In some embodiments, the sum of a, b, c, d, e, m, and n is an integer from 6 to 20. In some embodiments, the sum of a, b, c, d, e, i, m, and n is an integer from 6 to 20.
In some embodiments, the first synthetically derived pheromone has the following chemical structure:
a)
The insect pheromone composition of claim 32, wherein the first synthetically derived insect pheromone has the following chemical structure:
a)
and, wherein the positional isomer has the following chemical structure:
b)
In some embodiments, the insect pheromone composition further comprises a second synthetically derived insect pheromone having a chemical structure corresponding to the chemical structure of a naturally occurring insect pheromone produced by the target insect, wherein said second synthetically derived insect pheromone has the following chemical structure:
c)
and d) optionally, a positional isomer of said second synthetically derived insect pheromone, wherein said positional isomer is not naturally produced by the target insect.
In some embodiments, the positional isomer is present and has the following chemical structure:
The insect pheromone composition of claim 40, wherein the first synthetically derived insect pheromone has the following chemical structure:
a)
and, the positional isomer of the first synthetically derived insect pheromone has the following chemical structure:
b)
and wherein the second synthetically derived insect pheromone has the following chemical structure
c)
and
the positional isomer of the second synthetically derived insect pheromone is present and has the following chemical structure:
d)
In some embodiments, the positional isomer is not produced by the target insect. In some embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the order Lepidoptera. In some embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the family Noctuidae or Plutellidae. In some such embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the genus Helicoverpa, Plutella, Spodoptera, or Chrysodeixis. In further embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by an insect selected from the group consisting of Helicoverpa zea, Helicoverpa armigera, Plutella xylostella, Spodoptera frugiperda, and Chrysodeixis includens.
The following definitions and abbreviations are to be used for the interpretation of the disclosure.
As used herein, the term “a” as used herein to refer to noun can refer to the singular or the plural version. Thus, a reference to a pheromone can refer to one pheromone or a more than one pheromones.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having, “contains,” “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or.”
“About” in reference to a numerical value refers to the range of values somewhat less or greater than the stated value, as understood by one of skill in the art. For example, the term “about” could mean a value ranging from plus or minus a percentage (e.g., ±1%, ±2%, ±5%, or ±10%) of the stated value. Furthermore, since all numbers, values, and expressions referring to quantities used herein are subject to the various uncertainties of measurement encountered in the art, then unless otherwise indicated, all presented values may be understood as modified by the term “about.”
The terms “engineered enzyme” and “enzyme variant” include any enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different enzymes.
The terms “engineered heme enzyme” and “heme enzyme variant” include any heme-containing enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different heme-containing enzymes.
The terms “engineered cytochrome P450” and “cytochrome P450 variant” include any cytochrome P450 enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different cytochrome P450 enzymes.
The term “whole cell catalyst” includes microbial cells expressing hydroxylase enzymes, wherein the whole cell catalyst displays hydroxylation activity.
As used herein, the term “metathesis reaction” refers to a catalytic reaction which involves the interchange of alkylidene units (i.e., R2C=units) among compounds containing one or more carbon-carbon double bonds (e.g., olefinic compounds) via the formation and cleavage of the carbon-carbon double bonds. Metathesis can occur between two like molecules (often referred to as self-metathesis) and/or between two different molecules (often referred to as cross-metathesis). The product of a “metathesis reaction” can be referred to herein as a “metathesis product,” “olefinic substrate,” “unsaturated hydrocarbon” and derviation and variations thereof.
As used herein, the term “metathesis catalyst” refers to any catalyst or catalyst system that catalyzes a metathesis reaction. One of skill in the art will appreciate that a metathesis catalyst can participate in a metathesis reaction so as to increase the rate of the reaction, but is itself not consumed in the reaction.
As used herein, the term “metathesis product” refers to an olefin containing at least one double bond, the bond being formed via a metathesis reaction.
As used herein, the terms “microbial,” “microbial organism,” and “microorganism” include any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea, and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of a chemical.
As used herein, the term “non-naturally occurring,” when used in reference to a microbial organism or enzyme activity of the disclosure, is intended to mean that the microbial organism or enzyme has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous, or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary non-naturally occurring microbial organism or enzyme activity includes the hydroxylation activity described above.
As used herein, the term “natural pheromone” is intended to mean the volatile chemical or particular volatile chemical blend having a chemical structure corresponding to the chemical structure of a pheromone that is released by a particular insect for the function of chemical communication within the species. As used herein, the term “non-natural” or “non-naturally occurring,” when used in reference to a synthetic pheromone, is intended to mean a volatile chemical that is not produced by the particular insect species whose behavior is modified using said volatile chemical.
As used herein, the term “synthetically derived” when used in reference to a chemical compound is intended to indicate that the referenced chemical compound is transformed from starting material to product by human intervention. In some embodiments, a synthetically derived chemical compound can have a chemical structure corresponding an insect pheromone which is produced an insect species.
As used herein, the term “exogenous” is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The term as it is used in reference to expression of an encoding nucleic acid refers to the introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism.
The term “heterologous” as used herein with reference to molecules, and in particular enzymes and polynucleotides. indicates molecules that are expressed in an organism other than the organism from which they originated or are found in nature, independently of the level of expression that can be lower, equal or higher than the level of expression of the molecule in the native microorganism.
On the other hand, the terms “native” and/or “endogenous” as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicate molecules that are expressed in the organism in which they originated or are found in nature, independently of the level of expression that can be lower equal or higher than the level of expression of the molecule in the native microorganism. It is to be understood that expression of native enzymes or polynucleotides may be modified in recombinant microorganisms.
The term “homolog,” as used herein with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural, or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Homologs most often have functional, structural, or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homologs can be confirmed using functional assays and/or by genomic mapping of the genes.
A protein has “homology” or is “homologous” to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. Thus, the term “homologous proteins” is intended to mean that the two proteins have similar amino acid sequences. In certain instances, the homology between two proteins is indicative of its shared ancestry, related by evolution.
The terms “analog” and “analogous,” when used in reference to a nucleic acid or protein, include nucleic acid or protein sequences or protein structures that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure.
As used herein, the term “alkane” refers to a straight or branched, saturated, aliphatic hydrocarbon having the number of carbon atoms indicated. The term “alkyl” refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, such as C1-2, C1-3, C1-4, C1-5, C1-6, C1-7, C1-8, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. For example, C1-6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Alkanes and alkyl groups can be optionally substituted with one or more moieties selected from halo, alkenyl, and alkynyl.
As used herein, the term “alkene” and “olefin” refers to a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. The term “olefinic” refers to a composition derived from or including a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. A “terminal” alkene refers to an alkene wherein the double bond is between two carbon atoms at the end of the hydrocarbon chain (e.g., hex-1-ene). An “internal” alkene refers to an alkene wherein the double bond is between two carbon atoms that are not at the end of the hydrocarbon chain (e.g., (E)-hex-3-ene and (Z)-hex-3-ene). An “α,ω-alkenol” refers to a hydroxy-substituted terminal alkene having the formula (CH2═CH)(CH2)mOH, wherein m is an integer ranging from 1-30, such as 2-18. The term “alkenyl” refers to a straight chain or branched hydrocarbon radical having at least 2 carbon atoms and at least one double bond. Alkenyl can include any number of carbons, such as C2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8, C2-9, C2-10, C3, C3-4, C3-5, C3-6, C4, C4-5, C4-6, C5, C5-6, and C6. Alkenyl groups can have any suitable number of double bonds, including, but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenes and alkenyl groups can be optionally substituted with one or more moieties selected from halo, alkyl, and alkynyl.
As used herein, the term “selective” refers to preferential reaction of one site on a chemical compound over another site on the compound. As a non-limiting example, selectively hydroxylating hept-3-ene (an asymmetric alkene) refers to preferentially hydroxylating one end of the hept-3-ene to form more hept-3-en-1-ol than hept-4-en-1-ol (or forming exclusively hept-3-en-1-ol without forming hept-4-en-1-ol). Selectively hydroxylating the other end of hept-3-ene would result in the formation of more hept-4-en-1-ol than hept-3-en-1-ol (or the exclusive formation of hept-4-en-1-ol without formation of hept-3-en-1-ol).
As used herein, the term “alkyne” refers to either a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one triple bond. A “terminal” alkyne refers to an alkyne wherein the triple bond is between two carbon atoms at the end of the hydrocarbon chain (e.g., hex-1-yne). An “internal” alkyne refers to an alkyne wherein the triple bond is between two carbon atoms that are not at the end of the hydrocarbon chain (e.g., hex-3-yne). The term “alkynyl” refers to either a straight chain or branched hydrocarbon radical having at least 2 carbon atoms and at least one triple bond. Alkynyl can include any number of carbons, such as C2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8, C2-9, C2-10, C3, C3-4, C3-5, C3-6, C4, C4-5, C4-6, C5, C5-6, and C6. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynes and alkynyl groups can be optionally substituted with one or more moieties selected from halo, alkyl, and alkenyl.
As used herein, the term “isomer” refers to a molecule having the same chemical formula as another molecule, but with a different chemical structure. That is, isomers contain the same number of atoms of each element, but have different arrangements of their atoms. Isomers include “structural isomers” and “stereoisomers.” In “structural isomers” (also referred to as “constitutional isomers”), the atoms have a different bond-sequence. Structural isomers have different IUPAC names and may or may not belong to the same functional group. This type of isomer includes skeletal isomers wherein hydrocarbon chains have variable amounts of branching, and positional isomers, which deals with the position of a functional group on a chain; and functional group isomerism, in which the molecular formula is the same but the functional group is different.
As used herein, the term “positional isomer” refers to a first compound which has the same carbon skeleton and functional group as a second compound, but differs in the location of the functional group on or in the carbon skeleton. In a particular embodiment, a positional isomer can have a functional group (e.g., hydroxyl, aldehyde, and acetyl, etc.) located on the opposite terminus of a carbon skeleton compared to a naturally occurring compound. Thus, as used herein, a positional isomer of Z-hexadec-11-en-1-al is a Z-hexadec-5-en-1-al, because the Z-hexadec-11-en-1-al and Z-hexadec-5-en-1-al are produced via hydroxylation/oxidation of opposite termini on the Z-11-hexadecene carbon skeleton as shown in
In stereoisomers, the bond structure is the same, but the geometrical positioning of atoms and functional groups in space differs. This class of isomers includes enantiomers, which are isomers that are non-superimposable mirror-images of each other, and diastereomers, which are stereoisomers that are not mirror-images. Geometric isomers or cis/trans isomers are diastereomers that with a different stereochemical orientation at a bond. E/Z isomer, which are a subset of geometric isomers, are isomers with a different geometric arrangement at a double bond. Another type of isomer, conformational isomers (conformers), may be rotamers, diastereomers, or enantiomers depending on the exact compound.
The term “analog,” as used herein in reference to a chemical structure is intended to refer to compounds having a similar structure, but having a different molecular formula, e.g., a different or additional atom and/or functional group. By way of example, and not limitation, an analog of an insect pheromone can refer to molecule with two hydroxyl groups (as opposed to a single hydroxyl group required for a natural pheromone or precursor thereof) and/or an over-oxidized molecules with a carboxylic acid functional group (as opposed to an aldehyde functional group required for a natural pheromone).
An “effective amount” means that amount of the disclosed pheromone composition that is sufficient to affect desired results. An effective amount can be administered in one or more administrations. For example, an effective amount of the composition may refer to an amount of the pheromone composition that is sufficient to attract a given insect to a given locus. Further, an effective amount of the composition may refer to an amount of the pheromone composition that is sufficient to disrupt mating of a particular insect population of interest in a given locality.
The present disclosure addresses a need for a safe alternative to conventional insecticides. The present disclosure provides compositions and methods for modifying the behavior of an insect using a composition comprising a pheromone.
In some aspects, the composition comprises a pheromone chemically corresponding to the pheromone naturally produced by a given insect. In some aspects, the composition comprises a pheromone chemically corresponding to the pheromone naturally produced by a given insect, along with at least one isomer of said pheromone. In various aspects, the isomer of the naturally produced insect pheromone may be a positional isomer.
In some embodiments, the insect is a pest. As used herein, the term “pest” can refer to insects that cause damage to plants, other organisms or otherwise causes a nuisance. In some embodiments, an insect pest can be attracted to a pheromone composition, e.g., by flying toward the pheromone composition or interacting with an article treated with the pheromone composition.
In various aspects, the insect that is “attracted” to the compositions taught herein may, or may not, physically contact a locus containing said pheromone composition. That is, in some aspects, the compositions taught herein are able to attract a given insect within a close proximity to a locus containing the disclosed pheromone compositions, but do not entice said insect to physically contact the locus. However, in other aspects, the compositions taught herein do entice and/or attract an insect to physically come into contact with a locus containing said pheromone compositions. In this way, inter alia, the pheromone compositions taught herein are highly “tunable” and are able to modulate the behavior (e.g., degree of attracting an insect) of an insect to a high degree, which is not associated with pheromone compositions of the prior art. Thus, the pheromone compositions taught herein, which may contain a natural insect pheromone and at least one positional isomer of said pheromone, do not merely provoke a binary “attract or not attract” response in a given insect. Rather, the pheromone compositions of the present disclosure are able to modulate the degree to which an insect is attracted along a continuous scale, depending upon, among other things, the ratio of natural pheromone to its positional isomer.
Embodiments of the present disclosure are based on the inventors' discovery of a novel methodology for the synthesis of a pheromone. The novel method includes (1) metathesis of alpha olefins to form alkenes with an internal C═C bond, (2) biohydroxylation of the product alkene via an enzymatic reaction to generate an alkenol, and (3) modification of the alkenol to an aldehyde by oxidation (MBO) or to an acetate by esterification (MBE). This method is referred to herein as MBO and MBE. The inventors' discovered synthesis, performed according to the methodology disclosed herein, yields an isomeric mixture which includes the natural pheromone and at least one isomer, e.g., a positional isomer. An unexpected and surprising result of the application of pheromone compositions comprising a synthetically derived, natural pheromone and a non-natural positional isomer was that the presence of the non-natural positional isomer in the pheromone composition modulated the behavior of the target insect. That is, while long-range attraction was maintained (i.e., upwind orienting flight), close-range attraction was eliminated. This indicates a novel partial mimic/partial antagonist response to the non-natural position isomer. Thus, the disclosure provides for pheromone compositions that comprise a natural insect pheromone, which a given target insect has evolved to recognize, along with a positional isomer of said pheromone. The combination of a natural pheromone, which would be produced by a target male insect's female counterpart—along with a positional isomer of said pheromoneμleads to a composition with markedly different behavioral modification properties, as compared to the naturally produced pheromone composition of a female insect.
Thus, in some embodiments, the pheromone compositions taught herein, can elicit a markedly different response from a natural pheromone blend, as they possess functional attributes not found in natural pheromone compositions produced by female insects of a given target species.
Furthermore, the pheromone compositions taught herein, are structurally different than any naturally occurring pheromone composition produced by a female insect of a given target species, as the pheromone compositions taught herein provide for a combination of a natural pheromone along with its positional isomer. This combination of an insect pheromone and a positional isomer of said pheromone does not occur in nature.
Pheromone
As described above, one aspect of the disclosure is a pheromone composition which can modify the behavior of an insect. A pheromone is a secreted or excreted chemical factor that triggers a social response in members of the same species. Thus, pheromones are chemicals capable of acting outside the body of the secreting individual to impact the behavior of the receiving individual. There are, inter alia, alarm pheromones, food trail pheromones, sex pheromones, aggregation pheromones, epideictic pheromones, releaser pheromones, primer pheromones, and territorial pheromones, that affect behavior or physiology.
As used herein, a pheromone can be a chemical, or a set of chemicals, that attract at least one species of insect. In some embodiments, the pheromone is a sex pheromone which attracts one sex of at least one insect. A pheromone synthesized as disclosed herein can be chemically identical to the natural substance for the target insect or it can be an isomer (e.g., a positional isomer, a constitutional isomer, or a stereoisomer, e.g, conformational isomer, geometric isomer, diastereomer, or enantiomer, etc.) or an analog of the natural pheromone. As used herein, the term “positional isomer” refers to a first compound which has the same carbon skeleton and functional group as a second compound, but differs in the location of the functional group on or in the carbon skeleton. For example, a positional isomer can have an aldehyde functional group located on the opposite terminus of the carbon skeleton compared to a naturally occurring compound. Thus, as used herein, a positional isomer of Z-hexadec-11-en-1-al is a Z-hexadec-5-en-1-al, because the Z-hexadec-5-en-1-al and Z-hexadec-5-en-1-al are produced via hydroxylation/oxidation of the opposite terminus of the Z-5-hexadecene carbon skeleton as shown in
Pheromones described herein can be referred to using IUPAC nomenclature or various abbreviations and derivations. For example, (Z)-hexadec-11-en-1-al, can also be written as Z-11-hexadecen-1-al, Z-11-hexadecenal, or Z-x-y:Ald, wherein x represents the position of the double bond, and y represents the number of carbons in the hydrocarbon skeleton. Abbreviations used herein and known to those skilled art to identify functional groups on the hydrocarbon skeleton include “Ald,” indicating an aldehyde, “OH,” indicating an alcohol, and “Ac,” indicating an acetyl. Also, the number of carbons in the chain can be indicated using numerals rather than using the written name. Thus, as used herein, an unsaturated carbon chain comprised of sixteen carbons can be written as hexadecene or 16.
Non-limiting examples of C6-C20 linear insect pheromones that can be synthesized using the methodology disclosed herein are included in Table 1 below. Accordingly, a pheromone composition as described herein can include at least one of the pheromones listed in Table 1. Further, in some embodiments, the compositions taught herein comprise at least one of the pheromones listed in Table 1, along with at least one isomer thereof. In a particular embodiment, the compositions taught herein comprise at least one of the pheromones listed in Table 1, along with a positional isomer of at least one of the pheromones as listed in Table 1. In still further aspects of the disclosure, a composition may comprise only a positional isomer of a pheromone as listed in Table 1.
In some aspects, pheromone compositions taught in this disclosure comprise at least one pheromone listed in Table 2 and a positional isomer thereof to modulate the behavior of an insect listed in Table 2. By changing the ratios of a pheromone as listed in Table 2 and a positional isomer thereof in a given composition, the disclosure provides for a highly tunable insect behavior modifying composition.
Pheromones have the potential to challenge conventional approaches to agricultural insect control. Since their discovery in the late 1950s, these molecules have shown efficacy in reducing insect populations through sensory disruption and a subsequent reduction in mating frequency via a non-toxic mode of action.
Insect pheromones can be used in a variety of insect control strategies, including mating disruption, attract-and-kill, and mass trapping. These strategies have proven to be effective, selective (e.g., they do not harm beneficial insects, such as bees and lady bugs), and safe (e.g., the compounds are generally biodegradable and do not accumulate in the food chain).
The selectivity of pheromones allows farmers to control the population of the target pest causing minimal disruption to the ecology in the field. Because pheromones act via non-toxic mating disruption, they can be used to manage pests that have evolved resistance to chemical or transgenic insecticides.
This organic form of insect control has enjoyed success in permanent crops worldwide, particularly in Washington State apple orchards where adoption rates are greater than 90%. However, only <20 insect pests worldwide are currently controlled using pheromone solutions (e.g., mating disruption, attract-and-kill, mass trapping), and only 0.05% of global agricultural land employs pheromones. The limited use of pheromones is an unfortunate result of the high cost of synthesizing pheromones is very high, with industrial scale active ingredient (AI) prices ranging from $500 to $15,000 per kg, which prohibits widespread use of this sustainable technology beyond high-value crops.
Described herein are pheromone compositions synthesized using a novel enzymatic biohydroxylation step, which yields a pheromone at a fraction of the cost of conventional methodology. In some embodiments, the pheromone synthesized according to this methodology is a positional isomer of the natural pheromone. In one such embodiment, the positional isomer is not naturally produced by a female insect, but a male insect of the same species surprisingly responds to a composition which includes the positional isomer. This is surprising, given the fact that male insects are highly evolved to sense and respond to pheromone produced by their potential female mate. Accordingly, in some embodiments, a pheromone composition comprising: (a) an insect pheromone having a chemical structure identical to that of a pheromone produced by an insect pest, and (b) a positional isomer of said insect pheromone, can be used to modify the behavior of an insect pest.
The inclusion of a positional isomer in a pheromone composition comprising a synthetically derived natural pheromone can have at least four possible outcomes: (1) Inert—the isomer acts as a diluent, and the behavior of the target insect is not influenced; (2) Antagonist—the isomer acts as an inhibitor and blocks the response of the target insect to the natural pheromone; (3) Mimic—the isomer provides the same biological activity as the natural pheromone; and (4) Partial Mimic/Partial Antagonist—the isomer elicits an upwind flight response from the target insect, but the insect does not contact or land on a lure coated with the positional isomer.
In one embodiment, the target insect is member of the order Lepidoptera. In some embodiments, the composition is utilized to bring about mating disruption in the lepidopteran population, which subsequently leads to a decline in the population.
Lepidoptera
Lepidoptera is the second largest order in the class Insecta. The order Lepidoptera include the following families of butterflies: Nymphalidae, Danaidae, Pieridae, Papilionidae, Lycaenidae, Hesperiidae (e.g., Epargyreus clarus). The order also includes the following families of moths: Tineidae (e.g., (Tineola bisselliella, and Tinea pellionella), Gelechiidae (e.g., Sitotroga cerealella and Pectinophora gossypiella), Sesiidae (e.g., Synanthedon exitiosa and Melittia cucurbitae), Tortricidae (e.g., Cydia pomonella and Grapholita molesta). Pyralidae (e.g., Ostrinia nubilalis, Plodia interpunctella, and Galleria mellonella), Geometridae (e.g., Operophtera brumata and Alsophila pometaria), Lasiocampidae (e.g., Malacosoma Americana and Malacosoma disstria). Satumiidae (e.g., Hyalophora cecropiaa and Actias luna), Sphingidae (e.g., Manduca sexta and Manduca quinquemaculata), Arctiidae (e.g., Hyphantria cunea), Lymantriidae (e.g., Lymantria dispar and Euproctis chrysorrhoea), Noctuidae (e.g., Spodoptera frugiperda, Agrotis Ipsilon, Trichoplusia ni, Chrysodeixis includes, Helicoverpa zea, and Helicoverpa armigera), and Plutellidae (e.g., Plutella xylostella).
The larvae of many lepidopteran species, which are commonly referred to as caterpillars, are major pests in agriculture. In many lepidopteran species, the female may produce anywhere from 200 to 600 eggs; and some species produce up to 30,000 eggs in one day. Unmitigated, the larvae can affect acres of vegetation. In fact, the larvae of Lepidoptera are probably more destructive to agricultural crops and forest trees than any other group of insects.
Some of the major pests of the order Lepidoptera include members of the families Noctuidae and Plutellidae. The larvae of the Noctuidae genus Spodoptera (including armyworm), Helicoverpa (including corn earworm and cotton bollworm), Chrysodeixis (including soybean looper) and larvae of the Plutellidae genus Plutella (including diamondback moth) can cause extensive damage to valuable crops.
Helicoverpa zea is known as the corn earworm; the polyphagous larva are known to cause damage to a variety of crops, including: corn, tomato, artichoke, asparagus, cabbage, cantaloupe, collards, cowpea, cucumber, eggplant, lettuce, lime bean, melon, okra, pea, pepper, potato, pumpkin, snap bean, spinach, squash, sweet potato, watermelon, soybean, as non-limiting examples.
Helicoverpa armigera is commonly referred to as the cotton bollworm; the polyphagous larva are known to cause damage to a variety of crops, including: tomato, cotton, pigeon pea, chickpea, sorghum, cowpea, groundnut, okra, peas, field beans, soybeans, lucerne, a variety of legumes, tobacco, potatoes, maize, flax, Dianthus, Rosa, Pelargonium, Chrysanthemum, Lavandula angustifolia, fruit trees, forest trees, and a range of vegetable crops, as non-limiting examples.
Plutella xylostella is known as the diamondback moth and is a worldwide pest; it is known to feed on cruciferous vegetables, including: broccoli, Brussels sprouts, cabbage, Chinese cabbage, cauliflower, collard, kale, kohlrabi, mustard, radish, turnip, and watercress.
Spodoptera frugiperda, known as the fall armyworm, has been reported to damage field crops, including: alfalfa, barley, Bermuda grass, buckwheat, cotton, clover, corn, oat, millet, peanut, rice, ryegrass, sorghum, sugarbeet, sudangrass, soybean, sugarcane, timothy, tobacco, and wheat, sweet corn, apple, grape, orange, papaya, peach, strawberry and a number of flowers.
Chrysodeixis includens is a type of moth whose larva is known to damage crops, including: soybeans, goldenrod, lettuce, sweet potato, peanut, cotton, tomato, brassicas (cabbage, kale, broccoli), pea, tobacco, and cocklebur.
Today, lepidopteran pests are predominantly controlled by pyrethroid, organophosphate, and carbamate insecticide sprays. Organophosphates and carbamates have demonstrated carcinogenic and neurotoxic effects in humans, while pyrethroids and organophosphates may unintentionally harm beneficial insects or sensitive vertebrates like amphibians, and fish. Conversely, lepidopteran pheromones present no known risks to humans or the environment. These non-toxic compounds may serve as a substitute for conventional pesticides, reducing the amount of chemical exposure to consumers, farm laborers, and the environment.
Insects of the order Lepidoptera produce pheromones which generally consist of unbranched, oxyfunctionalized long-chain olefins containing one to three double bonds. Lepidopteran pheromones, which are naturally occurring compounds, or identical or substantially similar synthetic compounds, are designated by an unbranched aliphatic chain (between 9 and 18 carbons) ending in an alcohol, aldehyde, or acetate functional group and containing up to 3 double bonds in the aliphatic backbone. For examples, the sex pheromones of Helicoverpa zea, Helicoverpa armigera, Plutella xylostella, and Chrysodeixis includes insects typically include one or more aliphatic aldehyde compounds having from 10 to 16 carbon atoms (e.g., 7-hexadecenal, 11-hexadecenal, 13-octadecenal, and the like). Other insects, such as Spodoptera frugiperda, recognize pheromones that are aliphatic acetate compounds having from 10 to 16 carbon atoms (e.g., decyl acetate, decenyl acetate, decadienyl acetate, undecyl acetate, undecenyl acetate, dodecyl acetate, dodecenyl acetate, dodecadienyl acetate, tridecyl acetate, tridecenyl acetate, tridecadienyl acetate, tetradecyl acetate, tetradecenyl acetate, tetradecadienyl acetate, and the like).
Variation in the location, cis/trans selectivity, level of unsaturation along the chain, and chain length results in a diverse set of pheromones that facilitate species specific communication. These pheromones are used to attract a mate, sometimes at long distances.
The generally accepted natural pheromones produced by female Helicoverpa zea, Helicoverpa armigera, Plutella xylostella and Chrysodeixis includens are shown in the table below. Thus, aspects of the disclosure provide for pheromone compositions comprised of synthetically derived natural pheromone blends according to the table below along with various ratios of positional isomers. However, it should be noted that there can be other minor components produced by these insects as well. The below table merely lists the pheromone compositions produced by these insects, as they are commonly understood in the scientific literature. See, e.g., http://www.pherobase.com/database/species/species-Helicoverpa-zea.php; Halfhill, J. E. and McDonough, L. M. Southwest Entomol., 1985. 10; 176-180; Pope M M, Gaston L K, and Baker T C. (1984) Composition, quantification, and periodicity of sex pheromone volatiles from individual Heliothis zea females. J Insect Physiol 30:943-945; http://www.pherobase.com/database/species/species-Helicoverpa-armigera.php; Zhang, J. P., et al. J. Insect Physiol. (2012) 58:1209-1216; http://www.pherobase.com/database/species/species-Plutella-xylostella.php; Lin, Y. M., et al., Bull. Inst. Sool. Acad. Sin. 21:121-127; Chisholm M D, Underhill E W, and Steck W F. (1979) Field trapping of the diamondback moth Plutella xylostella using synthetic sex attractants. Environmental Entomology 8:516-518; http://www.pherobase.com/database/species/species-Spodoptera-frugiperda.php; Meagher, R. L. and Mitchell, E. R. Fla. Entomol., 1998. 81:556-559; Tumlinson J H, Mitchell E R, Teal P E A, Heath R R, and Mengelkoch L J (1986) Sex pheromone of fall armyworm, Spodoptera frugiperda (J. E. Smith). J Chem Ecol 12:1909-1926; http://www.pherobase.com/database/species/species-Pseudoplusia-includens.php; Tumlinson, J. H, et al., Environ. Entomol., 1972. 1:466-468; Linn C E, Du J, Hammond A, and Roelofs W L. (1987) Identification of unique pheromone components for soybean looper moth Pseudoplusia includens. J Chem Ecol 13:1351-1360; Cork A, Beevor P S, Hall D R, Nesbitt B F, Arida G S, and Mochida O. (1985). Components of the female sex pheromone of the yellow stem borer, Scirpophaga incertulas. Entomol. Exp. Appl. 37:149-153. Jiao X-G, Xuan W-J, Sheng C-F (2005) Mass trapping of the overwintering generation stripped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae) with the synthetic sex pheromone in northeastern China. Acta Entomologica Sinica 48:370-374; McLaughlin J and R Heath (1989) Field trapping and observations of male Velvetbean caterpillar moths and trapping of Mocis spp. (Lepidoptera: Noctuidae: Catacolinae) with calibrated formulations of sex pheromone. Environmental Entomology 18:933-938
†Ratios vary between regional populations and studies. The ratios reported here are based on either recent citations, more commonly cited blends, or historically accepted blends.
It is common for an individual pheromone to appear in multiple insects. For example, (Z)-11-hexadecenal is the main pheromone component for not only the corn earworm, but also the tobacco budworm, diamondback moth, and the rice stem borer.
As discussed above, pheromones can be used to manage pests. Accordingly, described herein are pheromone compositions and methods of use thereof to modulate the behavior of pests, e.g., by disrupting mating behavior.
In some embodiments, a pheromone described in Table 1 or Table 2 can be synthesized using the methods and synthetic schemes described herein. In aspects, positional isomers of the pheromones listed in Table 1 or Table 2 are produced by the synthetic schemes disclosed herein. Accordingly, a pheromone composition as described herein can include at least one of the pheromones listed in Table 1 or Table 2, along with at least one isomer thereof. In a particular embodiment, the compositions taught herein comprise at least one of the pheromones listed in Table 1 or Table 2, along with a positional isomer of at least one of the pheromones as listed in Table 1 or Table 2.
In exemplary embodiments, isomers of hexadecen-1-al can be synthesized for use in pheromone compositions. In the present disclosure, Z-hexadac-11-en-1-al, Z-11hexadacen-1-al, Z-11-hexadacenal, Z-hexadac-11-enal and Z-11-16:AL, are used synonymously, and similar variations can be used for other phenomes described herein. In exemplary embodiments, a Z-hexadac-11-en-1-al and a positional can be synthesized for use in pheromone compositions to modify the behavior of insect of the order Lepidoptera (e.g., Helicoverpa. zea,). In one such exemplary embodiment, the positional isomer of Z-hexadac-11-en-1-al is Z-hexadac-5-en-1-al (
The present disclosure is based in part on the inventors' unexpected discovery that a pheromone composition including a synthetically derived natural pheromone and a positional isomer thereof can be used to modulate the response of a target insect relative to the response of the target insect elicited by a natural pheromone or natural pheromone blend.
General Synthetic Route to Produce Pheromone Compositions
The present disclosure describes several methods for the synthesis of terminally oxyfunctionalized alkenes. Said methods are described in detail below and are generally applicable to the synthesis of various compounds, including but not limited to those shown in Table 1.
Some embodiments of the disclosure provide methods for synthesizing olefinic alcohol products wherein the olefinic alcohol product is a pheromone. In some embodiments, the olefinic alcohol product is selected from the alcohols in Table 1. Pheromones containing aldehyde functional groups can also be prepared using the olefinic alcohol products as intermediates. In such cases, the methods of the disclosure generally include oxidizing the olefin alcohol product to form an alcohol product. In some embodiments, the olefinic aldehyde product is selected from the aldehydes in Table 1.
Pheromones containing ester functional groups can also be prepared using the olefinic alcohol products as intermediates. In such cases, the methods of the disclosure generally include esterifying the olefinic alcohol product to form an olefinic ester product. In some embodiments, the olefinic ester product is an acetate ester. In some embodiments, the olefinic ester product is selected from the esters in Table 1 or Table 2.
Useful unsaturated fatty acids and related compounds can also be prepared using the olefinic alcohol products as intermediates. In such cases, the methods of the disclosure generally include oxidizing the olefinic alcohol product to form an olefinic acid product.
The synthetic strategies disclosed herein chiefly rely on the ability of hydroxylases to terminally hydroxylate hydrocarbon substrates such as linear alkenes. Linear alkenes and other hydrocarbon substrates can be synthesized via any route, including but not limited to olefin metathesis, Wittig olefination, or alkyne substitution followed by partial hydrogenation. The hydroxylation products can further be modified via any method, including—but not limited to—oxidation, esterification, and olefin metathesis, to produce the desired end products (Scheme 1). Deviations from this general scheme are also disclosed.
MBO or MBE Synthesis
In an exemplary embodiment, the synthesis route (Scheme 2) consists of (1) metathesis of alpha olefins to form alkenes with an internal C═C bond, (2) biohydroxylation of the product alkene via an enzymatic reaction to generate an alkenol, and (3) modification of the alkenol to an aldehyde by oxidation (MBO) or to an acetate by esterification (MBE). This short and concise route can potentially capture a large segment of all lepidopteran pheromones. Further, synthesis of any insect pheromone and its positional isomer can be achieved through altering the length of the alpha olefins used in the metathesis step and finding an enzyme catalyst capable of acting on a range of alkenes. Biohydroxylation of different terminal carbons on an olefinic substrate (and subsequent oxidation/esterification if necessary) will generate a mixture of pheromones having a chemical structure of an insect sex pheromone produced by an insect and positional isomers of said sex pheromone. Thus, the disclosure is not limited to producing compositions comprising lepidopteran pheromones and positional isomers thereof; rather, the methods of the disclosure can produce any insect pheromone and any positional isomers thereof, for utilization in the disclosed compositions.
Synthesis of Terminal Alkenols Via Metathesis and Hydroxylation
In one aspect, the disclosure provides a method for synthesizing an olefinic alcohol product that includes incubating an unsaturated hydrocarbon substrate with an enzyme capable of independently hydroxylating a first terminal carbon of a first unsaturated hydrocarbon substrate and a second terminal carbon of a second unsaturated hydrocarbon substrate to form a mixture of an unsaturated hydrocarbon alcohol. The hydrocarbon alcohol can be further converted via oxidation, acetylation, or esterification as disclosed herein or according to methods known to those skilled in the art.
Synthesis of Unsaturated Hydrocarbon Substrate for Subsequent Biohydroxylation to Produce Pheromone Composition
Hydroxylation of Asymmetric Alkenes
In some embodiments, the method for synthesizing an oxyfunctionalized alkene includes a combination of metathesis and terminal hydroxylation as shown in Scheme 3. In this process, terminal alkenes of different lengths are combined to generate asymmetric alkenes, which are then subjected to biohydroxylation conditions to afford the desired alkenol products.
Methods including hydroxylation of asymmetric alkenes can be conducted with alkenes of any suitable length. In some embodiments, the asymmetric olefinic alcohol product is a C4-C30 olefinic alcohol product. In such embodiments, the sum of the subscripts m and n shown in Scheme 3 will bring the total number of carbon atoms in a particular asymmetric olefinic alcohol product to 4-30, when added to the number of the non-subscripted carbon atoms shown in the structure for the asymmetric olefinic alcohol product. In such embodiments, for example, subscript m in Scheme 5 can be an integer from 8-18 and subscript n in Scheme 3 can be a different integer from 0-8, bringing the total number of the carbons in the asymmetric olefinic substrate to 4-30. When m is 9 and n is 3, the route depicted in Scheme 3 provides (E/Z)-hexadec-11-en-1-ol as the target product. In some embodiments, the asymmetric olefinic alcohol product is a C4-C20 olefinic alcohol product. The asymmetric olefinic alcohol can contain, for example, 4-20 carbon atoms, or 8-20 carbon atoms, or 12-20 carbon atoms, or 16-20 carbon atoms.
In some embodiments, for example, m is 0 and n is 4; or m is 1 and n is 3; or m is 3 and n is 1; or m is 4 and n is 0; or m is 0 and n is 5; or m is 1 and n is 4; or m is 2 and n is 3; or m is 3 and n is 2; or m is 4 and n is 1; or m is 5 and n is 0; or m is 0 and n is 6; or m is 1 and n is 5; or m is 2 and n is 4; or m is 4 and n is 2; or m is 5 and n is 1; or m is 6 and n is 0; or m is 0 and n is 7; or m is 1 and n is 6; or m is 2 and n is 5; or m is 3 and n is 4; or m is 4 and n is 3; or m is 5 and n is 2; or m is 6 and n is 1; or m is 7 and n is 0; or m is 0 and n is 8; or m is 1 and n is 7; or m is 2 and n is 6; or m is 3 and n is 5; or m is 5 and n is 3; or m is 6 and n is 2; or m is 7 and n is 1; or m is 8 and n is 0; or m is 0 and n is 9; or m is 1 and n is 8; or m is 2 and n is 7; or m is 3 and n is 6; or m is 4 and n is 5; or m is 5 and n is 4; or m is 6 and n is 3; or m is 7 and n is 2; or m is 8 and n is 1; or m is 9 and n is 0; or m is 0 and n is 10; or m is 1 and n is 9; or m is 2 and n is 8; or m is 3 and n is 7; or m is 4 and n is 6; or m is 6 and n is 4; or m is 7 and n is 3; or m is 8 and n is 2; or m is 9 and n is 1; or m is 10 and n is 0; or m is 0 and n is 11; or m is 1 and n is 10; or m is 2 and n is 9; or m is 3 and n is 8; or m is 4 and n is 7; or m is 5 and n is 6; or m is 6 and n is 5; or m is 7 and n is 4; or m is 8 and n is 3; or m is 9 and n is 2; or m is 10 and n is 1; or m is 11 and n is 0; or m is 0 and n is 12; or m is 1 and n is 11; or m is 2 and n is 10; or m is 3 and n is 9; or m is 4 and n is 8; or m is 5 and n is 7; or m is 7 and n is 5; or m is 8 and n is 4; or m is 9 and n is 3; or m is 10 and n is 2; or m is 11 and n is 1; or m is 12 and n is 0; or m is 0 and n is 13; or m is 1 and n is 12; or m is 2 and n is 11; or m is 3 and n is 10; or m is 4 and n is 9; or m is 5 and n is 8; or m is 6 and n is 7; or m is 7 and n is 6; or m is 8 and n is 5; or m is 9 and n is 4; or m is 10 and n is 3; or m is 11 and n is 2; or m is 12 and n is 1; or m is 13 and n is 0; or m is 0 and n is 14; or m is 1 and n is 13; or m is 2 and n is 12; or m is 3 and n is 11; or m is 4 and n is 10; or m is 5 and n is 9; or m is 6 and n is 8; or m is 8 and n is 6; or m is 9 and n is 5; or m is 10 and n is 4; or m is 11 and n is 3; or m is 12 and n is 2; or m is 13 and n is 1; or m is 14 and n is 0; or m is 0 and n is 15; or m is 1 and n is 14; or m is 2 and n is 13; or m is 3 and n is 12; or m is 4 and n is 11; or m is 5 and n is 10; or m is 6 and n is 9; or m is 7 and n is 8; or m is 8 and n is 7; or m is 9 and n is 6; or m is 10 and n is 5; or m is 11 and n is 4; or m is 12 and n is 3; or m is 13 and n is 2; or m is 14 and n is 1; or m is 15 and n is 0; or m is 0 and n is 16; or m is 1 and n is 15; or m is 2 and n is 14; or m is 3 and n is 13; or m is 4 and n is 12; or m is 5 and n is 11; or m is 6 and n is 10; or m is 7 and n is 9; or m is 9 and n is 7; or m is 10 and n is 6; or m is 11 and n is 5; or m is 12 and n is 4; or m is 13 and n is 3; or m is 14 and n is 2; or m is 15 and n is 1; or m is 16 and n is 0; or m is 1 and n is 16; or m is 2 and n is 15; or m is 3 and n is 14; or m is 4 and n is 13; or m is 5 and n is 12; or m is 6 and n is 11; or m is 7 and n is 10; or m is 8 and n is 9; or m is 9 and n is 8; or m is 10 and n is 7; or m is 11 and n is 6; or m is 12 and n is 5; or m is 13 and n is 4; or m is 14 and n is 3; or m is 15 and n is 2; or m is 16 and n is 1; or m is 17 and n is 0; or m is 0 and n is 18; or m is 1 and n is 17; or m is 2 and n is 16; or m is 3 and n is 15; or m is 4 and n is 14; or m is 5 and n is 13; or m is 6 and n is 12; or m is 7 and n is 11; or m is 8 and n is 10; or m is 10 and n is 8; or m is 11 and n is 7; or m is 12 and n is 6; or m is 13 and n is 5; or m is 14 and n is 4; or m is 15 and n is 3; or m is 16 and n is 2; or m is 17 and n is 1; or m is 18 and n is 0
Accordingly, some embodiments of the disclosure provide methods for preparing an olefinic alcohol product as described above, wherein the olefinic substrate is a metathesis product, and wherein the method includes: a) cross-metathesizing a first terminal olefin and a second different terminal olefin in the presence of a metathesis catalyst to form the metathesis product; and b) incubating the metathesis product with an enzyme capable of selectively hydroxylating one terminal carbon of the metathesis product to form an olefinic alcohol product.
In some embodiments, the first terminal olefin has the formula (CH2═CH)(CH2)mH, the second different terminal olefin has the formula (CH2═CH)(CH2)nH, the metathesis product has the formula H(CH2)m(CH═CH)(CH2)nH, the olefinic alcohol product has the formula HO(CH2)m(CH═CH)(CH2)nH, and m and n are different integers between 1 and 18. In some embodiments, the olefinic alcohol product has a chemical structure corresponding to an insect pheromone. In other embodiments, the olefinic alcohol product has a chemical structure corresponding to a precursor of a pheromone, wherein the precursor undergoes subsequent synthetic transformation, e.g., oxidation and/or acetylation, to produce a synthetically derived pheromone. In some embodiments, m and n are different integers between 1 and 9.
In some embodiments, methods described herein can be used to synthetically derive an olefinic alcohol which is an isomer of the olefinic alcohol product having a chemical structure corresponding to a pheromone or to a precursor of a pheromone. In some such embodiments, the pheromone isomer can be used in a pheromone composition.
In some embodiments, the olefinic alcohol product is a positional isomer of a pheromone which results from biohydroxylation of the terminal carbon on the n-end of the metathesis product, wherein the isomeric olefinic alcohol product has the formula H(CH2)m(CH═CH)(CH2)nOH, and m and n are different integers between 1 and 18. In some embodiments, m and n are different integers between 1 and 9. In other embodiments, the isomeric olefinic alcohol product can undergo a subsequent synthetic transformation, e.g., oxidation and/or acetylation, to produce a positional isomer of a pheromone.
In some embodiments, the olefinic alcohol product is a diol which results from biohydroxylation of the terminal carbon on the n-end and biohydroxylation of the terminal carbon on the m-end, the isomeric olefinic diol product has the formula HO(CH2)m(CH═CH)(CH2)nOH, and m and n are different integers between 1 and 18. In some embodiments, m and n are different integers between 1 and 9. In other embodiments, the olefinic diol product can undergo a subsequent synthetic transformation, e.g., oxidation and/or acetylation, to produce an analogue of a pheromone.
In some embodiments, the olefinic alcohol product is a positional isomer of a pheromone which results from biohydroxylation of a subterminal carbon on m-end of the metathesis produced, wherein the isomeric olefinic alcohol has the formula H(CH2)iCHOH(CH2)m-i-1(CH═CH)(CH2)nH. In some embodiments, the olefinic alcohol product is a positional isomer of a pheromone which results from biohydroxylation of a subterminal carbon on n-end of the metathesis produced, wherein the isomeric olefinic alcohol has the formula H(CH2)m(CH═CH)(CH2)n-i-1CHOH(CH2)H. In some embodiment, m, n and i are different integers between 1 and 17. In some embodiments, m, n, and i are different integers between 1 and 9.
The methods of the disclosure can also be conducted such that the biohydroxylation step is conducted prior to the metathesis step and/or other synthetic transformation steps. Accordingly, some embodiments of the disclosure provide methods wherein the olefinic substrate is a first terminal olefin, and wherein the method includes: a) incubating the first terminal olefin with an enzyme capable of selectively hydroxylating the terminal carbon of the terminal olefin to form an α,ω-alkenol; and b) metathesizing the α,ω-alkenol and a second terminal olefin in the presence of a metathesis catalyst to form the olefinic alcohol product.
The alcohol can be protected with a suitable protecting group if necessary. In some embodiments, the methods of the disclosure include: a) incubating the first terminal olefin with an enzyme capable of selectively hydroxylating the terminal carbon of the terminal olefin to form an α,ω-alkenol; b) protecting the α,ω-alkenol to form a protected α,ω-alkenol; c) metathesizing the protected α,ω-alkenol and a second terminal olefin in the presence of a metathesis catalyst to form a protected olefinic alcohol product; and d) deprotecting the protected olefinic alcohol product to form the olefinic alcohol product.
Any suitable alcohol protecting group can be used in the methods of the disclosure. Such protecting groups are well known to one of ordinary skill in the art, including those that are disclosed in Protective Groups in Organic Synthesis, 4th edition, T. W. Greene and P. G. M. Wuts, John Wiley & Sons, New York, 2006, which is incorporated herein by reference in its entirety. In some embodiments, the α,ω-alkenol is protected via esterification and the protected olefinic alcohol product is deprotected via hydrolysis. In some embodiments, the α,ω-alkenol is protected via esterification with an acid selected from the group consisting of formate and acetate.
Any suitable olefinic substrate can be used in methods where the biohydroxylation step is conducted prior to the metathesis step and/or other synthetic transformation steps. In some embodiments, the first terminal olefin has the formula (CH2═CH)(CH2)mH, the α,ω-alkenol has the formula (CH2═CH)(CH2)mOH, the second terminal olefin has the formula (CH2═CH)(CH2)nH, the olefinic alcohol product has the formula H(CH2)n(CH═CH)(CH2)mOH, and m and n are each independently selected from an integer between 1 and 17. In some embodiments, m and n are each independently selected from an integer between 1 and 9.
Hydroxylation of Asymmetric Alkenes Using Alkyne Starting Material
In some embodiments, the alkene is produced according to Scheme 4 (see, Oprean et al. (2006) for the acetylation step and Buck and Chong (2001) for the alkyne alkylation step), Scheme 5 (see, Buck and Chong (2001) regarding the alkyne alkylation step), Scheme 6a, or Scheme 6b. Scheme 6b shows Wittig reaction conditions that favor the formation of the Z-isomer according to Smith et al. (2000).
Accordingly, some embodiments of the disclosure provide a method for synthesizing an olefinic alcohol product wherein the method includes:
a) forming a reaction mixture comprising a terminal alkyne according to formula I
wherein n is an integer from 0 to 16,
and an alkyl halide according to formula II
wherein X is a halogen and m is an integer from 0 to 16,
under conditions sufficient to form a disubstituted alkyne according to formula III
b) reducing the disubstituted alkyne to form an olefin according to formula IVa or IVb
and
c) incubating the olefin with an enzyme capable of selectively hydroxylating one terminal carbon of the olefin to form the olefinic alcohol product.
The terminal alkyne, the alkyl halide, the disubstituted alkyne, the olefin, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.
In some embodiments, the disclosure includes:
a) forming a reaction mixture comprising a phosphonium salt according to formula XVI
wherein
and an aldehyde according to formula XVII
wherein m is an integer from 0 to 16,
under conditions sufficient to form an olefin according to formula XVIIIa or formula XVIIIb
and
b) incubating the olefin with an enzyme capable of selectively hydroxylating one terminal carbon of the olefin to form the olefinic alcohol product.
The phosphonium salt, the aldehyde, the olefin, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.
Metathesis Catalysts
In general, any metathesis catalyst stable under the reaction conditions and nonreactive with the functional groups present on the reactant shown in Schemes 3-6 may be used with the present disclosure. Such catalysts are, for example, those described by Grubbs (Grubbs, R. H., “Synthesis of large and small molecules using olefin metathesis catalysts.” PMSE Prepr., 2012), herein incorporated by reference in its entirety. Depending on the desired isomer of the olefin, as cis-selective metathesis catalyst may be used, for example one of those described by Shahane et al. (Shahane, S., et al. ChemCatChem, 2013. 5(12): p. 3436-3459), herein incorporated by reference in its entirety. Specific catalysts 1-5 exhibiting cis-selectivity are shown below (Scheme 7) and have been described previously (Khan, R. K., et al. J. Am. Chem. Soc., 2013. 135(28): p. 10258-61; Hartung, J. et al. J. Am. Chem. Soc., 2013. 135(28): p. 10183-5; Rosebrugh, L. E., et al. J. Am. Chem. Soc., 2013. 135(4): p. 1276-9; Marx, V. M., et al. J. Am. Chem. Soc., 2013. 135(1): p. 94-7; Herbert, M. B., et al. Angew. Chem. Int. Ed. Engl., 2013. 52(1): p. 310-4; Keitz, B. K., et al. J. Am. Chem. Soc., 2012. 134(4): p. 2040-3; Keitz, B. K., et al. J. Am. Chem. Soc., 2012. 134(1): p. 693-9; Endo, K. et al. J. Am. Chem. Soc., 2011. 133(22): p. 8525-7).
Additional Z-selective catalysts are described in (Cannon and Grubbs 2013; Bronner et al. 2014; Hartung et al. 2014; Pribisko et al. 2014; Quigley and Grubbs 2014) and are herein incorporated by reference in their entirety. Due to their excellent stability and functional group tolerance, in some embodiments metathesis catalysts include, but are not limited to, neutral ruthenium or osmium metal carbene complexes that possess metal centers that are formally in the +2 oxidation state, have an electron count of 16, are penta-coordinated, and are of the general formula LL′AA′M=CRbRc or LL′AA′M=(C═)nCRbRc (Pederson and Grubbs 2002); wherein
Other metathesis catalysts such as “well defined catalysts” can also be used. Such catalysts include, but are not limited to, Schrock's molybdenum metathesis catalyst, 2,6-diisopropylphenylimido neophylidenemolybdenum (VI) bis(hexafluoro-t-butoxide), described by Grubbs et al. (Tetrahedron 1998, 54: 4413-4450) and Basset's tungsten metathesis catalyst described by Couturier, J. L. et al. (Angew. Chem. Int. Ed. Engl. 1992, 31: 628).
Catalysts useful in the methods of the disclosure also include those described by Peryshkov, et al. J. Am. Chem. Soc. 2011, 133: 20754-20757; Wang, et al. Angewandte Chemie, 2013, 52: 1939-1943; Yu, et al. J. Am. Chem. Soc., 2012, 134: 2788-2799; Halford. Chem. Eng. News, 2011, 89 (45): 11; Yu, et al. Nature, 2011, 479: 88-93; Lee. Nature, 2011, 471: 452-453; Meek, et al. Nature, 2011: 471, 461-466; Flook, et al. J. Am. Chem. Soc. 2011, 133: 1784-1786; Zhao, et al. Org Lett., 2011, 13(4): 784-787; Ondi, et al. “High activity, stabilized formulations, efficient synthesis and industrial use of Mo- and W-based metathesis catalysts” XiMo Technology Updates, 2015: http://www.ximo-inc.com/files/ximo/uploads/download/Summary_3.11.15.pdf; Schrock, et al. Macromolecules, 2010: 43, 7515-7522; Peryshkov, et al. Organometallics 2013: 32, 5256-5259; Gerber, et al. Organometallics 2013: 32, 5573-5580; Marinescu, et al. Organometallics 2012: 31, 6336-6343; Wang, et al. Angew. Chem. Int. Ed. 2013: 52, 1939-1943; Wang, et al. Chem. Eur. J. 2013: 19, 2726-2740; and Townsend et al. J. Am. Chem. Soc. 2012: 134, 11334-11337.
Catalysts useful in the methods of the disclosure also include those described in International Pub. No. WO 2014/155185; International Pub. No. WO 2014/172534; U.S. Pat. Appl. Pub. No. 2014/0330018; International Pub. No. WO 2015/003815; and International Pub. No. WO 2015/003814.
Catalysts useful in the methods of the disclosure also include those described in U.S. Pat. No. 4,231,947; U.S. Pat. No. 4,245,131; U.S. Pat. No. 4,427,595; U.S. Pat. No. 4,681,956; U.S. Pat. No. 4,727,215; International Pub. No. WO 1991/009825; U.S. Pat. No. 5,087,710; U.S. Pat. No. 5,142,073; U.S. Pat. No. 5,146,033; International Pub. No. WO 1992/019631; U.S. Pat. No. 6,121,473; U.S. Pat. No. 6,346,652; U.S. Pat. No. 8,987,531; U.S. Pat. Appl. Pub. No. 2008/0119678; International Pub. No. WO 2008/066754; International Pub. No. WO 2009/094201; U.S. Pat. Appl. Pub. No. 2011/0015430; U.S. Pat. Appl. Pub. No. 2011/0065915; U.S. Pat. Appl. Pub. No. 2011/0077421; International Pub. No. WO 2011/040963; International Pub. No. WO 2011/097642; U.S. Pat. Appl. Pub. No. 2011/0237815; U.S. Pat. Appl. Pub. No. 2012/0302710; International Pub. No. WO 2012/167171; U.S. Pat. Appl. Pub. No. 2012/0323000; U.S. Pat. Appl. Pub. No. 2013/0116434; International Pub. No. WO 2013/070725; U.S. Pat. Appl. Pub. No. 2013/0274482; U.S. Pat. Appl. Pub. No. 2013/0281706; International Pub. No. WO 2014/139679; International Pub. No. WO 2014/169014; U.S. Pat. Appl. Pub. No. 2014/0330018; and U.S. Pat. Appl. Pub. No. 2014/0378637.
Catalysts useful in the methods of the disclosure also include those described in International Pub. No. WO 2007/075427; U.S. Pat. Appl. Pub. No. 2007/0282148; International Pub. No. WO 2009/126831; International Pub. No. WO 2011/069134; U.S. Pat. Appl. Pub. No. 2012/0123133; U.S. Pat. Appl. Pub. No. 2013/0261312; U.S. Pat. Appl. Pub. No. 2013/0296511; International Pub. No. WO 2014/134333; and U.S. Pat. Appl. Pub. No. 2015/0018557.
Catalysts useful in the methods of the disclosure also include those set forth in the following table:
Catalysts useful in the methods of the disclosure also include those described in U.S. Pat. Appl. Pub. No. 2008/0009598; U.S. Pat. Appl. Pub. No. 2008/0207911; U.S. Pat. Appl. Pub. No. 2008/0275247; U.S. Pat. Appl. Pub. No. 2011/0040099; U.S. Pat. Appl. Pub. No. 2011/0282068; and U.S. Pat. Appl. Pub. No. 2015/0038723.
Catalysts useful in the methods of the disclosure include those described in International Pub. No. WO 2007/140954; U.S. Pat. Appl. Pub. No. 2008/0221345; International Pub. No. WO 2010/037550; U.S. Pat. Appl. Pub. No. 2010/0087644; U.S. Pat. Appl. Pub. No. 2010/0113795; U.S. Pat. Appl. Pub. No. 2010/0174068; International Pub. No. WO 2011/091980; International Pub. No. WO 2012/168183; U.S. Pat. Appl. Pub. No. 2013/0079515; U.S. Pat. Appl. Pub. No. 2013/0144060; U.S. Pat. Appl. Pub. No. 2013/0211096; International Pub. No. WO 2013/135776; International Pub. No. WO 2014/001291; International Pub. No. WO 2014/067767; U.S. Pat. Appl. Pub. No. 2014/0171607; and U.S. Pat. Appl. Pub. No. 2015/0045558.
The catalyst is typically provided in the reaction mixture in a sub-stoichiometric amount (e.g., catalytic amount). In certain embodiments, that amount is in the range of about 0.001 to about 50 mol % with respect to the limiting reagent of the chemical reaction, depending upon which reagent is in stoichiometric excess. In some embodiments, the catalyst is present in less than or equal to about 40 mol % relative to the limiting reagent. In some embodiments, the catalyst is present in less than or equal to about 30 mol % relative to the limiting reagent. In some embodiments, the catalyst is present in less than about 20 mol %, less than about 10 mol %, less than about 5 mol %, less than about 2.5 mol %, less than about 1 mol %, less than about 0.5 mol %, less than about 0.1 mol %, less than about 0.015 mol %, less than about 0.01 mol %, less than about 0.0015 mol %, or less, relative to the limiting reagent. In some embodiments, the catalyst is present in the range of about 2.5 mol % to about 5 mol %, relative to the limiting reagent. In some embodiments, the reaction mixture contains about 0.5 mol % catalyst. In the case where the molecular formula of the catalyst complex includes more than one metal, the amount of the catalyst complex used in the reaction may be adjusted accordingly.
In some cases, the methods described herein can be performed in the absence of solvent (e.g., neat). In some cases, the methods can include the use of one or more solvents. Examples of solvents that may be suitable for use in the disclosure include, but are not limited to, benzene, p-cresol, toluene, xylene, diethyl ether, glycol, diethyl ether, petroleum ether, hexane, cyclohexane, pentane, methylene chloride, chloroform, carbon tetrachloride, dioxane, tetrahydrofuran (THF), dimethyl sulfoxide, dimethylformamide, hexamethyl-phosphoric triamide, ethyl acetate, pyridine, triethylamine, picoline, and the like, as well as mixtures thereof. In some embodiments, the solvent is selected from benzene, toluene, pentane, methylene chloride, and THF. In certain embodiments, the solvent is benzene.
In some embodiments, the method is performed under reduced pressure. This may be advantageous in cases where a volatile byproduct, such as ethylene, may be produced during the course of the metathesis reaction. For example, removal of the ethylene byproduct from the reaction vessel may advantageously shift the equilibrium of the metathesis reaction towards formation of the desired product. In some embodiments, the method is performed at a pressure of about less than 760 torr. In some embodiments, the method is performed at a pressure of about less than 700 torr. In some embodiments, the method is performed at a pressure of about less than 650 torr. In some embodiments, the method is performed at a pressure of about less than 600 torr. In some embodiments, the method is performed at a pressure of about less than 550 torr. In some embodiments, the method is performed at a pressure of about less than 500 torr. In some embodiments, the method is performed at a pressure of about less than 450 torr. In some embodiments, the method is performed at a pressure of about less than 400 torr. In some embodiments, the method is performed at a pressure of about less than 350 torr. In some embodiments, the method is performed at a pressure of about less than 300 torr. In some embodiments, the method is performed at a pressure of about less than 250 torr. In some embodiments, the method is performed at a pressure of about less than 200 torr. In some embodiments, the method is performed at a pressure of about less than 150 torr. In some embodiments, the method is performed at a pressure of about less than 100 torr. In some embodiments, the method is performed at a pressure of about less than 90 torr. In some embodiments, the method is performed at a pressure of about less than 80 torr. In some embodiments, the method is performed at a pressure of about less than 70 torr. In some embodiments, the method is performed at a pressure of about less than 60 torr. In some embodiments, the method is performed at a pressure of about less than 50 torr. In some embodiments, the method is performed at a pressure of about less than 40 torr. In some embodiments, the method is performed at a pressure of about less than 30 torr. In some embodiments, the method is performed at a pressure of about less than 20 torr. In some embodiments, the method is performed at a pressure of about 20 torr.
In some embodiments, the method is performed at a pressure of about 19 torr. In some embodiments, the method is performed at a pressure of about 18 torr. In some embodiments, the method is performed at a pressure of about 17 torr. In some embodiments, the method is performed at a pressure of about 16 torr. In some embodiments, the method is performed at a pressure of about 15 torr. In some embodiments, the method is performed at a pressure of about 14 torr. In some embodiments, the method is performed at a pressure of about 13 torr. In some embodiments, the method is performed at a pressure of about 12 torr. In some embodiments, the method is performed at a pressure of about 11 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 9 torr. In some embodiments, the method is performed at a pressure of about 8 torr. In some embodiments, the method is performed at a pressure of about 7 torr. In some embodiments, the method is performed at a pressure of about 6 torr. In some embodiments, the method is performed at a pressure of about 5 torr. In some embodiments, the method is performed at a pressure of about 4 torr. In some embodiments, the method is performed at a pressure of about 3 torr. In some embodiments, the method is performed at a pressure of about 2 torr. In some embodiments, the method is performed at a pressure of about 1 torr. In some embodiments, the method is performed at a pressure of less than about 1 torr.
In some embodiments, the two metathesis reactants are present in equimolar amounts. In some embodiments, the two metathesis reactants are not present in equimolar amounts. In certain embodiments, the two reactants are present in a molar ratio of about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In certain embodiments, the two reactants are present in a molar ratio of about 10:1. In certain embodiments, the two reactants are present in a molar ratio of about 7:1. In certain embodiments, the two reactants are present in a molar ratio of about 5:1. In certain embodiments, the two reactants are present in a molar ratio of about 2:1. In certain embodiments, the two reactants are present in a molar ratio of about 1:10. In certain embodiments, the two reactants are present in a molar ratio of about 1:7. In certain embodiments, the two reactants are present in a molar ratio of about 1:5. In certain embodiments, the two reactants are present in a molar ratio of about 1:2.
In general, the reactions with many of the metathesis catalysts disclosed herein provide yields better than 15%, better than 50%, better than 75%, or better than 90%. In addition, the reactants and products are chosen to provide at least a 5° C. difference, a greater than 20° C. difference, or a greater than 40° C. difference in boiling points. Additionally, the use of metathesis catalysts allows for much faster product formation than byproduct, it is desirable to run these reactions as quickly as practical. In particular, the reactions are performed in less than about 24 hours, less than 12 hours, less than 8 hours, or less than 4 hours.
One of skill in the art will appreciate that the time, temperature and solvent can depend on each other, and that changing one can require changing the others to prepare the pyrethroid products and intermediates in the methods of the disclosure. The metathesis steps can proceed at a variety of temperatures and times. In general, reactions in the methods of the disclosure are conducted using reaction times of several minutes to several days. For example, reaction times of from about 12 hours to about 7 days can be used. In some embodiments, reaction times of 1-5 days can be used. In some embodiments, reaction times of from about 10 minutes to about 10 hours can be used. In general, reactions in the methods of the disclosure are conducted at a temperature of from about 0° C. to about 200° C. For example, reactions can be conducted at 15-100° C. In some embodiments, reaction can be conducted at 20-80° C. In some embodiments, reactions can be conducted at 100-150° C.
Biohydroxylation to Produce a Pheromone and its Positional Isomer
As discussed above, an unsaturated hydrocarbon substrate can be subjected to biohydroxylation via an enzyme catalyst to thereby generate a mixture of a pheromone having a structure of an insect pheromone produced by a female insect and a positional isomer of the pheromone, which is not naturally produced by the female insect. The mixture of a pheromone and a positional isomer occurs through the enzyme catalyzing the hydrolysis of different carbons on the substrate, as shown in
Biohydroxylation Catalysts
Various enzymes and/or whole cells comprising enzymes can be used to catalyze hydroxylation reactions described above.
Known enzyme families with terminal hydroxylation activity for medium and long chain alkanes and fatty acids include AlkB, CYP52, CYP153, and LadA (Bordeaux et al., 2012, Angew. Chem.-Int. Edit. 51: 10712-10723; Ji et al., 2013, Front. Microbiol. 4). For example, Malca et al. describe terminal hydroxylation of mono-unsaturated fatty acid by cytochromes P450 of the CYP153 family (Malca et al., 2012, Chemical Communications 48: 5115-5117). Weissbart et al. describe the terminal hydroxylation of various cis and trans unsaturated lauric acid analogs (Weissbart et al., 1992, Biochimica et Biophysica Acta, Lipids and Lipid Metabolism 1124: 135-142). However, to date, none of these enzymes has been demonstrated to perform terminal hydroxylation of alkenes with internal olefins such as (E)-dec-5-ene. The presence of C═C bonds present competing sites of oxygen insertion and alters the 3-dimensional orientation of the molecule. The regioselectivity of these enzymes for the terminal C—H bond of alkanes and fatty acid substrate may not extend to alkenes with internal olefins for these reasons. For asymmetric substrates, obtaining hydroxylation at the desired terminal C—H bond presents additional challenges compared to symmetric substrates. Finally, controlling the reaction selectivity to produce a single terminal alcohol instead of α-ω diols, acids, or diacids is also a major concern.
In particular embodiments, the search for a terminal hydroxylase with activity for alkene with internal olefins starts with known terminal alkane and fatty acid hydroxylases. There are four families of enzymes with reported terminal alkane and fatty acid hydroxylation activity: (1) methane monooxygenases; (2) integral membrane diiron non-heme alkane hydroxylases (AlkB); (3) Cytochrome P450s (P450s); and (4) long chain alkane monooxygenases (LadA) (Bordeaux et al., 2012, Angew. Chem.-Int. Edit. 51: 10712-10723; Ji et al., 2013, Front. Microbiol. 4). Methane monooxygenases are difficult to express in heterologous non-methanotrophic hosts and generally prefer small substrate (<C4). Of the remaining three families, the substrate specificity based on substrate chain length of representative members is summarized below in Table 3.
P. putida GPo1
Biomol.
J. Bacteriol.
Chem. 9:
Org. Biomol.
Microb. Technol.
Biotechnol.
Chem. 9: 6727-6733)
Biochem. 68: 2171-2177)
Biochem
Biochem.
Acad. Sci. U.S.A.
Biochem Biophys.
Biophys. 328:
Biophys. 464:
In certain embodiments, depending on the chain length of the desired substrate, some members of these four enzyme families are better suited than others as candidates for evaluation. For C-10 substrates such as (E)-dec-5-ene, the substrate specificity of characterized CYP153 and AlkB enzymes makes them candidate enzymes. Likewise, for longer substrates such as (Z)-hexadec-11-ene, members of the LadA and CYP52 families appear to have the closest substrate profile.
The most widely characterized member of the AlkB family is obtained from the Alk system of Pseudomonas putida GPo1 (van Beilen and Funhoff, 2005, Curr. Opin. Biotechnol. 16: 308-314). In addition to the integral membrane diiron non-heme hydroxylase AlkB, a rubredoxin (AlkG) and a rubredoxin reductase (AlkT) are required for hydroxylation function. The entire Alk system of P. putida GPo1, alkBFGHJKL and alkST genes, which allows the strain to grown on alkanes as its sole carbon source, has been cloned into the broad host range vector pLAFR1 (pGEc47) and is available from DSMZ in the host E. Coli K12 Gec137 (Smits et al., 2001, Plasmid 46: 16-24). The other alk genes alkF, alkJ, alkH, alkK, alkL, and alkS encode an inactive rubredoxin, an alcohol dehydrogenase, an aldehyde dehydrogenase, an acyl-CoA synthase, an alkane transporter and a global pathway regulator, respectively (Smits et al., 2003, Antonie Van Leeuwenhoek 84: 193-200). These genes facilitate the use of the alcohol product from the AlkB reaction to generate the fatty acyl-CoA that is substrate for β-oxidation. To accumulate the alcohol product, a knockout strain of alkJ, E. coli GEC137 pGEc47ΔJ has been used in a whole-cell biotransformation to produce 1-dodecanol (Grant et al., 2011, Enzyme Microb. Technol. 48: 480-486). The presence of alkL appears to enhance substrate uptake and consequently improve the whole-cell activity for both Pseudomonas and E. coli (Cornelissen et al., 2013, Biotechnology and Bioengineering 110: 1282-1292; Julsing et al., 2012, Appl. Environ. Microbiol. 78: 5724-5733; Scheps et al., 2013, Microb. Biotechnol. 6: 694-707). A simplified version of pGEc47 containing only alkBFGST in the broad-host range vector pCOM10, pBT10, has also been used for the conversion of fatty-acid methyl esters to w-hydroxy fatty acid methyl esters in E. coli W3110 (Schrewe et al., 2011, Advanced Synthesis & Catalysis 353: 3485-3495).
CYP52 family members are membrane bound cytochrome P450s that require electron delivery from a reductase for function. CYP52 members have mainly been identified from alkane-degrading Candida species (Scheller et al., 1996, Arch. Biochem. Biophys. 328: 245-254; Craft et al., 2003, Appl. Environ. Microbiol. 69: 5983-5991; Scheller et al., 1998, J. Biol. Chem. 273: 32528-32534; Seghezzi et al., 1992, DNA Cell Biol. 11: 767-780; Zimmer et al., 1996, Biochem. Biophys. Res. Commun. 224: 784-789). Thus far, expression and characterization of CYP52 enzymes have been performed in the native Candida host and other yeast hosts. Gene knockouts of (1) the β-oxidation pathways, (2) alcohol dehydrogenases and (3) select native CYP52s has resulted in strains that can accumulate ω-hydroxy fatty acids when fatty acids are fed to the culture (Lu et al., 2010, J. Am. Chem. Soc. 132: 15451-15455). Of particular interest, DP428, DP522 and DP526 are C. tropicalis strains expressing a single CYP52 with the appropriate knockouts for catalyzing terminal hydroxylation of fatty acids (Lu et al., 2010, J. Am. Chem. Soc. 132: 15451-15455).
CYP153 family members are soluble and membrane associated cytochrome P450s that also depend on electron transfer from ferredoxin and ferredoxin reductase for function (Funhoff et al., 2007, Enzyme and Microbial Technology 40: 806-812). CYP153 members have been isolated from a range of alkane-degrading microorganisms. There are currently 56 annotated CYP153 sequences available from the Nelson P450 database, a BLAST search of CYP153A6 resulted in 221 identified homologs with >70% sequence identity. The use of CYP153 enzymes for terminal hydroxylation of octane and dodecanoic acid has been demonstrated with heterologous expression in E. coli. For the conversion of octane to octanol, the CYP153 operon from Mycobacterium sp. HXN-1500 was cloned into pET28b(+) and the biotransformation was performed in E. coli BL21(DE3) (Gudiminchi et al., 2012, Appl. Microbiol. Biotechnol. 96: 1507-1516). For the conversion of dodecanoic acid, an E. coli HMS174 strain containing a fusion of a CYP153AM.aq. mutant with the CYP102A1 reductase domain in pColaDuet-1 along with alkL was used for the transformation (Scheps et al., 2013, Microb. Biotechnol. 6: 694-707).
Long chain alkane monooxygenase, LadA, isolated from G. thermodenitrificants NG80-2 catalyzes the terminal hydroxylation of C15 to C36 alkanes with a metal-free flavoprotein mechanism that differs from AlkB and CYP enzymes (Dong et al., 2012, Appl. Microbiol. Biotechnol. 94: 1019-1029). The LadA reaction requires FMNH2 or NADPH and the native reductase partner has yet to be identified. Expression of the LadA gene in E. coli BL21 (DE3) using the pET-28a(+) plasmid yielded cell extracts with terminal hydroxylation activity for hexadecane (Dong et al., 2012, Appl. Microbiol. Biotechnol. 94: 1019-1029). Literature reports of LadA hydroxylation reactions have been performed using purified enzymes and examples of whole-cell biotransformation is lacking.
Coding sequences for enzymes that may be used herein may be derived from bacterial, fungal, or plant sources. Tables 3, 4, and 5 list enzymes for coding regions of representative non-heme diiron alkane monooxygenases, long-chain alkane hydroxylases, and cytochromes P450, respectively. Additional enzymes and their coding sequences may be identified by BLAST searching of public databases. Typically, BLAST searching of publicly available databases with known non-heme diiron alkane monooxygenases, cytochromes P450, and long-chain alkane hydroxylase sequences, such as those provided herein, is used to identify enzymes and their encoding sequences that may be used in the present disclosure. For example, enzymes having amino acid sequence identities of at least about 80-85%, 85%-90%, 90%-95%, or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the enzymes listed in Tables 3, 4, and 5 may be used. Hydroxylase enzymes can be codon-optimized for expression in certain desirable host organisms, such as yeast and E. coli.
In other embodiments, the sequences of the enzymes provided herein may be used to identify other homologs in nature. For example, each of the encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, (1) methods of nucleic acid hybridization, (2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker et al., Proc. Natl. Acad. Sci. USA, 89:392 (1992)), and (3) methods of library construction and screening by complementation.
Hydroxylase enzymes or whole cells expressing hydroxylase enzymes can be further engineered for use in the methods of the disclosure. Enzymes can be engineered for improved hydroxylation activity, improved Z:E selectivity, improved regioselectivity, improved selectivity for hydroxylation over epoxidation and/or improved selectivity for hydroxylation over dehalogenation. The term “improved hydroxylation activity” as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured in a comparable non-engineered hydroxylase enzyme of whole cells comprising a hydroxylase enzyme. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Mutations can be introduced into a hydroxylase enzyme resulting in engineered enzymes with improved hydroxylation activity. Methods to increase enzymatic activity are known to those skilled in the art. Such techniques can include increasing the expression of the enzyme by increasing plasmid copy number and/or use of a stronger promoter and/or use of activating riboswitches, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the KM for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press (2003).
Accordingly, some embodiments of the disclosure provide methods for synthesizing olefinic alcohol products as described above, wherein the enzyme is a non-heme diiron monooxygenase. In some embodiments, the non-heme diiron monooxygenase is selected from Table 4 or a variant thereof having at least 90% identity thereto.
Pseudomonas oleovorans
Pseudomonas mendocina (strain ymp)
Pseudomonas aeruginosa
Bacillus sp. BTRH40
Pseudomonas aeruginosa
Pseudomonas stutzeri (Pseudomonas
perfectomarina)
Pseudomonas aeruginosa
Pseudomonas chlororaphis subsp.
Arthrobacter sp. ITRH48
Streptomyces sp. ITRH51
Arthrobacter sp. ITRH49
Dietzia sp. ITRH56
Microbacterium sp. ITRH47
Pantoea sp. BTRH11
Pseudomonas sp. ITRI53
Pseudomonas sp. ITRI73
Pseudomonas sp. ITRH25
Pseudomonas sp. MIXRI75
Pseudomonas sp. MIXRI74
Rhodococcus sp. ITRH43
Ochrobactrum sp. ITRH1
Alcaligenaceae bacterium BTRH5
Pseudomonas sp. ITRH76
Pseudomonas sp. 7/156
Pseudomonas sp. ITRI22
Pseudomonas putida (Arthrobacter
siderocapsulatus)
Pseudomonas sp. G5(2012)
Alcanivorax dieselolei
Alcanivorax borkumensis
Alcanivorax sp. S17-16
Alcanivorax borkumensis
Xanthobacter flavus
Acidisphaera sp. C197
Kordiimonas gwangyangensis
Ralstonia sp. PT11
Marinobacter sp. P1-14D
Bradyrhizobium sp. DFCI-1
Thalassolituus oleivorans
Marinobacter sp. EVN1
Marinobacter hydrocarbonoclasticus
Alcanivorax borkumensis
Alcanivorax borkumensis (strain SK2/
Marinobacter aquaeolei (strain ATCC
Alcanivorax sp. 97CO-5
Marinobacter sp. C1S70
Marinobacter sp. EVN1
Pseudoxanthomonas spadix (strain BD-
Marinobacter sp. EN3
Marinobacter sp. ES-1
Oceanicaulis sp. HTCC2633
Citreicella sp. 357
Caulobacter sp. (strain K31)
Thalassolituus oleivorans MIL-1
Alcanivorax pacificus W11-5
Alcanivorax dieselolei
Alcanivorax sp. PN-3
Alcanivorax dieselolei (strain DSM 16502/
Alcanivorax dieselolei
Marinobacter sp. ELB17
Marinobacter sp. BSs20148
Pseudomonas alcaligenes NBRC 14159
Simiduia agarivorans (strain DSM 21679/
Limnobacter sp. MED105
Alcanivorax sp. R8-12
Alcanivorax hongdengensis A-11-3
Acidovorax sp. KKS102
Moritella sp. PE36
Moritella sp. PE36
Ahrensia sp. R2A130
Hoeflea phototrophica DFL-43
Curvibacter putative symbiont of Hydra
magnipapillata
Pseudovibrio sp. JE062
Ralstonia sp. AU12-08
Burkholderia phytofirmans (strain DSM
Pseudovibrio sp. (strain FO-BEG1)
Bradyrhizobium sp. DFCI-1
Alcanivorax dieselolei (strain DSM 16502/
Alcanivorax sp. PN-3
Alcanivorax dieselolei
Burkholderia thailandensis E444
Burkholderia thailandensis 2002721723
Burkholderia thailandensis H0587
Burkholderia thailandensis (strain E264/
Burkholderia pseudomallei 1026b
Burkholderia pseudomallei 1026a
Burkholderia pseudomallei MSHR305
Burkholderia pseudomallei 305
Burkholderia pseudomallei Pasteur 52237
Burkholderia pseudomallei (strain
Burkholderia pseudomallei (strain 1710b)
Burkholderia pseudomallei BPC006
Burkholderia pseudomallei 1710a
Burkholderia pseudomallei 1106b
Burkholderia pseudomallei (strain 1106a)
Burkholderia pseudomallei (strain 668)
Burkholderia pseudomallei NCTC 13178
Burkholderia pseudomallei MSHR1043
Burkholderia pseudomallei 354a
Burkholderia pseudomallei 354e
Burkholderia pseudomallei 1258b
Burkholderia pseudomallei 1258a
Burkholderia pseudomallei 576
Burkholderia pseudomallei 1655
Burkholderia pseudomallei S13
Burkholderia pseudomallei 406e
Burkholderia pseudomallei MSHR146
Burkholderia pseudomallei MSHR511
Burkholderia pseudomallei NAU20B-16
Burkholderia pseudomallei MSHR346
Burkholderia pseudomallei MSHR338
Burkholderia thailandensis MSMB43
Burkholderia sp. Ch1-1
Alcanivorax sp. R8-12
Alcanivorax pacificus W11-5
Actinoplanes sp. (strain ATCC 31044/
Alcanivorax sp. DG881
Methylibium sp. T29-B
Methylibium sp. T29
Burkholderia thailandensis MSMB121
Burkholderia sp. TJI49
Burkholderia mallei (strain ATCC 23344)
Burkholderia mallei (strain NCTC 10247)
Burkholderia mallei (strain NCTC 10229)
Burkholderia mallei (strain SAVP1)
Burkholderia mallei PRL-20
Burkholderia mallei GB8 horse 4
Burkholderia mallei ATCC 10399
Burkholderia mallei JHU
Burkholderia mallei FMH
Burkholderia mallei 2002721280
Burkholderia pseudomallei Pakistan 9
Burkholderia sp. (strain 383) (Burkholderia
cepacia (strain ATCC 17760/NCIB 9086/
Ralstonia sp. 5_2_56FAA
Ralstonia sp. 5_7_47FAA
Burkholderia cenocepacia (strain AU
Burkholderia cenocepacia (strain HI2424)
Burkholderia sp. KJ006
Burkholderia vietnamiensis (strain G4/
Burkholderia cenocepacia KC-01
Ralstonia pickettii (strain 12D)
Ralstonia pickettii (strain 12J)
Ralstonia pickettii OR214
Mycobacterium thermoresistibile ATCC
Burkholderia cenocepacia PC184
Parvularcula bermudensis (strain ATCC
Rhodococcus triatomae BKS 15-14
Alcanivorax hongdengensis A-11-3
Alcanivorax hongdengensis
Micromonospora sp. ATCC 39149
Micromonospora lupini str. Lupac 08
Patulibacter medicamentivorans
Burkholderia cenocepacia (strain ATCC
Burkholderia cenocepacia BC7
Burkholderia cenocepacia K56-2Valvano
Burkholderia cenocepacia H111
Burkholderia cepacia GG4
Burkholderia ambifaria IOP40-10
Burkholderia vietnamiensis AU4i
Burkholderia ambifaria MEX-5
Burkholderia cenocepacia (strain MC0-3)
Burkholderia cepacia (Pseudomonas
cepacia)
Burkholderia multivorans CGD1
Burkholderia multivorans (strain ATCC
Burkholderia multivorans (strain ATCC
Burkholderia multivorans CGD2M
Burkholderia multivorans CGD2
Burkholderia glumae (strain BGR1)
Burkholderia multivorans CF2
Burkholderia multivorans ATCC BAA-247
Mycobacterium xenopi RIVM700367
Alcanivorax sp. P2S70
Rhodococcus sp. p52
Rhodococcus pyridinivorans AK37
Micromonospora sp. M42
Actinoplanes missouriensis (strain ATCC
Mycobacterium thermoresistibile ATCC
Streptomyces collinus Tu 365
Mycobacterium smegmatis MKD8
Mycobacterium smegmatis (strain ATCC
Burkholderia gladioli (strain BSR3)
Nocardia cyriacigeorgica (strain GUH-2)
Mycobacterium sp. (strain Spyr1)
Mycobacterium gilvum (strain PYR-GCK)
Mycobacterium hassiacum DSM 44199
Mycobacterium phlei RIVM601174
Burkholderia ambifaria (strain MC40-6)
Conexibacter woesei (strain DSM 14684/
Burkholderia ambifaria (strain ATCC
Mycobacterium vaccae ATCC 25954
Streptomyces sp. AA4
Nocardia asteroides NBRC 15531
Hydrocarboniphaga effusa AP103
Mycobacterium sp. (strain Spyr1)
Rhodococcus sp. EsD8
Rhodococcus pyridinivorans SB3094
Dietzia sp. D5
Gordonia amarae NBRC 15530
gamma proteobacterium BDW918
Marinobacter sp. EVN1
Marinobacter santoriniensis NKSG1
Marinobacter sp. ES-1
gamma proteobacterium HdN1
Nocardia farcinica (strain IFM 10152)
Mycobacterium chubuense (strain NBB4)
Acinetobacter towneri DSM 14962 = CIP
Rhodococcus erythropolis CCM2595
Rhodococcus erythropolis (strain PR4/
Rhodococcus sp. P27
Rhodococcus erythropolis DN1
Rhodococcus erythropolis (Arthrobacter
picolinophilus)
Mycobacterium fortuitum subsp. fortuitum
Rhodococcus qingshengii BKS 20-40
Rhodococcus erythropolis (Arthrobacter
picolinophilus)
Rhodococcus sp. (strain RHA1)
Rhodococcus sp. JVH1
Rhodococcus wratislaviensis IFP 2016
Rhodococcus wratislaviensis
Rhodococcus sp. (strain Q15)
Rhodococcus opacus M213
Rhodococcus erythropolis (Arthrobacter
picolinophilus)
Streptomyces sp. AA4
Geobacillus sp. MH-1
Mycobacterium neoaurum VKM Ac-
Rhodococcus imtechensis RKJ300 = JCM
Prauserella rugosa
Rhodococcus erythropolis SK121
Amycolatopsis azurea DSM 43854
Mycobacterium rhodesiae (strain NBB3)
Rhodococcus ruber
Rhodococcus ruber BKS 20-38
Mycobacterium chubuense (strain NBB4)
Mycobacterium chubuense (strain NBB4)
Mycobacterium smegmatis JS623
Nocardia nova SH22a
Rhodococcus sp. BCP1
Saccharomonospora marina XMU15
Mycobacterium sp. (strain JLS)
Rhodococcus ruber
Mycobacterium tuberculosis BT2
Mycobacterium tuberculosis HKBS1
Mycobacterium tuberculosis EAI5
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium bovis (strain ATCC BAA-
Mycobacterium tuberculosis (strain ATCC
Mycobacterium tuberculosis str.
Mycobacterium bovis BCG str. Korea
Mycobacterium liflandii (strain 128FXT)
Mycobacterium tuberculosis (strain CDC
Mycobacterium canettii CIPT 140070017
Mycobacterium canettii CIPT 140070008
Mycobacterium canettii CIPT 140060008
Mycobacterium tuberculosis 7199-99
Mycobacterium tuberculosis KZN 605
Mycobacterium tuberculosis KZN 4207
Mycobacterium tuberculosis RGTB327
Mycobacterium tuberculosis (strain ATCC
Mycobacterium tuberculosis UT205
Mycobacterium bovis BCG str. Mexico
Mycobacterium tuberculosis CTRI-2
Mycobacterium canettii (strain CIPT
Mycobacterium canettii (strain CIPT
Mycobacterium africanum (strain
Mycobacterium tuberculosis (strain
Mycobacterium tuberculosis (strain
Mycobacterium tuberculosis (strain KZN
Mycobacterium bovis (strain BCG/Tokyo
Mycobacterium marinum (strain ATCC
Mycobacterium tuberculosis (strain F11)
Mycobacterium tuberculosis (strain ATCC
Mycobacterium tuberculosis str. Haarlem
Mycobacterium bovis (strain BCG/
Mycobacterium bovis 04-303
Mycobacterium bovis AN5
Mycobacterium tuberculosis GuangZ0019
Mycobacterium tuberculosis FJ05194
Mycobacterium tuberculosis ‘98-R604
Mycobacterium marinum str. Europe
Mycobacterium marinum MB2
Mycobacterium orygis 112400015
Mycobacterium tuberculosis NCGM2209
Mycobacterium bovis BCG str. Moreau
Mycobacterium tuberculosis W-148
Mycobacterium tuberculosis CDC1551A
Mycobacterium tuberculosis SUMu012
Mycobacterium tuberculosis SUMu011
Mycobacterium tuberculosis SUMu010
Mycobacterium tuberculosis SUMu009
Mycobacterium tuberculosis SUMu006
Mycobacterium tuberculosis SUMu005
Mycobacterium tuberculosis SUMu004
Mycobacterium tuberculosis SUMu003
Mycobacterium tuberculosis SUMu002
Mycobacterium tuberculosis SUMu001
Mycobacterium africanum K85
Mycobacterium tuberculosis T46
Mycobacterium tuberculosis T17
Mycobacterium tuberculosis GM 1503
Mycobacterium tuberculosis 02_1987
Mycobacterium tuberculosis EAS054
Mycobacterium tuberculosis T85
Mycobacterium tuberculosis T92
Mycobacterium tuberculosis C
Rhodococcus sp. EsD8
Amycolatopsis orientalis HCCB10007
Mycobacterium tuberculosis SUMu008
Mycobacterium tuberculosis SUMu007
Mycobacterium tuberculosis 94_M4241A
Gordonia amarae NBRC 15530
Rhodococcus rhodochrous ATCC 21198
Amycolatopsis decaplanina DSM 44594
Mycobacterium sp. 012931
Rhodococcus erythropolis (strain PR4/
Rhodococcus sp. (strain Q15)
Rhodococcus erythropolis CCM2595
Rhodococcus sp. P27
Rhodococcus erythropolis (Arthrobacter
picolinophilus)
Rhodococcus qingshengii BKS 20-40
Rhodococcus erythropolis SK121
Rhodococcus erythropolis DN1
Nocardia farcinica (strain IFM 10152)
Rhodococcus equi NBRC 101255 = C 7
Shewanella sp. NJ49
Mycobacterium canettii CIPT 140070010
Nocardia nova SH22a
Rhodococcus equi (strain 103S)
Gordonia terrae C-6
Nocardioides sp. (strain BAA-499/JS614)
Gordonia sp. TF6
Hydrocarboniphaga effusa AP103
Gordonia terrae NBRC 100016
Nocardia brasiliensis ATCC 700358
Amycolatopsis mediterranei RB
Amycolatopsis mediterranei (strain S699)
Amycolatopsis mediterranei (strain U-32)
Rhodococcus sp. p52
Rhodococcus pyridinivorans AK37
Rhodococcus pyridinivorans SB3094
Janibacter sp. HTCC2649
Gordonia sp. KTR9
Aeromicrobium marinum DSM 15272
Dietzia cinnamea P4
Micromonospora aurantiaca (strain ATCC
Dietzia sp. E1
Rhodococcus ruber BKS 20-38
Mycobacterium gilvum (strain PYR-GCK)
Nocardioidaceae bacterium Broad-1
Rhodococcus rhodochrous ATCC 21198
Salinisphaera shabanensis E1L3A
Rhodococcus erythropolis (strain PR4/
Corynebacterium falsenii DSM 44353
Rhodococcus erythropolis CCM2595
gamma proteobacterium BDW918
Rhodococcus sp. P27
Rhodococcus erythropolis DN1
Rhodococcus erythropolis SK121
Rhodococcus qingshengii BKS 20-40
In some embodiments, the disclosure provides methods for synthesizing olefinic alcohol products as described above, wherein the enzyme is a long-chain alkane hydroxylase. In some embodiments, the long-chain alkane hydroxylase is selected from Table 5 or a variant thereof having at least 90% identity thereto.
Geobacillus thermodenitrificans (strain NG80-2)
Geobacillus stearothermophilus (Bacillus
stearothermophilus)
Paenibacillus sp. JCM 10914
Bacillus methanolicus MGA3
Geobacillus sp. (strain Y4.1MC1)
Geobacillus thermoglucosidans TNO-09.020
Geobacillus thermoglucosidasius (strain C56-YS93)
Bacillus methanolicus PB1
Alicyclobacillus acidoterrestris ATCC 49025
Bhargavaea cecembensis DSE10
Bacillus sp. 1NLA3E
Burkholderia graminis C4D1M
Burkholderia thailandensis H0587
Planomicrobium glaciei CHR43
Burkholderia thailandensis E444
Burkholderia thailandensis 2002721723
Burkholderia pseudomallei (strain K96243)
Burkholderia mallei (strain ATCC 23344)
Burkholderia thailandensis (strain E264/ATCC 700388/
Burkholderia pseudomallei BPC006
Burkholderia pseudomallei 1106b
Burkholderia pseudomallei MSHR346
Burkholderia pseudomallei (strain 1106a)
Burkholderia mallei (strain NCTC 10247)
Burkholderia mallei (strain NCTC 10229)
Burkholderia pseudomallei MSHR338
Burkholderia mallei PRL-20
Burkholderia mallei GB8 horse 4
Burkholderia pseudomallei Pakistan 9
Burkholderia pseudomallei 576
Burkholderia pseudomallei S13
Burkholderia mallei ATCC 10399
Burkholderia pseudomallei Pasteur 52237
Burkholderia pseudomallei 406e
Burkholderia mallei JHU
Burkholderia mallei 2002721280
Alicyclobacillus acidoterrestris ATCC 49025
Burkholderia pseudomallei MSHR305
Burkholderia pseudomallei MSHR146
Burkholderia pseudomallei MSHR511
Burkholderia pseudomallei NAU20B-16
Burkholderia pseudomallei NCTC 13178
Burkholderia pseudomallei NCTC 13179
Burkholderia pseudomallei MSHR1043
Burkholderia pseudomallei 1655
Burkholderia pseudomallei 305
Segniliparus rugosus ATCC BAA-974
Burkholderia pseudomallei 1026b
Burkholderia pseudomallei 354a
Burkholderia pseudomallei 354e
Burkholderia pseudomallei 1026a
Burkholderia pseudomallei 1258b
Burkholderia pseudomallei 1258a
Pseudomonas putida (strain DOT-T1E)
Pseudomonas putida ND6
Pseudomonas putida TRO1
Pseudomonas putida LS46
Burkholderia graminis C4D1M
Burkholderia phytofirmans (strain DSM 17436/PsJN)
Bhargavaea cecembensis DSE10
Burkholderia thailandensis MSMB121
Burkholderia pseudomallei (strain 668)
Burkholderia pseudomallei (strain 1710b)
Burkholderia pseudomallei 1710a
Planomicrobium glaciei CHR43
Burkholderia thailandensis MSMB43
Pseudomonas sp. GM50
Pseudomonas fluorescens BBc6R8
Pseudomonas sp. Ag1
Pseudomonas sp. GM102
Pseudomonas fluorescens (strain SBW25)
Pseudomonas sp. (strain M1)
Pseudomonas sp. TKP
Pseudomonas putida (strain F1/ATCC 700007)
Pseudomonas putida (strain GB-1)
Azotobacter vinelandii CA6
Azotobacter vinelandii CA
Azotobacter vinelandii (strain DJ/ATCC BAA-1303)
Pseudomonas brassicacearum (strain NFM421)
Pseudomonas fluorescens Q8r1-96
Klebsiella oxytoca E718
Pseudomonas putida (strain KT2440)
Pseudomonas fluorescens BBc6R8
Pseudomonas fluorescens Q2-87
Pseudomonas sp. Ag1
Klebsiella oxytoca MGH 42
Klebsiella oxytoca 10-5245
Klebsiella oxytoca 10-5243
Klebsiella oxytoca (strain ATCC 8724/DSM 4798/
Streptomyces himastatinicus ATCC 53653
Klebsiella oxytoca MGH 28
Klebsiella oxytoca 10-5250
Klebsiella sp. OBRC7
Klebsiella oxytoca 10-5242
Pantoea ananatis LMG 5342
Pantoea ananatis PA13
Pantoea ananatis (strain AJ13355)
Pantoea ananatis (strain LMG 20103)
Pantoea ananatis BRT175
Segniliparus rotundus (strain ATCC BAA-972/CDC
Pantoea stewartii subsp. stewartii DC283
Pantoea stewartii subsp. stewartii DC283
Rhodococcus opacus M213
Klebsiella pneumoniae DMC0799
Klebsiella pneumoniae 700603
Klebsiella sp. MS 92-3
Klebsiella pneumoniae CG43
Klebsiella pneumoniae subsp. pneumoniae 1084
Klebsiella pneumoniae subsp. pneumoniae (strain
Klebsiella pneumoniae KCTC 2242
Klebsiella pneumoniae NB60
Klebsiella pneumoniae EGD-HP19-C
Escherichia coli ISC56
Klebsiella pneumoniae IS33
Klebsiella pneumoniae subsp. pneumoniae BJ1-GA
Klebsiella pneumoniae subsp. pneumoniae SA1
Klebsiella pneumoniae subsp. pneumoniae T69
Klebsiella pneumoniae MGH 18
Klebsiella pneumoniae MGH 17
Klebsiella pneumoniae MGH 21
Klebsiella pneumoniae MGH 19
Klebsiella pneumoniae MGH 32
Klebsiella pneumoniae MGH 30
Klebsiella pneumoniae MGH 40
Klebsiella pneumoniae MGH 36
Klebsiella pneumoniae BWH 28
Klebsiella pneumoniae BWH 30
Klebsiella pneumoniae UCICRE 2
Klebsiella pneumoniae UCICRE 7
Klebsiella pneumoniae UCICRE 6
Klebsiella pneumoniae BIDMC 21
Klebsiella pneumoniae BIDMC 22
Klebsiella pneumoniae BIDMC 24
Klebsiella pneumoniae BIDMC 25
Klebsiella pneumoniae BIDMC 40
Klebsiella pneumoniae BIDMC 36
Klebsiella pneumoniae BIDMC 41
Klebsiella pneumoniae BIDMC 12C
Klebsiella pneumoniae BIDMC 18C
Klebsiella pneumoniae BIDMC 16
Enterococcus gallinarum EGD-AAK12
Klebsiella pneumoniae subsp. pneumoniae MP14
Klebsiella pneumoniae subsp. pneumoniae
Klebsiella pneumoniae 120_1020
Klebsiella pneumoniae 140_1040
Klebsiella pneumoniae 280_1220
Klebsiella pneumoniae 160_1080
Klebsiella pneumoniae UHKPC06
Klebsiella pneumoniae UHKPC67
Klebsiella pneumoniae UHKPC02
Klebsiella pneumoniae UHKPC17
Klebsiella pneumoniae UHKPC31
Klebsiella pneumoniae UHKPC59
Klebsiella pneumoniae UHKPC18
Klebsiella pneumoniae UHKPC61
Klebsiella pneumoniae UHKPC07
Klebsiella pneumoniae DMC1316
Klebsiella pneumoniae UHKPC33
Klebsiella pneumoniae DMC1097
Klebsiella pneumoniae UHKPC96
Klebsiella pneumoniae UHKPC77
Klebsiella pneumoniae UHKPC28
Klebsiella pneumoniae UHKPC69
Klebsiella pneumoniae UHKPC47
Klebsiella pneumoniae UHKPC32
Klebsiella pneumoniae UHKPC48
Klebsiella pneumoniae DMC0526
Klebsiella pneumoniae VAKPC278
Klebsiella pneumoniae UHKPC29
Klebsiella pneumoniae UHKPC05
Klebsiella pneumoniae UHKPC45
Klebsiella pneumoniae UHKPC 52
Klebsiella pneumoniae 646_1568
Klebsiella pneumoniae 540_1460
Klebsiella pneumoniae 440_1540
Klebsiella pneumoniae 500_1420
Klebsiella pneumoniae VAKPC309
Klebsiella pneumoniae KP-11
Klebsiella pneumoniae 361_1301
Klebsiella pneumoniae VAKPC297
Klebsiella pneumoniae VAKPC270
Klebsiella pneumoniae VAKPC280
Klebsiella pneumoniae VAKPC276
Klebsiella pneumoniae VAKPC269
Klebsiella pneumoniae VAKPC254
Klebsiella pneumoniae UHKPC22
Klebsiella pneumoniae UHKPC04
Klebsiella pneumoniae VAKPC252
Klebsiella pneumoniae UHKPC26
Klebsiella pneumoniae UHKPC27
Klebsiella pneumoniae UHKPC24
Klebsiella pneumoniae UHKPC01
Klebsiella pneumoniae UHKPC81
Klebsiella pneumoniae UHKPC40
Klebsiella pneumoniae UHKPC09
Klebsiella pneumoniae KP-7
Klebsiella pneumoniae UHKPC23
Klebsiella pneumoniae subsp. pneumoniae KpMDU1
Klebsiella pneumoniae ATCC BAA-1705
Klebsiella pneumoniae ATCC BAA-2146
Klebsiella pneumoniae VA360
Klebsiella pneumoniae RYC492
Klebsiella pneumoniae RYC492
Klebsiella pneumoniae subsp. pneumoniae KpQ3
Klebsiella pneumoniae subsp. pneumoniae Ecl8
Klebsiella pneumoniae subsp. pneumoniae WGLW5
Klebsiella pneumoniae subsp. pneumoniae WGLW3
Klebsiella pneumoniae subsp. pneumoniae WGLW1
Klebsiella pneumoniae subsp. pneumoniae KPNIH23
Klebsiella pneumoniae subsp. pneumoniae KPNIH21
Klebsiella pneumoniae subsp. pneumoniae KPNIH18
Klebsiella pneumoniae subsp. pneumoniae KPNIH17
Klebsiella pneumoniae subsp. pneumoniae KPNIH9
Klebsiella pneumoniae subsp. pneumoniae KPNIH6
Klebsiella pneumoniae subsp. pneumoniae KPNIH1
Klebsiella pneumoniae subsp. pneumoniae KPNIH22
Klebsiella pneumoniae subsp. pneumoniae KPNIH19
Klebsiella pneumoniae subsp. pneumoniae KPNIH16
Klebsiella pneumoniae subsp. pneumoniae KPNIH14
Klebsiella pneumoniae subsp. pneumoniae KPNIH11
Klebsiella pneumoniae subsp. pneumoniae KPNIH2
Klebsiella pneumoniae subsp. pneumoniae KPNIH20
Klebsiella pneumoniae subsp. pneumoniae KPNIH12
Klebsiella pneumoniae subsp. pneumoniae KPNIH10
Klebsiella pneumoniae subsp. pneumoniae KPNIH8
Klebsiella pneumoniae subsp. pneumoniae KPNIH7
Klebsiella pneumoniae subsp. pneumoniae KPNIH5
Klebsiella pneumoniae subsp. pneumoniae KPNIH4
Klebsiella sp. 4_1_44FAA
Klebsiella pneumoniae JM45
Klebsiella pneumoniae subsp. pneumoniae Kp13
Klebsiella pneumoniae subsp. rhinoscleromatis ATCC
Klebsiella pneumoniae subsp. pneumoniae ST258-K26BO
Klebsiella variicola (strain At-22)
Klebsiella pneumoniae (strain 342)
Klebsiella pneumoniae MGH 20
Klebsiella pneumoniae UCICRE 10
Klebsiella sp. KTE92
Klebsiella pneumoniae hvKP1
Mycobacterium hassiacum DSM 44199
Klebsiella pneumoniae MGH 48
Pantoea vagans (strain C9-1) (Pantoea agglomerans
Klebsiella pneumoniae IS22
Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044
Burkholderia sp. CCGE1001
Microvirga lotononidis
Burkholderia phenoliruptrix BR3459a
Pseudomonas cichorii JBC1
Burkholderia sp. (strain CCGE1003)
Pseudomonas protegens CHA0
Herbaspirillum sp. CF444
Pseudomonas fluorescens (strain Pf-5/ATCC BAA-477)
Bacillus megaterium WSH-002
Pseudomonas sp. GM30
Pseudomonas sp. GM78
Pseudomonas sp. GM60
Pseudomonas sp. FH1
Pseudomonas sp. GM41(2012)
Pseudomonas sp. GM67
Pseudomonas fluorescens EGD-AQ6
Pseudomonas sp. CF161
Pseudomonas fluorescens BRIP34879
Pseudomonas sp. Lz4W
Collimonas fungivorans (strain Ter331)
Pseudomonas poae RE*1-1-14
Pseudomonas fluorescens BBc6R8
Pseudomonas sp. Lz4W
Pseudomonas sp. GM24
Pseudomonas sp. GM16
Rhizobium sp. CF080
Pseudomonas sp. FH1
Pseudomonas sp. GM25
Rhizobium leguminosarum bv. trifolii (strain WSM2304)
Pseudomonas sp. G5(2012)
Pseudomonas chlororaphis O6
Pseudomonas protegens CHA0
Pseudomonas fluorescens (strain Pf-5/ATCC BAA-477)
Rhizobium leguminosarum bv. trifolii WSM597
Bacillus megaterium (strain DSM 319)
Pseudomonas fluorescens WH6
Rhizobium sp. Pop5
Bacillus megaterium (strain ATCC 12872/QMB1551)
Pseudomonas cichorii JBC1
Pseudomonas sp. TKP
Pseudomonas aeruginosa C41
Pseudomonas aeruginosa 62
Pseudomonas aeruginosa BL19
Pseudomonas aeruginosa YL84
Pseudomonas aeruginosa SCV20265
Pseudomonas aeruginosa LES431
Pseudomonas aeruginosa MTB-1
Pseudomonas aeruginosa PA1R
Pseudomonas aeruginosa PA1
Pseudomonas aeruginosa PAO1-VE13
Pseudomonas aeruginosa PAO1-VE2
Pseudomonas aeruginosa c7447m
Pseudomonas aeruginosa RP73
Pseudomonas aeruginosa (strain ATCC 15692/PAO1/
Pseudomonas aeruginosa (strain UCBPP-PA14)
Pseudomonas aeruginosa B136-33
Pseudomonas aeruginosa DK2
Pseudomonas aeruginosa (strain LESB58)
Pseudomonas aeruginosa (strain PA7)
Pseudomonas aeruginosa (strain PA7)
Pseudomonas aeruginosa DHS29
Pseudomonas aeruginosa MH38
Pseudomonas aeruginosa VRFPA06
Pseudomonas aeruginosa VRFPA08
Pseudomonas aeruginosa DHS01
Pseudomonas aeruginosa VRFPA01
Pseudomonas aeruginosa HB15
Pseudomonas aeruginosa M8A.3
Pseudomonas aeruginosa CF27
Pseudomonas aeruginosa MSH10
Pseudomonas aeruginosa CF127
Pseudomonas aeruginosa CF5
Pseudomonas aeruginosa S54485
Pseudomonas aeruginosa BWHPSA007
Pseudomonas aeruginosa BWHPSA009
Pseudomonas aeruginosa BWHPSA008
Pseudomonas aeruginosa BWHPSA010
Pseudomonas aeruginosa BWHPSA015
Pseudomonas aeruginosa BWHPSA016
Pseudomonas aeruginosa BL03
Pseudomonas aeruginosa BL01
Pseudomonas aeruginosa BL02
Pseudomonas aeruginosa BL05
Pseudomonas aeruginosa BL06
Pseudomonas aeruginosa BL21
Pseudomonas aeruginosa BL23
Pseudomonas aeruginosa BL24
Pseudomonas aeruginosa M8A.4
Pseudomonas aeruginosa MSH3
Pseudomonas aeruginosa X24509
Pseudomonas aeruginosa UDL
Pseudomonas aeruginosa CF18
Pseudomonas aeruginosa 19660
Pseudomonas aeruginosa X13273
Pseudomonas aeruginosa S35004
Pseudomonas aeruginosa BWHPSA001
Pseudomonas aeruginosa BWHPSA003
Pseudomonas aeruginosa BWHPSA002
Pseudomonas aeruginosa BWHPSA004
Pseudomonas aeruginosa BWHPSA005
Pseudomonas aeruginosa BWHPSA011
Pseudomonas aeruginosa BWHPSA013
Pseudomonas aeruginosa BWHPSA012
Pseudomonas aeruginosa BWHPSA014
Pseudomonas aeruginosa BWHPSA017
Pseudomonas aeruginosa BWHPSA020
Pseudomonas aeruginosa BWHPSA019
Pseudomonas aeruginosa BWHPSA022
Pseudomonas aeruginosa BWHPSA023
Pseudomonas aeruginosa BWHPSA021
Pseudomonas aeruginosa BWHPSA025
Pseudomonas aeruginosa BWHPSA024
Pseudomonas aeruginosa BWHPSA027
Pseudomonas aeruginosa BL07
Pseudomonas aeruginosa BL04
Pseudomonas aeruginosa BL11
Pseudomonas aeruginosa BL10
Pseudomonas aeruginosa BL15
Pseudomonas aeruginosa BL16
Pseudomonas aeruginosa BL18
Pseudomonas aeruginosa M8A.2
Pseudomonas aeruginosa M8A.1
Pseudomonas aeruginosa M9A.1
Pseudomonas aeruginosa C20
Pseudomonas aeruginosa C23
Pseudomonas aeruginosa C40
Pseudomonas aeruginosa C48
Pseudomonas aeruginosa C51
Pseudomonas aeruginosa CF77
Pseudomonas aeruginosa C52
Pseudomonas aeruginosa CF614
Pseudomonas aeruginosa VRFPA04
Pseudomonas aeruginosa HB13
Pseudomonas aeruginosa MSH-10
Pseudomonas aeruginosa PA14
Pseudomonas aeruginosa PAK
Pseudomonas sp. P179
Pseudomonas aeruginosa str. Stone 130
Pseudomonas aeruginosa PA21_ST175
Pseudomonas aeruginosa E2
Pseudomonas aeruginosa ATCC 25324
Pseudomonas aeruginosa CI27
Pseudomonas aeruginosa ATCC 700888
Pseudomonas aeruginosa ATCC 14886
Pseudomonas aeruginosa PADK2_CF510
Pseudomonas aeruginosa MPAO1/P2
Pseudomonas aeruginosa MPAO1/P1
Pseudomonas sp. 2_1_26
Pseudomonas aeruginosa 2192
Pseudomonas aeruginosa C3719
Erwinia billingiae (strain Eb661)
Xanthomonas axonopodis pv. citri (strain 306)
Xanthomonas citri subsp. citri Aw12879
Xanthomonas axonopodis Xac29-1
Xanthomonas citri pv. mangiferaeindicae LMG 941
Xanthomonas axonopodis pv. punicae str. LMG 859
Leifsonia aquatica ATCC 14665
Serratia marcescens subsp. marcescens Db11
Pseudomonas aeruginosa VRFPA05
Pseudomonas aeruginosa BL22
Pseudomonas aeruginosa BL22
Xanthomonas axonopodis pv. malvacearum str.
Pseudomonas aeruginosa VRFPA07
Pseudomonas aeruginosa BL20
Pseudomonas aeruginosa BL25
Pseudomonas aeruginosa BL09
Serratia marcescens WW4
Serratia marcescens VGH107
Pseudomonas aeruginosa BWHPSA018
Pseudomonas aeruginosa M18
Pseudomonas aeruginosa BL12
Pseudomonas aeruginosa BWHPSA028
Pseudomonas aeruginosa WC55
Pseudomonas aeruginosa NCMG1179
Rhodococcus erythropolis SK121
Pseudomonas aeruginosa VRFPA03
Pseudomonas aeruginosa BL13
Serratia marcescens EGD-HP20
Pseudomonas aeruginosa NCGM2.S1
Pseudomonas aeruginosa 39016
Pseudomonas aeruginosa MH27
Pseudomonas aeruginosa JJ692
Pseudomonas aeruginosa 6077
Pseudomonas aeruginosa U2504
Pseudomonas aeruginosa BWHPSA006
Pseudomonas aeruginosa BL08
Pseudomonas aeruginosa BL14
Pseudomonas aeruginosa BL17
Pseudomonas aeruginosa PA45
Rhodococcus erythropolis CCM2595
Rhodococcus sp. P27
Kosakonia radicincitans DSM 16656
Rhodococcus erythropolis (strain PR4/NBRC 100887)
Klebsiella pneumoniae MGH 46
Klebsiella pneumoniae MGH 44
Klebsiella pneumoniae UCICRE 4
Klebsiella pneumoniae 303K
Klebsiella pneumoniae UHKPC179
Klebsiella pneumoniae UHKPC57
Klebsiella pneumoniae JHCK1
Klebsiella pneumoniae subsp. pneumoniae WGLW2
Klebsiella pneumoniae UCICRE 14
Rhodococcus qingshengii BKS 20-40
Pantoea sp. Sc1
Klebsiella sp. 1_1_55
Pantoea agglomerans Tx10
Escherichia coli 909957
Klebsiella pneumoniae KP-1
Rhodococcus erythropolis DN1
Klebsiella pneumoniae UCICRE 8
Brenneria sp. EniD312
Klebsiella pneumoniae BIDMC 23
Raoultella ornithinolytica B6
Klebsiella oxytoca 10-5246
Pantoea agglomerans 299R
Pantoea sp. aB
Pseudomonas sp. CFII64
Pseudomonas synxantha BG33R
Pseudomonas syringae pv. actinidiae ICMP 18801
Pseudomonas syringae pv. actinidiae ICMP 19072
Pseudomonas syringae pv. actinidiae ICMP 19073
Pseudomonas syringae pv. actinidiae ICMP 19071
Pseudomonas syringae pv. actinidiae ICMP 19104
Pseudomonas syringae pv. actinidiae ICMP 9855
Pseudomonas syringae pv. actinidiae ICMP 19102
Pseudomonas syringae pv. actinidiae ICMP 19068
Pseudomonas syringae pv. theae ICMP 3923
Pseudomonas syringae pv. actinidiae ICMP 19103
Rhizobium leguminosarum bv. viciae (strain 3841)
Pseudomonas sp. GM25
Herbaspirillum sp. YR522
Pseudomonas syringae pv. morsprunorum str. M302280
Pseudomonas fluorescens (strain Pf0-1)
Pseudomonas avellanae BPIC 631
Pseudomonas fluorescens R124
Pseudomonas syringae pv. syringae (strain B728a)
Pseudomonas syringae CC1557
Pseudomonas sp. GM80
Pseudomonas syringae pv. syringae SM
Pseudomonas syringae pv. avellanae str. ISPaVe037
Pseudomonas syringae pv. aceris str. M302273
Pseudomonas syringae pv. maculicola str. ES4326
Pseudomonas syringae BRIP39023
Pseudomonas syringae pv. aptata str. DSM 50252
Pseudomonas savastanoi pv. savastanoi NCPPB 3335
Pseudomonas syringae pv. aesculi str. 0893_23
Pseudomonas syringae BRIP34881
Pseudomonas syringae BRIP34876
Rhizobium leguminosarum bv. viciae WSM1455
Pseudomonas syringae Cit 7
Acinetobacter baumannii NIPH 410
Acinetobacter baumannii OIFC110
Acinetobacter baumannii WC-692
Pseudomonas sp. TKP
Pseudomonas syringae pv. syringae B64
Pseudomonas syringae pv. actinidiae ICMP 19094
Pseudomonas syringae pv. actinidiae ICMP 18883
Pseudomonas syringae pv. actinidiae ICMP 19095
Pseudomonas syringae pv. actinidiae ICMP 19099
Pseudomonas syringae pv. actinidiae ICMP 19100
Pseudomonas syringae pv. actinidiae ICMP 19098
In some embodiments, the disclosure provides methods for synthesizing olefinic alcohol products as described above, wherein the enzyme is a cytochrome P450. In some embodiments, the cytochrome P450 is selected from Table 6 or a variant thereof having at least 90% identity thereto. In some embodiments, the cytochrome P450 is a member of the CYP52 or CYP153 family. In some embodiments, the CYP52 enzyme is selected from CYP52A17, CYP52A13, and CYP52A12.
Candida tropicalis (Yeast)
Candida tropicalis (strain ATCC MYA-3404/
Candida tropicalis (Yeast)
Candida albicans (Yeast)
Candida maltosa (Yeast)
Candida dubliniensis (strain CD36/ATCC
Candida albicans (strain SC5314/ATCC MYA-
Candida albicans (strain SC5314/ATCC MYA-
Candida maltosa (strain Xu316) (Yeast)
Candida maltosa (Yeast)
Candida orthopsilosis (strain 90-125) (Yeast)
Candida parapsilosis (strain CDC 317/ATCC
Lodderomyces elongisporus (strain ATCC
Candida maltosa (Yeast)
Candida maltosa (Yeast)
Candida tropicalis (Yeast)
Debaryomyces hansenii (strain ATCC 36239/
Candida tropicalis (Yeast)
Candida maltosa (strain Xu316) (Yeast)
Spathaspora passalidarum (strain NRRL Y-
Scheffersomyces stipitis (strain ATCC 58785/
Candida parapsilosis (strain CDC 317/ATCC
Candida parapsilosis (strain CDC 317/ATCC
Candida tropicalis (Yeast)
Candida maltosa (strain Xu316) (Yeast)
Debaryomyces hansenii (Yeast) (Torulaspora hansenii)
Meyerozyma guilliermondii (strain ATCC 6260/
Debaryomyces hansenii (strain ATCC 36239/
Candida maltosa (Yeast)
Meyerozyma guilliermondii (strain ATCC 6260/
Debaryomyces hansenii (Yeast) (Torulaspora
hansenii)
Candida dubliniensis (strain CD36/ATCC
Meyerozyma guilliermondii (strain ATCC 6260/
Candida albicans (strain SC5314/ATCC MYA-
Candida albicans (strain WO-1) (Yeast)
Candida tropicalis (Yeast)
Candida tropicalis (Yeast)
Pichia sorbitophila (strain ATCC MYA-4447/
Candida parapsilosis (strain CDC 317/ATCC
Candida tropicalis (Yeast)
Candida tropicalis (Yeast)
Lodderomyces elongisporus (strain ATCC
Candida albicans (strain WO-1) (Yeast)
Candida albicans (strain SC5314/ATCC MYA-
Candida albicans (Yeast)
Candida maltosa (strain Xu316) (Yeast)
Scheffersomyces stipitis (strain ATCC 58785/
Lodderomyces elongisporus (strain ATCC
Candida tropicalis (strain ATCC MYA-3404/
Pichia sorbitophila (strain ATCC MYA-4447/
Candida parapsilosis (strain CDC 317/ATCC
Spathaspora passalidarum (strain NRRL Y-27907/11-Y1)
Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast)
Candida tropicalis (Yeast)
Candida parapsilosis (strain CDC 317/ATCC
Scheffersomyces stipitis (strain ATCC 58785/
Candida parapsilosis (strain CDC 317/ATCC
Candida maltosa (strain Xu316) (Yeast)
Candida orthopsilosis (strain 90-125) (Yeast)
Candida dubliniensis (strain CD36/ATCC
Pichia sorbitophila (strain ATCC MYA-4447/
Debaryomyces hansenii (strain ATCC 36239/
Candida maltosa (Yeast)
Scheffersomyces stipitis (strain ATCC 58785/
Spathaspora passalidarum (strain NRRL Y-
Candida tropicalis (strain ATCC MYA-3404/
Candida maltosa (Yeast)
Candida albicans (strain WO-1) (Yeast)
Candida tropicalis (strain ATCC MYA-3404/
Candida albicans (strain SC5314/ATCC MYA-
Candida tropicalis (Yeast)
Scheffersomyces stipitis (strain ATCC 58785/
Debaryomyces hansenii (strain ATCC 36239/
Candida tenuis (strain ATCC 10573/BCRC
Lodderomyces elongisporus (strain ATCC
Lodderomyces elongisporus (strain ATCC
Candida tropicalis (Yeast)
Candida tropicalis (Yeast)
Candida maltosa (Yeast)
Candida dubliniensis (strain CD36/ATCC
Candida maltosa (Yeast)
Candida tenuis (strain ATCC 10573/BCRC
Meyerozyma guilliermondii (Yeast) (Candida guilliermondii)
Spathaspora passalidarum (strain NRRL Y-
Candida tenuis (strain ATCC 10573/BCRC
Candida maltosa (strain Xu316) (Yeast)
Candida tropicalis (Yeast)
Clavispora lusitaniae (strain ATCC 42720)
Debaryomyces hansenii (strain ATCC 36239/
Candida tropicalis (Yeast)
Clavispora lusitaniae (strain ATCC 42720)
Meyerozyma guilliermondii (strain ATCC 6260/
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (strain CLIB 122/E 150)
Yarrowia lipolytica (strain CLIB 122/E 150)
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (strain CLIB 122/E 150)
Candida maltosa (Yeast)
Yarrowia lipolytica (strain CLIB 122/E 150)
Byssochlamys spectabilis (strain No. 5/NBRC
Aspergillus terreus (strain NIH 2624/FGSC A1156)
Neosartorya fischeri (strain ATCC 1020/DSM
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (strain CLIB 122/E 150)
Penicillium digitatum (strain PHI26/CECT
Penicillium digitatum (strain Pd1/CECT
Aspergillus niger (strain ATCC 1015/CBS
Aspergillus niger (strain CBS 513.88/FGSC
Tuber melanosporum (strain Mel28) (Perigord
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (strain CLIB 122/E 150)
Arthrobotrys oligospora (strain ATCC 24927/
Dactylellina haptotyla (strain CBS 200.50)
Yarrowia lipolytica (strain CLIB 122/E 150)
Aspergillus clavatus (strain ATCC 1007/CBS
Byssochlamys spectabilis (strain No. 5/NBRC
Aspergillus kawachii (strain NBRC 4308)
Aspergillus oryzae (strain 3.042) (Yellow koji mold)
Aspergillus flavus (strain ATCC 200026/FGSC
Aspergillus oryzae (strain ATCC 42149/RIB
Aspergillus oryzae (Yellow koji mold)
Candida tenuis (strain ATCC 10573/BCRC
Emericella nidulans (strain FGSC A4/ATCC
Talaromyces stipitatus (strain ATCC 10500/
Starmerella bombicola
Hordeum vulgare var. distichum (Two-rowed
Mycosphaerella graminicola (strain CBS 115943/
Neosartorya fumigata (strain ATCC MYA-4609/
Neosartorya fumigata (strain CEA10/CBS
Penicillium chrysogenum (strain ATCC 28089/
Clavispora lusitaniae (strain ATCC 42720)
Penicillium roqueforti
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (strain CLIB 122/E 150)
Candida tenuis (strain ATCC 10573/BCRC
Penicillium marneffei (strain ATCC 18224/
Yarrowia lipolytica (strain CLIB 122/E 150)
Candida apicola (Yeast)
Macrophomina phaseolina (strain MS6)
Cyphellophora europaea CBS 101466
Cochliobolus sativus (strain ND90Pr/ATCC
Cochliobolus sativus (strain ND90Pr/ATCC
Bipolaris victoriae FI3
Bipolaris zeicola 26-R-13
Cochliobolus heterostrophus (strain C5/ATCC
Cochliobolus heterostrophus (strain C4/ATCC
Pseudogymnoascus destructans (strain ATCC
Aspergillus terreus (strain NIH 2624/FGSC A1156)
Marssonina brunnea f. sp. multigermtubi (strain
Penicillium marneffei (strain ATCC 18224/
Neosartorya fumigata (strain CEA10/CBS
Candida apicola (Yeast)
Neosartorya fumigata (strain ATCC MYA-4609/
Neosartorya fischeri (strain ATCC 1020/DSM
Cordyceps militaris (strain CM01) (Caterpillar
Coniosporium apollinis (strain CBS 100218)
Penicillium chrysogenum (strain ATCC 28089/
Penicillium digitatum (strain Pd1/CECT
Penicillium digitatum (strain PHI26/CECT
Penicillium roqueforti
Marssonina brunnea f. sp. multigermtubi (strain
Botryotinia fuckeliana (strain BcDW1) (Noble
Botryotinia fuckeliana (strain T4) (Noble rot
Emericella nidulans (strain FGSC A4/ATCC
Candida maltosa (Yeast)
Phaeosphaeria nodorum (strain SN15/ATCC
Pyrenophora tritici-repentis (strain Pt-1C-BFP)
Pyrenophora teres f. teres (strain 0-1) (Barley net
Aspergillus niger (strain ATCC 1015/CBS
Bipolaris oryzae ATCC 44560
Cochliobolus heterostrophus (strain C4/ATCC
Cochliobolus heterostrophus (strain C5/ATCC
Botryosphaeria parva (strain UCR-NP2)
Ajellomyces capsulatus (strain H88) (Darling's
Bipolaris oryzae ATCC 44560
Dactylellina haptotyla (strain CBS 200.50)
Cladophialophora carrionii CBS 160.54
Exophiala dermatitidis (strain ATCC 34100/
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (strain CLIB 122/E 150)
Blumeria graminis f. sp. hordei (strain DH14)
Neosartorya fischeri (strain ATCC 1020/DSM
Dactylellina haptotyla (strain CBS 200.50)
Aspergillus kawachii (strain NBRC 4308)
Aspergillus niger (strain ATCC 1015/CBS
Aspergillus oryzae (strain 3.042) (Yellow koji
Aspergillus flavus (strain ATCC 200026/FGSC
Arthroderma gypseum (strain ATCC MYA-4604/
Arthroderma otae (strain ATCC MYA-4605/
Bipolaris victoriae FI3
Bipolaris oryzae ATCC 44560
Byssochlamys spectabilis (strain No. 5/NBRC
Bipolaris zeicola 26-R-13
Mycosphaerella fijiensis (strain CIRAD86)
Aspergillus terreus (strain NIH 2624/FGSC
Setosphaeria turcica (strain 28A) (Northern leaf
Colletotrichum graminicola (strain M1.001/M2/
Aspergillus clavatus (strain ATCC 1007/CBS
Ajellomyces capsulatus (strain G186AR/H82/
Aspergillus oryzae (strain ATCC 42149/RIB
Aspergillus oryzae (strain ATCC 42149/RIB
Aspergillus niger (strain CBS 513.88/FGSC
Penicillium marneffei (strain ATCC 18224/
Tuber melanosporum (strain Mel28) (Perigord
Colletotrichum higginsianum (strain IMI
Beauveria bassiana (strain ARSEF 2860) (White
Yarrowia lipolytica (strain CLIB 122/E 150)
Botryosphaeria parva (strain UCR-NP2)
Setosphaeria turcica (strain 28A) (Northern leaf
Aspergillus clavatus (strain ATCC 1007/CBS
Mycosphaerella fijiensis (strain CIRAD86)
Aspergillus oryzae (strain ATCC 42149/RIB
Aspergillus oryzae (strain 3.042) (Yellow koji
Aspergillus oryzae (Yellow koji mold)
Aspergillus flavus (strain ATCC 200026/FGSC
Candida tenuis (strain ATCC 10573/BCRC
Aspergillus niger (strain ATCC 1015/CBS
Aspergillus niger (strain CBS 513.88/FGSC
Pseudogymnoascus destructans (strain ATCC
Cladophialophora carrionii CBS 160.54
Candida albicans (strain WO-1) (Yeast)
Coccidioides posadasii (strain RMSCC 757/
Coccidioides posadasii (strain C735) (Valley
Candida maltosa (strain Xu316) (Yeast)
Metarhizium acridum (strain CQMa 102)
Bipolaris zeicola 26-R-13
Pyronema omphalodes (strain CBS 100304)
Bipolaris victoriae FI3
Botryotinia fuckeliana (strain T4) (Noble rot
Fusarium heterosporum
Cyphellophora europaea CBS 101466
Metarhizium acridum (strain CQMa 102)
Macrophomina phaseolina (strain MS6)
Colletotrichum graminicola (strain M1.001/M2/
Bipolaris zeicola 26-R-13
Cochliobolus heterostrophus (strain C5/ATCC
Bipolaris zeicola 26-R-13
Cochliobolus heterostrophus (strain C4/ATCC
Colletotrichum gloeosporioides (strain Cg-14)
Botryotinia fuckeliana (strain BcDW1) (Noble
Botryotinia fuckeliana (strain T4) (Noble rot
Sclerotinia sclerotiorum (strain ATCC 18683/
Penicillium digitatum (strain PHI26/CECT
Penicillium digitatum (strain Pd1/CECT
Metarhizium anisopliae (strain ARSEF 23/
Starmerella bombicola
Penicillium marneffei (strain ATCC 18224/
Metarhizium acridum (strain CQMa 102)
Mycosphaerella pini (strain NZE10/CBS
Aspergillus kawachii (strain NBRC 4308)
Aspergillus niger (strain CBS 513.88/FGSC
Aspergillus niger (strain ATCC 1015/CBS
Beauveria bassiana (strain ARSEF 2860) (White
Beauveria bassiana (White muscardine disease
Aspergillus oryzae (strain 3.042) (Yellow koji
Aspergillus flavus (strain ATCC 200026/FGSC
Aspergillus oryzae (strain ATCC 42149/RIB
Aspergillus oryzae (Yellow koji mold)
Endocarpon pusillum (strain Z07020/HMAS-L-
Sclerotinia sclerotiorum (strain ATCC 18683/
Pyrenophora tritici-repentis (strain Pt-1C-BFP)
Candida albicans (strain SC5314/ATCC MYA-
Candida albicans (Yeast)
Trichophyton verrucosum (strain HKI 0517)
Coccidioides immitis (strain RS) (Valley fever
Ajellomyces dermatitidis ATCC 26199
Ajellomyces dermatitidis (strain ATCC 18188/
Ajellomyces dermatitidis (strain SLH14081)
Ajellomyces dermatitidis (strain ER-3/ATCC
Coccidioides posadasii (strain C735) (Valley
Colletotrichum orbiculare (strain 104-T/ATCC
Coccidioides immitis (strain RS) (Valley fever
Talaromyces stipitatus (strain ATCC 10500/
Coccidioides posadasii (strain RMSCC 757/
Uncinocarpus reesii (strain UAMH 1704)
Starmerella bombicola
Pyrenophora tritici-repentis (strain Pt-1C-BFP)
Marssonina brunnea f. sp. multigermtubi (strain
Metarhizium anisopliae (strain ARSEF 23/
Macrophomina phaseolina (strain MS6)
Glarea lozoyensis (strain ATCC 20868/
Arthroderma otae (strain ATCC MYA-4605/
Trichophyton verrucosum (strain HKI 0517)
Hypocrea atroviridis (strain ATCC 20476/IMI
Glarea lozoyensis (strain ATCC 74030/
Ajellomyces capsulatus (strain NAm1/WU24)
Pyronema omphalodes (strain CBS 100304)
Endocarpon pusillum (strain Z07020/HMAS-L-
Penicillium marneffei (strain ATCC 18224/
Emericella nidulans (strain FGSC A4/ATCC
Botryotinia fuckeliana (strain T4) (Noble rot
Aspergillus terreus (strain NIH 2624/FGSC
Aspergillus niger (strain ATCC 1015/CBS
Aspergillus kawachii (strain NBRC 4308)
Beauveria bassiana (strain ARSEF 2860) (White
Sclerotinia sclerotiorum (strain ATCC 18683/
Beauveria bassiana (strain ARSEF 2860) (White
Beauveria bassiana (White muscardine disease
Cordyceps militaris (strain CM01) (Caterpillar
Penicillium chrysogenum (strain ATCC 28089/
notatum)
Mycosphaerella pini (strain NZE10/CBS
Mycosphaerella pini (strain NZE10/CBS
Aspergillus terreus (strain NIH 2624/FGSC
Arthroderma benhamiae (strain ATCC MYA-
Baudoinia compniacensis (strain UAMH 10762)
Candida tropicalis (strain ATCC MYA-3404/
Candida tropicalis (Yeast)
Metarhizium anisopliae (strain ARSEF 23/
Mycosphaerella graminicola (strain CBS 115943/
Cladophialophora carrionii CBS 160.54
Glarea lozoyensis (strain ATCC 20868/
Hypocrea virens (strain Gv29-8/FGSC 10586)
Marssonina brurnnea f. sp. multigermtubi (strain
Talaromyces stipitatus (strain ATCC 10500/
Arthroderma benhamiae (strain ATCC MYA-
Colletotrichum higginsianum (strain IMI
Trichophyton tonsurans (strain CBS 112818)
Marssonina brunnea f. sp. multigermtubi (strain
Aspergillus terreus (strain NIH 2624/FGSC
Claviceps purpurea (strain 20.1) (Ergot fungus)
Trichophyton rubrum (strain ATCC MYA-4607/
Setosphaeria turcica (strain 28A) (Northern leaf
Paracoccidioides brasiliensis (strain Pb03)
Arthroderma gypseum (strain ATCC MYA-4604/
Trichophyton equinum (strain ATCC MYA-
Talaromyces stipitatus (strain ATCC 10500/
Leptosphaeria maculans (strain JN3/isolate
Bipolaris victoriae FI3
Magnaporthe oryzae (strain Y34) (Rice blast
Fusarium oxysporum f. sp. cubense (strain race
Fusarium oxysporum f. sp. lycopersici (strain
Endocarpon pusillum (strain Z07020/HMAS-L-
Sphaerulina musiva (strain SO2202) (Poplar
Mycosphaerella graminicola (strain CBS 115943/
Penicillium oxalicum (strain 114-2/CGMCC
Mycosphaerella graminicola (strain CBS 115943/
Cladophialophora carrionii CBS 160.54
Togninia minima (strain UCR-PA7) (Esca
Fusarium oxysporum (strain Fo5176) (Fusarium
Gaeumannomyces graminis var. tritici (strain
Cochliobolus sativus (strain ND90Pr/ATCC
Neosartorya fumigata (strain CEA10/CBS
Neosartorya fumigata (strain ATCC MYA-4609/
Neosartorya fischeri (strain ATCC 1020/DSM
Hypocrea atroviridis (strain ATCC 20476/IMI
Candida orthopsilosis (strain 90-125) (Yeast)
Cyphellophora europaea CBS 101466
Penicillium oxalicum (strain 114-2/CGMCC
Arthroderma gypseum (strain ATCC MYA-4604/
Hypocrea virens (strain Gv29-8/FGSC 10586)
Botryotinia fuckeliana (strain BcDW1) (Noble
Botryosphaeria parva (strain UCR-NP2)
Cochliobolus sativus (strain ND90Pr/ATCC
Aspergillus niger (strain CBS 513.88/FGSC
Candida dubliniensis (strain CD36/ATCC
Cochliobolus heterostrophus (strain C4/ATCC
Cochliobolus heterostrophus (strain C5/ATCC
Aspergillus clavatus (strain ATCC 1007/CBS
Hypocrea jecorina (strain QM6a) (Trichoderma reesei)
Trichophyton tonsurans (strain CBS 112818)
Glarea lozoyensis (strain ATCC 20868/
Trichophyton rubrum (strain ATCC MYA-4607/
Leptosphaeria maculans (strain JN3/isolate
Cyphellophora europaea CBS 101466
Hypocrea jecorina (strain QM6a) (Trichoderma reesei)
Beauveria bassiana (strain ARSEF 2860) (White
Cordyceps militaris (strain CM01) (Caterpillar
Trichophyton rubrum (strain ATCC MYA-4607/
Botryotinia fuckeliana (strain BcDW1) (Noble
Magnaporthe oryzae (strain P131) (Rice blast
Magnaporthe oryzae (strain Y34) (Rice blast
Magnaporthe oryzae (strain 70-15/ATCC
Paracoccidioides lutzii (strain ATCC MYA-826/
Bipolaris zeicola 26-R-13
Verticillium dahliae (strain VdLs.17/ATCC
Trichophyton verrucosum (strain HKI 0517)
Arthroderma benhamiae (strain ATCC MYA-
Chaetomium globosum (strain ATCC 6205/
Magnaporthe poae (strain ATCC 64411/73-15)
Hypocrea atroviridis (strain ATCC 20476/IMI
Colletotrichum orbiculare (strain 104-T/ATCC
Penicillium chrysogenum (strain ATCC 28089/
Ophiocordyceps sinensis (strain Co18/CGMCC
Pyrenophora teres f. teres (strain 0-1) (Barley net
Baudoinia compniacensis (strain UAMH 10762)
Podospora anserina (strain S/ATCC MYA-
Aspergillus terreus (strain NIH 2624/FGSC
Hypocrea jecorina (strain QM6a) (Trichoderma reesei)
Claviceps purpurea (strain 20.1) (Ergot fungus)
Aspergillus flavus (strain ATCC 200026/FGSC
Mycosphaerella fijiensis (strain CIRAD86)
Grosmannia clavigera (strain kw1407/UAMH
Lodderomyces elongisporus (strain ATCC
Candida tropicalis (strain ATCC MYA-3404/
Coniosporium apollinis (strain CBS 100218)
Candida parapsilosis (strain CDC 317/ATCC
Aspergillus niger (strain CBS 513.88/FGSC
Baudoinia compniacensis (strain UAMH 10762)
Candida tropicalis (Yeast)
Aspergillus kawachii (strain NBRC 4308)
Colletotrichum gloeosporioides (strain Cg-14)
Colletotrichum gloeosporioides (strain Cg-14)
Endocarpon pusillum (strain Z07020/HMAS-L-
Arthroderma gypseum (strain ATCC MYA-4604/
Botryotinia fuckeliana (strain T4) (Noble rot
Exophiala dermatitidis (strain ATCC 34100/
Aspergillus oryzae (Yellow koji mold)
Aspergillus oryzae (strain ATCC 42149/RIB
Neurospora tetrasperma (strain FGSC 2508/
Sordaria macrospora (strain ATCC MYA-333/
Neurospora crassa (strain ATCC 24698/74-
Eutypa lata (strain UCR-EL1) (Grapevine
Neurospora tetrasperma (strain FGSC 2509/
Setosphaeria turcica (strain 28A) (Northern leaf
Pyrenophora tritici-repentis (strain Pt-1C-BFP)
Paracoccidioides lutzii (strain ATCC MYA-826/
Neosartorya fischeri (strain ATCC 1020/DSM
Sphaerulina musiva (strain SO2202) (Poplar
Emericella nidulans (strain FGSC A4/ATCC
Candida orthopsilosis (strain 90-125) (Yeast)
Aspergillus oryzae (strain 3.042) (Yellow koji
Aspergillus flavus (strain ATCC 200026/FGSC
Candida parapsilosis (strain CDC 317/ATCC
Aspergillus oryzae (strain ATCC 42149/RIB
Aspergillus oryzae (Yellow koji mold)
Neosartorya fumigata (strain ATCC MYA-4609/
Botryotinia fuckeliana (strain T4) (Noble rot
Sclerotinia sclerotiorum (strain ATCC 18683/
Pyronema omphalodes (strain CBS 100304)
Thielavia heterothallica (strain ATCC 42464/
Pestalotiopsis fici W106-1
Eutypa lata (strain UCR-EL1) (Grapevine
Colletotrichum orbiculare (strain 104-T/ATCC
Colletotrichum graminicola (strain M1.001/M2/
Trichophyton verrucosum (strain HKI 0517)
Sphaerulina musiva (strain SO2202) (Poplar
Nectria haematococca (strain 77-13-4/ATCC
Coniosporium apollinis (strain CBS 100218)
Gaeumannomyces graminis var. tritici (strain
Fusarium pseudograminearum (strain CS3096)
Magnaporthe oryzae (strain P131) (Rice blast
Magnaporthe oryzae (strain Y34) (Rice blast
Magnaporthe oryzae (strain 70-15/ATCC
Thielavia terrestris (strain ATCC 38088/NRRL
Gibberella fujikuroi (strain CBS 195.34/IMI
Pyronema omphalodes (strain CBS 100304)
Gibberella moniliformis (strain M3125/FGSC
Magnaporthe oryzae (strain P131) (Rice blast
Magnaporthe oryzae (strain 70-15/ATCC
Fusarium oxysporum f. sp. cubense (strain race
Chaetomium thermophilum (strain DSM 1495/
Botryotinia fuckeliana (strain BcDW1) (Noble
Verticillium alfalfae (strain VaMs.102/ATCC
Arthroderma gypseum (strain ATCC MYA-4604/
Uncinocarpus reesii (strain UAMH 1704)
Bipolaris oryzae ATCC 44560
Paracoccidioides brasiliensis (strain Pb03)
Paracoccidioides brasiliensis (strain Pb18)
Neosartorya fumigata (strain ATCC MYA-4609/
Neosartorya fumigata (strain CEA10/CBS
Aspergillus niger (strain ATCC 1015/CBS
Coniosporium apollinis (strain CBS 100218)
Aspergillus niger (strain CBS 513.88/FGSC
Aspergillus niger (strain ATCC 1015/CBS
Neosartorya fischeri (strain ATCC 1020/DSM
Aspergillus oryzae (strain ATCC 42149/RIB
Aspergillus oryzae (strain 3.042) (Yellow koji
Aspergillus oryzae (Yellow koji mold)
Magnaporthe poae (strain ATCC 64411/73-15)
Cladophialophora carrionii CBS 160.54
Podospora anserina (strain S/ATCC MYA-
Gibberella zeae (strain PH-1/ATCC MYA-
Colletotrichum orbiculare (strain 104-T/ATCC
Neosartorya fischeri (strain ATCC 1020/DSM
Trichophyton verrucosum (strain HKI 0517)
Botryotinia fuckeliana (strain T4) (Noble rot
Trichophyton rubrum (strain ATCC MYA-4607/
Botryotinia fuckeliana (strain T4) (Noble rot
Setosphaeria turcica (strain 28A) (Northern leaf
Bipolaris victoriae FI3
Bipolaris zeicola 26-R-13
Podospora anserina (strain S/ATCC MYA-
Sporothrix schenckii (strain ATCC 58251/de
Exophiala dermatitidis (strain ATCC 34100/
Colletotrichum gloeosporioides (strain Cg-14)
Arthroderma benhamiae (strain ATCC MYA-
Macrophomina phaseolina (strain MS6)
Trichophyton tonsurans (strain CBS 112818)
Trichophyton equinum (strain ATCC MYA-
Arthroderma benhamiae (strain ATCC MYA-
Arthroderma otae (strain ATCC MYA-4605/
Aspergillus flavus (strain ATCC 200026/FGSC
Mycosphaerella graminicola (strain CBS 115943/
Penicillium chrysogenum (strain ATCC 28089/
Alternaria solani
Colletotrichum higginsianum (strain IMI
Thielavia heterothallica (strain ATCC 42464/
Togninia minima (strain UCR-PA7) (Esca
Ophiostoma piceae (strain UAMH 11346) (Sap
Cladophialophora carrionii CBS 160.54
Botryotinia fuckeliana (strain BcDW1) (Noble
Mycobacterium sp. HXN-1500
Gordonia amicalis NBRC 100051 = JCM 11271
Mycobacterium austroafricanum
Mycobacterium sp. ENV421
Polaromonas sp. (strain JS666/ATCC BAA-500)
Parvibaculum sp. S13-6
Parvibaculum sp. S13-5
Tistrella mobilis
Parvibaculum sp. S13-6
Parvibaculum sp. S13-6
Parvibaculum sp. S13-5
Parvibaculum sp. S18-4
Parvibaculum sp. S18-4
Parvibaculum sp. S13-5
Parvibaculum sp. S18-4
Caulobacter sp. (strain K31)
Erythrobacter sp. S11-13
Parvibaculum sp. S13-5
Erythrobacter flavus
Sphingobium sp. S13-2
Sphingopyxis sp. S16-14
Parvibaculum sp. S13-6
Erythrobacter sp. S17-1
Erythrobacter flavus
Bradyrhizobium sp. CCGE-LA001
Caulobacter sp. AP07
Parvibaculum lavamentivorans (strain DS-1/
Erythrobacter flavus
Erythrobacter sp. S2-1
Erythrobacter citreus
Erythrobacter citreus
Erythrobacter flavus
Erythrobacter sp. S14-1
Sphingopyxis macrogoltabida (Sphingomonas
macrogoltabidus)
Afipia broomeae ATCC 49717
Parvibaculum sp. S18-4
Parvibaculum lavamentivorans (strain DS-1/
Caulobacter crescentus (strain NA1000/
Caulobacter crescentus (strain ATCC 19089/
Parvibaculum lavamentivorans (strain DS-1/
Caulobacter segnis (strain ATCC 21756/DSM
Novosphingobium sp. PP1Y
Parvibaculum sp. S18-4
Bradyrhizobium sp. STM 3843
Bradyrhizobium sp. (strain ORS278)
Bradyrhizobium sp. (strain BTAi1/ATCC
Caulobacter crescentus OR37
Afipia broomeae ATCC 49717
Afipia clevelandensis ATCC 49720
Novosphingobium pentaromativorans US6-1
Sphingopyxis macrogoltabida (Sphingomonas
macrogoltabidus)
Bradyrhizobium sp. ORS 375
Bradyrhizobium sp. ORS 285
Bradyrhizobium sp. STM 3809
Rhodopseudomonas palustris (strain BisA53)
Bradyrhizobium sp. YR681
Bradyrhizobium sp. STM 3843
Rhodopseudomonas palustris (strain BisB18)
Caulobacter sp. (strain K31)
Sphingopyxis macrogoltabida (Sphingomonas
macrogoltabidus)
Bradyrhizobium oligotrophicum S58
Bradyrhizobium diazoefficiens (strain JCM
Bradyrhizobium oligotrophicum S58
Erythrobacter litoralis (strain HTCC2594)
Erythrobacter sp. SD-21
Bradyrhizobium sp. DFCI-1
Bradyrhizobium sp. DFCI-1
Bradyrhizobium diazoefficiens (strain JCM
Rhodopseudomonas palustris (strain TIE-1)
Bradyrhizobium sp. CCGE-LA001
Parvibaculum lavamentivorans (strain DS-1/
Rhodopseudomonas palustris (strain ATCC
Bradyrhizobium sp. S23321
Bradyrhizobium sp. ORS 285
Bradyrhizobium sp. ORS 375
Bradyrhizobium sp. (strain BTAi1/ATCC
Bradyrhizobium japonicum USDA 6
Afipia sp. P52-10
Afipia sp. P52-10
Afipia sp. P52-10
Bradyrhizobium japonicum USDA 6
Bradyrhizobium sp. WSM471
Bradyrhizobium sp. S23321
Rhodopseudomonas palustris (strain DX-1)
Bradyrhizobium sp. STM 3809
Bradyrhizobium sp. (strain ORS278)
Rhodopseudomonas palustris (strain HaA2)
Rhodopseudomonas palustris (strain BisB5)
Phenylobacterium zucineum (strain HLK1)
Bradyrhizobium sp. WSM1253
Bradyrhizobium sp. WSM471
Bradyrhizobium sp. WSM1253
Bradyrhizobium japonicum USDA 6
Bradyrhizobium sp. YR681
Afipia sp. P52-10
Bradyrhizobium sp. CCGE-LA001
Congregibacter litoralis KT71
Bradyrhizobium diazoefficiens (strain JCM
Bradyrhizobium japonicum
Pseudomonas sp. 19-rlim
Bradyrhizobium sp. WSM1253
Bradyrhizobium sp. WSM471
Afipia sp. P52-10
Glaciecola psychrophila 170
Congregibacter litoralis KT71
Marinobacter santoriniensis NKSG1
Alcanivorax hongdengensis
Alcanivorax sp. DG881
Ochrobactrum anthropi
Bradyrhizobium sp. DFCI-1
Burkholderia xenovorans (strain LB400)
Alcanivorax sp. P2S70
Marinobacter hydrocarbonoclasticus ATCC 49840
Marinobacter sp. EVN1
Marinobacter adhaerens (strain HP15)
Alcanivorax hongdengensis
Hyphomonas neptunium (strain ATCC 15444)
Alcanivorax dieselolei (strain DSM 16502/
Alcanivorax hongdengensis A-11-3
Alcanivorax dieselolei
Alcanivorax pacificus W11-5
Marinobacter sp. ES-1
Limnobacter sp. MED105
Marinobacter aquaeolei (strain ATCC 700491/
Marinobacter sp. EVN1
Marinobacter sp. EN3
Marinobacter manganoxydans MnI7-9
Marinobacter hydrocarbonoclasticus ATCC
Marinobacter hydrocarbonoclasticus
Patulibacter medicamentivorans
Acinetobacter baumannii WC-141
Saccharomonospora marina XMU15
Mycobacterium marinum (strain ATCC BAA-
Mycobacterium abscessus 3A-0930-R
Mycobacterium abscessus 3A-0930-S
Mycobacterium abscessus 3A-0731
Mycobacterium abscessus 3A-0119-R
Mycobacterium abscessus 6G-0728-R
Mycobacterium abscessus subsp. bolletii 1S-
Mycobacterium abscessus 6G-0728-5
Mycobacterium abscessus 3A-0810-R
Mycobacterium abscessus 3A-0122-S
Mycobacterium abscessus 3A-0122-R
Mycobacterium abscessus 6G-0212
Mycobacterium abscessus subsp. bolletii 1S-
Mycobacterium abscessus subsp. bolletii 1S-
Mycobacterium abscessus subsp. bolletii 1S-
Mycobacterium abscessus 6G-1108
Mycobacterium abscessus 6G-0125-S
Mycobacterium abscessus 6G-0125-R
Mycobacterium abscessus subsp. bolletii 2B-
Mycobacterium abscessus subsp. bolletii 2B-
Mycobacterium abscessus subsp. bolletii 2B-
Mycobacterium abscessus subsp. bolletii 2B-
Mycobacterium abscessus subsp. bolletii 2B-
Mycobacterium abscessus subsp. bolletii 2B-
Parvibaculum lavamentivorans (strain DS-1/
Alcanivorax hongdengensis
Alcanivorax sp. DG881
Marinobacter sp. C1S70
Alcanivorax sp. P2S70
Marinobacter goseongensis
Hirschia baltica (strain ATCC 49814/DSM
Acinetobacter indicus CIP 110367
Acinetobacter indicus ANC 4215
Acinetobacter sp. OC4
Acinetobacter baumannii NIPH 527
Acinetobacter sp. CIP 102129
Acinetobacter sp. NIPH 809
Acinetobacter baumannii OIFC0162
Acinetobacter sp. EB104
Dietzia cinnamea P4
Acinetobacter sp. WC-743
Acinetobacter baumannii WC-348
Acinetobacter baumannii WC-141
Acinetobacter baumannii WC-323
Gordonia malaquae NBRC 108250
Rhodococcus erythropolis SK121
Acinetobacter sp. COS3
Acinetobacter guillouiae MSP4-18
Acinetobacter gyllenbergii MTCC 11365
Acinetobacter gyllenbergii CIP 110306
Acinetobacter sp. CIP 110321
Acinetobacter pittii ANC 3678
Acinetobacter beijerinckii CIP 110307
Acinetobacter beijerinckii CIP 110307
Acinetobacter guillouiae CIP 63.46
Acinetobacter sp. NIPH 236
Acinetobacter radioresistens DSM 6976 =
Acinetobacter sp. NBRC 100985
Williamsia sp. D3
Rhodococcus ruber BKS 20-38
Gordonia neofelifaecis NRRL B-59395
Nocardioidaceae bacterium Broad-1
Rhodococcus erythropolis DN1
Rhodococcus erythropolis (strain PR4/NBRC
Rhodococcus erythropolis DN1
Alcanivorax dieselolei
Alcanivorax borkumensis
Alcanivorax sp. 97CO-5
Alcanivorax borkumensis (strain SK2/ATCC
Alcanivorax borkumensis
Amycolicicoccus subflavus (strain DSM 45089/
Dietzia cinnamea P4
Rhodococcus sp. R04
Dietzia sp. DQ12-45-1b
Gordonia terrae C-6
Gordonia rubripertincta NBRC 101908
Gordonia polyisoprenivorans NBRC 16320
Gordonia amicalis NBRC 100051 = JCM 11271
Nocardia cyriacigeorgica (strain GUH-2)
Mycobacterium gilvum (strain PYR-GCK)
Acinetobacter sp. ANC 3862
Rhodococcus erythropolis (strain PR4/NBRC
Mycobacterium rhodesiae (strain NBB3)
Rhodococcus wratislaviensis IFP 2016
Nocardioides sp. CF8
Rhodococcus sp. AW25M09
Mycobacterium sp. (strain MCS)
Mycobacterium sp. (strain JLS)
Mycobacterium sp. (strain KMS)
Mycobacterium intracellulare MOTT-02
Mycobacterium abscessus subsp. bolletii str. GO
Mycobacterium abscessus (strain ATCC 19977/
Mycobacterium abscessus V06705
Mycobacterium abscessus M94
Mycobacterium avium subsp. hominissuis 10-4249
Mycobacterium parascrofulaceum ATCC BAA-
Rhodococcus sp. AW25M09
Nocardia asteroides NBRC 15531
Aeromicrobium marinum DSM 15272
Mycobacterium abscessus MAB_091912_2446
Mycobacterium abscessus MAB_082312_2258
Mycobacterium abscessus 47J26
Nocardioides sp. CF8
Gordonia polyisoprenivorans NBRC 16320
Gordonia araii NBRC 100433
Gordonia paraffinivorans NBRC 108238
Planctomyces maris DSM 8797
Amycolicicoccus subflavus (strain DSM 45089/
Candidatus Microthrix parvicella RN1
Gordonia paraffinivorans NBRC 108238
Nocardioides sp. CF8
Mycobacterium chubuense (strain NBB4)
Gordonia polyisoprenivorans (strain DSM 44266/
Aeromicrobium marinum DSM 15272
Gordonia rubripertincta NBRC 101908
Gordonia namibiensis NBRC 108229
Gordonia sp. KTR9
Gordonia terrae NBRC 100016
Gordonia alkanivorans NBRC 16433
Gordonia alkanivorans
Gordonia sp. TF6
Alcanivorax borkumensis (strain SK2/ATCC
Gordonia malaquae NBRC 108250
Oceanicola batsensis HTCC2597
Sphingobium baderi LL03
Erythrobacter litoralis (strain HTCC2594)
Erythrobacter sp. SD-21
Novosphingobium nitrogenifigens DSM 19370
Sphingopyxis macrogoltabida (Sphingomonas
macrogoltabidus)
Sphingopyxis alaskensis (strain DSM 13593/
Sphingopyxis macrogoltabida (Sphingomonas
macrogoltabidus)
Novosphingobium aromaticivorans (strain DSM 12444)
Dickeya dadantii (strain Ech586)
Sphingopyxis sp. MC1
Dietzia sp. D5
Sphingobium indicum B90A
Sphingobium chinhatense IP26
Sphingobium sp. HDIP04
Erythrobacter sp. NAP1
Dickeya dadantii (strain 3937) (Erwinia
chrysanthemi (strain 3937))
Sphingomonas sanxanigenens DSM 19645 = NX02
Sphingopyxis sp. MC1
Dickeya sp. D s0432-1
Novosphingobium aromaticivorans (strain DSM 12444)
Erythrobacter litoralis (strain HTCC2594)
Parvibaculum lavamentivorans (strain DS-1/
Novosphingobium pentaromativorans US6-1
In some embodiments, the disclosure provide methods for synthesizing olefinic alcohol products as described above, wherein the enzyme is selected from AlkB, AlkB P1, and AlkB1 AB. In some embodiments, the enzyme is selected from CYP153 M. sp; CYP153A M. aq; CYP153A M. aq. (G307A); Cyp153A M. aq. (G307A)-CPRBM3; Cyp153A P.sp.-CPRBM3; CYP153A13N2; CYP153A13N3; CYP153A13P2; and CYP153A7. In some embodiments, the enzyme is selected from CYP52A13 and CYP52A3.
In a related aspect, the disclosure provides a whole cell catalyst comprising an enzyme capable of selectively hydroxylating one terminal carbon of an unsaturated or saturated hydrocarbon substrate. In some embodiments, the cell is a microbial cell. In some embodiments, the enzyme is selected from the group consisting of a non-heme diiron monooxygenase, a long-chain alkane hydroxylase, a cytochrome P450, and combinations thereof. In some embodiments, the enzyme is selected from Table 4, Table 5, Table 6, or a variant thereof having at least 90% identity thereto.
E. coli K12 GEc137
E. coli W3110
E. coli W3110
E. coli BL21(DE3)
E. coli BL21(DE3)
E. coli JM109
E. coli HMS174
E. coli HMS174
E. coli HMS174
C. tropicalis DP522
C. tropicalis DP526
C. tropicalis DP428
The methods of the disclosure allow for the production of terminal alcohols with controlled regioselectivity, while disfavoring the formation of unwanted species such as epoxides or elimination products. The stereochemistry of an olefinic alcohol product will depend on factors including the structure of the particular olefinic substrate used in a particular reaction, as well as the identity of the enzyme. The methods of the disclosure can be conducted with enzymes that are selective for particular substrates (e.g., cis or Z alkenes vs. trans or E alkenes), as well as with enzymes that demonstrate terminal selectivity (e.g., hydroxylation of one end of an asymmetric alkene vs. the other end of the asymmetric alkene).
In certain instances, a hydroxylase enzyme will exhibit catalytic efficiency with one isomer of an internal alkene (e.g., the cis or Z isomer of an internal alkene) that is greater than the catalytic efficiency exhibited with the other isomer of the same internal alkene (e.g., the trans or E isomer of an internal alkene). In some embodiments, the disclosure provides methods wherein the catalytic efficiency of the hydroxylase enzyme is at least about 2-fold greater with one isomer of an internal alkene than with the other isomer of the internal alkene. The catalytic efficiency exhibited by a hydroxylase with one isomer of an internal alkene can be, for example, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or at least about 500-fold greater than the catalytic efficiency exhibited by the hydroxylase with the other isomer of the internal alkene.
A particular enzyme can therefore produce Z product over E product from a mixture of Z and E isomeric substrates or enrich the Z product over the E product. In certain embodiments, the disclosure provides methods for preparing olefinic alcohol products wherein the Z:E (cis:trans) isomeric ratio of the olefinic alcohol product is different from the Z:E (cis:trans) isomeric ratio of the olefinic substrate. The Z:E isomeric ratio of the olefinic alcohol product can be, for example, around 2 times greater than the Z:E isomeric ratio of the olefinic substrate. The Z:E isomeric ratio of the olefinic alcohol product can be, for example, around 1.25 times, 1.5 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, or 40 times greater than the Z:E isomeric ratio of the olefinic substrate.
In some embodiments, the disclosure provides methods for preparing olefinic alcohol products wherein the E:Z (trans:cis) isomeric ratio of the olefinic alcohol product is different from the E:Z (trans:cis) isomeric ratio of the olefinic substrate. The E:Z isomeric ratio of the olefinic alcohol product can be, for example, around 2 times greater than the E:Z isomeric ratio of the olefinic substrate. The E:Z isomeric ratio of the olefinic alcohol product can be, for example, around 1.25 times, 1.5 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, or 40 times greater than the E:Z isomeric ratio of the olefinic substrate.
In some embodiments, the Z:E isomeric ratio of the olefinic alcohol is about 1.25 times greater than the Z:E isomeric ratio of the olefinic substrate. In some embodiments, the E:Z isomeric ratio of the olefinic alcohol is about 1.25 times greater than the E:Z isomeric ratio of the olefinic substrate.
In certain instances, the biohydroxylation reactions in the methods of the disclosure have the potential to form a mixture of two or more products from the same substrate. When an olefinic substrate is asymmetric, for example, hydroxylation of one end/terminus of the substrate leads to one product while hydroxylation of the other end/terminus of the substrate leads to a different product. A reaction could therefore result in a mixture of two olefinic alcohol products. The terminal isomer ratio of an asymmetric olefinic alcohol product can range from about 1:99 to about 99:1. The terminal isomer ratio can be, for example, from about 1:99 to about 1:75, or from about 1:75 to about 1:50, or from about 1:50 to about 1:25, or from about 99:1 to about 75:1, or from about 75:1 to about 50:1, or from about 50:1 to about 25:1. The terminal isomer ratio can be from about 1:80 to about 1:20, or from about 1:60 to about 1:40, or from about 80:1 to about 20:1 or from about 60:1 to about 40:1. The terminal isomer ratio can be about 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, or about 1:95. The terminal isomer ratio can be about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, or about 95:1.
The distribution of a product mixture can be expressed as a regioselectivity percentage (“regioselectivity %”). Taking the reaction in
In some embodiments, the regioselectivity % is at least about 60%. In some embodiments, the regioselectivity % is at least about 60% and the Z:E isomeric ratio of the olefinic alcohol is about 1.25 times greater than the Z:E isomeric ratio of the olefinic substrate.
In certain instances, varying levels of olefin epoxidation will occur during the biohydroxylation reactions used in the methods of the disclosure. See, e.g., Scheme 7. Epoxidation of terminal alkenes, in particular, can occur when certain hydroxylase enzymes are used. It is often desirable to minimize such epoxidation or avoid the formation of epoxides altogether. Typically, methods of the disclosure are conducted with hydroxylase enzymes that produce product mixtures with alcohol product:epoxide ratios of at least 1:1. The alcohol product:epoxide ratio can range from about 1:1 to about 99:1. The alcohol:epoxide ratio can be, for example, from about 99:1 to about 75:1, or from about 75:1 to about 50:1, or from about 50:1 to about 25:1. The alcohol:epoxide ratio can be from about 80:1 to about 20:1 or from about 60:1 to about 40:1. The alcohol:epoxide ratio can be about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, or about 95:1.
In some embodiments, methods are conducted using an enzyme that produces an olefinic alcohol product:epoxide product ratio of greater than 1:1. In some embodiments, the enzyme produces an olefinic alcohol product:epoxide product ratio of greater than 2:1.
The distribution of a product mixture can be expressed as a percent selectivity for hydroxylation vs. epoxidation. Taking the reaction in Scheme 7a as a non-limiting example, the percent selectivity for hydroxylation vs. epoxidation of a terminal alkene can be calculated using the formula: selectivity %=[(χH)/(χH+χE)]×100%, wherein χH is the mole fraction for the hydroxylation product (i.e., the terminal olefinic alcohol) and wherein χE is the mole fraction for the epoxidation product (i.e., the terminal epoxide). In general, the percent selectivity for hydroxylation vs. epoxidation ranges from about 1% to about 99%. The percent selectivity for hydroxylation vs. epoxidation can be from about 1% to about 99%, or from about 20% to about 80%, or from about 40% to about 60%, or from about 1% to about 25%, or from about 25% to about 50%, or from about 50% to about 75%. The percent selectivity for hydroxylation vs. epoxidation can be about 5% 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
When halogen-substituted substrates are used in the methods of the disclosure, varying levels of dehalogenation can occur during hydroxylation. Dehalogenation typically results in the formation of aldehyde byproduct. Preferably, dehalogenation is minimized or avoided during the hydroxylation reactions. Typically, methods of the disclosure are conducted with hydroxylase enzymes that produce product mixtures with alcohol:aldehyde ratios of at least 1:1. The alcohol:aldehyde ratio of the product can range from about 1:1 to about 99:1. The alcohol:aldehyde ratio can be, for example, from about 99:1 to about 75:1, or from about 75:1 to about 50:1, or from about 50:1 to about 25:1. The alcohol:aldehyde ratio can be from about 80:1 to about 20:1 or from about 60:1 to about 40:1. The alcohol:aldehyde ratio can be about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, or about 95:1.
The distribution of a product mixture can be expressed as a percent selectivity for hydroxylation vs. dehalogenation. The percent selectivity for hydroxylation vs. dehalogenation of a halogen-substituted substrate can be calculated using the formula: selectivity %=[(χH)/(χH+χA)]×100%, wherein χH is the mole fraction for the hydroxylation product and wherein χA is the mole fraction for the aldehyde product. In general, the percent selectivity for hydroxylation vs. dehalogenation ranges from about 1% to about 99%. The percent selectivity for hydroxylation vs. dehalogenation can be from about 1% to about 99%, or from about 20% to about 80%, or from about 40% to about 60%, or from about 1% to about 25%, or from about 25% to about 50%, or from about 50% to about 75%. The percent selectivity for hydroxylation vs. dehalogenation can be about 5% 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
Biohydroxylation of Subterminal Carbon
In some embodiments, biohydroxylation can occur on an subterminal carbon as shown in Scheme 8. Accordingly, in some embodiments, the disclosure provides for isomers of a sex pheromone which include an subterminal alcohol functional group, an subterminal acetyl, or an subterminal ketone, provided, however, that the subterminal ketone is not located on the same carbon that forms a double bond with an adjacent carbon.
In some embodiments, for example, n is 0, m is 1, i is 3; or n is 1, m is 1, and i is 2; or n is 2, m is 1, and i is 1; or n is 0, m is 2, i is 2; or n is 1, m is 2, and i is 1; or n is 0, m is 3, i is 1; and positional isomers thereof.
In some embodiments, for example, n is 0, m is 1, and i is 4; or n is 1, m is 1; and i is 3; or n is 2, m is 1, and i is 2; or n is 3, m is 1, and i is 1; n is 0, m is 2, i is 3; or n is 1, m is 2, and i is 2; or n is 2, m is 2, and i is 1; n is 0, nm is 3, i is 2; or n is 1, m is 3, and i is 1; n is 0, m is 4, i is 1; and positional isomers thereof.
In some embodiments, for example, n is 0, m is 1, and i is 5; or n is 1, m is 1, and i is 4; or n is 2, m is 1, and i is 3; or n is 3, m is 1, i is 2; or n is 4, m is 1, and i is 1; or n is 0, m is 2, and i is 4; or n is 1, m is 2, and i is 3; or n is 2, m is 2, and i is 2; or n is 3, m is 2; and i is 1; or n is 0, m is 3, and i is 3; or n is 1, n is 3 and i is 2; or n is 2, m is 3, and i is 1; or n is 0, m is 4, and i is 2; or n is 1, m is 4 and i is 1; or n is 0, m is 5, and i is 1; and positional isomers thereof.
In some embodiments, for example, n is 0, m is 1 and i is 6; or n is 1, m is 1, and i is 5; or n is 2, m is 1, and i is 4; or n is 3, m is 1, and i is 3; or n is 4, m is 1; and i is 2; or n is 5, m is 1, and i is 1; or n is 0, m is 2 and i is 5; or n is 1, m is 2, i and is 4; or n is 2, m is 2, and i is 3; or n is 3, m is 2, and i is 2; or n is 4, m is 2, and i is 1; or n is 0, m is 3 and i is 4; or n is 1, m is 3, i is 3; or n is 2, m is 3, and i is 2; or n is 3, m is 3, and i is 1; or n is 0, m is 4 and i is 3; or n is 1, m is 4, i is 2; or n is 2, m is 4, and i is 1; or n is 0, m is 5 and i is 2; or n is 1, m is 5, i is 1; or n is 0, m is 6, and i is 1; and positional isomers thereof.
In some embodiments, for example, n is 0, m is 1, and i is 7; or n is 1 and m is 1, and i is 6; or n is 2, m is 1, and i is 5; or n is 3, m is 1, and i is 4; or n is 4, m is 1, and i is 3; or n is 5, m is 1, and i is 2; or n is 6, m is 1; and i is 1; n is 0, m is 2, and i is 6; or n is 1 and m is 2, and i is 5; or n is 2, m is 2, and i is 4; or n is 3, m is 2, and i is 3; or n is 4, m is 2, and i is 2; or n is 5, m is 2, and i is 1; n is 0, m is 3, and i is 5; or n is 1, and m is 3, and i is 4; or n is 2, m is 3, and i is 3; or n is 3, m is 3, and i is 2; or n is 4, m is 3, and i is 1; n is 0, m is 4, and i is 4; or n is 1 and m is 4, and i is 3; or n is 2, m is 4, and i is 2; or n is 3, m is 4, and i is 1; n is 0, m is 5, and i is 3; or n is 1 and m is 5, and i is 2; or n is 2, m is 5, and i is 1; n is 0, m is 6, and i is 2; or n is 1 and m is 6, and i is 1; n is 0, m is 7, and i is 1; and positional isomers thereof.
In some embodiments, for example, n is 0, m is 1; and i is 8; or n is 1, m is 1, and i is 7; or n is 2, m is 1, i is 6; or n is 3, m is 1, i is 5; or n is 4, m is 1, i is 4; or n is 5, m is 1, i is 3; or n is 6, m is 1, i is 2 or n is 7, m is 1, and i is 1; n is 0, m is 2; and i is 7; or n is 1, m is 2, and i is 6; or n is 2, m is 2, i is 5; or n is 3, m is 2, i is 4; or n is 4, m is 2, i is 3; or n is 5, m is 2, i is 2; or n is 6, m is 2, i is 1; n is 0, m is 3; and i is 6; or n is 1, m is 3, and i is 5; or n is 2, m is 3, i is 4; or n is 3, m is 3, i is 3; or n is 4, m is 3, i is 2; or n is 5, m is 3, i is 1; n is 0, m is 4; and i is 5; or n is 1, m is 4, and i is 4; or n is 2, m is 4, i is 3; or n is 3, m is 4, i is 2; or n is 4, m is 4, i is 1; n is 0, m is 5; and i is 4; or n is 1, m is 5, and i is 3; or n is 2, m is 5, i is 2; or n is 3, m is 5, i is 1; n is 0, m is 6; and i is 3; or n is 1, m is 6, and i is 2; or n is 2, m is 6, i is 1; n is 0, m is 7; and i is 2; or n is 1, m is 7, and i is 1; or n is 0, m is 8, i is 1; and positional isomers thereof.
In some embodiments, for example, n is 0, m is 1, and i is 9; or n is 1, m is 1, i is 8; or n is 2, m is 1, and i is 7; or n is 3, m is 1, and i is 6; or n is 4, m is 1, and i is 5; or n is 5, m is 1, and i is 4; or n is 6, m is 1, and i is 3; or n is 7, m is 1, and i is 2; or n is 8, m is 1 and i is 1; n is 0, m is 2, and i is 8; or n is 1, m is 2, i is 7; or n is 2, m is 2, and i is 6; or n is 3, m is 2, and i is 5; or n is 4, m is 2, and i is 4; or n is 5, m is 2, and i is 3; or n is 6, m is 2, and i is 2; or n is 7, m is 2, and i is 1; n is 0, m is 3, and i is 7; or n is 1, m is 3, i is 6; or n is 2, m is 3, and i is 5; or n is 3, m is 3, and i is 4; or n is 4, m is 3, and i is 3; or n is 5, m is 3, and i is 2; or n is 6, m is 3, and i is 1; n is 0, m is 4, and i is 6; or n is 1, m is 4, i is 5; or n is 2, m is 4, and i is 4; or n is 3, m is 4, and i is 3; or n is 4, m is 4, and i is 2; or n is 5, m is 4, and i is 1; n is 0, m is 5, and i is 5; or n is 1, m is 5, i is 4; or n is 2, m is 5, and i is 3; or n is 3, m is 5, and i is 2; or n is 4, m is 5, and i is 1; n is 0, m is 6, and i is 4; or n is 1, m is 6, i is 3; or n is 2, m is 6, and i is 2; or n is 3, m is 6, and i is 1; n is 0, m is 7, and i is 3; or n is 1, m is 7, i is 2; or n is 2, m is 7, and i is 1; n is 0, m is 8, and i is 2; or n is 1, m is 8, i is 1; n is 0, n is 9, and i is 1; and positional isomers thereof.
In some embodiments, for example, n is 0, m is 1, and i is 10; or n is 1, m is 1, and i is 9; or n is 2, m is 1, and i is 8; or n is 3, m is 1, and i is 7; or n is 4, m is 1, and i is 6; or n is 5, n is 1, i is 5; or n is 6, m is 1, and i is 4; or n is 7, m is 1 and i is 3; or n is 8, m is 1, i is 2; or n is 9, m is 1, and i is 1; or n is 0, m is 2, and i is 9; or n is 1, m is 2, and i is 8; or n is 2, m is 2, and i is 7; or n is 3, m is 2, and i is 6; or n is 4, m is 2, and i is 5; or n is 5, m is 2, i is 4; or n is 6, m is 2, and i is 3; or n is 7, m is 2 and i is 2; or n is 8, m is 2, i is 1; n is 0, m is 3, and i is 8; or n is 1, m is 3, and i is 7; or n is 2, m is 3, and i is 6; or n is 3, m is 3, and i is 5; or n is 4, m is 3, and i is 4; or n is 5, m is 3, i is 3; or n is 6, m is 3, and i is 2; or n is 7, m is 3 and i is 1; n is 0, m is 4, and i is 7; or n is 1, m is 4, and i is 6; or n is 2, m is 4, and i is 5; or n is 3, m is 4, and i is 4; or n is 4, m is 4, and i is 3; or n is 5, m is 4, i is 2; or n is 6, m is 4, and i is 1; n is 0, m is 5, and i is 6; or n is 1, m is 5, and i is 5; or n is 2, m is 5, and i is 4; or n is 3, m is 5, and i is 3; or n is 4, m is 5, and i is 2; or n is 5, m is 5, i is 1; n is 0, m is 6, and i is 5; or n is 1, m is 6, and i is 4; or n is 2, m is 6, and i is 3; or n is 3, m is 6, and i is 2; or n is 4, m is 6, and i is 1; n is 0, m is 7, and i is 4; or n is 1, m is 7, and i is 3; or n is 2, m is 7, and i is 2; or n is 3, m is 7, and i is 1; n is 0, m is 8, and i is 3; or n is 1, m is 8, and i is 2; or n is 2, m is 8, and i is 1; n is 0, m is 7, and i is 4; or n is 1, m is 7, and i is 3; or n is 2, m is 7, and i is 2; or n is 3, m is 7, and i is 1; n is 0, m is 9, and i is 2; or n is 1, m is 9, and i is 1; or n is 0, m is 10, and i is 1; and positional isomers thereof.
In some embodiments, for example, n is 0, m is 1, and i is 11; or n is 1, m is 1, and i is 10; or n is 2, m is 1, and i is 9; or n is 3, m is 1, and i is 8; or n is 4, m is 1, and i is 7; or n is 5, m is 1, and i is 6; or n is 6, m is 1 and i is 5; or n is 7, m is 1 and i is 4; or n is 8, m is 1, and i is 3; or n is 9, m is 1, and i is 2; or n is 10, m is 1 and i is 1; n is 0, m is 2, and i is 10; or n is 1, m is 2, and i is 9; or n is 2, m is 2, and i is 8; or n is 3, m is 2, and i is 7; or n is 4, m is 2, and i is 6; or n is 5, m is 2, and i is 5; or n is 6, m is 2 and i is 4; or n is 7, m is 2 and i is 3; or n is 8, m is 2, and i is 2; or n is 9, m is 2 and i is 1; n is 0, m is 3, and i is 9; or n is 1, m is 3, and i is 8; or n is 2, m is 3, and i is 7; or n is 3, m is 3, and i is 6; or n is 4, m is 3, and i is 5; or n is 5, m is 3, and i is 4; or n is 6, m is 3 and i is 3; or n is 7, m is 3 and i is 2; or n is 8, m is 3, and i is 1; n is 0, m is 4, and i is 8; or n is 1, m is 4, and i is 7; or n is 2, m is 4, and i is 6; or n is 3, m is 4, and i is 5; or n is 4, m is 4, and i is 4; or n is 5, m is 4, and i is 3; or n is 6, m is 4 and i is 2; or n is 7, m is 4 and i is 1; n is 0, m is 5, and i is 7; or n is 1, m is 5, and i is 6; or n is 2, m is 5 and i is 5; or n is 3, m is 5, and i is 4; or n is 4, m is 5, and i is 3; or n is 5, m is 5, and i is 2; or n is 6, m is 5 and i is 1; n is 0, m is 6, and i is 6; or n is 1, m is 6, and i is 5; or n is 2, m is 6 and i is 4; or n is 3, m is 6, and i is 3; or n is 4, m is 6, and i is 2; or n is 5, m is 6, and i is 1; n is 0, m is 7, and i is 5; or n is 1, m is 7, and i is 4; or n is 2, m is 7 and i is 3; or n is 3, m is 7, and i is 2; or n is 4, m is 7, and i is 1; n is 0, m is 8, and i is 4; or n is 1, m is 8, and i is 3; or n is 2, m is 8 and i is 2; or n is 3, m is 8, and i is 1; n is 0, m is 9, and i is 3; or n is 1, m is 9, and i is 2; or n is 2, m is 9 and i is 1; n is 0, m is 10, and i is 2; or n is 1, m is 10, and i is 1; or n is 0, m is 11 and i is 1; and positional isomers thereof.
In some embodiments, for example, n is 0, m is 1, and i is 12; or n is 1, m is 1, and i is 11; or n is 2, m is 1, and i is 10; or n is 3, m is 1, and i is 9; or n is 4, m is 1, and i is 8; or n is 5, m is 1, and i is 7; or n is 6, m is 1, and i is 6; or n is 7, m is 1, and i is 5; or n is 8, m is 1, and i is 4; or n is 9, m is 1, and i is 3; or n is 10, m is 1, and i is 2; or n is 11, m is 1 and i is 1; n is 0, m is 2, and i is 11; or n is 1, m is 2, and i is 10; or n is 2, m is 2, and i is 9; or n is 3, m is 2, and i is 8; or n is 4, m is 2, and i is 7; or n is 5, m is 2, and i is 6; or n is 6, m is 2, and i is 5; or n is 7, m is 2, and i is 4; or n is 8, m is 2, and i is 3; or n is 9, m is 2, and i is 2; or n is 10, m is 2, and i is 1; n is 0, m is 2, and i is 11; or n is 1, m is 2, and i is 10; or n is 2, m is 2, and i is 9; or n is 3, m is 2, and i is 8; or n is 4, m is 2, and i is 7; or n is 5, m is 2, and i is 6; or n is 6, m is 2, and i is 5; or n is 7, m is 2, and i is 4; or n is 8, m is 2, and i is 3; or n is 9, m is 2, and i is 2; or n is 10, m is 2, and i is 1; n is 0, m is 3, and i is 10; or n is 1, m is 3, and i is 9; or n is 2, m is 3, and i is 8; or n is 3, m is 3, and i is 7; or n is 4, m is 3, and i is 6, or n is 5, m is 3, and i is 5; or n is 6, m is 3, and i is 4; or n is 7, m is 3, and i is 3; or n is 8, m is 3, and i is 2; or n is 9, m is 3, and i is 1; n is 0, m is 4, and i is 9; or n is 1, m is 4, and i is 8; or n is 2, m is 4, and i is 7; or n is 3, m is 4, and i is 6; or n is 4, m is 4, and i is 5, or n is 5, m is 4, and i is 4; or n is 6, m is 4, and i is 3; or n is 7, m is 4, and i is 2; or n is 8, m is 4, and i is 1; n is 0, m is 5, and i is 8; or n is 1, m is 5, and i is 7; or n is 2, m is 5, and i is 6; or n is 3, m is 5, and i is 7; or n is 4, m is 5, and i is 4, or n is 5, m is 5, and i is 3; or n is 6, m is 5, and i is 2; or n is 7, m is 5, and i is 1; n is 0, m is 6, and i is 7; or n is 1, m is 6, and i is 6; or n is 2, m is 6, and i is 5; or n is 3, m is 6, and i is 4; or n is 4, m is 6, and i is 3, or n is 5, m is 6, and i is 2; or n is 6, m is 6, and i is 1; n is 0, m is 7, and i is 6; or n is 1, m is 7, and i is 5; or n is 2, m is 7, and i is 4; or n is 3, m is 7, and i is 3; or n is 4, m is 7, and i is 2, or n is 5, m is 7, and i is 1; n is 0, m is 8, and i is 5; or n is 1, m is 8, and i is 4; or n is 2, m is 8, and i is 3; or n is 3, m is 8, and i is 2; or n is 4, m is 8, and i is 1; n is 0, m is 9, and i is 4; or n is 1, m is 9, and i is 3; or n is 2, m is 9, and i is 2; or n is 3, m is 8, and i is 1; n is 0, m is 10, and i is 3; or n is 1, m is 10, and i is 2; or n is 2, m is 10, and i is 1; n is 0, m is 11, and i is 2; or n is 1, m is 11, and i is 1; or n is 0, m is 12, and i is 1; and positional isomers thereof.
In some embodiments, for example, n is 0, m is 1, and i is 13; or n is 1, m is 1, and i is 12; or n is 2, m is 1, and i is 11; or n is 3, m is 1, and i is 10; or n is 4, m is 1, and i is 9; or n is 5, m is 1, and i is 8; or n is 6, m is 1, and i is 7; or n is 8, m is 1, and i is 5; or n is 9, m is 1, and i is 4; or n is 10, m is 1, and i is 3; or n is 11, m is 1, and i is 2; or n is 12, m is 1, and i is 1; n is 0, m is 2, and i is 12; or n is 1, m is 2, and i is 11; or n is 2, m is 2, and i is 10; or n is 3, m is 2, and i is 9; or n is 4, m is 2, and i is 8; or n is 5, m is 2, and i is 7; or n is 6, m is 2, and i is 6; or n is 7, m is 2, and i is 5, n is 8, m is 2, and i is 4; or n is 9, m is 2, and i is 3; or n is 10, m is 2, and i is 2; or n is 11, m is 2, and i is 1; n is 0, m is 3, and i is 11; or n is 1, m is 3, and i is 10; or n is 2, m is 3, and i is 9; or n is 3, m is 3, and i is 8; or n is 4, m is 3, and i is 7; or n is 5, m is 3, and i is 6; or n is 6, m is 3, and i is 5; or n is 7, m is 3, and i is 4; or n is 8, m is 3, and i is 3; or n is 9, m is 3, and i is 2; or n is 10, m is 3, and i is 1; n is 0, m is 4, and i is 10; or n is 1, m is 4, and i is 9; or n is 2, m is 4, and i is 8; or n is 3, m is 4, and i is 7; or n is 4, m is 4, and i is 6; or n is 5, m is 4, and i is 5; or n is 6, m is 4, and i is 4; or n is 7, m is 4, and i is 3; or n is 8, m is 4, and i is 2; or n is 9, m is 4, and i is 1; n is 0, m is 5, and i is 9; or n is 1, m is 5, and i is 8; or n is 2, m is 5, and i is 7; or n is 3, m is 5, and i is 6; or n is 4, m is 5, and i is 5; or n is 5, m is 5, and i is 4; or n is 6, m is 5, and i is 3; or n is 7, m is 5, and i is 2; or n is 8, m is 5, and i is 1; n is 0, m is 6, and i is 8; or n is 1, m is 6, and i is 7; or n is 2, m is 6, and i is 6; or n is 3, m is 6, and i is 5; or n is 4, m is 6, and i is 4; or n is 5, m is 6, and i is 3; or n is 6, m is 6, and i is 2; or n is 7, m is 6, and i is 1; n is 0, m is 7, and i is 7; or n is 1, m is 7, and i is 6; or n is 2, m is 7, and i is 5; or n is 3, m is 7, and i is 4; or n is 4, m is 7, and i is 3; or n is 5, m is 7, and i is 2; or n is 6, m is 7, and i is 1; n is 0, m is 8, and i is 6; or n is 1, m is 8, and i is 5; or n is 2, m is 8, and i is 4; or n is 3, m is 8, and i is 3; or n is 4, m is 8, and i is 2; or n is 5, m is 8, and i is 1; n is 0, m is 9, and i is 5; or n is 1, m is 9, and i is 4; or n is 2, m is 9, and i is 3; or n is 3, m is 9, and i is 2; or n is 4, m is 9, and i is 1; n is 0, m is 10, and i is 4; or n is 1, m is 10, and i is 3; or n is 2, m is 10, and i is 2; or n is 3, m is 10, and i is 1; n is 0, m is 11, and i is 3; or n is 1, m is 11, and i is 2; or n is 2, m is 11, and i is 1; n is 0, m is 12, and i is 2; or n is 1, m is 12, and i is 1; or n is 0, m is 13, and i is 1; and positional isomers thereof.
In some embodiments, for example, n is 0, m is 1, and i is 14; or n is 1, m is 1, and i is 13; or n is 2, m is 1, and i is 12; or n is 3, m is 1, or i is 11; or n is 4, m is 1, and i is 10; or n is 5, m is 1, and i is 9; or n is 6, m is 1, and i is 8; or n is 7, m is 1, and i is 7; or n is 8, m is 1, and i is 6; or n is 9, m is 1, and i is 5; or n is 10, m is 1, and i is 4; or n is 11, m is 1 and i is 3; or n is 12, m is 1, and i is 2; or n is 13 and m is 1, and i is 1; n is 0, m is 2, and i is 13; or n is 1, m is 2, and i is 12; or n is 2, m is 2, and i is 11; or n is 3, m is 2, or i is 10; or n is 4, m is 2, and i is 9; or n is 5, m is 2, and i is 8; or n is 6, m is 2, and i is 7; or n is 7, m is 2, and i is 6; or n is 8, m is 2, and i is 5; or n is 9, m is 2, and i is 4; or n is 10, m is 2, and i is 3; or n is 11, m is 2 and i is 2; n is 12, m is 2 and i is 1; or n is 0, m is 3, and i is 12; or n is 1, m is 3, and i is 11; or n is 2, m is 3, and i is 10; or n is 3, m is 3, or i is 9; or n is 4, m is 3, and i is 8; or n is 5, m is 3, and i is 7; or n is 6, m is 3, and i is 6; or n is 7, m is 3, and i is 5; or n is 8, m is 3, and i is 4; or n is 9, m is 3, and i is 3; or n is 10, m is 3, and i is 2; or n is 11, m is 3 and i is 1; n is 0, m is 4, and i is 11; or n is 1, m is 4, and i is 10; or n is 2, m is 4, and i is 9; or n is 3, m is 4, or i is 8; or n is 4, m is 4, and i is 7; or n is 5, m is 4, and i is 6; or n is 6, m is 4, and i is 5; or n is 7, m is 4, and i is 4; or n is 8, m is 4, and i is 3; or n is 9, m is 4, and i is 2; or n is 10, m is 4, and i is 1; n is 0, m is 5, and i is 10; or n is 1, m is 5, and i is 9; or n is 2, m is 5, and i is 8; or n is 3, m is 5, or i is 7; or n is 4, m is 5, and i is 6; or n is 5, m is 5, and i is 5; or n is 6, m is 5, and i is 4; or n is 7, m is 5, and i is 3; or n is 8, m is 5, and i is 2; or n is 9, m is 5, and i is 1; n is 0, m is 6, and i is 9; or n is 1, m is 6, and i is 8; or n is 2, m is 6, and i is 7; or n is 3, m is 6, or i is 6; or n is 4, m is 6, and i is 5; or n is 5, m is 6, and i is 4; or n is 6, m is 6, and i is 3; or n is 7, m is 6, and i is 2; or n is 8, m is 6, and i is 1; n is 0, m is 7, and i is 8; or n is 1, m is 7, and i is 7; or n is 2, m is 7, and i is 6; or n is 3, m is 7, or i is 5; or n is 4, m is 7, and i is 4; or n is 5, m is 7, and i is 3; or n is 6, m is 7, and i is 2; or n is 7, m is 7, and i is 1; n is 0, m is 8, and i is 7; or n is 1, m is 8, and i is 6; or n is 2, m is 8, and i is 5; or n is 3, m is 8, or i is 4; or n is 4, m is 8, and i is 3; or n is 5, m is 8, and i is 2; or n is 6, m is 8, and i is 1; n is 0, m is 9, and i is 6; or n is 1, m is 9, and i is 5; or n is 2, m is 9, and i is 4; or n is 3, m is 9, or i is 3; or n is 4, m is 9, and i is 2; or n is 5, m is 9, and i is 1; n is 0, m is 10, and i is 5; or n is 1, m is 10, and i is 4; or n is 2, m is 10, and i is 3; or n is 3, m is 10, or i is 2; or n is 4, m is 10, and i is 1; n is 0, m is 11, and i is 4; or n is 1, m is 11, and i is 3; or n is 2, m is 11, and i is 2; or n is 3, m is 11, or i is 1; n is 0, m is 12, and i is 3; or n is 1, m is 12, and i is 2; or n is 2, m is 12, and i is 1; n is 0, m is 13, and i is 2; or n is 1, m is 13, and i is 1; n is 0, m is 14, and i is 1;
In some embodiments, for example, n is 0, m is 1, and i is 15; or n is 1, m is 1, and i is 14; or n is 2, m is 1 and i is 13; or n is 3, m is 1, and i is 12; or n is 4, m is 1, and i is 11; or n is 5, m is 1, and i is 10; or n is 6, m is 1 and i is 9; or n is 7, m is 1, and i is 8; or n is 9, m is 1, and i is 6; or n is 10, m is 1, and i is 5; or n is 11, m is 1, and i is 4; or n is 12, m is 1, and i is 3; or n is 13, m is 1, and i is 2; or n is 14, m is 1, and i is 1; n is 0, m is 2, and i is 14; or n is 1, m is 2, and i is 13; or n is 2, m is 2 and i is 12; or n is 3, m is 2, and i is 11; or n is 4, m is 2, and i is 10; or n is 5, m is 2, and i is 9; or n is 6, m is 2 and i is 8; or n is 7, m is 2, and i is 7; or n is 9, m is 2, and i is 5; or n is 10, m is 2, and i is 4; or n is 11, m is 2, and i is 3; or n is 12, m is 2, and i is 2; or n is 13, m is 1, and i is 1; n is 0, m is 3, and i is 13; or n is 1, m is 3, and i is 12; or n is 2, m is 3 and i is 11; or n is 3, m is 3, and i is 10; or n is 4, m is 3, and i is 9; or n is 5, m is 3, and i is 8; or n is 6, m is 3 and i is 7; or n is 7, m is 3, and i is 6; or n is 9, m is 3, and i is 4; or n is 10, m is 3, and i is 3; or n is 11, m is 3, and i is 2; or n is 12, m is 3, and i is 1; n is 0, m is 4, and i is 12; or n is 1, m is 4, and i is 11; or n is 2, m is 4 and i is 10; or n is 3, m is 4, and i is 9; or n is 4, m is 4, and i is 8; or n is 5, m is 4, and i is 7; or n is 6, m is 4 and i is 6; or n is 7, m is 4, and i is 5; or n is 8, m is 4, and i is 4; or n is 9, m is 4, and i is 3; or n is 10, m is 4, and i is 2; or n is 11, m is 4, and i is 1; n is 0, m is 5, and i is 11; or n is 1, m is 5, and i is 10; or n is 2, m is 5 and i is 9; or n is 3, m is 5, and i is 8; or n is 4, m is 5, and i is 7; or n is 5, m is 5, and i is 6; or n is 6, m is 5 and i is 5; or n is 7, m is 5, and i is 4; or n is 8, m is 5, and i is 3; or n is 9, m is 5, and i is 2; or n is 10, m is 5, and i is 1; n is 0, m is 6, and i is 10; or n is 1, m is 6, and i is 9; or n is 2, m is 6 and i is 8; or n is 3, m is 6, and i is 7; or n is 4, m is 6, and i is 6; or n is 5, m is 6, and i is 5; or n is 6, m is 6 and i is 4; or n is 7, m is 6, and i is 3; or n is 8, m is 6, and i is 2; or n is 9, m is 6, and i is 1; n is 0, m is 7, and i is 9; or n is 1, m is 7, and i is 8; or n is 2, m is 7 and i is 7; or n is 3, m is 7, and i is 6; or n is 4, m is 7, and i is 5; or n is 5, m is 7, and i is 4; or n is 6, m is 7 and i is 3; or n is 7, m is 7, and i is 2; or n is 8, m is 7, and i is 1; n is 0, m is 8, and i is 8; or n is 1, m is 8, and i is 7; or n is 2, m is 8 and i is 6; or n is 3, m is 8, and i is 5; or n is 4, m is 8, and i is 4; or n is 5, m is 8, and i is 3; or n is 6, m is 8 and i is 2; or n is 7, m is 8, and i is 1; n is 0, m is 9, and i is 7; or n is 1, m is 9, and i is 6; or n is 2, m is 9 and i is 5; or n is 3, m is 9, and i is 4; or n is 4, m is 9, and i is 3; or n is 5, m is 9, and i is 2; or n is 6, m is 9 and i is 1; n is 0, m is 10, and i is 6; or n is 1, m is 10, and i is 5; or n is 2, m is 10 and i is 4; or n is 3, m is 10, and i is 3; or n is 4, m is 10, and i is 2; or n is 5, m is 10, and i is 1; n is 0, m is 11, and i is 5; or n is 1, m is 11, and i is 4; or n is 2, m is 11 and i is 3; or n is 3, m is 11, and i is 2; or n is 4, m is 11, and i is 1; n is 0, m is 12, and i is 4; or n is 1, m is 12, and i is 3; or n is 2, m is 12 and i is 2; or n is 3, m is 12, and i is 1; n is 0, m is 13, and i is 3; or n is 1, m is 13, and i is 2; or n is 2, m is 13 and i is 1; n is 0, m is 14, and i is 2; or n is 1, m is 14, and i is 1; n is 0, m is 15, and i is 1; and positional isomers thereof.
In some embodiments, for example, n is 0, m is 1, and i is 16, or n is 1, m is 1, and i is 15; or n is 2, m is 1, and i is 14; or n is 3, m is 1, and i is 13; or n is 4, m is 1, and i is 12; or n is 5, m is 1 and i is 11; or n is 6, m is 1, and i is 10; or n is 7, m is 1, and i is 9; or n is 8, m is 1, and i is 8; or n is 9, m is 1, and i is 7; or n is 10, m is 1, and i is 6; or n is 11, m is 1, and i is 5; or n is 12, m is 1; and i is 4; or n is 13, m is 1, and i is 3; or n is 14, m is 1, and i is 2; or n is 15, m is 1 and i is 1; n is 0, m is 2, and i is 15, or n is 1, m is 2, and i is 14; or n is 2, m is 2, and i is 13; or n is 3, m is 2, and i is 12; or n is 4, m is 2, and i is 11; or n is 5, m is 2 and i is 10; or n is 6, m is 2, and i is 9; or n is 7, m is 2, and i is 8; or n is 8, m is 2, and i is 7; or n is 9, m is 2, and i is 6; or n is 10, m is 2, and i is 5; or n is 11, m is 2, and i is 4; or n is 12, m is 2; and i is 3; or n is 13, m is 2, and i is 2; or n is 14, m is 1, and i is 1; n is 0, m is 3, and i is 14, or n is 1, m is 3, and i is 13; or n is 2, m is 3, and i is 12; or n is 3, m is 3, and i is 11; or n is 4, m is 3, and i is 10; or n is 5, m is 3 and i is 9; or n is 6, m is 3, and i is 8; or n is 7, m is 3, and i is 7; or n is 8, m is 3, and i is 6; or n is 9, m is 3, and i is 5; or n is 10, m is 3, and i is 4; or n is 11, m is 3, and i is 3; or n is 12, m is 3; and i is 2; or n is 13, m is 3, and i is 1; n is 0, m is 4, and i is 13, or n is 1, m is 4, and i is 12; or n is 2, m is 4, and i is 11; or n is 3, m is 4, and i is 10; or n is 4, m is 4, and i is 9; or n is 5, m is 4 and i is 8; or n is 6, m is 4, and i is 7; or n is 7, m is 4, and i is 6; or n is 8, m is 4, and i is 5; or n is 9, m is 4, and i is 4; or n is 10, m is 4, and i is 3; or n is 11, m is 4, and i is 2; or n is 12, m is 4; and i is 1; n is 0, m is 5, and i is 12, or n is 1, m is 5, and i is 11; or n is 2, m is 5, and i is 10; or n is 3, m is 5, and i is 9; or n is 4, m is 5, and i is 8; or n is 5, m is 5 and i is 7; or n is 6, m is 5, and i is 6; or n is 7, m is 5, and i is 5; or n is 8, m is 5, and i is 4; or n is 9, m is 5, and i is 3; or n is 10, m is 5, and i is 2; or n is 11, m is 5, and i is 1; n is 0, m is 6, and i is 11, or n is 1, m is 6, and i is 10; or n is 2, m is 6, and i is 9; or n is 3, m is 6, and i is 8; or n is 4, m is 6, and i is 7; or n is 5, m is 6 and i is 6; or n is 6, m is 6, and i is 5; or n is 7, m is 6, and i is 4; or n is 8, m is 6, and i is 3; or n is 9, m is 6, and i is 2; or n is 10, m is 6, and i is 1; n is 0, m is 7, and i is 10, or n is 1, m is 7, and i is 9; or n is 2, m is 7, and i is 8; or n is 3, m is 7, and i is 7; or n is 4, m is 7, and i is 6; or n is 5, m is 7 and i is 5; or n is 6, m is 7, and i is 4; or n is 7, m is 7, and i is 3; or n is 8, m is 7, and i is 2; or n is 9, m is 7, and i is 1; n is 0, m is 8, and i is 9, or n is 1, m is 8, and i is 8; or n is 2, m is 8, and i is 7; or n is 3, m is 8, and i is 6; or n is 4, m is 8, and i is 5; or n is 5, m is 8 and i is 4; or n is 6, m is 8, and i is 3; or n is 7, m is 8, and i is 2; or n is 8, m is 8, and i is 1; n is 0, m is 9, and i is 8, or n is 1, m is 9, and i is 7; or n is 2, m is 9, and i is 6; or n is 3, m is 9, and i is 5; or n is 4, m is 9, and i is 4; or n is 5, m is 9 and i is 3; or n is 6, m is 9, and i is 2; or n is 7, m is 9, and i is 1; n is 0, m is 10, and i is 7, or n is 1, m is 10, and i is 6; or n is 2, m is 10, and i is 5; or n is 3, m is 10, and i is 4; or n is 4, m is 10, and i is 3; or n is 5, m is 10 and i is 2; or n is 6, m is 10, and i is 1; n is 0, m is 11, and i is 6, or n is 1, m is 11, and i is 5; or n is 2, m is 11, and i is 4; or n is 3, m is 11, and i is 3; or n is 4, m is 11, and i is 2; or n is 5, m is 11 and i is 1; n is 0, m is 12, and i is 5, or n is 1, m is 12, and i is 4; or n is 2, m is 12, and i is 3; or n is 3, m is 12, and i is 2; or n is 4, m is 12, and i is 1; n is 0, m is 13, and i is 4, or n is 1, m is 13, and i is 3; or n is 2, m is 13, and i is 2; or n is 3, m is 13, and i is 1; n is 0, m is 14, and i is 3, or n is 1, m is 14, and i is 2; or n is 2, m is 14, and i is 1; n is 0, m is 15, and i is 2, or n is 1, m is 15, and i is 1; or n is 0, m is 16, and i is 1; or positional isomers thereof.
Accordingly, some embodiments of the disclosure provide methods for preparing an olefinic alcohol product as described above, wherein the olefinic substrate is a metathesis product, and wherein the method includes: a) cross-metathesizing a first terminal olefin and a second different terminal olefin in the presence of a metathesis catalyst to form the metathesis product; and b) incubating the metathesis product with an enzyme capable of hydroxylating an subterminal carbon of the metathesis product to form an olefinic alcohol product.
In some embodiments, the first terminal olefin has the formula (CH2═CH)(CH2)mH, the second different terminal olefin has the formula (CH2═CH)(CH2)nH, the metathesis product has the formula H(CH2)m(CH═CH)(CH2)nH, the olefinic alcohol product has the formula H(CH2)iCHOH(CH2)m-i-1(CH═CH)(CH2)nH or H(CH2)m(CH═CH)(CH2)n-i-1CHOH(CH2)iH, and m, n and i are different integers between 1 and 17. In some embodiments, m, n, and i are different integers between 1 and 9.
The methods of the disclosure can also be conducted such that the biohydroxylation step is conducted prior to the metathesis step and/or other synthetic transformation steps. Accordingly, some embodiments of the disclosure provide methods wherein the olefinic substrate is a first terminal olefin, and wherein the method includes: a) incubating the first terminal olefin with an enzyme capable of hydroxylating an subterminal carbon of the terminal olefin to form an alkenol; and b) metathesizing the alkenol and a second terminal olefin in the presence of a metathesis catalyst to form the olefinic alcohol product.
The alcohol can be protected with a suitable protecting group if necessary. In some embodiments, the methods of the disclosure include: a) incubating the first terminal olefin with an enzyme capable of selectively hydroxylating an subterminal carbon of the terminal olefin to form an alkenol; b) protecting the alkenol to form a protected alkenol; c) metathesizing the protected alkenol and a second terminal olefin in the presence of a metathesis catalyst to form a protected olefinic alcohol product; and d) deprotecting the protected olefinic alcohol product to form the olefinic alcohol product.
Any suitable alcohol protecting group can be used in the methods of the disclosure. Such protecting groups are well known to one of ordinary skill in the art, including those that are disclosed in Protective Groups in Organic Synthesis, 4th edition, T. W. Greene and P. G. M. Wuts, John Wiley & Sons, New York, 2006, which is incorporated herein by reference in its entirety. In some embodiments, the α,ω-alkenol is protected via esterification and the should alkenol is protected via esterification with an acid selected from the group consisting of formate and acetate.
Synthesis of Terminal Alkenals
As indicated above, the alcohol moiety generated via hydroxylation can be further modified to generate alkenals or acetate esters.
Oxidation of Fatty Alcohols
Oxidation of fatty alcohols is often achieved via selective oxidation via pyridinium chlorochromate (PCC) (Scheme 9).
Alternatively, TEMPO (TEMPO=2,2,6,6-tetramethylpiperidinyl-N-oxyl) and related catalyst systems can be used to selectively oxidize alcohols to aldehydes. These methods are described in Ryland and Stahl (2014), herein incorporated by reference in its entirety.
Bio-Oxidation of Terminal Alcohols
Many insect pheromones are fatty aldehydes or comprise a fatty aldehyde component. As such, the conversion of the fatty alcohol produced via terminal hydroxylation to the fatty aldehyde is required to produce certain pheromones. The conversion of a fatty alcohol to a fatty aldehyde is known to be catalyzed by alcohol dehydrogenases (ADH) and alcohol oxidases (AOX). Additionally, the conversion of a length Cn fatty acid to a Cn-1 fatty aldehyde is catalyzed by plant α-dioxygenases (α-DOX) (Scheme 10).
The present disclosure describes enzymes that oxidize fatty alcohols to fatty aldehydes.
In some embodiments, an alcohol oxidase (AOX) is used to catalyze the conversion of a fatty alcohol to a fatty aldehyde. Alcohol oxidases catalyze the conversion of alcohols into corresponding aldehydes (or ketones) with electron transfer via the use of molecular oxygen to form hydrogen peroxide as a by-product. AOX enzymes utilize flavin adenine dinucleotide (FAD) as an essential cofactor and regenerate with the help of oxygen in the reaction medium. Catalase enzymes may be coupled with the AOX to avoid accumulation of the hydrogen peroxide via catalytic conversion into water and oxygen.
Based on the substrate specificities, AOXs may be categorized into four groups: (a) short chain alcohol oxidase, (b) long chain alcohol oxidase, (c) aromatic alcohol oxidase, and (d) secondary alcohol oxidase (Goswami et al. 2013). Depending on the chain length of the desired substrate, some member of these four groups are better suited than others as candidates for evaluation.
Short chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.13, Table 7) catalyze the oxidation of lower chain length alcohol substrates in the range of C1-C8 carbons (van der Klei et al. 1991) (Ozimek et al. 2005). Aliphatic alcohol oxidases from methylotrophic yeasts such as Candida boidinii and Komagataella pastoris (formerly Pichia pastoris) catalyze the oxidation of primary alkanols to the corresponding aldehydes with a preference for unbranched short-chain aliphatic alcohols. The most broad substrate specificity is found for alcohol oxidase from the Pichia pastoris including propargyl alcohol, 2-chloroethanol, 2-cyanoethanol (Dienys et al. 2003). The major challenge encountered in alcohol oxidation is the high reactivity of the aldehyde product. Utilization of a two liquid phase system (water/solvent) can provide in-situ removal of the aldehyde product from the reaction phase before it is further converted to the acid. For example, hexanal production from hexanol using Pichia pastoris alcohol oxidase coupled with bovine liver catalase was achieved in a bi-phasic system by taking advantage of the presence of a stable alcohol oxidase in aqueous phase (Karra-Chaabouni et al. 2003). For example, alcohol oxidase from Pichia pastoris was able to oxidize aliphatic alcohols of C6 to C11 when used biphasic organic reaction system (Murray and Duff 1990). Methods for using alcohol oxidases in a biphasic system according to (Karra-Chaabouni et al. 2003) and (Murray and Duff 1990) are incorporated by reference in their entirety.
Long chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.20; Table 8) include fatty alcohol oxidases, long chain fatty acid oxidases, and long chain fatty alcohol oxidases that oxidize alcohol substrates with carbon chain length of greater than six (Goswami et al. 2013). Banthorpe et al. reported a long chain alcohol oxidase purified from the leaves of Tanacetum vulgare that was able to oxidize saturated and unsaturated long chain alcohol substrates including hex-trans-2-en-1-ol and octan-1-ol (Banthorpe 1976) (Cardemil 1978). Other plant species, including Simmondsia chinensis (Moreau, R. A., Huang 1979), Arabidopsis thaliana (Cheng et al. 2004), and Lotus japonicas (Zhao et al. 2008) have also been reported as sources of long chain alcohol oxidases. Fatty alcohol oxidases are mostly reported from yeast species (Hommel and Ratledge 1990) (Vanhanen et al. 2000) (Hommel et al. 1994) (Kemp et al. 1990) and these enzymes play an important role in long chain fatty acid metabolism (Cheng et al. 2005). Fatty alcohol oxidases from yeast species that degrade and grow on long chain alkanes and fatty acid catalyze the oxidation of fatty alcohols. Fatty alcohol oxidase from Candida tropicalis has been isolated as microsomal cell fractions and characterized for a range of substrates (Eirich et al. 2004) (Kemp et al. 1988) (Kemp et al. 1991) (Mauersberger et al. 1992). Significant activity is observed for primary alcohols of length C8 to C16 with reported KM in the 10-50 μM range (Eirich et al. 2004). Alcohol oxidases described may be used for the conversion of medium chain aliphatic alcohols to aldehydes as described, for example, for whole-cells Candida boidinii (Gabelman and Luzio 1997), and Pichia pastoris (Duff and Murray 1988) (Murray and Duff 1990). Long chain alcohol oxidases from filamentous fungi were produced during growth on hydrocarbon substrates (Kumar and Goswami 2006) (Savitha and Ratledge 1991). The long chain fatty alcohol oxidase (LjFAO1) from Lotus japonicas has been heterologously expressed in E. coli and exhibited broad substrate specificity for alcohol oxidation including 1-dodecanol and 1-hexadecanol (Zhao et al. 2008).
Komagataella pastoris (strain ATCC 76273/
Komagataella pastoris (strain GS115/ATCC
Komagataella pastoris (strain ATCC 76273/
Komagataella pastoris (strain GS115/ATCC
Candida boidinii (Yeast)
Pichia angusta (Yeast) (Hansenula polymorpha)
Thanatephorus cucumeris (strain AG1-IB/
Thanatephorus cucumeris (strain AG1-IB/
Thanatephorus cucumeris (strain AG1-IB/
Thanatephorus cucumeris (strain AG1-IB/
Thanatephorus cucumeris (strain AG1-IB/
Thanatephorus cucumeris (strain AG1-IB/
Thanatephorus cucumeris (strain AG1-IB/
Thanatephorus cucumeris (strain AG1-IB/
Thanatephorus cucumeris (strain AG1-IB/
Thanatephorus cucumeris (strain AG1-IB/
Ogataea henricii
Candida methanosorbosa
Candida methanolovescens
Candida succiphila
Aspergillus niger (strain CBS 513.88/FGSC
Aspergillus niger (strain CBS 513.88/FGSC
Moniliophthora perniciosa (Witches'-broom
Candida cariosilignicola
Candida pignaliae
Candida pignaliae
Candida sonorensis
Candida sonorensis
Pichia naganishii
Ogataea minuta
Ogataea philodendri
Ogataea wickerhamii
Kuraishia capsulata
Talaromyces stipitatus (strain ATCC 10500/
Talaromyces stipitatus (strain ATCC 10500/
Talaromyces stipitatus (strain ATCC 10500/
Talaromyces stipitatus (strain ATCC 10500/
Ogataea glucozyma
Ogataea parapolymorpha (strain DL-1/ATCC
polymorpha)
Gloeophyllum trabeum (Brown rot fungus)
Pichia angusta (Yeast) (Hansenula polymorpha)
Pichia trehalophila
Pichia angusta (Yeast) (Hansenula polymorpha)
Pichia angusta (Yeast) (Hansenula polymorpha)
Ixodes scapularis (Black-legged tick) (Deer
Lotus japonicus (Lotus corniculatus var.
japonicus)
Arabidopsis thaliana (Mouse-ear cress)
Lotus japonicus (Lotus corniculatus var.
japonicus)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Microbotryum violaceum (strain p1A1
violacea)
Ajellomyces dermatitidis ATCC 26199
Gibberella zeae (strain PH-1/ATCC
Pichia sorbitophila (strain ATCC MYA-
Emericella nidulans (strain FGSC A4/
Pyrenophora tritici-repentis (strain Pt-
Paracoccidioides lutzii (strain ATCC
brasiliensis)
Candida parapsilosis (strain CDC 317/
parapsilosis)
Pseudozyma brasiliensis (strain
Candida parapsilosis (strain CDC 317/
parapsilosis)
Sclerotinia borealis F-4157
Sordaria macrospora (strain ATCC
Sordaria macrospora (strain ATCC
Meyerozyma guilliermondii (strain
Trichophyton rubrum CBS 202.88
Arthrobotrys oligospora (strain ATCC
Scheffersomyces stipitis (strain ATCC
Scheffersomyces stipitis (strain ATCC
Aspergillus oryzae (strain 3.042)
Fusarium oxysporum (strain Fo5176)
Rhizopus delemar (strain RA 99-880/
Rhizopus delemar (strain RA 99-880/
Fusarium oxysporum (strain Fo5176)
Penicillium roqueforti
Aspergillus clavatus (strain ATCC 1007/
Arthroderma otae (strain ATCC MYA-
canis)
Trichophyton tonsurans (strain CBS
Colletotrichum higginsianum (strain IMI
Ajellomyces capsulatus (strain H143)
capsulatum)
Trichophyton rubrum (strain ATCC
Cochliobolus heterostrophus (strain C5/
Candida orthopsilosis (strain 90-125)
Candida orthopsilosis (strain 90-125)
Candida orthopsilosis (strain 90-125)
Pseudozyma aphidis DSM 70725
Coccidioides posadasii (strain C735)
Magnaporthe oryzae (strain P131) (Rice
Neurospora tetrasperma (strain FGSC
Hypocrea virens (strain Gv29-8/FGSC
Hypocrea virens (strain Gv29-8/FGSC
Aspergillus niger (strain CBS 513.88/
Verticillium dahliae (strain VdLs.17/
Ustilago maydis (strain 521/FGSC
Fusarium oxysporum f. sp. lycopersici
Fusarium oxysporum f. sp. lycopersici
Candida tropicalis (Yeast)
Magnaporthe oryzae (strain 70-15/
Candida tropicalis (Yeast)
Candida tropicalis (Yeast)
Phaeosphaeria nodorum (strain SN15/
nodorum)
Candida tropicalis (Yeast)
Pestalotiopsis fici W106-1
Magnaporthe oryzae (strain Y34) (Rice
Pseudogymnoascus destructans (strain
Pseudogymnoascus destructans (strain
Mycosphaerella fijiensis (strain
Bipolaris oryzae ATCC 44560
Cladophialophora psammophila CBS
Fusarium oxysporum f. sp. melonis
Fusarium oxysporum f. sp. melonis
Cyphellophora europaea CBS 101466
Aspergillus kawachii (strain NBRC
awamori var. kawachi)
Aspergillus terreus (strain NIH 2624/
Coccidioides immitis (strain RS) (Valley
Ajellomyces dermatitidis (strain ER-3/
dermatitidis)
Fusarium oxysporum f. sp. cubense
Rhodotorula glutinis (strain ATCC
Aspergillus niger (strain ATCC 1015/
Candida cloacae
Candida cloacae
Fusarium oxysporum f. sp. cubense
Candida albicans (strain SC5314/
Candida albicans (strain SC5314/
Chaetomium thermophilum (strain DSM
Mucor circinelloides f. circinelloides
Mucor circinelloides f. circinelloides
Mucor circinelloides f. circinelloides
Botryotinia fuckeliana (strain BcDW1)
Podospora anserina (strain S/ATCC
Neosartorya fumigata (strain ATCC
Fusarium oxysporum f. sp. vasinfectum
Fusarium oxysporum f. sp. vasinfectum
Trichophyton interdigitale H6
Beauveria bassiana (strain ARSEF 2860)
Fusarium oxysporum f. sp. radicis-
lycopersici 26381
Fusarium oxysporum f. sp. radicis-
lycopersici 26381
Neurospora tetrasperma (strain FGSC
Pseudozyma hubeiensis (strain SY62)
Lodderomyces elongisporus (strain
Malassezia globosa (strain ATCC MYA-
Byssochlamys spectabilis (strain No. 5/
Ajellomyces capsulatus (strain H88)
capsulatum)
Trichosporon asahii var. asahii (strain
Penicillium oxalicum (strain 114-2/
decumbens)
Fusarium oxysporum f. sp. conglutinans
Fusarium oxysporum f. sp. conglutinans
Fusarium oxysporum f. sp. raphani
Fusarium oxysporum f. sp. raphani
Metarhizium acridum (strain CQMa
Arthroderma benhamiae (strain ATCC
Fusarium oxysporum f. sp. cubense
Fusarium oxysporum f. sp. cubense
Cochliobolus heterostrophus (strain C4/
Trichosporon asahii var. asahii (strain
Mycosphaerella graminicola (strain CBS
Botryotinia fuckeliana (strain T4)
Metarhizium anisopliae (strain ARSEF
Cladophialophora carrionii CBS 160.54
Coccidioides posadasii (strain RMSCC
Rhodosporidium toruloides (strain
Puccinia graminis f. sp. tritici (strain
Trichophyton rubrum CBS 288.86
Colletotrichum fioriniae PJ7
Trichophyton rubrum CBS 289.86
Cladophialophora yegresii CBS 114405
Colletotrichum orbiculare (strain 104-T/
lagenarium)
Drechslerella stenobrocha 248
Neosartorya fumigata (strain CEA10/
Thielavia terrestris (strain ATCC 38088/
alabamense)
Gibberella fujikuroi (strain CBS 195.34/
fujikuroi)
Gibberella fujikuroi (strain CBS 195.34/
fujikuroi)
Aspergillus flavus (strain ATCC 200026/
Togninia minima (strain UCR-PA7)
Ajellomyces dermatitidis (strain ATCC
dermatitidis)
Macrophomina phaseolina (strain MS6)
Neurospora crassa (strain ATCC 24698/
Neosartorya fischeri (strain ATCC 1020/
Fusarium pseudograminearum (strain
Spathaspora passalidarum (strain NRRL
Spathaspora passalidarum (strain NRRL
Trichophyton verrucosum (strain HKI
Arthroderma gypseum (strain ATCC
Hypocrea jecorina (strain QM6a)
Trichophyton rubrum MR1448
Aspergillus ruber CBS 135680
Glarea lozoyensis (strain ATCC 20868/
Setosphaeria turcica (strain 28A)
Paracoccidioides brasiliensis (strain
Fusarium oxysporum Fo47
Fusarium oxysporum Fo47
Trichophyton rubrum MR1459
Penicillium marneffei (strain ATCC
Sphaerulina musiva (strain SO2202)
musiva)
Gibberella moniliformis (strain M3125/
Gibberella moniliformis (strain M3125/
Pseudozyma antarctica (strain T-34)
Paracoccidioides brasiliensis (strain
Rhizophagus irregularis (strain DAOM
Penicillium chrysogenum (strain ATCC
Baudoinia compniacensis (strain UAMH
Hypocrea atroviridis (strain ATCC
atroviride)
Colletotrichum gloeosporioides (strain
Cordyceps militaris (strain CM01)
Pyronema omphalodes (strain CBS
Colletotrichum graminicola (strain
graminicola)
Glarea lozoyensis (strain ATCC 74030/
Fusarium oxysporum f. sp. cubense
Fusarium oxysporum f. sp. cubense
Cochliobolus sativus (strain ND90Pr/
sorokiniana)
Mixia osmundae (strain CBS 9802/
Mycosphaerella pini (strain NZE10/
Grosmannia clavigera (strain kw1407/
Fusarium oxysporum FOSC 3-a
Fusarium oxysporum FOSC 3-a
Fusarium oxysporum FOSC 3-a
Nectria haematococca (strain 77-13-4/
Nectria haematococca (strain 77-13-4/
Tuber melanosporum (strain Mel28)
Ajellomyces dermatitidis (strain
Chaetomium globosum (strain ATCC
Candida tenuis (strain ATCC 10573/
Trichophyton rubrum CBS 100081
Pyrenophora teres f. teres (strain 0-1)
teres f. teres)
Colletotrichum gloeosporioides (strain
Gibberella zeae (Wheat head blight
Trichophyton soudanense CBS 452.61
Sclerotinia sclerotiorum (strain ATCC
Fusarium oxysporum f. sp. pisi HDV247
Fusarium oxysporum f. sp. pisi HDV247
Ustilago hordei (strain Uh4875-4)
Sporisorium reilianum (strain SRZ2)
Bipolaris zeicola 26-R-13
Melampsora larici-populina (strain
Fusarium oxysporum f. sp. lycopersici
Fusarium oxysporum f. sp. lycopersici
Bipolaris victoriae FI3
Debaryomyces hansenii (strain ATCC
hansenii)
Clavispora lusitaniae (strain ATCC
Candida albicans (strain WO-1) (Yeast)
Trichophyton rubrum MR850
Candida dubliniensis (strain CD36/
Starmerella bombicola
Thielavia heterothallica (strain ATCC
Claviceps purpurea (strain 20.1) (Ergot
Aspergillus oryzae (strain ATCC 42149/
Dictyostelium discoideum (Slime mold)
Triticum urartu (Red wild einkorn)
Solanum tuberosum (Potato)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Zea mays (Maize)
Citrus clementina
Citrus clementina
Citrus clementina
Citrus clementina
Morus notabilis
Morus notabilis
Medicago truncatula (Barrel medic)
Arabidopsis thaliana (Mouse-ear cress)
Medicago truncatula (Barrel medic)
Simmondsia chinensis (Jojoba) (Buxus
chinensis)
Prunus persica (Peach) (Amygdalus
persica)
Aphanomyces astaci
Aphanomyces astaci
Aphanomyces astaci
Aphanomyces astaci
Aphanomyces astaci
Aphanomyces astaci
Phaeodactylum tricornutum (strain
Hordeum vulgare var. distichum (Two-
Hordeum vulgare var. distichum (Two-
Hordeum vulgare var. distichum (Two-
Hordeum vulgare var. distichum (Two-
Hordeum vulgare var. distichum (Two-
Ricinus communis (Castor bean)
Brassica rapa subsp. pekinensis (Chinese
Ricinus communis (Castor bean)
Brassica rapa subsp. pekinensis (Chinese
Brassica rapa subsp. pekinensis (Chinese
Brassica rapa subsp. pekinensis (Chinese
Brassica rapa subsp. pekinensis (Chinese
Ricinus communis (Castor bean)
Zea mays (Maize)
Oryza glaberrima (African rice)
Zea mays (Maize)
Zea mays (Maize)
Aegilops tauschii (Tausch's goatgrass)
Solanum habrochaites (Wild tomato)
Physcomitrella patens subsp. patens
Physcomitrella patens subsp. patens
Physcomitrella patens subsp. patens
Solanum pennellii (Tomato)
Vitis vinifera (Grape)
Vitis vinifera (Grape)
Vitis vinifera (Grape)
Vitis vinifera (Grape)
Capsella rubella
Capsella rubella
Capsella rubella
Capsella rubella
Capsella rubella
Eutrema salsugineum (Saltwater cress)
Eutrema salsugineum (Saltwater cress)
Eutrema salsugineum (Saltwater cress)
Eutrema salsugineum (Saltwater cress)
Eutrema salsugineum (Saltwater cress)
Selaginella moellendorffii (Spikemoss)
Selaginella moellendorffii (Spikemoss)
Selaginella moellendorffii (Spikemoss)
Selaginella moellendorffii (Spikemoss)
Sorghum bicolor (Sorghum) (Sorghum
vulgare)
Sorghum bicolor (Sorghum) (Sorghum
vulgare)
Sorghum bicolor (Sorghum) (Sorghum
vulgare)
Sorghum bicolor (Sorghum) (Sorghum
vulgare)
Sorghum bicolor (Sorghum) (Sorghum
vulgare)
Solanum pimpinellifolium (Currant
pimpinellifolium)
Phaseolus vulgaris (Kidney bean)
Phaseolus vulgaris (Kidney bean)
Phaseolus vulgaris (Kidney bean)
Solanum tuberosum (Potato)
Solanum tuberosum (Potato)
Solanum tuberosum (Potato)
Glycine max (Soybean) (Glycine
hispida)
Glycine max (Soybean) (Glycine
hispida)
Populus trichocarpa (Western balsam
trichocarpa)
Picea sitchensis (Sitka spruce) (Pinus
sitchensis)
Populus trichocarpa (Western balsam
trichocarpa)
Populus trichocarpa (Western balsam
trichocarpa)
Glycine max (Soybean) (Glycine
hispida)
Glycine max (Soybean) (Glycine
hispida)
Setaria italica (Foxtail millet) (Panicum
italicum)
Solanum lycopersicum (Tomato)
Setaria italica (Foxtail millet) (Panicum
italicum)
Solanum lycopersicum (Tomato)
Solanum lycopersicum (Tomato)
Solanum lycopersicum (Tomato)
Solanum lycopersicum (Tomato)
Setaria italica (Foxtail millet) (Panicum
italicum)
Setaria italica (Foxtail millet) (Panicum
italicum)
Mimulus guttatus (Spotted monkey
Mimulus guttatus (Spotted monkey
Mimulus guttatus (Spotted monkey
Mimulus guttatus (Spotted monkey
Mimulus guttatus (Spotted monkey
Musa acuminata subsp. malaccensis
Musa acuminata subsp. malaccensis
Musa acuminata subsp. malaccensis
Saprolegnia diclina VS20
Brachypodium distachyon (Purple false
Brachypodium distachyon (Purple false
Brachypodium distachyon (Purple false
Oryza sativa subsp. indica (Rice)
Oryza sativa subsp. indica (Rice)
Oryza sativa subsp. indica (Rice)
Oryza sativa subsp. indica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Arabidopsis lyrata subsp. lyrata (Lyre-
Arabidopsis lyrata subsp. lyrata (Lyre-
Arabidopsis lyrata subsp. lyrata (Lyre-
Arabidopsis lyrata subsp. lyrata (Lyre-
In some embodiments, an alcohol dehydrogenase (ADH, Table 9) is used to catalyze the conversion of a fatty alcohol to a fatty aldehyde. A number of ADHs identified from alkanotrophic organisms, Pseudomonas fluorescens NRRL B-1244 (Hou et al. 1983), Pseudomonas butanovora ATCC 43655 (Vangnai and Arp 2001), and Acinetobacter sp. strain M-1 (Tani et al. 2000), have shown to be active on short to medium-chain alkyl alcohols (C2 to C14). Additionally, commercially available ADHs from Sigma, Horse liver ADH and Baker's yeast ADH have detectable activity for substrates with length C10 and greater. The reported activities for the longer fatty alcohols may be impacted by the difficulties in solubilizing the substrates. For the yeast ADH from Sigma, little to no activity is observed for C12 to C14 aldehydes by (Tani et al. 2000), however, activity for C12 and C16 hydroxy-ω-fatty acids has been observed (Lu et al. 2010). Recently, two ADHs were characterized from Geobacillus thermodenitrificans NG80-2, an organism that degrades Cis to C36 alkanes using the LadA hydroxylase. Activity was detected from methanol to 1-triacontanol (C30) for both ADHs, with 1-octanol being the preferred substrate for ADH2 and ethanol for ADH1 (Liu et al. 2009).
The use of ADHs in whole-cell bioconversions has been mostly focused on the production of chiral alcohols from ketones (Ernst et al. 2005) (Schroer et al. 2007). Using the ADH from Lactobacillus brevis and coupled cofactor regeneration with isopropanol, Schroer et al. reported the production of 797 g of (R)-methyl-3 hydroxybutanoate from methyl acetoacetate, with a space time yield of 29 g/L/h (Schroer et al. 2007). Examples of aliphatic alcohol oxidation in whole-cell transformations have been reported with commercially obtained S. cerevisiae for the conversion of hexanol to hexanal (Presecki et al. 2012) and 2-heptanol to 2-heptanone (Cappaert and Larroche 2004).
Bactrocera oleae (Olive fruit fly) (Dacus oleae)
Cupriavidus necator (Alcaligenes eutrophus)
Drosophila adiastola (Fruit fly) (Idiomyia
adiastola)
Drosophila affinidisjuncta (Fruit fly) (Idiomyia
affinidisjuncta)
Drosophila ambigua (Fruit fly)
Drosophila borealis (Fruit fly)
Drosophila differens (Fruit fly)
Drosophila equinoxialis (Fruit fly)
Drosophila flavomontana (Fruit fly)
Drosophila guanche (Fruit fly)
Drosophila hawaiiensis (Fruit fly)
Drosophila heteroneura (Fruit fly)
Drosophila immigrans (Fruit fly)
Drosophila insularis (Fruit fly)
Drosophila lebanonensis (Fruit fly)
Drosophila mauritiana (Fruit fly)
Drosophila madeirensis (Fruit fly)
Drosophila mimica (Fruit fly) (Idiomyia mimica)
Drosophila nigra (Fruit fly) (Idiomyia nigra)
Drosophila orena (Fruit fly)
Drosophila pseudoobscura bogotana (Fruit fly)
Drosophila picticornis (Fruit fly) (Idiomyia
picticornis)
Drosophila planitibia (Fruit fly)
Drosophila paulistorum (Fruit fly)
Drosophila silvestris (Fruit fly)
Drosophila subobscura (Fruit fly)
Drosophila teissieri (Fruit fly)
Drosophila tsacasi (Fruit fly)
Fragaria ananassa (Strawberry)
Malus domestica (Apple) (Pyrus malus)
Scaptomyza albovittata (Fruit fly)
Scaptomyza crassifemur (Fruit fly) (Drosophila
crassifemur)
Sulfolobus sp. (strain RC3)
Zaprionus tuberculatus (Vinegar fly)
Geobacillus stearothermophilus (Bacillus
stearothermophilus)
Drosophila mayaguana (Fruit fly)
Drosophila melanogaster (Fruit fly)
Drosophila pseudoobscura pseudoobscura (Fruit
Drosophila simulans (Fruit fly)
Drosophila yakuba (Fruit fly)
Drosophila ananassae (Fruit fly)
Drosophila erecta (Fruit fly)
Drosophila grimshawi (Fruit fly) (Idiomyia
grimshawi)
Drosophila willistoni (Fruit fly)
Drosophila persimilis (Fruit fly)
Drosophila sechellia (Fruit fly)
Cupriavidus necator (strain ATCC 17699/H16/
Mycobacterium tuberculosis (strain CDC 1551/
Staphylococcus aureus (strain MW2)
Mycobacterium tuberculosis (strain ATCC 25618/
Staphylococcus aureus (strain N315)
Staphylococcus aureus (strain bovine RF122/
Sulfolobus acidocaldarius (strain ATCC 33909/
Staphylococcus aureus (strain COL)
Staphylococcus aureus (strain NCTC 8325)
Staphylococcus aureus (strain MRSA252)
Staphylococcus aureus (strain MSSA476)
Staphylococcus aureus (strain USA300)
Staphylococcus aureus (strain Mu50/ATCC
Staphylococcus epidermidis (strain ATCC 12228)
Staphylococcus epidermidis (strain ATCC 35984/
Sulfolobus solfataricus (strain ATCC 35092/DSM
Sulfolobus tokodaii (strain DSM 16993/JCM
Anas platyrhynchos (Domestic duck) (Anas
boschas)
Apteryx australis (Brown kiwi)
Ceratitis capitata (Mediterranean fruit fly)
Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra)
Gallus gallus (Chicken)
Columba livia (Domestic pigeon)
Coturnix coturnix japonica (Japanese quail)
Drosophila hydei (Fruit fly)
Drosophila montana (Fruit fly)
Drosophila mettleri (Fruit fly)
Drosophila mulleri (Fruit fly)
Drosophila navojoa (Fruit fly)
Geomys attwateri (Attwater's pocket gopher)
Geomys bursarius (Plains pocket gopher)
Geomys knoxjonesi (Knox Jones's pocket gopher)
Hordeum vulgare (Barley)
Kluyveromyces marxianus (Yeast) (Candida kefyr)
Zea mays (Maize)
Mesocricetus auratus (Golden hamster)
Pennisetum americanum (Pearl millet) (Pennisetum
glaucum)
Petunia hybrida (Petunia)
Oryctolagus cuniculus (Rabbit)
Solanum tuberosum (Potato)
Struthio camelus (Ostrich)
Trifolium repens (Creeping white clover)
Zea luxurians (Guatemalan teosinte) (Euchlaena
luxurians)
Saccharomyces cerevisiae (strain ATCC 204508/
Arabidopsis thaliana (Mouse-ear cress)
Schizosaccharomyces pombe (strain 972/ATCC
Drosophila lacicola (Fruit fly)
Mus musculus (Mouse)
Peromyscus maniculatus (North American deer
Rattus norvegicus (Rat)
Drosophila virilis (Fruit fly)
Scheffersomyces stipitis (strain ATCC 58785/
Aspergillus flavus (strain ATCC 200026/FGSC
Neurospora crassa (strain ATCC 24698/74-OR23-
Candida albicans (Yeast)
Oryza sativa subsp. japonica (Rice)
Drosophila mojavensis (Fruit fly)
Kluyveromyces lactis (strain ATCC 8585/CBS
Oryza sativa subsp. indica (Rice)
Pongo abelii (Sumatran orangutan) (Pongo
pygmaeus abelii)
Homo sapiens (Human)
Macaca mulatta (Rhesus macaque)
Pan troglodytes (Chimpanzee)
Papio hamadryas (Hamadryas baboon)
Homo sapiens (Human)
Homo sapiens (Human)
Papio hamadryas (Hamadryas baboon)
Ceratitis capitata (Mediterranean fruit fly)
Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra)
Ceratitis rosa (Natal fruit fly) (Pterandrus rosa)
Drosophila arizonae (Fruit fly)
Drosophila buzzatii (Fruit fly)
Drosophila hydei (Fruit fly)
Drosophila montana (Fruit fly)
Drosophila mulleri (Fruit fly)
Drosophila wheeleri (Fruit fly)
Entamoeba histolytica
Hordeum vulgare (Barley)
Kluyveromyces marxianus (Yeast) (Candida kefyr)
Zea mays (Maize)
Oryza sativa subsp. indica (Rice)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Solanum tuberosum (Potato)
Scheffersomyces stipitis (strain ATCC 58785/
Arabidopsis thaliana (Mouse-ear cress)
Candida albicans (strain SC5314/ATCC MYA-
Oryza sativa subsp. japonica (Rice)
Drosophila mojavensis (Fruit fly)
Kluyveromyces lactis (strain ATCC 8585/CBS
Oryctolagus cuniculus (Rabbit)
Oryctolagus cuniculus (Rabbit)
Hordeum vulgare (Barley)
Solanum tuberosum (Potato)
Kluyveromyces lactis (strain ATCC 8585/CBS
Saccharomyces cerevisiae (strain ATCC 204508/
Homo sapiens (Human)
Mus musculus (Mouse)
Rattus norvegicus (Rat)
Struthio camelus (Ostrich)
Kluyveromyces lactis (strain ATCC 8585/CBS
Schizosaccharomyces pombe (strain 972/ATCC
Saccharomyces cerevisiae (strain YJM789)
Saccharomyces cerevisiae (strain ATCC 204508/
Saccharomyces pastorianus (Lager yeast)
eubayanus)
Bos taurus (Bovine)
Equus caballus (Horse)
Mus musculus (Mouse)
Rattus norvegicus (Rat)
Oryctolagus cuniculus (Rabbit)
Homo sapiens (Human)
Dictyostelium discoideum (Slime mold)
Saccharomyces cerevisiae (strain ATCC 204508/
Homo sapiens (Human)
Peromyscus maniculatus (North American deer
Pongo abelii (Sumatran orangutan) (Pongo
pygmaeus abelii)
Rattus norvegicus (Rat)
Homo sapiens (Human)
Rattus norvegicus (Rat)
Mus musculus (Mouse)
Mycobacterium tuberculosis (strain CDC 1551/
Rhizobium meliloti (strain 1021) (Ensifer meliloti)
Mycobacterium tuberculosis (strain ATCC 25618/
Zymomonas mobilis subsp. mobilis (strain ATCC
Mycobacterium bovis (strain ATCC BAA-935/
Mycobacterium tuberculosis (strain CDC 1551/
Mycobacterium tuberculosis (strain ATCC 25618/
Zymomonas mobilis subsp. mobilis (strain ATCC
Zymomonas mobilis subsp. mobilis (strain ATCC
Mycobacterium tuberculosis (strain CDC 1551/
Mycobacterium tuberculosis (strain ATCC 25618/
Clostridium acetobutylicum (strain ATCC 824/
Escherichia coli (strain K12)
Escherichia coli O157:H7
Rhodobacter sphaeroides (strain ATCC 17023/
Oryza sativa subsp. indica (Rice)
Escherichia coli (strain K12)
Geobacillus stearothermophilus (Bacillus
stearothermophilus)
Emericella nidulans (strain FGSC A4/ATCC
Emericella nidulans (strain FGSC A4/ATCC
Emericella nidulans (strain FGSC A4/ATCC
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Zea mays (Maize)
Drosophila melanogaster (Fruit fly)
Bacillus subtilis (strain 168)
Caenorhabditis elegans
Oryza sativa subsp. japonica (Rice)
Mycobacterium tuberculosis (strain ATCC 25618/
Caenorhabditis elegans
Caenorhabditis elegans
Pseudomonas sp.
Escherichia coli (strain K12)
Moraxella sp. (strain TAE123)
Alligator mississippiensis (American alligator)
Catharanthus roseus (Madagascar periwinkle)
Gadus morhua subsp. callarias (Baltic cod) (Gadus
callarias)
Naja naja (Indian cobra)
Pisum sativum (Garden pea)
Pelophylax perezi (Perez's frog) (Rana perezi)
Saara hardwickii (Indian spiny-tailed lizard)
Saara hardwickii (Indian spiny-tailed lizard)
Equus caballus (Horse)
Equus caballus (Horse)
Geobacillus stearothermophilus (Bacillus
stearothermophilus)
Gadus morhua (Atlantic cod)
Gadus morhua (Atlantic cod)
Myxine glutinosa (Atlantic hagfish)
Octopus vulgaris (Common octopus)
Pisum sativum (Garden pea)
Saara hardwickii (Indian spiny-tailed lizard)
Scyliorhinus canicula (Small-spotted catshark)
Sparus aurata (Gilthead sea bream)
In some embodiments, an α-dioxygenase is used to catalyze the conversion of a fatty acid to a fatty aldehyde (Hamberg et al. 2005). Alpha-dioxygenases catalyze the conversion of a Cn fatty acid to a Cn-1 aldehyde and may serve as an alternative to both ADH and AOX for fatty aldehyde production if a fatty acid is used as a biotransformation substrate. Due to the chain shortening of the dioxygenase reaction, this route requires a different synthesis pathway compared to the ADH and AOX routes. Biotransformations of E. coli cells expressing a rice α-dioxygenase exhibited conversion of C10, C12, C14 and C16 fatty acids to the corresponding Cn-1 aldehydes. With the addition of the detergent Triton X 100, 3.7 mM of pentadecanal (0.8 g/L) was obtained after 3 hours from hexadecanoic acid with 74% conversion (Kaehne et al. 2011). Exemplary α-dioxygenases are shown in Table 10.
Arabidopsis thaliana
Arabidopsis thaliana
Homo sapiens (Human)
Solanum lycopersicum (Tomato)
Solanum lycopersicum (Tomato)
Solanum lycopersicum (Tomato)
Arabidopsis lyrata subsp. lyrata
Ectocarpus siliculosus
Nicotiana attenuata
An enzyme's total turnover number (or TTN) refers to the maximum number of molecules of a substrate that the enzyme can convert before becoming inactivated. In general, the TTN for the hydroxylases and other enzymes used in the methods of the disclosure range from about 1 to about 100,000 or higher. For example, the TTN can be from about 1 to about 1,000, or from about 1,000 to about 10,000, or from about 10,000 to about 100,000, or from about 50,000 to about 100,000, or at least about 100,000. In particular embodiments, the TTN can be from about 100 to about 10,000, or from about 10,000 to about 50,000, or from about 5,000 to about 10,000, or from about 1,000 to about 5,000, or from about 100 to about 1,000, or from about 250 to about 1,000, or from about 100 to about 500, or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, or more.
When whole cells expressing a hydroxylase are used to carry out a hydroxylation reaction, the turnover can be expressed as the amount of substrate that is converted to product by a given amount of cellular material. In general, in vivo hydroxylation reactions exhibit turnovers from at least about 0.01 to at least about 10 mmol·gcdw−1, wherein gnaw is the mass of cell dry weight in grams. When whole cells expressing a hydroxylase are used to carry out a hydroxylation reaction, the activity can further be expressed as a specific productivity, e.g., concentration of product formed by a given concentration of cellular material per unit time, e.g., in g/L of product per g/L of cellular material per hour (g gcdw−1 h−1). In general, in vivo hydroxylation reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g·gcdw−1 h−1, wherein gnaw is the mass of cell dry weight in grams.
The TTN for heme enzymes, in particular, typically ranges from about 1 to about 100,000 or higher. For example, the TTN can be from about 1 to about 1,000, or from about 1,000 to about 10,000, or from about 10,000 to about 100,000, or from about 50,000 to about 100,000, or at least about 100,000. In particular embodiments, the TTN can be from about 100 to about 10,000, or from about 10,000 to about 50,000, or from about 5,000 to about 10,000, or from about 1,000 to about 5,000, or from about 100 to about 1,000, or from about 250 to about 1,000, or from about 100 to about 500, or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, or more. In certain embodiments, the variant or chimeric heme enzymes of the present disclosure have higher TTNs compared to the wild-type sequences. In some instances, the variant or chimeric heme enzymes have TTNs greater than about 100 (e.g., at least about 100, 150, 200, 250, 300, 325, 350, 400, 450, 500, or more) in carrying out in vitro hydroxylation reactions. In other instances, the variant or chimeric heme enzymes have TTNs greater than about 1000 (e.g., at least about 1000, 2500, 5000, 10,000, 25,000, 50,000, 75,000, 100,000, or more) in carrying out in vivo whole cell hydroxylation reactions.
When whole cells expressing a heme enzyme are used to carry out a hydroxylation reaction, the turnover can be expressed as the amount of substrate that is converted to product by a given amount of cellular material. In general, in vivo hydroxylation reactions exhibit turnovers from at least about 0.01 to at least about 10 mmol·gcdw−1, wherein gnaw is the mass of cell dry weight in grams. For example, the turnover can be from about 0.1 to about 10 mmol·gcdw−1, or from about 1 to about 10 mmol·gcdw−1, or from about 5 to about 10 mmol·gcdw−1, or from about 0.01 to about 1 mmol·gcdw−1, or from about 0.01 to about 0.1 mmol·gcdw−1, or from about 0.1 to about 1 mmol·gcdw−1, or greater than 1 mmol·gcdw−1. The turnover can be about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or about 10 mmol·gcdw−1.
When whole cells expressing a heme enzyme are used to carry out a hydroxylation reaction, the activity can further be expressed as a specific productivity, e.g., concentration of product formed by a given concentration of cellular material per unit time, e.g., in g/L of product per g/L of cellular material per hour (g·gcdw−1 h−1). In general, in vivo hydroxylation reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g·gcdw−1 h−1, wherein gnaw is the mass of cell dry weight in grams. For example, the specific productivity can be from about 0.01 to about 0.1 g·gcdw−1 h−1, or from about 0.1 to about 0.5 g·gcdw−1 h−1, or greater than 0.5 g·gcdw−1 h−1. The specific productivity can be about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or about 0.5 g·gcdw−1 h−1.
In certain embodiments, mutations can be introduced into the target gene using standard cloning techniques (e.g., site-directed mutagenesis) or by gene synthesis to produce the hydroxylases (e.g., cytochrome P450 variants) of the present disclosure. The mutated gene can be expressed in a host cell (e.g., bacterial cell) using an expression vector under the control of an inducible promoter or by means of chromosomal integration under the control of a constitutive promoter. Hydroxylation activity can be screened in vivo or in vitro by following product formation by GC or HPLC as described herein.
The expression vector comprising a nucleic acid sequence that encodes a heme enzyme of the disclosure can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage (e.g., a bacteriophage P1-derived vector (PAC)), a baculovirus vector, a yeast plasmid, or an artificial chromosome (e.g., bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a mammalian artificial chromosome (MAC), and human artificial chromosome (HAC)). Expression vectors can include chromosomal, non-chromosomal, and synthetic DNA sequences. Equivalent expression vectors to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.
The expression vector can include a nucleic acid sequence encoding a heme enzyme that is operably linked to a promoter, wherein the promoter comprises a viral, bacterial, archaeal, fungal, insect, or mammalian promoter. In certain embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In other embodiments, the promoter is a tissue-specific promoter or an environmentally regulated or a developmentally regulated promoter.
It is to be understood that affinity tags may be added to the N- and/or C-terminus of a heme enzyme expressed using an expression vector to facilitate protein purification. Non-limiting examples of affinity tags include metal binding tags such as His6-tags and other tags such as glutathione S-transferase (GST).
Non-limiting expression vectors for use in bacterial host cells include pCWori, pET vectors such as pET22 (EMD Millipore), pBR322 (ATCC37017), pQE™ vectors (Qiagen), pBluescript™ vectors (Stratagene), pNH vectors, lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), pRSET, pCR-TOPO vectors, pET vectors, pSyn_1 vectors, pChlamy_1 vectors (Life Technologies, Carlsbad, Calif.), pGEM1 (Promega, Madison, Wis.), and pMAL (New England Biolabs, Ipswich, Mass.). Non-limiting examples of expression vectors for use in eukaryotic host cells include pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia), pcDNA3.3, pcDNA4/TO, pcDNA6/TR, pLenti6/TR, pMT vectors (Life Technologies), pKLAC1 vectors, pKLAC2 vectors (New England Biolabs), pQE™ vectors (Qiagen), BacPak baculoviral vectors, pAdeno-X™ adenoviral vectors (Clontech), and pBABE retroviral vectors. Any other vector may be used as long as it is replicable and viable in the host cell.
The host cell can be a bacterial cell, an archaeal cell, a fungal cell, a yeast cell, an insect cell, or a mammalian cell.
Suitable bacterial host cells include, but are not limited to, BL21 E. coli, DE3 strain E. coli, E. coli M15, DH5α, DH10β, HB101, T7 Express Competent E. coli (NEB), B. subtilis cells, Pseudomonas fluorescens cells, and cyanobacterial cells such as Chlamydomonas reinhardtii cells and Synechococcus elongates cells. Non-limiting examples of archaeal host cells include Pyrococcus furiosus, Metallosphera sedula, Thermococcus litoralis, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Pyrococcus abyssi, Sulfolobus solfataricus, Pyrococcus woesei, Sulfolobus shibatae, and variants thereof. Fungal host cells include, but are not limited to, yeast cells from the genera Saccharomyces (e.g., S. cerevisiae), Pichia (P. Pastoris), Candida (C. tropicalis), Kluyveromyces (e.g., K. lactis), Hansenula and Yarrowia, and filamentous fungal cells from the genera Aspergillus, Trichoderma, and Myceliophthora. Suitable insect host cells include, but are not limited to, Sf9 cells from Spodoptera frugiperda, Sf21 cells from Spodoptera frugiperda, Hi-Five cells, BTI-TN-5B1-4 Trichophusia ni cells, and Schneider 2 (S2) cells and Schneider 3 (S3) cells from Drosophila melanogaster. Non-limiting examples of mammalian host cells include HEK293 cells, HeLa cells, CHO cells, COS cells, Jurkat cells, NS0 hybridoma cells, baby hamster kidney (BHK) cells, MDCK cells, NIH-3T3 fibroblast cells, and any other immortalized cell line derived from a mammalian cell.
In certain embodiments, the present disclosure provides heme enzymes such as the P450 variants described herein that are active hydroxylation catalysts inside living cells. As a non-limiting example, bacterial cells (e.g., E. coli) can be used as whole cell catalysts for the in vivo hydroxylation reactions of the present disclosure. In some embodiments, whole cell catalysts containing P450 enzymes with the equivalent C400X mutation are found to significantly enhance the total turnover number (TTN) compared to in vitro reactions using isolated P450 enzymes.
Biohydroxylation Reaction Conditions
The methods of the disclosure include forming reaction mixtures that contain the hydroxylases described herein. The hydroxylases can be, for example, purified prior to addition to a reaction mixture or secreted by a cell present in the reaction mixture. The reaction mixture can contain a cell lysate including the enzyme, as well as other proteins and other cellular materials. Alternatively, a hydroxylase can catalyze the reaction within a cell expressing the hydroxylase. Any suitable amount of hydroxylase can be used in the methods of the disclosure. In general, hydroxylation reaction mixtures contain from about 0.01 weight % (wt %) to about 100 wt % hydroxylase with respect to the hydrocarbon substrate. The reaction mixtures can contain, for example, from about 0.01 wt % to about 0.1 wt % hydroxylase, or from about 0.1 wt % to about 1 wt % hydroxylase, or from about 1 wt % to about 10 wt % hydroxylase, or from about 10 wt % to about 100 wt % hydroxylase. The reaction mixtures can contain from about 0.05 wt % to about 5 wt % hydroxylase, or from about 0.05 wt % to about 0.5 wt % hydroxylase. The reaction mixtures can contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, or about 3 wt % hydroxylase. One of skill in the art will understand how to convert wt % values to mol % values with respect to the hydroxylase and/or substrate concentrations set forth herein.
If the hydroxylase catalyses the reaction within a cell expressing the hydroxylase then any suitable amount of cells can be used in the methods of the disclosure. In general, hydroxylation whole-cell reaction mixtures contain from about 1 weight % to about 10,000 wt % of cells on a cell dry weight basis with respect to the hydrocarbon substrate. The whole-cell reaction mixtures can contain, for example, from about 1 wt % to about 10 wt % cells, or from about 10 wt % to about 100 wt % cells, or from about 100 wt % to about 1000 wt % cells, or from about 1000 wt % cells to about 2500 wt % cells, or from about 2500 wt % cells to about 5000 wt % cells, or from about 5000 wt % cells to about 7500 wt % cells, or from about 7500 wt % cells to about 10000 wt % cells with respect to the hydrocarbon substrate. The whole-cell reaction mixtures can contain from about 2 wt % to about 1000 wt % cells, or from about 5 wt % to about 500 wt % cells with respect to the hydrocarbon substrate. The whole-cell reaction mixtures can contain about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 wt % cells with respect to the hydrocarbon substrate.
The concentration of a saturated or unsaturated hydrocarbon substrate is typically in the range of from about 100 μM to about 1 M. The concentration can be, for example, from about 100 μM to about 1 mM, or about from 1 mM to about 100 mM, or from about 100 mM to about 500 mM, or from about 500 mM to 1 M. The concentration can be from about 500 μM to about 500 mM, 500 μM to about 50 mM, or from about 1 mM to about 50 mM, or from about 15 mM to about 45 mM, or from about 15 mM to about 30 mM. The concentration of the saturated or unsaturated hydrocarbon substrate can be, for example, about 100, 200, 300, 400, 500, 600, 700, 800, or 900 μM. The concentration of the saturated or unsaturated hydrocarbon substrate can be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM.
Reaction mixtures can contain additional reagents. As non-limiting examples, the reaction mixtures can contain buffers (e.g., 2-(N-morpholino)ethanesulfonic acid (MES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 3-morpholinopropane-1-sulfonic acid (MOPS), 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), potassium phosphate, sodium phosphate, phosphate-buffered saline, sodium citrate, sodium acetate, and sodium borate), cosolvents (e.g., dimethylsulfoxide, dimethylformamide, ethanol, methanol, isopropanol, glycerol, tetrahydrofuran, acetone, acetonitrile, and acetic acid), salts (e.g., NaCl, KCl, CaCl2, and salts of Mn2+ and Mg2+), denaturants (e.g., urea and guandinium hydrochloride), detergents (e.g., sodium dodecylsulfate and Triton-X 100), chelators (e.g., ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 2-({2-[Bis(carboxymethyl)amino]ethyl} (carboxymethyl)amino)acetic acid (EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA)), sugars (e.g., glucose, sucrose, and the like), and reducing agents (e.g., sodium dithionite, NADPH, dithiothreitol (DTT), β-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP)). Buffers, cosolvents, salts, denaturants, detergents, chelators, sugars, and reducing agents can be used at any suitable concentration, which can be readily determined by one of skill in the art. In general, buffers, cosolvents, salts, denaturants, detergents, chelators, sugars, and reducing agents, if present, are included in reaction mixtures at concentrations ranging from about 1 μM to about 1 M. For example, a buffer, a cosolvent, a salt, a denaturant, a detergent, a chelator, a sugar, or a reducing agent can be included in a reaction mixture at a concentration of about 1 μM, or about 10 μM, or about 100 μM, or about 1 mM, or about 10 mM, or about 25 mM, or about 50 mM, or about 100 mM, or about 250 mM, or about 500 mM, or about 1 M. Cosolvents, in particular, can be included in the reaction mixtures in amounts ranging from about 1% v/v to about 75% v/v, or higher. A co-solvent can be included in the reaction mixture, for example, in an amount of about 5, 10, 20, 30, 40, or 50% (v/v).
Reactions are conducted under conditions sufficient to catalyze the formation of a hydroxylation product. The reactions can be conducted at any suitable temperature. In general, the reactions are conducted at a temperature of from about 4° C. to about 40° C. The reactions can be conducted, for example, at about 25° C. or about 37° C. The reactions can be conducted at any suitable pH. In general, the reactions are conducted at a pH of from about 3 to about 10. The reactions can be conducted, for example, at a pH of from about 6.5 to about 9. The reactions can be conducted for any suitable length of time. In general, the reaction mixtures are incubated under suitable conditions for anywhere between about 1 minute and several hours. The reactions can be conducted, for example, for about 1 minute, or about 5 minutes, or about 10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, or about 4 hours, or about 8 hours, or about 12 hours, or about 24 hours, or about 48 hours, or about 72 hours, or about 96 hours, or about 120 hours, or about 144 hours, or about 168 hours, or about 192 hours. In general, reactions are conducted under aerobic conditions. In some embodiments, the solvent forms a second phase, and the hydroxylation occurs in the aqueous phase. In some embodiments, the hydroxylases is located in the aqueous layer whereas the substrates and/or products occur in an organic layer. Other reaction conditions may be employed in the methods of the disclosure, depending on the identity of a particular hydroxylase, or olefinic substrate.
Reactions can be conducted in vivo with intact cells expressing a hydroxylase of the disclosure. The in vivo reactions can be conducted with any of the host cells used for expression of the hydroxylases, as described herein. A suspension of cells can be formed in a suitable medium supplemented with nutrients (such as mineral micronutrients, glucose and other fuel sources, and the like). Hydroxylation yields from reactions in vivo can be controlled, in part, by controlling the cell density in the reaction mixtures. Cellular suspensions exhibiting optical densities ranging from about 0.1 to about 50 at 600 nm can be used for hydroxylation reactions. Other densities can be useful, depending on the cell type, specific hydroxylases, or other factors.
Pheromones, Precursors, Positional Isomers, and Analogs Comprising More than One C═C Double Bond
In some embodiments, an olefinic product described herein can have more than one carbon-carbon (C═C) double bond. In some embodiments, such olefinic products can be used to synthetically derive pheromones with more than one double bond.
In some embodiments, conjugated and unconjugated alkenes can be biohydroxylated to generate corresponding conjugated and unconjugated alkenols as illustrated in Scheme 11 below.
In some embodiments, biohydroxylation occurs on the terminal carbon an m-end of a carbon-carbon double bond in an unsaturated hydrocarbon substrate to produce first synthetically derived insect pheromone having a chemical structure corresponding to the chemical structure of a naturally occurring insect pheromone produced by the target insect. In some embodiments, biohydroxylation occurs on a terminal carbon of an n-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate a positional isomer of said first synthetically derived insect pheromone. In some embodiments, biohydroxylation occurs on a subterminal carbon on the m-end or the n-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate thereby forming an i-end, wherein the i-end comprises a terminal carbon of the unsaturated hydrocarbon substrate. In some embodiments, biohydroxylation and subsequent oxidation produces a pheromone or precursor that is over-oxidized, e.g., by hydroxylation of both terminal carbons (i.e., the m-end and the n-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate) and/or oxidation to a carboxylic acid.
The substrate, the pheromone, the positional isomer, and the analog can have any suitable combination of subscripts a, b, c, d, e, i, m, and n. In some embodiments, a, c, and e are independently integers from 0 to 1, provided that at least one of a, c, or e is 1. In some embodiments, m and n are integers independently selected from 1 to 15. In some embodiments, m, n, and i are integers independently selected from 1 to 15. In some embodiments, b and d are integers independently selected from 1 to 10. In some embodiments, the sum of a, b, c, d, e, m, and n is an integer that results in a total number of carbons from 6 to 20. In some embodiments, the sum of a, b, c, d, e, i, m, and n is an integer that results in a total number of carbons from 6 to 20. In some embodiments, each R is independently —OH, ═O, or —OAc. In some embodiments, each R′ is independently —OH, ═O, —OAc, or —OOH.
Pheromone Compositions
In some embodiments, the present disclosure provides for a pheromone and its positional isomer. For example, in some embodiments, the present disclosure provides for the synthesis of (E/Z)-hexadecen-1-al. Depending on the synthetic route and starting material, embodiments described herein, a variety of isomers of Z-hexadecen-1-al can be synthesized. Accordingly, a pheromone composition prepared as described herein can include a mixture of two or more of the following isomers: Z-hexadec-2-en-1-al, Z-hexadec-3-en-1-al, Z-hexadec-4-en-1-al, Z-hexadec-5-en-1-al, Z-hexadec-6-en-1-al, Z-hexadec-7-en-1-al, Z-hexadec-8-en-1-al, Z-hexadec-9-en-1-al, Z-hexadec-10-en-1-al, Z-hexadec-11-en-1-al, Z-hexadec-12-en-1-al, Z-hexadec-13-en-1-al, Z-hexadec-14-en-1-al, and Z-hexadec-15-en-1-al. Thus, in some embodiments, a pheromone composition as described herein can include at least one isomer of a natural pheromone or a mixture of isomers.
In some embodiments, the isomer is a positional isomer. The positional isomer produced using the methodology disclosed herein occurs via biohydroxylation of a location on the carbon skeleton which is different from location required to produce the natural pheromone for an insect. Accordingly, in some embodiments, a pheromone composition as described herein can include a natural pheromone produced by an insect and least one positional isomer of the natural pheromone. In some embodiments, the positional isomer is not produced by the insect whose behavior is modified by the pheromone composition. Thus, in some embodiments, the pheromone composition can include a first insect pheromone having a chemical structure of an insect sex pheromone produced by a member of the order Lepidoptera and a positional isomer of said first insect pheromone. In one such embodiment, the positional isomer is not produced by a member of the order Lepidoptera.
Mixtures of a pheromone with its positional isomer, as disclosed herein, can be used modulate the behavior of Lepidopteran species in a controllable or tunable manner. Although positional isomers of a pheromone, which are contained in various compositions of the disclosure, may not be emitted by a female Lepidoptera, its presence in mixtures with the authentic pheromone elicits a mating response from male Lepidoptera. The mating response of the male insects differ with different ratios of the positional isomer to the authentic pheromone, which indicates that the use of pheromones in mixtures of its positional isomers enables modulation of a male insect response that cannot be obtained with pure pheromone alone. For example, mixtures of (Z)-5-hexadecenal with the natural (Z)-11hexadecenal are able to elicit a mating response from H. zea males, even though (Z)-11-hexadecenal is emitted by the female Lepidoptera. The addition of (Z)-9-hexadecenal to mixtures of (Z)-5-hexadecenal and (Z)-11-hexadecenal may also act an as insect pheromone attractant. Accordingly, embodiments of the disclosure provide for mixtures of a natural pheromone with its positional isomer to elicit altered insect responses. The elicited response can be tuned depending on the ratio of the positional isomer to the natural pheromone. Thus, the amount of the positional isomer present in the mixture can be used to attenuate the mating response of an insect, e.g., male Lepidoptera, thereby eliciting a response which would not be possible with the natural pheromone.
In an exemplary embodiment, the pheromone compositions can include at least one synthetically derived natural pheromone and its synthetically derived positional isomer. In some embodiments, the pheromone composition includes Z-11-hexadecenal and it positional isomer Z-5-hexadecenal. In some embodiments, the pheromone composition includes a synthetically derived natural blend of Z-11-hexadecenal/Z-9-hexadecenal and the synthetically derived positional isomer of Z-11-hexadecenal—Z-5-hexadecenal, which is not produced by the target insect. In a further embodiment, the pheromone composition can also include the synthetically derived positional isomer of Z-9-hexadecenal—Z-7-hexadecenal, which is not produced by the target insect.
Thus, in some such embodiments, the pheromone composition can include compounds selected from the group consisting of Z-11-hexadecenal, Z-5-hexadecenal, Z-9-hexadecenal, Z-7-hexadecenal and combinations thereof. In other exemplary embodiments, the pheromone composition can include at least one of following combinations of synthetically derived natural pheromones and its positional isomer: Z-11-hexadecenal and Z-5-hexadecenal, or Z-9-hexadecenal and Z-7-hexadecenal.
In some embodiments, the pheromone composition includes a mixture of Z-11-hexadecenal and Z-5-hexadecenal. In some such embodiments, the percent of Z-11-hexadecenal to the percent of Z-5-hexadecenal in the composition is about 99.9% to about 0.1%, about 99.8% to about 0.2%, about 99.7% to about 0.3%, about 99.6% to about 0.4%, about 99.5% to about 0.5%, about 99.4% to about 0.6%, about 99.3% to about 0.7%, about 99.2% to about 0.8%, or about 99.1% to about 0.9%, including all values and subranges in between. In other embodiments, the ratio of Z-11-hexadecenal to Z-5-hexadecenal in the composition is about 99% to about 1.0%, 98% to about 2.0%, about 97% to about 3.0%, about 96% to about 4.0%, about 94% to about 6.0%, about 93% to about 7.0%, about 92% to about 8.0%, about 91% to about 9%, about 90% to about 10%, about 85% to about 15%, about 80% to about 20%, about 75% to about 25%, about 70% to about 30%, about 65% to about 35%, about 60% to about 40%, about 55% to about 45%, about 50% to about 50%, including all values and subranges in between.
In some such embodiments, the percent of Z-5-hexadecenal to the percent of Z-11-hexadecenal in the composition is about 99.9% to about 0.1%, about 99.8% to about 0.2%, about 99.7% to about 0.3%, about 99.6% to about 0.4%, about 99.5% to about 0.5%, about 99.4% to about 0.6%, about 99.3% to about 0.7%, about 99.2% to about 0.8%, or about 99.1% to about 0.9%, include all values and subranges in between. In some such embodiments, the percent of Z-5-hexadecenal to the percent of Z-11-hexadecenal in the composition is about 99% to about 1.0%, about 98% to about 2.0%, about 97% to about 3.0%, about 96% to about 4.0%, about 94% to about 6.0%, about 93% to about 7.0%, about 92% to about 8.0%, about 91% to about 9%, about 90% to about 10%, about 85% to about 15%, about 80% to about 20%, about 75% to about 25%, about 70% to about 30%, about 65% to about 35%, about 60% to about 40%, about 55% to about 45%, about 50% to about 50%, including all values and subranges in between.
In some embodiments, Z-11-hexadecenal is present in the composition at a percent of from about 99% mol to about 1 mol %, about 95 mol % to about 5 mol %, about 90 mol % to about 10 mol %, about 85 mol % to about 15 mol %, about 80 mol % to about 20 mol %, about 75 mol % to about 25 mol %, about 70 mol % to about 30 mol %, about 65 mol % to about 35 mol %, about 60 mol % to about 40 mol %, about 55 mol % to about 45 mol %, including all values and subranges in between. In some embodiments, Z-11-hexadecenal is present in the composition at a percent of about 97 mol % or less.
In some embodiments, the Z-11-hexadecenal is present in the composition in an amount of from about 99.9% w/w to about 0.1% w/w, about 99% to about 1% w/w, about 98% w/w to about 2% w/w, about 97% w/w to about 3% w/w, about 96% w/w to about 4% w/w, about 95% w/w to about 5% w/w, about 90% w/w to about 10% w/w, about 80% w/w to about 20% w/w, about 70% w/w to about 30% w/w, about 60% w/w to about 40% w/w, or about 50% w/w. In some embodiments, Z-11-hexadecenal is present in the composition at a percent of about 97% or less.
In some embodiments, Z-5-hexadecenal is present at a percent of from about 99.9 mol % to about 0.1 mol %, about 99 mol % to about 1 mol %, about 95 mol % to about 5 mol %, about 90 mol % to about 10 mol %, about 85 mol % to about 15 mol %, about 80 mol % to about 20 mol %, about 75 mol % to about 25 mol %, about 70 mol % to about 30 mol %, about 65 mol % to about 35 mol %, about 60 mol % to about 40 mol %, about 55 mol % to about 45 mol %, including all values and subranges in between. In other embodiments, Z-5-hexadecenal is present in the composition at percent of about 100 mol % or less, about 50 mol % or less, or about 5 mol % or less.
In some embodiments, the Z-5-hexadecenal is present in the composition in an amount of from about 99.9% w/w to about 0.1% w/w, 99% w/w to about 1% w/w, about 98% w/w to about 2% w/w, about 97% w/w to about 3% w/w, about 96% w/w to about 4% w/w, about 95% w/w to about 5% w/w, about 90% w/w to about 10% w/w, about 80% w/w to about 20% w/w, about 70% w/w to about 30% w/w, about 60% w/w to about 40% w/w, or about 50% w/w. In other embodiments, Z-5-hexadecenal is present in the composition in an amount about 100% w/w or less, about 50% w/w or less, about 5% w/w or less, or about 0.5% or less.
In some embodiments, the Z-9-hexadecenal is present in the composition at a percent of from about 99.9 mol % to about 0.1 mol %, about 99 mol % to about 1 mol %, about 95 mol % to about 5 mol %, about 90 mol % to about 10 mol %, about 85 mol % to about 15 mol %, about 80 mol % to about 20 mol %, about 75 mol % to about 25 mol %, about 70 mol % to about 30 mol %, about 65 mol % to about 35 mol %, about 60 mol % to about 40 mol %, about 55 mol % to about 45 mol %, including all values and subranges in between. In some embodiments, Z-9-hexadecenal is present in the composition at less than or equal to about 50 mol %, less than or equal to about 40 mol %, less than or equal to about 30 mol %, less than or equal to about 25 mol %, less than or equal to about 20 mol %, less than or equal to about 15 mol %, or less than or equal to about 10 mol %. In some embodiments, Z-9-hexadecenal is present at less than or equal to about 10 mol %, less than or equal to about 9 mol %, less than or equal to about 8 mol %, less than or equal to about 7 mol %, less than or equal to about 6 mol %, less than or equal to about 5 mol %, less than or equal to about 4 mol %, less than or equal to about 3 mol %, less than or equal to about 2 mol %, or less than or equal to 1 mol %. In some embodiments, the Z-9-hexadecenal is present at about 3 mol % or less, about 2.5 mol %, at about 2 mol %, at about 1.5 mol %, at about 1 mol %, or at about 0.5 mol % or less.
In some embodiments, the Z-9-hexadecenal is present in the composition in an about amount of from about 99.9% w/w to about 0.1% w/w, about 99% w/w to about 1% w/w, about 98% to about 2% w/w, about 97% to about 3% w/w, about 96% to about 4% w/w, about 95% to about 5% w/w, about 90% to about 10% w/w, about 80% to about 20% w/w, about 70% to about 30% w/w, about 60% to about 40% w/w, or about 50% w/w. In some embodiments, the Z-9-hexadecenal is present in the composition in an amount of 3% w/w or less.
In some embodiments, the Z-7-hexadecenal is present at a percent of from about 99.9 mol % to about 0.1 mol %, about 99 mol % to about 1 mol %, about 95 mol % to about 5 mol %, about 90 mol % to about 10 mol %, about 85 mol % to about 15 mol %, about 80 mol % to about 20 mol %, about 75 mol % to about 25 mol %, about 70 mol % to about 30 mol %, about 65 mol % to about 35 mol %, about 60 mol % to about 40 mol %, about 55 mol % to about 45 mol %, including all values and subranges in between. In some embodiments, Z-9-hexadecenal is preset less than or equal to about 50 mol %, less than or equal to about 40 mol %, less than or equal to about 30 mol %, less than or equal to about 25 mol %, less than or equal to about 20 mol %, less than or equal to about 15 mol %, or less than or equal to about 10 mol %. In some embodiments, Z-7-hexadecenal is preset less than or equal to about 10 mol %, less than or equal to about 9 mol %, less than or equal to about 8 mol %, less than or equal to about 7 mol %, less than or equal to about 6 mol %, less than or equal to about 5 mol %, less than or equal to about 4 mol %, less than or equal to about 3 mol %, less than or equal to about 2 mol %, or less than or equal to 1 mol %. In some embodiments, the Z-7-hexadecenal is present at about 3 mol %, about 2.5 mol %, at about 2 mol %, at about 1.5 mol %, at about 1 mol %, or at about 0.5 mol % or less.
In some embodiments, the Z-7-hexadecenal is present in the composition in an amount of from about 99.9% w/w to about 0.1% w/w, about 99% w/w to about 1% w/w, about 98% w/w to about 2% w/w, about 97% w/w to about 3% w/w, about 96% w/w to about 4% w/w, about 95% w/w to about 5% w/w, about 90% w/w to about 10% w/w, about 80% w/w to about 20% w/w, about 70% w/w to about 30% w/w, about 60% w/w to about 40% w/w, or about 50% w/w.
In some embodiments, the pheromone composition comprises about 97% Z-11-hexadecenal and about 3% Z-9-hexadecenal. In further embodiments, Z-5-hexadecenal is added to the composition comprising 97/3 Z-11-hexadecenal to Z-9-hexadecenal such that the Z-5-hexadecenal constitutes about 0.5 mol %, about 1 mol %, about 5 mol %, about 10 mol %, about 15 mol %, about 20 mol %, about 25 mol %, about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, about 50 mol %, about 55 mol %, about 60 mol %, about 65 mol %, about 70 mol %, about 75 mol %, about 80 mol %, about 85 mol %, about 90 mol %, about 95 mol % or about 99 mol %, of the resulting composition.
By varying the ratio of the synthetically derived sex pheromone to its positional isomer, embodiments described herein create a tunable pheromone composition which can be used to modulate the response of the target insect species. In some embodiments, the ratio of the sex pheromone to the positional isomer can varied by selecting and/or engineering the biocatalyst. The insect that is “attracted” to the compositions taught herein may, or may not, physically contact a locus containing said pheromone composition. That is, in some aspects, the compositions taught herein are able to attract a given insect within a close proximity to a locus containing the disclosed pheromone compositions, but do not entice said insect to physically contact the locus. However, in other aspects, the compositions taught herein do entice and/or attract an insect to physically come into contact with a locus containing said pheromone compositions. In this way, inter alia, the pheromone compositions taught herein are highly “tunable” and are able to modulate the behavior (e.g., degree of attracting an insect) of an insect to a high degree, which is not associated with pheromone compositions of the prior art (i.e., compositions including only the natural pheromone). Accordingly, the pheromone compositions of the present disclosure are able to modulate the degree to which an insect is attracted along a continuous scale, depending upon, among other things, the ratio of natural pheromone to its positional isomer.
Agricultural Compositions
As described above, a variety of pheromones can be synthesized according to the MBO or MBE method. Further, utilization of the aforementioned synthesis methods can produce positional isomers of said pheromones via biohydroxylation of an alternative location of the carbon skeleton. The pheromone and its positional isomer, prepared according to these methods, can be formulated for use in compositions which modify the behavior of insects, e.g., by applying the pheromone composition to a locus thereby attracting a target insect. Pheromone compositions can contain at least one pheromone and optionally adjuvants and other compounds provided that such compounds do not substantially interfere with the activity of the composition.
In some embodiments, the agricultural compositions of the present disclosure may include, but are not limited to: wetters, compatibilizing agents (also referred to as “compatibility agents”), antifoam agents, cleaning agents, sequestering agents, drift reduction agents, neutralizing agents and buffers, corrosion inhibitors, dyes, odorants, spreading agents (also referred to as “spreaders”), penetration aids (also referred to as “penetrants”), sticking agents (also referred to as “stickers” or “binders”), dispersing agents, thickening agents (also referred to as “thickeners”), stabilizers, emulsifiers, freezing point depressants, antimicrobial agents, and the like.
Carriers
In some embodiments, a pheromone composition can include a carrier. The carrier can be, but is not limited to, an inert liquid or solid.
Examples of solid carriers include but are not limited to fillers such as kaolin, bentonite, dolomite, calcium carbonate, talc, powdered magnesia, Fuller's earth, wax, gypsum, diatomaceous earth, rubber, plastic, China clay, mineral earths such as silicas, silica gels, silicates, attaclay, limestone, chalk, loess, clay, dolomite, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, products of vegetable origin such as cereal meals, tree bark meal, wood meal and nutshell meal, cellulose powders, attapulgites, montmorillonites, mica, vermiculites, synthetic silicas and synthetic calcium silicates, or compositions of these.
Examples of liquid carriers include, but are not limited to, water; alcohols, such as ethanol, butanol or glycol, as well as their ethers or esters, such as methylglycol acetate; ketones, such as acetone, cyclohexanone, methylethyl ketone, methylisobutylketone, or isophorone; alkanes such as hexane, pentane, or heptanes; aromatic hydrocarbons, such as xylenes or alkyl naphthalenes; mineral or vegetable oils; aliphatic chlorinated hydrocarbons, such as trichloroethane or methylene chloride; aromatic chlorinated hydrocarbons, such as chlorobenzenes; water-soluble or strongly polar solvents such as dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone; liquefied gases; waxes, such as beeswax, lanolin, shellac wax, carnauba wax, fruit wax (such as bayberry or sugar cane wax) candelilla wax, other waxes such as microcrystalline, ozocerite, ceresin, or montan; salts such as monoethanolamine salt, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium hydrogen sulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammonium hydrogen diphosphate, ammonium dihydrogen monophosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate or ammonium carbamateand mixtures thereof. Baits or feeding stimulants can also be added to the carrier.
Synergist
In some embodiments, the pheromone composition is combined with an active chemical agent such that a synergistic effect results. The synergistic effect obtained by the taught methods can be quantified according to Colby's formula (i.e. (E)=X+Y−(X*Y/100). See Colby, R. S., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations”, 1967 Weeds, vol. 15, pp. 20-22, incorporated herein by reference in its entirety. Thus, by “synergistic” is intended a component which, by virtue of its presence, increases the desired effect by more than an additive amount. The pheromone compositions and adjuvants of the present methods can synergistically increase the effectiveness of agricultural active compounds and also agricultural auxiliary compounds.
Thus, in some embodiments, a pheromone composition can be formulated with a synergist. The term, “synergist,” as used herein, refers to a substance that can be used with a pheromone for reducing the amount of the pheromone dose or enhancing the effectiveness of the pheromone for attracting at least one species of insect. The synergist may or may not be an independent attractant of an insect in the absence of a pheromone.
In some embodiments, the synergist is a volatile phytochemical that attracts at least one species of Lepidoptera. The term, “phytochemical,” as used herein, means a compound occurring naturally in a plant species. In a particular embodiment, the synergist is selected from the group comprising β-caryophyllene, iso-caryophyllene, α-humulene, inalool, Z3-hexenol/yl acetate, β-farnesene, benzaldehyde, phenylacetaldehyde, and combinations thereof.
The pheromone composition can contain the pheromone and the synergist in a mixed or otherwise combined form, or it may contain the pheromone and the synergist independently in a non-mixed form.
Insecticide
The pheromone composition can include one or more insecticides. In one embodiment, the insecticides are chemical insecticides known to one skilled in the art. Examples of the chemical insecticides include one or more of pyrethoroid or organophosphorus insecticides, including but are not limited to, cyfluthrin, permethrin, cypermethrin, bifinthrin, fenvalerate, flucythrinate, azinphosmethyl, methyl parathion, buprofezin, pyriproxyfen, flonicamid, acetamiprid, dinotefuran, clothianidin, acephate, malathion, quinolphos, chloropyriphos, profenophos, bendiocarb, bifenthrin, chlorpyrifos, cyfluthrin, diazinon, pyrethrum, fenpropathrin, kinoprene, insecticidal soap or oil, neonicotinoids, diamides, avermectin and derivatives, spinosad and derivatives, azadirachtin, pyridalyl, and mixtures thereof.
In another embodiment, the insecticides are one or more biological insecticides known to one skilled in the art. Examples of the biological insecticides include, but are not limited to, azadirachtin (neem oil), toxins from natural pyrethrins, Bacillus thuringiencis and Beauveria bassiana, viruses (e.g., CYD-X™, CYD-X HP™, Germstar™ Madex H P™ and Spod-X™), peptides (Spear-T™, Spear-P™, and Spear-C™)
In another embodiment, the insecticides are insecticides that target the nerve and muscle. Examples include acetylcholinesterase (AChE) inhibitors, such as carbamates (e.g., methomyl and thiodicarb) and organophosphates (e.g., chlorpyrifos) GABA-gated chloride channel antagonists, such as cyclodiene organochlorines (e.g., endosulfan) and phenylpyrazoles (e.g., fipronil), sodium channel modulators, such as pyrethrins and pyrethroids (e.g., cypermethrin and λ-cyhalothrin), nicotinic acetylcholine receptor (nAChR) agonists, such as neonicotinoids (e.g., acetamiprid, tiacloprid, thiamethoxam), nicotinic acetylcholine receptor (nAChR) allosteric modulators, such as spinosyns (e.g., spinose and spinetoram), chloride channel activators, such as avermectins and milbemycins (e.g., abamectin, emamectin benzoate), Nicotinic acetylcholine receptor (nAChR) blockers, such as bensultap and cartap, voltage dependent sodium channel blockers, such as indoxacarb and metaflumizone, ryanodine receptor modulator, such as diamides (e.g. dhlorantraniliprole and flubendiamide). In another embodiment, the insecticides are insecticides that target respiration. Examples include chemicals that uncouple oxidative phosphorylation via disruption of the proton gradient, such as chlorfenapyr, and mitochondrial complex I electron transport inhibitors.
In another embodiment, the insecticides are insecticides that target midgut. Examples include microbial disruptors of insect midgut membranes, such as Bacillus thuringiensis and Bacillus sphaericus.
In another embodiment, the insecticides are insecticides that target growth and development. Examples include juvenile hormone mimics, such as juvenile hormone analogues (e.g. fenoxycarb), inhibitors of chitin biosynthesis, Type 0, such as benzoylureas (e.g., flufenoxuron, lufenuron, and novaluron), and ecdysone receptor agonists, such as diacylhydrazines (e.g., methoxyfenozide and tebufenozide)
Stabilizer
According to another embodiment of the disclosure, the pheromone composition may include one or more additives that enhance the stability of the composition. Examples of additives include, but are not limited to, fatty acids and vegetable oils, such as for example olive oil, soybean oil, corn oil, safflower oil, canola oil, and combinations thereof.
Filler
According to another embodiment of the disclosure, the pheromone composition may include one or more fillers. Examples of fillers include, but are not limited to, one or more mineral clays (e.g., attapulgite). In some embodiments, the attractant-composition may include one or more organic thickeners. Examples of such thickeners include, but are not limited to, methyl cellulose, ethyl cellulose, and any combinations thereof.
Solvent
According to another embodiment, the pheromone compositions of the present disclosure can include one or more solvents. Compositions containing solvents are desirable when a user is to employ liquid compositions which may be applied by brushing, dipping, rolling, spraying, or otherwise applying the liquid compositions to substrates on which the user wishes to provide a pheromone coating (e.g., a lure). In some embodiments, the solvent(s) to be used is/are selected so as to solubilize, or substantially solubilize, the one or more ingredients of the pheromone composition. Examples of solvents include, but are not limited to, water, aqueous solvent (e.g., mixture of water and ethanol), ethanol, methanol, chlorinated hydrocarbons, petroleum solvents, turpentine, xylene, and any combinations thereof.
In some embodiments, the pheromone compositions of the present disclosure comprise organic solvents. Organic solvents are used mainly in the formulation of emulsifiable concentrates, ULV formulations, and to a lesser extent granular formulations. Sometimes mixtures of solvents are used. In some embodiments, the present disclosure teaches the use of solvents including aliphatic paraffinic oils such as kerosene or refined paraffins. In other embodiments, the present disclosure teaches the use of aromatic solvents such as xylene and higher molecular weight fractions of C9 and C10 aromatic solvents. In some embodiments, chlorinated hydrocarbons are useful as co-solvents to prevent crystallization when the formulation is emulsified into water. Alcohols are sometimes used as co-solvents to increase solvent power.
Solubilizing Agent
In some embodiments, the pheromone compositions of the present disclosure comprise solubilizing agents. A solubilizing agent is a surfactant, which will form micelles in water at concentrations above the critical micelle concentration. The micelles are then able to dissolve or solubilize water-insoluble materials inside the hydrophobic part of the micelle. The types of surfactants usually used for solubilization are non-ionics: sorbitan monooleates; sorbitan monooleate ethoxylates; and methyl oleate esters.
Binder
According to another embodiment of the disclosure, the pheromone composition may include one or more binders. Binders can be used to promote association of the pheromone composition with the surface of the material on which said composition is coated. In some embodiments, the binder can be used to promote association of another additive (e.g., insecticide, insect growth regulators, and the like) to the pheromone composition and/or the surface of a material. For example, a binder can include a synthetic or natural resin typically used in paints and coatings. These may be modified to cause the coated surface to be friable enough to allow insects to bite off and ingest the components of the composition (e.g., insecticide, insect growth regulators, and the like), while still maintaining the structural integrity of the coating.
Non-limiting examples of binders include polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethylcellulose, starch, vinylpyrrolidone/vinyl acetate copolymers and polyvinyl acetate, or compositions of these; lubricants such as magnesium stearate, sodium stearate, talc or polyethylene glycol, or compositions of these; antifoams such as silicone emulsions, long-chain alcohols, phosphoric esters, acetylene diols, fatty acids or organofluorine compounds, and complexing agents such as: salts of ethylenediaminetetraacetic acid (EDTA), salts of trinitrilotriacetic acid or salts of polyphosphoric acids, or compositions of these.
In some embodiments, the binder also acts a filler and/or a thickener. Examples of such binders include, but are not limited to, one or more of shellac, acrylics, epoxies, alkyds, polyurethanes, linseed oil, tung oil, and any combinations thereof.
Surface-Active Agents
In some embodiments, the pheromone compositions comprise surface-active agents. In some embodiments, the surface-active agents are added to liquid agricultural compositions. In other embodiments, the surface-active agents are added to solid formulations, especially those designed to be diluted with a carrier before application. Thus, in some embodiments, the pheromone compositions comprise surfactants. Surfactants are sometimes used, either alone or with other additives, such as mineral or vegetable oils as adjuvants to spray-tank mixes to improve the biological performance of the pheromone on the target. The surface-active agents can be anionic, cationic, or nonionic in character, and can be employed as emulsifying agents, wetting agents, suspending agents, or for other purposes. In some embodiments, the surfactants are non-ionics such as: alky ethoxylates, linear aliphatic alcohol ethoxylates, and aliphatic amine ethoxylates. Surfactants conventionally used in the art of formulation and which may also be used in the present formulations are described, in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, N.J., 1998, and in Encyclopedia of Surfactants, Vol. I-III, Chemical Publishing Co., New York, 1980-81. In some embodiments, the present disclosure teaches the use of surfactants including alkali metal, alkaline earth metal or ammonium salts of aromatic sulfonic acids, for example, ligno-, phenol-, naphthalene- and dibutylnaphthalenesulfonic acid, and of fatty acids of arylsulfonates, of alkyl ethers, of lauryl ethers, of fatty alcohol sulfates and of fatty alcohol glycol ether sulfates, condensates of sulfonated naphthalene and its derivatives with formaldehyde, condensates of naphthalene or of the naphthalenesulfonic acids with phenol and formaldehyde, condensates of phenol or phenolsulfonic acid with formaldehyde, condensates of phenol with formaldehyde and sodium sulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-, octyl- or nonylphenol, tributylphenyl polyglycol ether, alkylaryl polyether alcohols, isotridecyl alcohol, ethoxylated castor oil, ethoxylated triarylphenols, salts of phosphated triarylphenolethoxylates, lauryl alcohol polyglycol ether acetate, sorbitol esters, lignin-sulfite waste liquors or methylcellulose, or compositions of these.
In some embodiments, the present disclosure teaches other suitable surface-active agents, including salts of alkyl sulfates, such as diethanolammonium lauryl sulfate; alkylarylsulfonate salts, such as calcium dodecylbenzenesulfonate; alkylphenol-alkylene oxide addition products, such as nonylphenol-C18 ethoxylate; alcohol-alkylene oxide addition products, such as tridecyl alcohol-C16 ethoxylate; soaps, such as sodium stearate; alkylnaphthalene-sulfonate salts, such as sodium dibutyl-naphthalenesulfonate; dialkyl esters of sulfosuccinate salts, such as sodium di(2-ethylhexyl)sulfosuccinate; sorbitol esters, such as sorbitol oleate; quaternary amines, such as lauryl trimethylammonium chloride; polyethylene glycol esters of fatty acids, such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; salts of mono and dialkyl phosphate esters; vegetable oils such as soybean oil, rapeseed/canola oil, olive oil, castor oil, sunflower seed oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil and the like; and esters of the above vegetable oils, particularly methyl esters.
Wetting Agents
In some embodiments, the pheromone compositions comprise wetting agents. A wetting agent is a substance that when added to a liquid increases the spreading or penetration power of the liquid by reducing the interfacial tension between the liquid and the surface on which it is spreading. Wetting agents are used for two main functions in agrochemical formulations: during processing and manufacture to increase the rate of wetting of powders in water to make concentrates for soluble liquids or suspension concentrates; and during mixing of a product with water in a spray tank or other vessel to reduce the wetting time of wettable powders and to improve the penetration of water into water-dispersible granules. In some embodiments, examples of wetting agents used in the pheromone compositions of the present disclosure, including wettable powders, suspension concentrates, and water-dispersible granule formulations are: sodium lauryl sulphate; sodium dioctyl sulphosuccinate; alkyl phenol ethoxylates; and aliphatic alcohol ethoxylates.
Dispersing Agent
In some embodiments, the pheromone compositions of the present disclosure comprise dispersing agents. A dispersing agent is a substance which adsorbs onto the surface of particles and helps to preserve the state of dispersion of the particles and prevents them from reaggregating. In some embodiments, dispersing agents are added to pheromone compositions of the present disclosure to facilitate dispersion and suspension during manufacture, and to ensure the particles redisperse into water in a spray tank. In some embodiments, dispersing agents are used in wettable powders, suspension concentrates, and water-dispersible granules. Surfactants that are used as dispersing agents have the ability to adsorb strongly onto a particle surface and provide a charged or steric barrier to re-aggregation of particles. In some embodiments, the most commonly used surfactants are anionic, non-ionic, or mixtures of the two types.
In some embodiments, for wettable powder formulations, the most common dispersing agents are sodium lignosulphonates. In some embodiments, suspension concentrates provide very good adsorption and stabilization using polyelectrolytes, such as sodium naphthalene sulphonate formaldehyde condensates. In some embodiments, tristyrylphenol ethoxylated phosphate esters are also used. In some embodiments, such as alkylarylethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionics as dispersing agents for suspension concentrates.
Polymeric Surfactant
In some embodiments, the pheromone compositions of the present disclosure comprise polymeric surfactants. In some embodiments, the polymeric surfactants have very long hydrophobic ‘backbones’ and a large number of ethylene oxide chains forming the ‘teeth’ of a ‘comb’ surfactant. In some embodiments, these high molecular weight polymers can give very good long-term stability to suspension concentrates, because the hydrophobic backbones have many anchoring points onto the particle surfaces. In some embodiments, examples of dispersing agents used in pheromone compositions of the present disclosure are: sodium lignosulphonates; sodium naphthalene sulphonate formaldehyde condensates; tristyrylphenol ethoxylate phosphate esters; aliphatic alcohol ethoxylates; alky ethoxylates; EO-PO block copolymers; and graft copolymers.
Emulsifying Agent
In some embodiments, the pheromone compositions of the present disclosure comprise emulsifying agents. An emulsifying agent is a substance, which stabilizes a suspension of droplets of one liquid phase in another liquid phase. Without the emulsifying agent the two liquids would separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier blends include alkylphenol or aliphatic alcohol with 12 or more ethylene oxide units and the oil-soluble calcium salt of dodecylbenzene sulphonic acid. A range of hydrophile-lipophile balance (“HLB”) values from 8 to 18 will normally provide good stable emulsions. In some embodiments, emulsion stability can sometimes be improved by the addition of a small amount of an EO-PO block copolymer surfactant.
Gelling Agent
In some embodiments, the pheromone compositions comprise gelling agents. Thickeners or gelling agents are used mainly in the formulation of suspension concentrates, emulsions, and suspoemulsions to modify the rheology or flow properties of the liquid and to prevent separation and settling of the dispersed particles or droplets. Thickening, gelling, and anti-settling agents generally fall into two categories, namely water-insoluble particulates and water-soluble polymers. It is possible to produce suspension concentrate formulations using clays and silicas. In some embodiments, the pheromone compositions comprise one or more thickeners including, but not limited to: montmorillonite, e.g. bentonite; magnesium aluminum silicate; and attapulgite. In some embodiments, the present disclosure teaches the use of polysaccharides as thickening agents. The types of polysaccharides most commonly used are natural extracts of seeds and seaweeds or synthetic derivatives of cellulose. Some embodiments utilize xanthan and some embodiments utilize cellulose. In some embodiments, the present disclosure teaches the use of thickening agents including, but are not limited to: guar gum; locust bean gum; carrageenam; alginates; methyl cellulose; sodium carboxymethyl cellulose (SCMC); hydroxyethyl cellulose (HEC). In some embodiments, the present disclosure teaches the use of other types of anti-settling agents such as modified starches, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.
Anti-Foam Agent
In some embodiments, the presence of surfactants, which lower interfacial tension, can cause water-based formulations to foam during mixing operations in production and in application through a spray tank. Thus, in some embodiments, in order to reduce the tendency to foam, anti-foam agents are often added either during the production stage or before filling into bottles/spray tanks. Generally, there are two types of anti-foam agents, namely silicones and nonsilicones. Silicones are usually aqueous emulsions of dimethyl polysiloxane, while the nonsilicone anti-foam agents are water-insoluble oils, such as octanol and nonanol, or silica. In both cases, the function of the anti-foam agent is to displace the surfactant from the air-water interface.
Preservative
In some embodiments, the pheromone compositions comprise a preservative.
Additional Active Agent
According to another embodiment of the disclosure, the pheromone composition may include one or more insect feeding stimulants. Examples of insect feeding stimulants include, but are not limited to, crude cottonseed oil, fatty acid esters of phytol, fatty acid esters of geranyl geraniol, fatty acid esters of other plant alcohols, plant extracts, and combinations thereof.
According to another embodiment of the disclosure, the pheromone composition may include one or more insect growth regulators (“IGRs”). IGRs may be used to alter the growth of the insect and produce deformed insects. Examples of insect growth regulators include, for example, dimilin.
According to another embodiment of the disclosure, the attractant-composition may include one or more insect sterilants that sterilize the trapped insects or otherwise block their reproductive capacity, thereby reducing the population in the following generation. In some situations allowing the sterilized insects to survive and compete with non-trapped insects for mates is more effective than killing them outright.
Sprayable Compositions
In some embodiments, the pheromone compositions disclosed herein can be formulated as a sprayable composition (i.e., a sprayable pheromone composition). An aqueous solvent can be used in the sprayable composition, e.g., water or a mixture of water and an alcohol, glycol, ketone, or other water-miscible solvent. In some embodiments, the water content of such mixture is at least about 10%, at least about 20%, at least about 30%, at least about 40%, 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In some embodiments, the sprayable composition is concentrate, i.e. a concentrated suspension of the pheromone, and other additives (e.g., a waxy substance, a stabilizer, and the like) in the aqueous solvent, and can be diluted to the final use concentration by addition of solvent (e.g., water).
In some embodiments, the a waxy substance can be used as a carrier for the pheromone and its positional isomer in the sprayable composition. The waxy substance can be, e.g., a biodegradable wax, such as bees wax, carnauba wax and the like, candelilla wax (hydrocarbon wax), montan wax, shellac and similar waxes, saturated or unsaturated fatty acids, such as lauric, palmitic, oleic or stearic acid, fatty acid amides and esters, hydroxylic fatty acid esters, such as hydroxyethyl or hydroxypropyl fatty acid esters, fatty alcohols, and low molecular weight polyesters such as polyalkylene succinates.
In some embodiments, a stabilizer can be used with the sprayable pheromone compositions. The stabilizer can be used to regulate the particle size of concentrate and/or to allow the preparation of a stable suspension of the pheromone composition. In some embodiments, the stabilizer is selected from hydroxylic and/or ethoxylated polymers. Examples include ethylene oxide and propylene oxide copolymer, polyalcohols, including starch, maltodextrin and other soluble carbohydrates or their ethers or esters, cellulose ethers, gelatin, polyacrylic acid and salts and partial esters thereof and the like. In other embodiments, the stabilizer can include polyvinyl alcohols and copolymers thereof, such as partly hydrolyzed polyvinyl acetate. The stabilizer may be used at a level sufficient to regulate particle size and/or to prepare a stable suspension, e.g., between 0.1% and 15% of the aqueous solution.
In some embodiments, a binder can be used with the sprayable pheromone compositions. In some embodiments, the binder can act to further stabilize the dispersion and/or improve the adhesion of the sprayed dispersion to the target locus (e.g., trap, lure, plant, and the like). The binder can be polysaccharide, such as an alginate, cellulose derivative (acetate, alkyl, carboxymethyl, hydroxyalkyl), starch or starch derivative, dextrin, gum (arabic, guar, locust bean, tragacanth, carrageenan, and the like), sucrose, and the like. The binder can also be a non-carbohydrate, water-soluble polymer such as polyvinyl pyrrolidone, or an acidic polymer such as polyacrylic acid or polymethacrylic acid, in acid and/or salt form, or mixtures of such polymers.
Microencapsulated Pheromones
In some embodiments, the pheromone compositions disclosed herein can be formulated as a microencapsulated pheromone, such as disclosed in Ill'lchev, A L et al., J. Econ. Entomol. 2006; 99(6):2048-54; and Stelinki, L L et al., J. Econ. Entomol. 2007; 100(4):1360-9. Microencapsulated pheromones (MECs) are small droplets of pheromone enclosed within polymer capsules. The capsules control the release rate of the pheromone into the surrounding environment, and are small enough to be applied in the same method as used to spray insecticides. The effective field longevity of the microencapsulated pheromone formulations can range from a few days to slightly more than a week, depending on inter alia climatic conditions, capsule size and chemical properties.
Slow-Release Formulation
Pheromone compositions can be formulated so as to provide slow release into the atmosphere, and/or so as to be protected from degradation following release. For example, the pheromone compositions can be included in carriers such as microcapsules, biodegradable flakes and paraffin wax-based matrices. Alternatively, the pheromone composition can be formulated as a slow release sprayable.
In certain embodiments, the pheromone composition may include one or more polymeric agents known to one skilled in the art. The polymeric agents may control the rate of release of the composition to the environment. In some embodiments, the polymeric attractant-composition is impervious to environmental conditions. The polymeric agent may also be a sustained-release agent that enables the composition to be released to the environment in a sustained manner.
Examples of polymeric agents include, but are not limited to, celluloses, proteins such as casein, fluorocarbon-based polymers, hydrogenated rosins, lignins, melamine, polyurethanes, vinyl polymers such as polyvinyl acetate (PVAC), polycarbonates, polyvinylidene dinitrile, polyamides, polyvinyl alcohol (PVA), polyamide-aldehyde, polyvinyl aldehyde, polyesters, polyvinyl chloride (PVC), polyethylenes, polystyrenes, polyvinylidene, silicones, and combinations thereof. Examples of celluloses include, but are not limited to, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate-butyrate, cellulose acetate-propionate, cellulose propionate, and combinations thereof.
Other agents which can be used in slow-release or sustained-release formulations include fatty acid esters (such as a sebacate, laurate, palmitate, stearate or arachidate ester) or a fatty alcohols (such as undecanol, dodecanol, tridecanol, tridecenol, tetradecanol, tetradecenol, tetradecadienol, pentadecanol, pentadecenol, hexadecanol, hexadecenol, hexadecadienol, octadecenol and octadecadienol).
Administration of Pheromone Composition
Lures
The pheromone compositions of the present disclosure may be coated on or sprayed on a lure, or the lure may be otherwise impregnated with a pheromone composition.
Traps
The pheromone compositions of the disclosure may be used in traps, such as those commonly used to attract any insect species, e.g., insects of the order Lepidoptera. Such traps are well known to one skilled in the art, and are commonly used in many states and countries in insect eradication programs. In one embodiment, the trap includes one or more septa, containers, or storage receptacles for holding the pheromone composition. Thus, in some embodiments, the present disclosure provides a trap loaded with at least one pheromone composition. Thus, the pheromone compositions of the present disclosure can be used in traps for example to attract insects as part of a strategy for insect monitoring, mass trapping, mating disruption, or lure/attract and kill for example by incorporating a toxic substance into the trap to kill insects caught.
Mass trapping involves placing a high density of traps in a crop to be protected so that a high proportion of the insects are removed before the crop is damaged. Lure/attract-and-kill techniques are similar except once the insect is attracted to a lure, it is subjected to a killing agent. Where the killing agent is an insecticide, a dispenser can also contain a bait or feeding stimulant that will entice the insects to ingest an effective amount of an insecticide. The insecticide may be an insecticide known to one skilled in the art. The insecticide may be mixed with the attractant-composition or may be separately present in a trap. Mixtures may perform the dual function of attracting and killing the insect.
Such traps may take any suitable form, and killing traps need not necessarily incorporate toxic substances, the insects being optionally killed by other means, such as drowning or electrocution. Alternatively, the traps can contaminate the insect with a fungus or virus that kills the insect later. Even where the insects are not killed, the trap can serve to remove the male insects from the locale of the female insects, to prevent breeding.
It will be appreciated by a person skilled in the art that a variety of different traps are possible. Suitable examples of such traps include water traps, sticky traps, and one-way traps. Sticky traps come in many varieties. One example of a sticky trap is of cardboard construction, triangular or wedge-shaped in cross-section, where the interior surfaces are coated with a non-drying sticky substance. The insects contact the sticky surface and are caught. Water traps include pans of water and detergent that are used to trap insects. The detergent destroys the surface tension of the water, causing insects that are attracted to the pan, to drown in the water. One-way traps allow an insect to enter the trap but prevent it from exiting. The traps of the disclosure can be colored brightly, to provide additional attraction for the insects.
In some embodiments, the pheromone traps containing the composition may be combined with other kinds of trapping mechanisms. For example, in addition to the pheromone composition, the trap may include one or more florescent lights, one or more sticky substrates and/or one or more colored surfaces for attracting moths. In other embodiments, the pheromone trap containing the composition may not have other kinds of trapping mechanisms.
The trap may be set at any time of the year in a field. Those of skill in the art can readily determine an appropriate amount of the compositions to use in a particular trap, and can also determine an appropriate density of traps/acre of crop field to be protected.
The trap can be positioned in an area infested (or potentially infested) with insects. Generally, the trap is placed on or close to a tree or plant. The aroma of the pheromone attracts the insects to the trap. The insects can then be caught, immobilized and/or killed within the trap, for example, by the killing agent present in the trap.
Traps may also be placed within an orchard to overwhelm the pheromones emitted by the females, so that the males simply cannot locate the females. In this respect, a trap need be nothing more than a simple apparatus, for example, a protected wickable to dispense pheromone.
The traps of the present disclosure may be provided in made-up form, where the compound of the disclosure has already been applied. In such an instance, depending on the half-life of the compound, the compound may be exposed, or may be sealed in conventional manner, such as is standard with other aromatic dispensers, the seal only being removed once the trap is in place.
Alternatively, the traps may be sold separately, and the compound of the disclosure provided in dispensable format so that an amount may be applied to trap, once the trap is in place. Thus, the present disclosure may provide the compound in a sachet or other dispenser.
Dispenser
Pheromone compositions can be used in conjunction with a dispenser for release of the composition in a particular environment. Any suitable dispenser known in the art can be used. Examples of such dispensers include but are not limited to, aerosol emitters, hand-applied dispensers, bubble caps comprising a reservoir with a permeable barrier through which pheromones are slowly released, pads, beads, tubes rods, spirals or balls composed of rubber, plastic, leather, cotton, cotton wool, wood or wood products that are impregnated with the pheromone composition. For example, polyvinyl chloride laminates, pellets, granules, ropes or spirals from which the pheromone composition evaporates, or rubber septa. One of skill in the art will be able to select suitable carriers and/or dispensers for the desired mode of application, storage, transport or handling.
In another embodiment, a device may be used that contaminates the male insects with a powder containing the pheromone substance itself. The contaminated males then fly off and provide a source of mating disruption by permeating the atmosphere with the pheromone substance, or by attracting other males to the contaminated males, rather than to real females.
Behavior Modification
Pheromone compositions prepared according to the methods disclosed herein can be used to control or modulate the behavior of insects. In some embodiments, the behavior of the target insect can be modulated in a tunable manner inter alia by varying the ratio of the pheromone to the positional isomer in the composition such that the insect is attracted to a particular locus but does not contact said locus or such the insect in fact contacts said locus. Thus, in some embodiments, the pheromones can be used to attract insects away from vulnerable crop areas. Accordingly, the disclosure also provides a method for attracting insects to a locus. The method includes administering to a the locus an effective amount of the pheromone composition.
The method of mating disruption may include periodically monitoring the total number or quantity of the trapped insects. The monitoring may be performed by counting the number of insects trapped for a predetermined period of time such as, for example, daily, Weekly, bi-Weekly, monthly, once-in-three months, or any other time periods selected by the monitor. Such monitoring of the trapped insects may help estimate the population of insects for that particular period, and thereby help determine a particular type and/or dosage of pest control in an integrated pest management system. For example, a discovery of a high insect population can necessitate the use of methods for removal of the insect. Early warning of an infestation in a new habitat can allow action to be taken before the population becomes unmanageable. Conversely, a discovery of a low insect population can lead to a decision that it is sufficient to continue monitoring the population. Insect populations can be monitored regularly so that the insects are only controlled when they reach a certain threshold. This provides cost-effective control of the insects and reduces the environmental impact of the use of insecticides.
Mating Disruption
Pheromones prepared according to the methods of the disclosure can also be used to disrupt mating. Mating disruption is a pest management technique designed to control insect pests by introducing artificial stimuli (e.g., a pheromone composition as disclosed herein) that confuses the insects and disrupts mating localization and/or courtship, thereby preventing mating and blocking the reproductive cycle.
In many insect species of interest to agriculture, such as those in the order Lepidoptera, females emit an airborne trail of a specific chemical blend constituting that species' sex pheromone. This aerial trail is referred to as a pheromone plume. Males of that species use the information contained in the pheromone plume to locate the emitting female (known as a “calling” female). Mating disruption exploits the male insects' natural response to follow the plume by introducing a synthetic pheromone into the insects' habitat, which is designed to mimic the sex pheromone produced by the female insect. Thus, in some embodiments, the synthetic pheromone utilized in mating disruption is a synthetically derived pheromone composition comprising a pheromone having a chemical structure of a sex pheromone and a positional isomer thereof which is not produced by the target insect.
The general effect of mating disruption is to confuse the male insects by masking the natural pheromone plumes, causing the males to follow “false pheromone trails” at the expense of finding mates, and affecting the males' ability to respond to “calling” females. Consequently, the male population experiences a reduced probability of successfully locating and mating with females, which leads to the eventual cessation of breeding and collapse of the insect infestation
Strategies of mating disruption include confusion, trail-masking and false-trail following. Constant exposure of insects to a high concentration of a pheromone can prevent male insects from responding to normal levels of the pheromone released by female insects. Trail-masking uses a pheromone to destroy the trail of pheromones released by females. False-trail following is carried out by laying numerous spots of a pheromone in high concentration to present the male with many false trails to follow. When released in sufficiently high quantities, the male insects are unable to find the natural source of the sex pheromones (the female insects) so that mating cannot occur.
In some embodiments, a wick or trap may be adapted to emit a pheromone for a period at least equivalent to the breeding season(s) of the midge, thus causing mating disruption. If the midge has an extended breeding season, or repeated breeding season, the present disclosure provides a wick or trap capable of emitting pheromone for a period of time, especially about two weeks, and generally between about 1 and 4 weeks and up to 6 weeks, which may be rotated or replaced by subsequent similar traps. A plurality of traps containing the pheromone composition may be placed in a locus, e.g., adjacent to a crop field. The locations of the traps, and the height of the traps from ground may be selected in accordance with methods known to one skilled in the art.
Alternatively, the pheromone composition may be dispensed from formulations such as microcapsules or twist-ties, such as are commonly used for disruption of the mating of insect pests.
A variety of pheromones, including those set forth in Table 1 can be prepared according to the methods and formulations as described above. For example, the methods can be used to synthesize a corn earworm (H. zea) sex pheromone blend, which is generally understood in the art to entail a mixture of (Z)-hexadeca-9-en-1-al (3%) and (Z)-hexadeca-11-en-1-al (97%). However, as disclosed herein, the pheromone blend can be doped with (Z)-hexadeca-5-en-1-al to tunably elicit a response in the male corn earworms. Thus, the corn earworm sex pheromone can be used in conjunction with a sustained pheromone release device having a polymer container containing a mixture of the sex pheromone and a fatty acid ester (such as a sebacate, laurate, palmitate, stearate or arachidate ester) or a fatty alcohol (such as undecanol, dodecanol, tridecanol, tridecenol, tetradecanol, tetradecenol, tetradecadienol, pentadecanol, pentadecenol, hexadecanol, hexadecenol, hexadecadienol, octadecenol and octadecadienol). The polymer container can be a tube, an ampule, or a bag made of a polyolefin or an olefin component-containing copolymer. Sex pheromones of other pest insects, such as, but not limited to, the cotton bollworm (Helicoverpa armigera), fall army worm (Spodoptera frugiperda), oriental fruit moth (Grapholita molesta), peach twig borer (Anarsia lineatella), diamondback moth (Plutella xylostella), soybean looper (Chrysodeixis includes) and leaf roller (Tortricidae) can be used in this type of sustained pheromone release device.
As will be apparent to one of skill in the art, the amount of a pheromone or pheromone composition used for a particular application can vary depending on several factors such as the type and level of insect infestation; the type of composition used; the concentration of the active components; how the composition is provided, for example, the type of dispenser used; the type of location to be treated; the length of time the method is to be used for; and environmental factors such as temperature, wind speed and direction, rainfall and humidity. Those of skill in the art will be able to determine an effective amount of a pheromone or pheromone composition for use in a given application.
The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the disclosure in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.
The purpose of this example is to illustrate the biocatalytic hydroxylation of (Z)-5-hexadecene by members of the CYP52 family.
Two P450 cytochromes of the CYP52 family were integrated into the P. pastoris CBS7435 Muts genome along with their corresponding cytochrome P450 reductases (CPR). Biotransformations were performed with these strains to determine whether these P450s hydroxylate (Z)-5-hexadecene. Strains and oligonucleotides disclosed in this example are listed in Tables 11 and 12.
P. pastoris CBS7435
P. pastoris CBS7435
Gene sequences for C. tropicalis CYP52A13 (Accession No. AA073953.1), C. tropicalis CPR (Accession No. P37201.1), C. maltosa CYP52A3 (Accession No. P24458.1), as well as the C. maltosa CPR (Accession No. P50126.1), were ordered as synthetic genes (DNA 2.0, Menlo Park, Calif., USA), and cloned into the pT4_S vector using EcoRI/NotI restriction sites for directional cloning. The plasmid containing the expression cassettes for CYP52A3/CPR and CYP52A13/CPR under the control of an AOX promoter and terminator were linearized using the restriction enzyme SmiI and purified. Next, 500 ng of the linearized DNA sequences for expressing CYP52A3/CPR (SEQ ID NO:4) and CYP52A13/CPR (SEQ ID NO:5) were used to transform P. pastoris CBS7435 Muts. The parent strain and the generation of the pT4 S plasmid used to generate the subsequent constructs are described by Gudiminchi et al. (Biotechnology Journal, 2013, 8(1), 146-52).
Colony PCR of the obtained P. pastoris strains was performed to verify the P450 enzymes CYP52A3 and CYP52A13 were present using the Failsafe™ PCR Kit (EPICENTRE® Biotechnologies, Madison, Wis; Catalog #FS99060) using Premix D and primers shown in Table 21 according to the manufactures recommendations.
Shake flask cultivations of the strains SPV048 and SPV051 were started from single colonies derived from an YBD agar plate (10 g/L Bacto™ yeast extract, 20 g/L Bacto™ peptone, 20 g/L D (+) glucose, 15 g/L agar) containing 100 mg/L Zeocin™. A volume of 45 mL of BMD1 medium (BMD1(1 L): 10 g/L D (+) glucose autoclaved, 200 mL 10×PPB (10×PPB: 30.0 g/L K2HPO4, 118 g/L KH2PO4, pH 6.0, autoclaved), 100 mL 10×YNB (10×YNB: 134 g/L Difco™ yeast nitrogen base without amino acids, autoclaved), 2 mL 500×buffer B (buffer B:10 mg/50 mL d-Biotin, filter sterilized), add autoclaved H2O to 1 L) was inoculated with a single colony and incubated for approximately 63 h at 28° C. to 30° C. and 130 rpm in a 250 mL baffled Erlenmeyer flask. After the initial 63 h incubation 5 mL of BMM10 medium (BMM10 (1 L): 50 mL methanol, 200 mL 10×PPB (10×PPB: 30.0 g/L K2HPO4, 118 g/L KH2PO4, pH 6.0, autoclaved), 100 mL 10×YNB (10×YNB: 134 g/L Difco™ yeast nitrogen base without amino acids, autoclaved), 2 mL 500×buffer B (buffer B:10 mg/50 mL d-Biotin, filter sterilized), add autoclaved H2O to 1 L) was added. The cultivations were incubated for 12 h at 28° C. to 30° C., 130 rpm. After 12 hours incubation 0.4 mL of methanol was added to induce expression of the P450 enzymes and their corresponding CPR's and incubated for 12 h at 28° C. to 30° C., 130 rpm. Thereafter, 0.4 mL of methanol was added every 12 h and incubated at 28° C. to 30° C., 130 rpm. Cells were harvested after induction for approximately 72 h to 80 h and a total cultivation time of approximately 132 h to 143 h.
As control a volume of 45 mL of BMD1 medium (BMD1 (1 L): 10 g/L D (+) glucose autoclaved, 200 mL 10×PPB (10×PPB: 30.0 g/L K2HPO4, 118 g/L KH2PO4, pH 6.0, autoclaved), 100 mL 10×YNB (10×YNB: 134 g/L Difco™ yeast nitrogen base without amino acids, autoclaved), 2 mL 500×buffer B (buffer B:10 mg/50 mL d-Biotin, filter sterilized), add autoclaved H2O to 1 L) was inoculated with a single colony of strain SPV051 incubated for approximately 63 h at 28° C. to 30° C. and 130 rpm in a 250 mL baffled Erlenmeyer flask. After the initial 63 h incubation 5 mL of BMM10 medium without methanol (BMM10 without methanol (1 L): 200 mL 10×PPB (10×PPB: 30.0 g/L K2HPO4, 118 g/L KH2PO4, pH 6.0, autoclaved), 100 mL 10×YNB (10×YNB: 134 g/L Difco™ yeast nitrogen base without amino acids, autoclaved), 2 mL 500×buffer B (buffer B:10 mg/50 mL d-Biotin, filter sterilized), add autoclaved H2O to 1 L) was added. The cultivations were incubated for additional 60 h to 68 h at 28° C. to 30° C., 130 rpm. Cells were harvested after a total cultivation time of approximately 132 h to 143 h.
Cultivations were harvested in 50 mL Falcon tubes via centrifugation at 3000×rcf for 5 min at 4° C. The supernatant was discarded. The pellet was resuspended in 5 mL 100 mM PPB (mix stock solutions: 80.2 mL of 1M K2HPO4 (174.18 g/L) with 19.8 mL of 1M KH2PO4 (136.09 g/L) autoclaved, add autoclaved H2O to 1 L and adjust pH 7.4), containing 20% glycerol, pH 7.4 and centrifuged again at 3000×rcf for 5 min at 4° C. (washing step). The supernatant was discarded and the Falcon tube was carefully patted on a Kimwipe to remove excess buffer. Each pellet was weighed to determine the cell wet weight (cww) of the cultures. The washed pellet was resuspended in bioconversion buffer (100 mM PPB (mix stock solutions: 80.2 mL of 1M K2HPO4 (174.18 g/L) with 19.8 mL of 1M KH2PO4 (136.09 g/L) autoclaved, add autoclaved H2O to 1 L and adjust to pH 7.4), 20% glycerol, 0.2% Emulgen 913 (Kao Chemicals, Japan), pH 7.4) targeting a final cell density of ˜200 g cww/L.
1 ml of the resuspended cultivation (200 g cww/L) was dispensed in a 50 mL Falcon tube. 125 μL neat substrate was added to each culture to initiate the bioconversion reactions. The bioconversion reactions were incubated at 30° C. and 200 rpm for 40 h to 48 h. The samples were stored at −80° C. until extraction and analysis of the respective product formation.
250 μL of 3 M HCl was added to each of the frozen samples. After addition of HCl samples were extracted twice with 1×1 mL or 2×2 mL diethyl ether. 10 μL of 10 mg/mL 1-Heptanol or 10 μL of 10 mg/mL 1-Tetradecanol was added to the sample as internal standard. Upon addition of diethyl ether and internal standard the sample was vortexed for 5 min. The entire sample was transferred to new reaction tubes and centrifuged for 10 min/8000×rcf at room temperature. The organic upper phase was transferred to a glass vial and air dried. The sample was resuspended to a final volume of 100 μL to 150 μL using Methyl Tertiary Butyl Ether (MTBE) or resuspended to a final volume of 200 μL using Tetrahydrofuran (THF) and analyzed via gas chromatography (GC).
An Agilent 6890 equipped with an FID detector and a J&W DB-23 column (length: 30 m, I.D. 25 mm, film 25 μm) was used to analyze the samples using the following program: Split ratio of 1:10. 240° C. for the injector inlet: 240° C. for the detector. H2 at 40.0 mL/min, Air at 450 mL/min, Makeup flow (He) at 45 mL/min. Carrier He at 1.1 ml/min and 13 psi. 45° C. oven for 0.5 min; 5° C./min gradient to 50° C. then hold at 50° C. for 0.5 min; 30° C./min gradient to 220° C., then hold at 220° C. for 3.33 min. Analysis was performed in triplicate using authentic standards (obtained from Sigma-Aldrich or Bedoukian Research).
Results are shown in Table 13 and
The results indicate that the biohydroxylation catalyst can functionalize an unsaturated hydrocarbon substrate on different termini to generate a mixture which includes a pheromone having a chemical structure corresponding to that of a natural insect pheromone produced by a given target member of the order Lepidoptera and a positional isomer of said sex pheromone, which is not naturally produced by said target insect.
Z-5-Hexadecene:
The cross metathesis reactions of 1-hexene and dodec-1-ene is carried out in a 250 mL three-necked round-bottomed flask fitted with a condenser, thermometer and septum. The dodec-1-ene (20 mL) is transferred to the reaction flask along with 4 mole equivalent of 1-hexene and the mixture is heated to the desired reaction temperature (ranging from 30 to 100° C.) using an oil bath on a controlled hotplate magnetic stirrer. Thereafter 0.5 mol % of the catalyst is added to the flask and the reaction mixture is continuously stirred with a magnetic stirrer bar until the formation of the primary metathesis products is completed. The progress of the reaction is monitored by GC/FID. The sample is prepared for GC analysis by diluting an aliquot (0.3 mL) of the sample, taken at various reaction time intervals, with 0.3 mL toluene and quenched with 2 drops of tert-butyl hydrogen peroxide prior to analysis. Once dodec-1-ene is completely consumed, the reaction is quenched with tert-butyl hydrogen peroxide and filtered through a plug of silica using hexane as eluent. The hexane filtrate is concentrated and the Z-5-hexadecene is isolated by distillation.
Z-11-Hexadecen-1-ol:
Z-5-Hexadecene is subjected to biohydroxylation according to the process disclosed in Example 1 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent, concentrate and silica-gel chromatography.
1-Dodecyne:
The synthesis of 1-dodecyne is carried out according to the protocol described in Oprean, Ioan et al. Studia Universitatis Babes-Bolyai, Chemia, 2006, 51, 33.
5-Hexadecyne:
To a −78° C. solution of 1-dodecyne (5 mmol) in THF (20 mL), 2.5M n-BuLi (5 mmol) in hexane is added dropwise via a syringe. A solution of 1-bromobutane (5 mmol) and TBAI (0.2 mmol) dissolve in THF is then dropwise added to the reaction mixture. The reaction mixture is allowed to warm to room temperature and then heat at 70° C. for 24 hours. The reaction is quenched with 5 mL of 1M NH4Cl and extract with hexanes (3×). The organic fractions are combined, dry with anhydrous MgSO4 and concentrate under reduced pressure. The resulting residue is purified by silica gel flash chromatography using 60:1/hexane:ethyl acetate as mobile phase. Fractions containing the desired product are pulled and concentrate. 5-Hexadecyne is further purified by distillation.
Z-5-Hexadecene:
With stirring, a mixture of Lindlar's catalyst (40 mg) in pentane (10 mL) is put under a balloon of hydrogen for 90 min at 0° C. Quinoline (1 mg) is then added and the mixture is allowed to stir at 0° C. for another 30 min. A solution of Z-5-hexadecene (55 mg) in 2 mL of pentane is then added to the reaction mixture via a syringe. The reaction is allowed to warm to room temperature and the progress of the reaction is monitored by GC. After 18 hours of reaction time, the reaction mixture is filtered through a No. 4 Whatman filter paper and the filtrate is concentrated under reduced pressure to afford the desired product, Z-5-hexadecene, which can be further purified by distillation.
Z-11-Hexadecen-1-ol:
Z-5-hexadecene is then subject to biohydroxylation according to the process disclosed in Example 1 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with ethyl acetate and further purified by distillation.
1-Hexyne:
The synthesis of 1-hexyne is carried out according to the protocol described in Oprean, Joan et al. Studia Universitatis Babes-Bolyai, Chemia, 2006, 51, 33.
5-Hexadecyne:
To a −78° C. solution of 1-hexyne (5 mmol) in THF (20 mL), 2.5M n-BuLi (5 mmol) in hexane is added dropwise via a syringe. A solution of 1-bromodecane (5 mmol) and n-Bu4NI (TBAI) (0.2 mmol) dissolve in THF is then dropwise added to the reaction mixture. The reaction mixture is allowed to warm to room temperature and then heat at 70° C. for 24 hours. The reaction is quenched with 5 mL of 1M NH4Cl and extract with hexanes (3×). The organic fractions are combined, dry with anhydrous MgSO4 and concentrate under reduced pressure. The resulting residue is purified by silica gel flash chromatography using 60:1/hexane:ethyl acetate as mobile phase. Fractions containing the desired product are pulled and concentrate.
Z-5-Hexadecene:
With stirring, a mixture of Lindlar's catalyst (40 mg) in pentane (10 mL) is put under a balloon of hydrogen for 90 min at 0° C. Quinoline (1 mg) is then added and the mixture is allowed to stir at 0° C. for another 30 min. A solution of Z-5-hexadecene (55 mg) in 2 mL of pentane is then added to the reaction mixture via a syringe. The reaction is allowed to warm to room temperature and the progress of the reaction is monitored by GC. After 18 hours of reaction time, the reaction mixture is filtered through a No. 4 Whatman filter paper and the filtrate is concentrated under reduced pressure to afford Z-5-hexadecene, which can be further purified by distillation.
Z-11-Hexadecen-1-ol:
Z-5-Hexadecene is then subjected to biohydroxylation according to the process disclosed in Example 1 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and further purified by distillation.
5-Hexadecyne:
To a −78° C. solution of 1-hexyne (0.383 g, 4.67 mmol) in THF (20 mL), 2.5 M n-BuLi (1.87 mL, 4.67 mmol) in hexane is added dropwise via a syringe. A solution of 1-bromodecane (4.67 mmol) and n-Bu4NI (TBAI, 57 mg, 0.16 mmol) dissolved in THF is then dropwise added to the reaction mixture. The reaction mixture is allowed to warm to room temperature and then heat at 70° C. for 24 hours. The reaction is quenched with 5 mL of 1M NH4Cl and extract with hexanes (3×). The organic fractions are combined, dried with anhydrous MgSO4, and concentrated under reduced pressure. The resulting residue is purified by silica gel flash chromatography using 60:1 hexane:ethyl acetate as the mobile phase. Fractions containing the desired product, 5-hexadecyne, are pooled and concentrated.
Z-5-Hexadecene:
With stirring, a mixture of Lindlar's catalyst (40 mg) in pentane (10 mL) is put under a balloon of hydrogen for 90 min at 0° C. Quinoline (1 mg) is then added and the mixture is allowed to stir at 0° C. for another 30 min. A solution of Z-5-hexadecene (55 mg) in 2 mL of pentane is then added to the reaction mixture via a syringe. The reaction is allowed to warm to room temperature and the progress of the reaction is monitored by GC. After 18 hours of reaction time, the reaction mixture is filtered through a No. 4 Whatman filter paper and the filtrate is concentrated under reduced pressure to afford the desired product, Z-5-hexadecene.
Z-11-Hexadecen-1-ol:
Z-5-Hexadecene is then subjected to biohydroxylation according to the process disclosed in Example 1 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.
Z-5-Hexadecene:
Into an oven-dried three-neck RBF, N-amyl triphenylphosphnium bromide (13.98 g, 33.83 mmol) is dissolved in anhydrous toluene (30 mL). The mixture is allowed to stir via a magnetic stir bar at ambient temperature until complete dissolution of the alkyl phosphonium bromide salt is achieved. A solution of 6.57 g of potassium bis(trimethylsilyl)amide (KHMDS) in anhydrous toluene (30 mL) is then dropwise added to the reaction mixture. Upon complete addition of KHMDS solution to the reaction mixture, the reaction solution is allowed to stir for another 15 minutes, and is then cooled to −78° C. in an acetone and dry ice bath.
A solution of undecanal (4.59 mL, 22.28 mmol) in toluene (40 mL) is then drop-wise added to the reaction mixture via an addition funnel. The reaction is stirred at −78° C. for 20 minutes, then allowed to warm at room temperature with stirring for another 30 minutes. The reaction is terminated by addition of methanol (40 mL) and then concentrated under reduced pressure. The resulting residue is triturated with hexanes and white precipitate, triphenyl phosphine oxide, is removed by filtration. The process is repeated until triphenyl phosphine oxide is no longer precipitated out of the solution. The remnant triphenyl phosphine oxide is removed by passing the crude reaction product through a short bed of silica using hexane as a mobile phase. Z-5-hexadecane is obtained as a colorless oil.
Z-11-hexadecen-1-ol:
Z-5-Hexadecene is subjected to biohydroxylation according to the process disclosed in Example 1 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.
As proof of principal that a synthetically derived pheromone composition comprised of a synthetically derived sex pheromone and a positional isomer can be used to modulate the behavior of a target insect (H. zea), the Z-5-hexadecene was subject to biohydroxylation and oxidation as described above. A mixture of Z-hexadec-11-enal and Z-hexadec-5-enal was produced as shown below.
Four separate experiments were conducted with the moth Helicoverpa zea and its respective pheromone components (Z-11-hexadecenal, Z-9-hexadecenal). Further, the addition of Z-5-hexadecenal, the positional isomer of the natural insect Z-11-hexadecenal pheromone, was also added to the Z-11 and Z-9 blends and tested. Upwind flight and lure location of male moths were compared for: natural ratios of pheromone (97% Z-11-hexadecenal with 3% of Z-9-hexadecenal) with and without addition of the Z-5-hexadecenal positional isomer at various ratios.
Methods
Moths (4-6 day-old males in second half of scotophase) were flown in a glass wind tunnel (120×30×30 cm). A fan pushed air into the wind tunnel at 0.4 m sec−1 and a second fan exhausted air at a similar rate. To provide visual cues for navigation, the floor was covered with light colored construction paper on which small (2-5 cm) circles were drawn with marker (Experiment 1) or light colored circles that were cut from light colored construction paper were placed on the floor (Experiments 2-4). Lures were made with Soxhlet-extracted, grey-rubber septa from West. Compounds were added to the septa in 50 μL of hexane and lures were dried in a fume hood for 1 h before use. Our lures (Experiments 1 and 2) were loaded with 5 μg of the pheromone (Z-11-hexadecenal with 3% of Z-9-hexadecenal) and this treatment was compared to lures with 50, 5 or 0.5% added Z-5-hexadecenal.
Conditions in the wind tunnel (27° C., 50% relative humidity) were similar to the air in the room during bioassays and to conditions under which moths were kept before assays were run.
A moth in a release cage was held on a platform 20 cm above the floor for 15 s in the plume of pheromone and then released by turning open end of the cage toward upwind. The lure was at the same height as the moth and 90 cm upwind. Moths were given 5 min to locate the lure. Data collected were: 1) whether or not a moth contacted the pheromone lure, 2) whether or not a moth nearly contacted the lure (hovering downwind within 10 cm of the lure without contact: “close but no contact”), and 3) time until contact. Flights were recorded on video and data were collected from videos.
We ran 2 experiments:
Over the course of the 4 experiments, occasionally lures with no pheromone (50 uL clean hexane) were included as negative controls (n=35). Conditions in the wind tunnel were the same for all experiments.
This experiment was performed to evaluate the response of the moths to pheromone coated lures with and without high concentrations of the Z-5-hexadecenal positional isomer
With the treatments, the moths flew upwind, although relatively few moths located the pheromone lure, and there were no significant differences between numbers of moths that flew close to the lure but did not make contact (4, 6, and 5 respectively); however, significantly more moths contacted the lure (43%) with the natural pheromone blend (0% added Z5-hexadecacenal) than with 5% added Z5-hexadecacenal (Table 15; 11%; χ2=9.07, P<0.01) or 50% added Z11-hexadecacenal (Table 15; 14%; χ2=8.04, P<0.01). There were no significant differences among treatments in any experiment in latency (time from the start of the bioassay and contact with the lure).
To the surprise of the inventors, the results from Experiment 8.1 indicate that the number of Helicoverpa zea moths finding the pheromone composition that includes the Z-5-hexadecenal positional isomer was significantly reduced relative to the natural pheromone blend. The moth is unexpectedly responsive to pheromone compositions including the Z-5-hexadecenal positional isomer despite the structural difference compared to Z-11-hexadecenal as the moth species flew upwind to interact with a plume in the presence and absence of the Z-5-hexadecenal positional isomer. That is, the natural pheromone blend elicited an flew upwind flight and contact response, whereas, in the presence of the Z-5-hexadecenal positional isomer, the moths flew upwind but did not contact the lures.
This experiment was performed to assess the response of the target moth to lower concentrations of the Z-5-hexadecenal in the natural pheromone blend.
There were no significant differences between treatments in number of moths that flew close to the lure but did not make contact (Table 16; χ2=3.98, P>0.05). Significantly more moths contacted the lure with 0% Z5-hexadecenal (38%) than with 5% added Z5-hexadecacenal (Table 16; 17%; χ2=4.11, P<0.05), but there was no difference in numbers contacting the lures with 0 or 0.5% Z5-hexadecacenal (38 and 44% respectively; χ2=0.33, P>0.05).
The results of Experiment 8.2 indicate that while normal upwind flight seems to occur when the Z-5-hexadecenal positional isomer is included in the composition, contact with the lure is reduced. Furthermore, the results indicate that the amount of the Z-5-hexadecenal positional isomer can be varied to modulate attraction and landing as more moths landed on the lure in the presence of 0% or only 0.5% Z-5 hexadecenal compared to 5% Z-5 hexadecenal, whereas more moths came close to but did not contact the lure coated with 5% Z-5 hexadecenal compared to 0% or only 0.5% Z-5 hexadecenal. Thus, the pheromone compositions described herein can be used to elicit a tunable response in a target insect by inter alia varying the ratio of the positional isomer to the natural pheromone to thereby modulate attraction and/or landing.
Conclusions from 8.1-8.2
Across all treatments, nearly all moths flew upwind and most interacted with the plume in some way. Thus, the Z-5-hexadecedenal positional isomer triggers a similar upwind flight response in male moths. In general, contact was lower for treatments with added Z-5-hexadecenal.
These results indicate that the presence of the Z-5-hexadecenal positional isomer in the natural pheromone blend reduced the number of moths contacting the lures while still maintaining upwind orientation similar to the physiological responses to the natural pheromone blend alone.
The moths therefore can respond to the Z-5-hexadecenal positional isomer, which indicates that a positional isomer has valuable applications in modulating insect behavior. Thus, the presence of the positional isomer can be used to elicit a tunable response from target insects. That is, the amount of the he Z-5-hexadecenal positional isomer in the pheromone composition can be varied to either cause the moths to fly toward the lure but not land or to land on the lure.
Some moths flew upwind when an unbaited lure was present (15 of 35) but none of these exhibited plume-oriented flight or lure contact.
Thus, a surprising and unexpected result of including the Z-5-hexadecenal positional isomer in the pheromone composition was that the number of moths finding the lures was reduced while upwind orientation was maintained relative to the natural pheromone blend (i.e., in the absence of Z-5-hexadecenal). Although the number of moths finding the lures was reduced, the ability to attract the moth species using a pheromone composition containing a positional isomer indicates that the compositions taught herein can be used to modify insect behavior. Further, because the response elicited was dependent on the relative amount of the Z-5-hexadecenal positional isomer, these results indicate the response of the targeted insects can be tuned, e.g., to attract a target insect or to cause the target insect to land, by varying the amount of the positional isomer.
As shown below, Z-9-tetradecenal is a naturally produced sex pheromone for various lepidopteran species. Using the biohydroxylation methodology disclosed herein, a pheromone composition comprising Z-9-tetradecenal and its positional isomer Z-5-tetradecenal can be prepared as shown below.
Wind tunnel experiments will be performed as described above using pheromone compositions comprising synthetically derived sex pheromone and a positional isomer. The inventors expect the positional isomer Z-5-tetradecenal to elicit an upwind flight response.
The blend of sex pheromones produced by female Spodoptera frugiperda (Fall armyworm) includes an 96.4/3.6 mixture of Z-9-tetradecenyl acetate and Z-7 dodecenyl acetate.
The Z-9-tetradecenyl acetate sex pheromone produced by female Spodoptera frugiperda (Fall armyworm) is shown below. Using the biohydroxylation methodology disclosed herein, a pheromone composition comprising Z-9-tetradecenyl acetate and its positional isomer Z-5-tetradecenyl acetate can be prepared as shown below.
Wind tunnel experiments will be performed as described above using pheromone compositions comprising the synthetically derived sex pheromone and a positional isomer. The inventors expect the positional isomer Z-5-tetradecenyl acetate in a composition with Z-9-tetradecenyl acetate to elicit response physiological response.
Any of the pheromones listed in Table 1 above can be synthesized as described herein to produce a pheromone composition comprising synthetically derived mixture of a natural pheromone and a positional isomer.
Wind tunnel experiments can be performed using a pheromone composition comprising synthetically derived mixture of a natural pheromone and a positional isomer to modulate the behavior of the target insect.
Based on the inventors' unexpected and first reported discovery that enzyme catalysts can be used to hydroxylate an unsaturated hydrocarbon substrate, thereby creating olefins with a terminal alcohol, the inventor propose using different biohydroxylation catalysts to hydroxylate carbon atoms within the carbon skeleton (i.e., subterminal carbons). A variety of P450 enzyme are known to catalyze hydroxylation of subterminal carbons to produce secondary alcohols. See, e.g., Greer et al., Plant Physiology. 2007; 143(3):653-667. It is also possible that the hydroxylase enzymes disclosed herein catalyze the formation of subterminal hydroxyl groups in low yields. Further, it is also possible to engineer an enzyme to selectively catalyze hydroxylation of an subterminal carbon.
As shown below, the hydroxyl group can be inserted on an subterminal carbon of an unsaturated hydrocarbon substrate to produce an olefinic alcohol. Subsequent oxidation, acetylation, or esterification can generate a positional isomer of a sex pheromone naturally produced by an insect species.
Based on the wind tunnel results discussed above with terminally functionalized positional isomer thereof, the inventors expect that isomers with a subterminal hydroxy group will similarly modulate the behavior of an insect.
All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.
Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. All publications, patents, patent applications, and sequence accession numbers cited herein are hereby incorporated by reference in their entirety for all purposes.
The present application claims the benefit of priority to U.S. Provisional Application No. 62/255,215, filed on Nov. 13, 2015, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4216202 | Klun et al. | Aug 1980 | A |
| 4219542 | Klun | Aug 1980 | A |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20170135343 A1 | May 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62255215 | Nov 2015 | US |
