Patent Grant
10202620
The Substitute Sequence Listing written in file SecondSubstituteSequenceListing_095864-0957387.txt created on Nov. 14, 2017, 359,804 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.
Insect pheromones can be used in a variety of insect control strategies that include mating disruption and attract-and-kill, as well as 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). Even the very stringent USDA Organic Program lists insect pheromones as one of the few synthetic organic compounds allowed in organic crop production, another important recognition of the high safety of these products. Accordingly, pheromones already form the basis of integrated pest management (IPM) practices in fruit production on the U.S. west coast, and their use in organic farming is growing worldwide.
Despite these advantages, pheromones are not widely used today because of the high cost of about $500 to $14,000 per kg of active ingredient (AI). Even though thousands of insect pheromones have been identified, less than about twenty insect pests worldwide are currently controlled using pheromone strategies, and only 0.05% of global agricultural land employs pheromones.
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.
The present invention provides methods by which lepidopteran insect pheromones as well as structurally related compounds are prepared using synthetic strategies that are enabled by a biocatalytic step.
In a first aspect, the invention provides a method for synthesizing an olefinic alcohol product that includes incubating an unsaturated hydrocarbon substrate with an enzyme capable of selectively hydroxylating one terminal carbon of the unsaturated hydrocarbon substrate to form an unsaturated hydrocarbon alcohol. In some embodiments, the unsaturated hydrocarbon alcohol is the olefinic alcohol product. In some embodiments, the method further includes converting the unsaturated hydrocarbon alcohol to the olefinic alcohol product.
In some embodiments, the unsaturated hydrocarbon substrate is an olefinic substrate. The olefinic substrate can be prepared via olefin metathesis and other routes including alkylation and reduction of alkynes, as well as Wittig-type reaction of aldehydes with phosphine reagents.
In some embodiments, the unsaturated hydrocarbon substrate is an alkyne. In some embodiments, the unsaturated hydrocarbon substrate is an alkenyl halide.
In a related aspect, the invention provides a method for synthesizing an olefinic alcohol product that includes incubating a saturated hydrocarbon substrate with an enzyme capable of selectively hydroxylating one terminal carbon of the saturated hydrocarbon substrate to form a saturated hydrocarbon alcohol, and converting the saturated hydrocarbon alcohol to the olefinic alcohol product.
In some embodiments, the saturated hydrocarbon substrate is an alkane substrate. In some embodiments, the method includes incubating the alkane substrate with an enzyme capable of selectively hydroxylating both terminal carbons of the alkane substrate to form a terminal diol. The terminal diol can be converted to the olefinic alcohol product in one or more subsequent steps.
In some embodiments, the saturated hydrocarbon substrate is an alkyl halide. In some embodiments, the method includes incubating the alkyl halide with an enzyme capable of selectively hydroxylating one terminal carbon of the alkyl halide to form a halogen-substituted alkanol. The halogen-substituted alkanol can be converted to the olefinic alcohol product in one or more subsequent steps.
In some embodiments, the saturated hydrocarbon substrate is a fatty acid. In some embodiments, the method includes incubating the fatty acid with an enzyme capable of selectively hydroxylating the terminal carbon of the fatty acid to form a terminal hydroxy fatty acid. The terminal hydroxy fatty acid can be converted to the olefinic alcohol product in one or more subsequent steps.
In some embodiments, the enzyme used in the methods of the invention is a non-heme diiron monooxygenase. In some embodiments, the enzyme is a long-chain alkane hydroxylase. In some embodiments, the enzyme is a cytochrome P450. In some embodiments, the cytochrome P450 is a member of the CYP52 or CYP153 family.
In certain embodiments, the olefinic alcohol product prepared according to the method of the invention is a pheromone. In particular embodiments, the pheromone is a lepidopteran insect pheromone.
In another aspect, the invention 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.
Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.
The present invention provides a method of producing terminally oxyfunctionalized alkenes and alkynes. In certain aspects, the method includes contacting an unsaturated or saturated hydrocarbon substrate with a hydroxylase enzyme in the presence of molecular oxygen, reducing equivalents, and optionally redox partners under conditions sufficient to provide a terminally hydroxylated alkene or alkyne. The unsaturated or saturated hydrocarbon substrates can be prepared using the methods described herein. Relevant terminal hydroxylases useful for carrying out this method exhibit strong selectivity towards the terminal carbon of an alkyl chain and include, but are not limited to, non-heme diiron alkane monooxygenases, cytochromes P450 (e.g., cytochromes P450 of the CYP52 and CYP153 family), as well as long chain alkane hydroxylases. In certain embodiments, the terminally hydroxylated alkene or alkyne is further converted to a terminal alkenal. The terminal alkenal can be obtained by chemically or enzymatically oxidizing the terminally hydroxylated alkene or alkyne. Alcohol oxidases, alcohol dehydrogenases, and alpha-dioxygenases can be used for the enzymatic oxidation. In certain embodiments, terminally hydroxylated alkenes and alkynes are useful as insect pheromones which modify insect behavior. In some embodiments, terminally hydroxylated alkenes and alkynes are useful intermediates for producing pheromones via acetylation or oxidation of the alcohol moiety.
The following definitions and abbreviations are to be used for the interpretation of the invention. The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment but encompasses all possible embodiments.
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.”
The terms “about” and “around,” as used herein to modify a numerical value, indicate a close range surrounding that explicit value. If “X” were the value, “about X” or “around X” would indicate a value from 0.9X to 1.1X, and more preferably, a value from 0.95X to 1.05X. Any reference to “about X” or “around X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, “about X” and “around X” are intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”
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).
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 invention, 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 “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” 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” refers to 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 “aryl” refers to an aromatic carbon ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups can include any suitable number of carbon ring atoms, such as, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ring members. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group. Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. Aryl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.
As used herein, the terms “halo” and “halogen” refer to fluorine, chlorine, bromine and iodine.
As used herein, the term “carboxy” refers to a moiety —C(O)OH. The carboxy moiety can be ionized to form the carboxylate anion.
As used herein, the term “hydroxy” refers to a moiety —OH.
As used herein, the term “amino” refers to a moiety —NR3, wherein each R group is H or alkyl. An amino moiety can be ionized to form the corresponding ammonium cation.
As used herein, the term “amido” refers to a moiety —NRC(O)R or —C(O)NR2, wherein each R group is H or alkyl.
As used herein, the term “nitro” refers to the moiety —NO2.
As used herein, the term “oxo” refers to an oxygen atom that is double-bonded to a compound (i.e., O═).
As used herein, the term “cyano” refers to the moiety —CN.
Traditionally, straight chain monoene alcohols, acetates, and aldehydes are synthesized via multi-step syntheses. Scheme 1 represents an example of such synthesis.
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 invention 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 invention generally include oxidizing the olefinic alcohol product to form an olefinic aldehyde product. In some of these 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 invention 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.
Agrotis
segetum sex pheromone component
Anarsia
lineatella sex pheromone component
Anarsia
lineatella sex pheromone component
Pseudoplusia
includens sex pheromone
Agrotis
segetum sex
Grapholitha
molesta, Ecdytolophaaurantiana sex
Grapholitha
molesta, Ecdytolophaaurantiana sex pheromone component
Grapholitha
molesta sex pheromone component
Eupoecilia
ambiguella sex pheromone
Pandemis
pyrusana, Narangaaenescens, Agrotissegetum
Pandemis
pyrusana, Choristoneuraroseceana sex
Choristoneura
roseceana, Crocidolomiapavonana sex
Diatraea
considerata sex pheromone component
Helicoverpa
zea, Helicoverpaarmigera, Heliothisvirescens
Naranga
aenescens sex pheromone component
Platyptila
carduidactyla, Heliothisvirescens sex
Helicoverpa
zea, Helicoverpa
armigera, Plutellaxylostella,
Diatraea
considerate,
Diatraea
grandiosella,
Diatraea
saccharalis,
Acrolepiopsis
assectella sex
Discestra
trifolii sex pheromone
Heliothis
virescens, Plutella
xylostella, Acrolepiopsis
assectella, Crocidolomia
pavonana, Narangaaenescens
Diatraea
considerata, Diatraeagrandiosella sex
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 invention 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 2). Deviations from this general scheme are also disclosed.
Synthesis of Terminal Alkenols Via Metathesis and Hydroxylation
In a first aspect, the invention provides a method for synthesizing an olefinic alcohol product that includes incubating an unsaturated hydrocarbon substrate with an enzyme capable of selectively hydroxylating one terminal carbon of the unsaturated hydrocarbon substrate to form an unsaturated hydrocarbon alcohol. In some embodiments, the unsaturated hydrocarbon alcohol is the olefinic alcohol product. In some embodiments, the method further includes converting the unsaturated hydrocarbon alcohol to the olefinic alcohol product. In some embodiments, the unsaturated hydrocarbon substrate is an olefinic substrate. In some embodiments, the olefinic substrate is a metathesis product.
In some embodiments, the method for synthesizing an oxyfunctionalized alkene includes a combination of metathesis and terminal hydroxylation as shown in Scheme 3.
According to Scheme 3, an E-selective metathesis catalyst or a Z-selective metathesis catalyst is used to convert a linear, terminal alkene of chain length m to the respective linear symmetric alkene. Following the metathesis step, a terminal hydroxylation biocatalyst is used to convert the symmetric alkene to the terminal alkenol. Optionally, this scheme can be expanded by an additional metathesis step that replaces the non-hydroxylated end of the terminal alkenol with one of another chain length (Scheme 4a).
In some instances, Scheme 4a can be further modified to protect the alcohol prior to metathesis. For example, the alcohol can be esterified prior to the metathesis step (Scheme 4b). The esterification is typically performed with formate or acetate, resulting in R═H or CH3, respectively. The ester intermediates or final products can be hydrolysed to yield alcohol products.
The methods can be conducted with alkenes of any suitable length, which will depend on factors such as the desired olefinic alcohol product and the enzyme used in the biohydroxylation step. In some embodiments, the olefinic alcohol product is a C4-C20 olefinic alcohol product. The 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 such embodiments, the sum of the subscripts m, n, and y shown in Scheme 3 and Scheme 4 will bring the total number of carbon atoms in a particular olefinic alcohol product to 4-20, when added to the number of the non-subscripted carbon atoms shown in the structure for the olefinic alcohol product. In such embodiments, for example, subscript m in Scheme 3 can be an integer from 0-8, bringing the total number of the carbons in the symmetric olefinic substrate to 4-20. When m is 3, the route depicted in Scheme 3 provides (E/Z)-5-decen-1-ol.
Accordingly, some embodiments of the invention 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) self-metathesizing a 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 terminal olefin has the formula (CH2═CH)(CH2)mH, the metathesis product has the formula H(CH2)m(CH═CH)(CH2)mH, the olefinic alcohol product has the formula H(CH2)m(CH═CH)(CH2)mOH, and m is selected from an integer between 1 and 17. In some embodiments, m is selected from an integer between 1 and 9. In some embodiments, for example, m is 1, 2, 3, 4, 5, 6, 7, or 8. It is to be understood that any range disclosed in the present specification and recited in the claims includes the endpoints the endpoints of the range, unless explicitly stated otherwise. As a non-limiting example, integers in the range “between 1 and 17” include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.
In some embodiments, the method for synthesizing an oxyfunctionalized alkene includes a combination of metathesis and terminal hydroxylation as shown in Scheme 5. 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 5 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 5 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 5 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 17; 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 invention 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 H(CH2)m(CH═CH)(CH2)nOH, and m and n are different integers between 1 and 17. In some embodiments, m and n are different integers between 1 and 9.
The methods of the invention 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 invention 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 invention 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 invention. 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 Alkanes to Terminal Diols Followed by Monodehydration and Metathesis
Saturated hydrocarbon substrates can also be used in the methods of the invention. Accordingly, another aspect of the invention provides a method for synthesizing an olefinic alcohol product that includes: incubating a saturated hydrocarbon substrate with an enzyme capable of selectively hydroxylating one terminal carbon of the saturated hydrocarbon substrate to form a saturated hydrocarbon alcohol; and converting the saturated hydrocarbon alcohol to the olefinic alcohol product.
In some embodiments, terminal alkenols are synthesized according to Scheme 6a.
In this synthesis scheme, the terminal hydroxylation occurs on both ends of a linear alkane, resulting in a diol. The diol is then selectively monodehydrated through various chemical processes, including but not limited to those described in the literature (Sato et al., 2013) to generate a terminal alkenol. Coupling of the terminal alkenol with other alkenes via an olefin metathesis process allows for the synthesis of various pheromones.
In some instances, Scheme 6a can be further modified to protect the alcohol prior to metathesis. For example, the alcohol can be esterified prior to the metathesis step (Scheme 6b). The esterification is typically performed with formate or acetate, resulting in R═H or CH3, respectively. The ester intermediates or final products can be hydrolysed to yield the alcohol products.
Accordingly, some embodiments of the invention provide a method wherein the saturated hydrocarbon substrate is an alkane substrate, and wherein the method includes:
Accordingly, some embodiments of the invention provide a method wherein the saturated hydrocarbon substrate is an alkane substrate, and wherein the method includes:
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.
In some embodiments, the alkane substrate has the formula H(CH2)mH, the terminal diol has the formula HO(CH2)mOH, the α,ω-alkenol has the formula (CH2═CH)(CH2)m-2OH, the terminal olefin has the formula (CH2═CH) (CH2)nH, the olefinic alcohol product has the formula H(CH2)n(CH═CH)(CH2)m-2OH, m is an integer between 3 and 17, and n is an integer between 1 and 17. The alkane substrate, the terminal diol, the α,ω-alkenol, the terminal 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, terminal alkenols are synthesized according to Scheme 7a.
In a particular embodiment, the hydroxylase enzyme favors hydroxylation of the terminal CH3 group over epoxidation of the C═C double bond.
In some instances, Scheme 7a can be further modified to protect the alcohol prior to metathesis. For example, the alcohol can be esterified prior to the metathesis step (Scheme 7b). The esterification is typically performed with formate or acetate, resulting in R═H or CH3, respectively. The ester intermediates or final products can be hydrolysed to yield alcohol products.
The alkenes 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, terminal alkenols are synthesized according to Scheme 8a. In this Scheme, a terminal olefin forming fatty acid decarboxylase is used to convert a ω-hydroxy fatty acid into a fatty alkene.
In some instances, Scheme 8a can be further modified to protect the alcohol prior to metathesis. For example, the alcohol can be esterified prior to the metathesis step (Scheme 8b). The esterification is typically performed with formate or acetate, resulting in R═H or CH3, respectively. The ester intermediates or final products can be hydrolysed to yield alcohol products.
The fatty acid substrate, the terminal hydroxy fatty acid, the alkenes, 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.
Alternative Synthesis of Terminal Alkenols Via Hydroxylation
In some embodiments, symmetric or asymmetric alkenes are hydroxylated according to Schemes 3 and 4, respectively to produce symmetric or asymmetric alkenols. However, in this embodiment, the alkene is produced according to Scheme 9 (see, Oprean et al. (2006) for the acetylation step and Buck and Chong (2001) for the alkyne alkylation step), Scheme 10 (see, Buck and Chong (2001) regarding the alkyne alkylation step), Scheme 11a, or Scheme 11b. Scheme 11b shows Wittig reaction conditions that favor the formation of the Z-isomer according to Smith et al. (2000).
Accordingly, some embodiments of the invention 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
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 invention includes:
a) forming a reaction mixture comprising a phosphonium salt according to formula XVI
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.
In some embodiments, terminal alkenols are synthesized according to Scheme 12a. In certain embodiments, the Wittig reaction favors formation of the Z-olefin (Scheme 12b; see, Smith et al. 2000).
Accordingly, some embodiments of the invention provide methods that include:
The alkane, the diol, the aldehyde, the phosphonium salt, 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, terminal alkenols are synthesized by first hydroxylating terminal alkynes to their corresponding alkynols. Upon protection of the hydroxyl functional group, the alkynols can be further functionalized by alkylation with other haloalkanes to generate disubstituted alkynols and partial hydrogenation to afford the desired alkene products, as shown in Scheme 13.
Accordingly, some embodiments of the invention provide a method wherein the hydrocarbon substrate is an alkyne and the method includes:
a) incubating a terminal alkyne according to formula V
b) forming a reaction mixture comprising the unsaturated hydrocarbon alcohol and an alkyl halide according to formula VII
In some embodiments, the unsaturated hydrocarbon alcohol according to formula VI is protected, and then the resulting protected unsaturated hydrocarbon alcohol is combined with an alkyl halide according to formula VII under conditions sufficient to form a protected disubstituted alkyne. The protected disubstituted alkyne can be reduced and deprotected to provide an olefinic alcohol product according to formula IXa or IXb.
The terminal alkyne, the unsaturated hydrocarbon alcohol, the alkyl halide, the disubstituted alkyne, 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, terminal halogenated alkanes can be biohydroxylated to generate α,ω-halogenated alcohols. Upon protection of the alcohol moiety, the substrate can be coupled with a terminal alkyne to afford an internal alkyne product, which can be partially reduced via known chemical processes to generate either the cis- or trans-alkenes that can be readily converted to insect pheromones, as illustrated in Scheme 14.
Accordingly, some embodiments of the invention provide a method wherein the saturated hydrocarbon substrate is an alkyl halide, and wherein the method includes:
a) incubating an alkyl halide according to formula X
b) converting the halogen-substituted alkanol to a protected halogen-substituted alkanol according to formula XII
d) reducing the disubstituted alkyne to form a protected olefinic alcohol according to formula XVa or formula XVb
The alkyl halide, the halogen-substituted alkanol, the protected halogen-substituted alkanol, the terminal alkyne, the disubstituted alkyne, 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, halogenated alkenes can be biohydroxylated to generate corresponding alkenols. Upon protection of the alcohol moiety, the substrate is then coupled with haloalkanes, alkenes, or alkynes to provide suitable intermediates for synthesis of insect pheromones as illustrated in Scheme 15 below.
Accordingly, some embodiments of the invention provide a method wherein the unsaturated hydrocarbon substrate is an alkenyl halide, and wherein the method includes:
a) incubating an alkenyl halide according to formula X′
b) converting the halogen-substituted unsaturated hydrocarbon alcohol to a protected halogen-substituted unsaturated hydrocarbon alcohol according to formula XII′
In some embodiments, terminal alkenols are synthesized according to Scheme 16.
Accordingly, some embodiments of the invention provide a method which includes:
a) incubating an alkane according to formula XXIX
b) converting the diol to an alkyne according to formula XXXI
c) forming a reaction mixture comprising the alkyne and an alkyl halide according to formula XXXII
The alkane, the diol, the alkyne, the alkyl halide, the disubstituted alkyne, 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, terminal alkenols are synthesized according to Scheme 17a. Under certain conditions, the Wittig reaction favors the formation of the Z-isomer (Scheme 17b; see, Smith et al. 2000).
Accordingly, some embodiments of the invention provide a method wherein the saturated hydrocarbon substrate is a fatty acid, and wherein the method includes:
In some embodiments, the invention provides a method wherein the saturated hydrocarbon substrate is a fatty acid, and wherein the method includes:
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.
In some embodiments wherein the saturated hydrocarbon substrate is a fatty acid, the fatty acid has the formula H(CH2)mCO2H, the terminal hydroxy fatty acid has the formula HO(CH2)mCO2H, the α,ω-alkenol has the formula (CH2═CH)(CH2)m-2OH, the terminal olefin has the formula (CH2═CH)(CH2)nH, the olefinic alcohol product has the formula H(CH2)n(CH═CH)(CH2)m-2OH, and m and n are each independently selected from an integer between 3 and 17.
The fatty acid, the terminal hydroxy fatty acid, the α,ω-alkenol, the terminal 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 different integers between 1 and 9.
In some embodiments, the invention provides a method wherein the saturated hydrocarbon substrate is a fatty acid, and wherein the method includes:
a) incubating a fatty acid according to formula XXIV
b) reducing the terminal hydroxy fatty acid to form an aldehyde according to formula XXVI
The alkane, fatty acid, the terminal hydroxy fatty acid, the aldehyde, the phosponium salt, 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.
Synthesis of Oxyfunctionalized Olefins Comprising More than One C═C Double Bond
In some embodiments, conjugated and unconjugated alkenes can be biohydroxylated to generate corresponding conjugated and unconjugated alkenols. The resulting products are then coupled with haloalkanes, haloalkenes, or alkynes to provide suitable intermediates for synthesis of insect pheromones as illustrated in Scheme 18 below.
Accordingly, some embodiments of the invention provide a method wherein the unsaturated hydrocarbon substrate is an alkenyl halide, and wherein the method includes:
a) incubating an alkenyl halide according to formula X″
b) converting the halogen-substituted unsaturated hydrocarbon alcohol to a protected halogen-substituted unsaturated hydrocarbon alcohol according to formula XII″
The alkenyl halide, the halogen-substituted unsaturated hydrocarbon alcohol, the protected halogen-substituted unsaturated hydrocarbon alcohol, the alkyl halide according to formula XIIa″, the elongated protected unsaturated hydrocarbon alcohol, and the olefinic alcohol product can have any suitable combination of subscripts a, b, c, d, e, and f. In some embodiments, a, b, c, d, e, and f are integers independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9. In some embodiments, the sum of a, b, c, d, e, and f is an integer from 1 to 14. In some embodiments, the sum of a, b, c, d, e, and f is an integer from 1 to 8.
In some embodiments, conjugated and unconjugated alkene-alkyne bi-functional substrates can be biohydroxylated to generate alkene-alkynols. The resulting biohydroxylation products can then be used for coupling with haloalkanes, alkenes, or alkynes to provide suitable intermediates for synthesis of insect pheromones as illustrated in Scheme 19 below.
Accordingly, some embodiments of the invention provide a method which includes:
a) incubating an unsaturated halogen-substituted hydrocarbon according to formula X′″
b) converting the halogen-substituted unsaturated hydrocarbon alcohol to a protected halogen-substituted unsaturated hydrocarbon alcohol according to formula XII′″
The unsaturated halogen-substituted hydrocarbon, the halogen-substituted unsaturated hydrocarbon alcohol, the protected halogen-substituted unsaturated hydrocarbon alcohol, the alkyl halide according to formula XIIa′″, the elongated protected unsaturated hydrocarbon alcohol, and the olefinic alcohol product can have any suitable combination of subscripts a, b, c, d, e, and f. In some embodiments, a, b, c, d, e, and f are integers independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9. In some embodiments, the sum of a, b, c, d, e, and f is an integer from 1 to 14. In some embodiments, the sum of a, b, c, d, e, and f is an integer from 1 to 8.
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 through 8 may be used with the present invention. 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. Chem Cat Chem, 2013. 5(12): p. 3436-3459), herein incorporated by reference in its entirety. Specific catalysts 1-5 exhibiting cis-selectivity are shown below (Scheme 19a) 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, preferred 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 invention 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 invention 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 invention 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 invention 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 invention also include those set forth in the following table:
Catalysts useful in the methods of the invention 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 invention 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 invention 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%, preferably better than 50%, more preferably better than 75%, and most preferably better than 90%. In addition, the reactants and products are chosen to provide at least a 5° C. difference, preferably a greater than 20° C. difference, and most preferably 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, preferably less than 12 hours, more preferably less than 8 hours, and most preferably 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 invention. The metathesis steps can proceed at a variety of temperatures and times. In general, reactions in the methods of the invention 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 invention 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.
Hydroxylation 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 monoxxygenases; (2) integral membrane diiron non-heme alkane hydroxylases (AlkB); (3) Cytochrome P450s (P450s); and (4) long chain alkane monoxygenases (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 2.
P. putida
Gordonia
Proc.
Natl.
Arch.
Arch.
Arch.
Org.
Org.
Enzyme
Acad.
Biochem
Biochem
Biochem.
Biomol.
Biomol.
Microb.
Biotechnol.
Sci. U.S.A.
Biophys.
Biophys.
Biophys.
Bacteriol.
Chem. 9:
Chem. 9:
Technol. 16:
Biochem. 68:
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 ω-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. thermodemtrificants 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. Microbial. 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 invention. 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 invention. 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 invention 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 3 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. aureofaciens
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
Pseudomonas sp. ITRH76
Pseudomonas sp. 7/156
Pseudomonas sp. ITRI22
Pseudomonas putida (Arthrobacter siderocapsulatus)
Pseudomonas sp. G5(2012)
Alcanivorax dieselolei
Alcanivorax borkumensis
Marinobacter sp. S17-4
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 aquaeolei (strain ATCC 700491/DSM
Marinobacter hydrocarbonoclasticus ATCC 49840
Alcanivorax borkumensis
Alcanivorax borkumensis (strain SK2/ATCC
Marinobacter aquaeolei (strain ATCC 700491/DSM
Alcanivorax sp. 97CO-5
Marinobacter sp. C1S70
Marinobacter sp. EVN1
Pseudoxanthomonas spadix (strain BD-a59)
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/CGMCC
Alcanivorax dieselolei
Marinobacter sp. ELB17
Marinobacter sp. BSs20148
Pseudomonas alcaligenes NBRC 14159
Simiduia agarivorans (strain DSM 21679/JCM 13881/
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
Methylibium petroleiphilum (strain PM1)
Ralstonia sp. AU12-08
Burkholderia phytofirmans (strain DSM 17436/PsJN)
Pseudovibrio sp. (strain FO-BEG1)
Bradyrhizobium sp. DFCI-1
Alcanivorax dieselolei (strain DSM 16502/CGMCC
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 K96243)
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 xenovorans (strain LB400)
Burkholderia thailandensis MSMB43
Burkholderia sp. Ch1-1
Alcanivorax sp. R8-12
Alcanivorax pacificus W11-5
Actinoplanes sp. (strain ATCC 31044/CBS 674.73/
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
Ralstonia sp. 5_2_56FAA
Ralstonia sp. 5_7_47FAA
Burkholderia cenocepacia (strain AU 1054)
Burkholderia cenocepacia (strain HI2424)
Burkholderia sp. KJ006
Burkholderia vietnamiensis (strain G4/LMG 22486)
Burkholderia cenocepacia KC-01
Ralstonia pickettii (strain 12D)
Ralstonia pickettii (strain 12J)
Ralstonia pickettii OR214
Mycobacterium thermoresistibile ATCC 19527
Burkholderia cenocepacia PC184
Parvularcula bermudensis (strain ATCC BAA-594/
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 BAA-245/DSM
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 17616/249)
Burkholderia multivorans (strain ATCC 17616/249)
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
Nocardia nova SH22a
Actinoplanes missouriensis (strain ATCC 14538/DSM
Mycobacterium thermoresistibile ATCC 19527
Streptomyces collinus Tu 365
Mycobacterium smegmatis MKD8
Mycobacterium smegmatis (strain ATCC 700084/
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/JCM 11494/
Burkholderia ambifaria (strain ATCC BAA-244/
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
Marinobacter sp. EVN1
Marinobacter santoriniensis NKSG1
Marinobacter sp. ES-1
Nocardia farcinica (strain IFM 10152)
Mycobacterium chubuense (strain NBB4)
Acinetobacter towneri DSM 14962 = CIP 107472
Rhodococcus erythropolis CCM2595
Rhodococcus erythropolis (strain PR4/NBRC 100887)
Rhodococcus sp. P27
Rhodococcus erythropolis DN1
Rhodococcus erythropolis (Arthrobacter picolinophilus)
Mycobacterium fortuitum subsp. fortuitum DSM 46621
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-1815D
Rhodococcus imtechensis RKJ300 = JCM 13270
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 BT1
Mycobacterium tuberculosis BT2
Mycobacterium tuberculosis HKBS1
Mycobacterium tuberculosis EAI5
Mycobacterium tuberculosis EAI5/NITR206
Mycobacterium tuberculosis CAS/NITR204
Mycobacterium bovis (strain ATCC BAA-935/
Mycobacterium tuberculosis (strain ATCC 25618/
Mycobacterium tuberculosis str. Beijing/NITR203
Mycobacterium bovis BCG str. Korea 1168P
Mycobacterium liflandii (strain 128FXT)
Mycobacterium tuberculosis (strain CDC 1551/
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 35801/TMC
Mycobacterium tuberculosis UT205
Mycobacterium bovis BCG str. Mexico
Mycobacterium tuberculosis CTRI-2
Mycobacterium canettii (strain CIPT 140010059)
Mycobacterium canettii (strain CIPT 140010059)
Mycobacterium africanum (strain GM041182)
Mycobacterium tuberculosis (strain CCDC5180)
Mycobacterium tuberculosis (strain CCDC5079)
Mycobacterium tuberculosis (strain KZN 1435/MDR)
Mycobacterium bovis (strain BCG/Tokyo 172/ATCC
Mycobacterium marinum (strain ATCC BAA-535/M)
Mycobacterium tuberculosis (strain F11)
Mycobacterium tuberculosis (strain ATCC 25177/
Mycobacterium tuberculosis str. Haarlem
Mycobacterium bovis (strain BCG/Pasteur 1173P2)
Mycobacterium bovis 04-303
Mycobacterium bovis AN5
Mycobacterium tuberculosis GuangZ0019
Mycobacterium tuberculosis FJ05194
Mycobacterium tuberculosis ‘98-R604 INH-RIF-EM’
Mycobacterium marinum str. Europe
Mycobacterium marinum MB2
Mycobacterium orygis 112400015
Mycobacterium tuberculosis NCGM2209
Mycobacterium bovis BCG str. Moreau RDJ
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 CPHL_A
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/NBRC 100887)
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) (Corynebacterium equi)
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) (Nocardia
mediterranei)
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 27029/DSM
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/NBRC 100887)
Corynebacterium falsenii DSM 44353
Rhodococcus erythropolis CCM2595
Rhodococcus sp. P27
Rhodococcus erythropolis DN1
Rhodococcus erythropolis SK121
Rhodococcus qingshengii BKS 20-40
In some embodiments, the invention 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 4 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 invention 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 5 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.
Candida tropicalis (Yeast)
Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast)
Candida tropicalis (Yeast)
Candida albicans (Yeast)
Candida maltosa (Yeast)
Candida dubliniensis (strain CD36/ATCC MYA-646/
Candida albicans (strain SC5314/ATCC MYA-2876)
Candida albicans (strain SC5314/ATCC MYA-2876)
Candida maltosa (strain Xu316) (Yeast)
Candida maltosa (Yeast)
Candida orthopsilosis (strain 90-125) (Yeast)
Candida parapsilosis (strain CDC 317/ATCC MYA-4646)
Lodderomyces elongisporus (strain ATCC 11503/CBS
Candida maltosa (Yeast)
Candida maltosa (Yeast)
Candida tropicalis (Yeast)
Debaryomyces hansenii (strain ATCC 36239/CBS 767/
hansenii)
Candida tropicalis (Yeast)
Candida maltosa (strain Xu316) (Yeast)
Spathaspora passalidarum (strain NRRL Y-27907/11-Y1)
Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/
Candida parapsilosis (strain CDC 317/ATCC MYA-4646)
Candida parapsilosis (strain CDC 317/ATCC MYA-4646)
Candida tropicalis (Yeast)
Candida maltosa (strain Xu316) (Yeast)
Debaryomyces hansenii (Yeast) (Torulaspora hansenii)
Meyerozyma guilliermondii (strain ATCC 6260/CBS 566/
Debaryomyces hansenii (strain ATCC 36239/CBS 767/
hansenii)
Candida maltosa (Yeast)
Meyerozyma guilliermondii (strain ATCC 6260/CBS 566/
Debaryomyces hansenii (Yeast) (Torulaspora hansenii)
Candida dubliniensis (strain CD36/ATCC MYA-646/
Meyerozyma guilliermondii (strain ATCC 6260/CBS 566/
Candida albicans (strain SC5314/ATCC MYA-2876)
Candida albicans (strain WO-1) (Yeast)
Candida tropicalis (Yeast)
Candida tropicalis (Yeast)
Pichia sorbitophila (strain ATCC MYA-4447/BCRC
Candida parapsilosis (strain CDC 317/ATCC MYA-
Candida tropicalis (Yeast)
Candida tropicalis (Yeast)
Lodderomyces elongisporus (strain ATCC 11503/CBS
Candida albicans (strain WO-1) (Yeast)
Candida albicans (strain SC5314/ATCC MYA-2876)
Candida albicans (Yeast)
Candida maltosa (strain Xu316) (Yeast)
Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/
Lodderomyces elongisporus (strain ATCC 11503/CBS
Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast)
Pichia sorbitophila (strain ATCC MYA-4447/BCRC
Candida parapsilosis (strain CDC 317/ATCC MYA-4646)
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 MYA-4646)
Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/
Candida parapsilosis (strain CDC 317/ATCC MYA-4646)
Candida maltosa (strain Xu316) (Yeast)
Candida orthopsilosis (strain 90-125) (Yeast)
Candida dubliniensis (strain CD36/ATCC MYA-646/
Pichia sorbitophila (strain ATCC MYA-4447/BCRC
Debaryomyces hansenii (strain ATCC 36239/CBS 767/
hansenii)
Candida maltosa (Yeast)
Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/
Spathaspora passalidarum (strain NRRL Y-27907/11-Y1)
Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast)
Candida maltosa (Yeast)
Candida albicans (strain WO-1) (Yeast)
Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast)
Candida albicans (strain SC5314/ATCC MYA-2876)
Candida tropicalis (Yeast)
Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/
Debaryomyces hansenii (strain ATCC 36239/CBS 767/
hansenii)
Candida tenuis (strain ATCC 10573/BCRC 21748/CBS
Lodderomyces elongisporus (strain ATCC 11503/CBS
Lodderomyces elongisporus (strain ATCC 11503/CBS
Candida tropicalis (Yeast)
Candida tropicalis (Yeast)
Candida maltosa (Yeast)
Candida dubliniensis (strain CD36/ATCC MYA-646/
Candida maltosa (Yeast)
Candida tenuis (strain ATCC 10573/BCRC 21748/CBS
Meyerozyma guilliermondii (Yeast) (Candida
guilliermondii)
Spathaspora passalidarum (strain NRRL Y-27907/11-Y1)
Candida tenuis (strain ATCC 10573/BCRC 21748/CBS
Candida maltosa (strain Xu316) (Yeast)
Candida tropicalis (Yeast)
Clavispora lusitaniae (strain ATCC 42720) (Yeast)
Debaryomyces hansenii (strain ATCC 36239/CBS 767/
hansenii)
Candida tropicalis (Yeast)
Clavispora lusitaniae (strain ATCC 42720) (Yeast)
Meyerozyma guilliermondii (strain ATCC 6260/CBS 566/
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast)
Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast)
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast)
Candida maltosa (Yeast)
Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast)
Byssochlamys spectabilis (strain No. 5/NBRC 109023)
Byssochlamys spectabilis (strain No. 5/NBRC 109023)
Aspergillus terreus (strain NIH 2624/FGSC A1156)
Neosartorya fischeri (strain ATCC 1020/DSM 3700/
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast)
Penicillium digitatum (strain PHI26/CECT 20796)
Penicillium digitatum (strain Pd1/CECT 20795) (Green
Aspergillus niger (strain ATCC 1015/CBS 113.46/
Aspergillus niger (strain CBS 513.88/FGSC A1513)
Tuber melanosporum (strain Mel28) (Perigord black
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast)
Arthrobotrys oligospora (strain ATCC 24927/CBS
Dactylellina haptotyla (strain CBS 200.50) (Nematode-
Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast)
Aspergillus clavatus (strain ATCC 1007/CBS 513.65/
Byssochlamys spectabilis (strain No. 5/NBRC 109023)
Aspergillus kawachii (strain NBRC 4308) (White koji
Aspergillus oryzae (strain 3.042) (Yellow koji mold)
Aspergillus flavus (strain ATCC 200026/FGSC A1120/
Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow
Aspergillus oryzae (Yellow koji mold)
Candida tenuis (strain ATCC 10573/BCRC 21748/CBS
Emericella nidulans (strain FGSC A4/ATCC 38163/
Talaromyces stipitatus (strain ATCC 10500/CBS 375.48/
Starmerella bombicola
Hordeum vulgare var. distichum (Two-rowed barley)
Mycosphaerella graminicola (strain CBS 115943/
Neosartorya fumigata (strain ATCC MYA-4609/Af293/
Neosartorya fumigata (strain CEA10/CBS 144.89/
Penicillium chrysogenum (strain ATCC 28089/DSM
Clavispora lusitaniae (strain ATCC 42720) (Yeast)
Penicillium roqueforti
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast)
Candida tenuis (strain ATCC 10573/BCRC 21748/CBS
Penicillium marneffei (strain ATCC 18224/CBS 334.59/
Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast)
Candida apicola (Yeast)
Macrophomina phaseolina (strain MS6) (Charcoal rot
Cyphellophora europaea CBS 101466
Cochliobolus sativus (strain ND90Pr/ATCC 201652)
sorokiniana)
Cochliobolus sativus (strain ND90Pr/ATCC 201652)
sorokiniana)
Bipolaris victoriae FI3
Bipolaris zeicola 26-R-13
Cochliobolus heterostrophus (strain C5/ATCC 48332/
maydis)
Cochliobolus heterostrophus (strain C4/ATCC 48331/
maydis)
Pseudogymnoascus destructans (strain ATCC MYA-4855/
destructans)
Aspergillus terreus (strain NIH 2624/FGSC A1156)
Marssonina brunnea f. sp. multigermtubi (strain MB_m1)
Penicillium marneffei (strain ATCC 18224/CBS 334.59/
Neosartorya fumigata (strain CEA10/CBS 144.89/
Candida apicola (Yeast)
Neosartorya fumigata (strain ATCC MYA-4609/Af293/
Neosartorya fischeri (strain ATCC 1020/DSM 3700/
Cordyceps militaris (strain CM01) (Caterpillar fungus)
Coniosporium apollinis (strain CBS 100218) (Rock-
Penicillium chrysogenum (strain ATCC 28089/DSM
Penicillium digitatum (strain Pd1/CECT 20795) (Green
Penicillium digitatum (strain PHI26/CECT 20796)
Penicillium roqueforti
Marssonina brunnea f. sp. multigermtubi (strain MB_m1)
Botryotinia fuckeliana (strain BcDW1) (Noble rot fungus)
Botryotinia fuckeliana (strain T4) (Noble rot fungus)
Emericella nidulans (strain FGSC A4/ATCC 38163/
Candida maltosa (Yeast)
Phaeosphaeria nodorum (strain SN15/ATCC MYA-4574/
Pyrenophora tritici-repentis (strain Pt-1C-BFP) (Wheat tan
Pyrenophora teres f. teres (strain 0-1) (Barley net blotch
Aspergillus niger (strain ATCC 1015/CBS 113.46/
Bipolaris oryzae ATCC 44560
Cochliobolus heterostrophus (strain C4/ATCC 48331/
maydis)
Cochliobolus heterostrophus (strain C5/ATCC 48332/
maydis)
Botryosphaeria parva (strain UCR-NP2) (Grapevine
Ajellomyces capsulatus (strain H88) (Darling's disease
Bipolaris oryzae ATCC 44560
Dactylellina haptotyla (strain CBS 200.50) (Nematode-
Cladophialophora carrionii CBS 160.54
Exophiala dermatitidis (strain ATCC 34100/CBS 525.76/
Yarrowia lipolytica (Candida lipolytica)
Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast)
Blumeria graminis f. sp. hordei (strain DH14) (Barley
Neosartorya fischeri (strain ATCC 1020/DSM 3700/
Dactylellina haptotyla (strain CBS 200.50) (Nematode-
Aspergillus kawachii (strain NBRC 4308) (White koji
Aspergillus niger (strain ATCC 1015/CBS 113.46/
Aspergillus oryzae (strain 3.042) (Yellow koji mold)
Aspergillus flavus (strain ATCC 200026/FGSC A1120/
Arthroderma gypseum (strain ATCC MYA-4604/CBS
Arthroderma otae (strain ATCC MYA-4605/CBS
Bipolaris victoriae FI3
Bipolaris oryzae ATCC 44560
Byssochlamys spectabilis (strain No. 5/NBRC 109023)
Bipolaris zeicola 26-R-13
Mycosphaerella fijiensis (strain CIRAD86) (Black leaf
Aspergillus terreus (strain NIH 2624/FGSC A1156)
Setosphaeria turcica (strain 28A) (Northern leaf blight
Colletotrichum graminicola (strain M1.001/M2/FGSC
graminicola)
Aspergillus clavatus (strain ATCC 1007/CBS 513.65/
Ajellomyces capsulatus (strain G186AR/H82/ATCC
Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow
Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow
Aspergillus niger (strain CBS 513.88/FGSC A1513)
Penicillium marneffei (strain ATCC 18224/CBS 334.59/
Tuber melanosporum (strain Mel28) (Perigord black
Colletotrichum higginsianum (strain IMI 349063)
Beauveria bassiana (strain ARSEF 2860) (White
Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast)
Botryosphaeria parva (strain UCR-NP2) (Grapevine
Setosphaeria turcica (strain 28A) (Northern leaf blight
Aspergillus clavatus (strain ATCC 1007/CBS 513.65/
Mycosphaerella fijiensis (strain CIRAD86) (Black leaf
Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow
Aspergillus oryzae (strain 3.042) (Yellow koji mold)
Aspergillus oryzae (Yellow koji mold)
Aspergillus flavus (strain ATCC 200026/FGSC A1120/
Candida tenuis (strain ATCC 10573/BCRC 21748/CBS
Aspergillus niger (strain ATCC 1015/CBS 113.46/
Aspergillus niger (strain CBS 513.88/FGSC A1513)
Pseudogymnoascus destructans (strain ATCC MYA-4855/
destructans)
Cladophialophora carrionii CBS 160.54
Candida albicans (strain WO-1) (Yeast)
Coccidioides posadasii (strain RMSCC 757/Silveira)
Coccidioides posadasii (strain C735) (Valley fever fungus)
Candida maltosa (strain Xu316) (Yeast)
Metarhizium acridum (strain CQMa 102)
Bipolaris zeicola 26-R-13
Pyronema omphalodes (strain CBS 100304) (Pyronema
confluens)
Bipolaris victoriae FI3
Botryotinia fuckeliana (strain T4) (Noble rot fungus)
Fusarium heterosporum
Cyphellophora europaea CBS 101466
Metarhizium acridum (strain CQMa 102)
Macrophomina phaseolina (strain MS6) (Charcoal rot
Colletotrichum graminicola (strain M1.001/M2/FGSC
graminicola)
Bipolaris zeicola 26-R-13
Cochliobolus heterostrophus (strain C5/ATCC 48332/
maydis)
Bipolaris zeicola 26-R-13
Cochliobolus heterostrophus (strain C4/ATCC 48331/
maydis)
Colletotrichum gloeosporioides (strain Cg-14)
Botryotinia fuckeliana (strain BcDW1) (Noble rot fungus)
Botryotinia fuckeliana (strain T4) (Noble rot fungus)
Sclerotinia sclerotiorum (strain ATCC 18683/1980/Ss-1)
Penicillium digitatum (strain PHI26/CECT 20796)
Penicillium digitatum (strain Pd1/CECT 20795) (Green
Metarhizium anisopliae (strain ARSEF 23/ATCC MYA-3075)
Starmerella bombicola
Penicillium marneffei (strain ATCC 18224/CBS 334.59/
Metarhizium acridum (strain CQMa 102)
Mycosphaerella pini (strain NZE10/CBS 128990) (Red
Aspergillus kawachii (strain NBRC 4308) (White koji
Aspergillus niger (strain CBS 513.88/FGSC A1513)
Aspergillus niger (strain ATCC 1015/CBS 113.46/
Beauveria bassiana (strain ARSEF 2860) (White
Beauveria bassiana (White muscardine disease fungus)
Aspergillus oryzae (strain 3.042) (Yellow koji mold)
Aspergillus flavus (strain ATCC 200026/FGSC A1120/
Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow
Aspergillus oryzae (Yellow koji mold)
Endocarpon pusillum (strain Z07020/HMAS-L-300199)
Sclerotinia sclerotiorum (strain ATCC 18683/1980/Ss-1)
Pyrenophora tritici-repentis (strain Pt-1C-BFP) (Wheat tan
Candida albicans (strain SC5314/ATCC MYA-2876)
Candida albicans (Yeast)
Trichophyton verrucosum (strain HKI 0517)
Coccidioides immitis (strain RS) (Valley fever fungus)
Ajellomyces dermatitidis ATCC 26199
Ajellomyces dermatitidis (strain ATCC 18188/CBS
Ajellomyces dermatitidis (strain SLH14081) (Blastomyces
dermatitidis)
Ajellomyces dermatitidis (strain ER-3/ATCC MYA-2586)
Coccidioides posadasii (strain C735) (Valley fever fungus)
Colletotrichum orbiculare (strain 104-T/ATCC 96160/
Coccidioides immitis (strain RS) (Valley fever fungus)
Talaromyces stipitatus (strain ATCC 10500/CBS 375.48/
Coccidioides posadasii (strain RMSCC 757/Silveira)
Uncinocarpus reesii (strain UAMH 1704)
Starmerella bombicola
Pyrenophora tritici-repentis (strain Pt-1C-BFP) (Wheat tan
Marssonina brunnea f. sp. multigermtubi (strain MB_m1)
Metarhizium anisopliae (strain ARSEF 23/ATCC MYA-3075)
Macrophomina phaseolina (strain MS6) (Charcoal rot
Glarea lozoyensis (strain ATCC 20868/MF5171)
Arthroderma otae (strain ATCC MYA-4605/CBS
Trichophyton verrucosum (strain HKI 0517)
Hypocrea atroviridis (strain ATCC 20476/IMI 206040)
Glarea lozoyensis (strain ATCC 74030/MF5533)
Ajellomyces capsulatus (strain NAm1/WU24) (Darling's
Pyronema omphalodes (strain CBS 100304) (Pyronema
confluens)
Endocarpon pusillum (strain Z07020/HMAS-L-300199)
Penicillium marneffei (strain ATCC 18224/CBS 334.59/
Emericella nidulans (strain FGSC A4/ATCC 38163/
Botryotinia fuckeliana (strain T4) (Noble rot fungus)
Aspergillus terreus (strain NIH 2624/FGSC A1156)
Aspergillus niger (strain ATCC 1015/CBS 113.46/
Aspergillus kawachii (strain NBRC 4308) (White koji
Beauveria bassiana (strain ARSEF 2860) (White
Sclerotinia sclerotiorum (strain ATCC 18683/1980/Ss-1)
Beauveria bassiana (strain ARSEF 2860) (White
Beauveria bassiana (White muscardine disease fungus)
Cordyceps militaris (strain CM01) (Caterpillar fungus)
Penicillium chrysogenum (strain ATCC 28089/DSM
Mycosphaerella pini (strain NZE10/CBS 128990) (Red
Mycosphaerella pini (strain NZE10/CBS 128990) (Red
Aspergillus terreus (strain NIH 2624/FGSC A1156)
Arthroderma benhamiae (strain ATCC MYA-4681/CBS
Baudoinia compniacensis (strain UAMH 10762) (Angels'
Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast)
Candida tropicalis (Yeast)
Metarhizium anisopliae (strain ARSEF 23/ATCC MYA-3075)
Mycosphaerella graminicola (strain CBS 115943/
Cladophialophora carrionii CBS 160.54
Glarea lozoyensis (strain ATCC 20868/MF5171)
Hypocrea virens (strain Gv29-8/FGSC 10586)
Marssonina brunnea f. sp. multigermtubi (strain MB_m1)
Talaromyces stipitatus (strain ATCC 10500/CBS 375.48/
Arthroderma benhamiae (strain ATCC MYA-4681/CBS
Colletotrichum higginsianum (strain IMI 349063)
Trichophyton tonsurans (strain CBS 112818) (Scalp
Marssonina brunnea f. sp. multigermtubi (strain MB_m1)
Aspergillus terreus (strain NIH 2624/FGSC A1156)
Claviceps purpurea (strain 20.1) (Ergot fungus) (Sphacelia
segetum)
Trichophyton rubrum (strain ATCC MYA-4607/CBS
Setosphaeria turcica (strain 28A) (Northern leaf blight
Paracoccidioides brasiliensis (strain Pb03)
Arthroderma gypseum (strain ATCC MYA-4604/CBS
Trichophyton equinum (strain ATCC MYA-4606/CBS
Talaromyces stipitatus (strain ATCC 10500/CBS 375.48/
Leptosphaeria maculans (strain JN3/isolate v23.1.37 race
Bipolaris victoriae FI3
Magnaporthe oryzae (strain Y34) (Rice blast fungus)
Fusarium oxysporum f. sp. cubense (strain race 1)
Fusarium oxysporum f. sp. lycopersici (strain 4287/CBS
Endocarpon pusillum (strain Z07020/HMAS-L-300199)
Sphaerulina musiva (strain SO2202) (Poplar stem canker
Mycosphaerella graminicola (strain CBS 115943/
Penicillium oxalicum (strain 114-2/CGMCC 5302)
Mycosphaerella graminicola (strain CBS 115943/
Cladophialophora carrionii CBS 160.54
Togninia minima (strain UCR-PA7) (Esca disease fungus)
Fusarium oxysporum (strain Fo5176) (Fusarium vascular
Gaeumannomyces graminis var. tritici (strain R3-111a-1)
Cochliobolus sativus (strain ND90Pr/ATCC 201652)
sorokiniana)
Neosartorya fumigata (strain CEA10/CBS 144.89/
Neosartorya fumigata (strain ATCC MYA-4609/Af293/
Neosartorya fischeri (strain ATCC 1020/DSM 3700/
Hypocrea atroviridis (strain ATCC 20476/IMI 206040)
Candida orthopsilosis (strain 90-125) (Yeast)
Cyphellophora europaea CBS 101466
Penicillium oxalicum (strain 114-2/CGMCC 5302)
Penicillium chrysogenum (strain ATCC 28089/DSM
Arthroderma gypseum (strain ATCC MYA-4604/CBS
Hypocrea virens (strain Gv29-8/FGSC 10586)
Botryotinia fuckeliana (strain BcDW1) (Noble rot fungus)
Botryosphaeria parva (strain UCR-NP2) (Grapevine
Cochliobolus sativus (strain ND90Pr/ATCC 201652)
sorokiniana)
Aspergillus niger (strain CBS 513.88/FGSC A1513)
Candida dubliniensis (strain CD36/ATCC MYA-646/
Cochliobolus heterostrophus (strain C4/ATCC 48331/
maydis)
Cochliobolus heterostrophus (strain C5/ATCC 48332/
maydis)
Aspergillus clavatus (strain ATCC 1007/CBS 513.65/
Hypocrea jecorina (strain QM6a) (Trichoderma reesei)
Trichophyton tonsurans (strain CBS 112818) (Scalp
Glarea lozoyensis (strain ATCC 20868/MF5171)
Trichophyton rubrum (strain ATCC MYA-4607/CBS
Leptosphaeria maculans (strain JN3/isolate v23.1.37 race
Cyphellophora europaea CBS 101466
Hypocrea jecorina (strain QM6a) (Trichoderma reesei)
Beauveria bassiana (strain ARSEF 2860) (White
Cordyceps militaris (strain CM01) (Caterpillar fungus)
Trichophyton rubrum (strain ATCC MYA-4607/CBS
Botryotinia fuckeliana (strain BcDW1) (Noble rot fungus)
Magnaporthe oryzae (strain P131) (Rice blast fungus)
Magnaporthe oryzae (strain Y34) (Rice blast fungus)
Magnaporthe oryzae (strain 70-15/ATCC MYA-4617/
Paracoccidioides lutzii (strain ATCC MYA-826/Pb01)
Bipolaris zeicola 26-R-13
Verticillium dahliae (strain VdLs.17/ATCC MYA-4575/
Trichophyton verrucosum (strain HKI 0517)
Arthroderma benhamiae (strain ATCC MYA-4681/CBS
Chaetomium globosum (strain ATCC 6205/CBS 148.51/
Magnaporthe poae (strain ATCC 64411/73-15)
Hypocrea atroviridis (strain ATCC 20476/IMI 206040)
Colletotrichum orbiculare (strain 104-T/ATCC 96160/
Penicillium chrysogenum (strain ATCC 28089/DSM
Ophiocordyceps sinensis (strain Co18/CGMCC 3.14243)
Pyrenophora teres f. teres (strain 0-1) (Barley net blotch
Baudoinia compniacensis (strain UAMH 10762) (Angels'
Podospora anserina (strain S/ATCC MYA-4624/DSM
Aspergillus terreus (strain NIH 2624/FGSC A1156)
Hypocrea jecorina (strain QM6a) (Trichoderma reesei)
Claviceps purpurea (strain 20.1) (Ergot fungus) (Sphacelia
segetum)
Aspergillus flavus (strain ATCC 200026/FGSC A1120/
Mycosphaerella fijiensis (strain CIRAD86) (Black leaf
Grosmannia clavigera (strain kw1407/UAMH 11150)
Lodderomyces elongisporus (strain ATCC 11503/CBS
Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast)
Coniosporium apollinis (strain CBS 100218) (Rock-
Candida parapsilosis (strain CDC 317/ATCC MYA-4646)
Aspergillus niger (strain CBS 513.88/FGSC A1513)
Baudoinia compniacensis (strain UAMH 10762) (Angels'
Candida tropicalis (Yeast)
Aspergillus kawachii (strain NBRC 4308) (White koji
Colletotrichum gloeosporioides (strain Cg-14)
Colletotrichum gloeosporioides (strain Cg-14)
Endocarpon pusillum (strain Z07020/HMAS-L-300199)
Arthroderma gypseum (strain ATCC MYA-4604/CBS
Botryotinia fuckeliana (strain T4) (Noble rot fungus)
Exophiala dermatitidis (strain ATCC 34100/CBS 525.76/
Aspergillus oryzae (Yellow koji mold)
Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow
Neurospora tetrasperma (strain FGSC 2508/ATCC
Sordaria macrospora (strain ATCC MYA-333/DSM 997/
Neurospora crassa (strain ATCC 24698/74-OR23-1A/
Eutypa lata (strain UCR-EL1) (Grapevine dieback disease
Neurospora tetrasperma (strain FGSC 2509/P0656)
Setosphaeria turcica (strain 28A) (Northern leaf blight
Pyrenophora tritici-repentis (strain Pt-1C-BFP) (Wheat tan
Paracoccidioides lutzii (strain ATCC MYA-826/Pb01)
Neosartorya fischeri (strain ATCC 1020/DSM 3700/
Sphaerulina musiva (strain SO2202) (Poplar stem canker
Emericella nidulans (strain FGSC A4/ATCC 38163/
Candida orthopsilosis (strain 90-125) (Yeast)
Aspergillus oryzae (strain 3.042) (Yellow koji mold)
Aspergillus flavus (strain ATCC 200026/FGSC A1120/
Candida parapsilosis (strain CDC 317/ATCC MYA-4646)
Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow
Aspergillus oryzae (Yellow koji mold)
Neosartorya fumigata (strain ATCC MYA-4609/Af293/
Botryotinia fuckeliana (strain T4) (Noble rot fungus)
Sclerotinia sclerotiorum (strain ATCC 18683/1980/Ss-1)
Pyronema omphalodes (strain CBS 100304) (Pyronema
confluens)
Thielavia heterothallica (strain ATCC 42464/BCRC
Pestalotiopsis fici W106-1
Eutypa lata (strain UCR-EL1) (Grapevine dieback disease
Colletotrichum orbiculare (strain 104-T/ATCC 96160/
Colletotrichum graminicola (strain M1.001/M2/FGSC
graminicola)
Trichophyton verrucosum (strain HKI 0517)
Sphaerulina musiva (strain SO2202) (Poplar stem canker
Nectria haematococca (strain 77-13-4/ATCC MYA-4622/
Coniosporium apollinis (strain CBS 100218) (Rock-
Gaeumannomyces graminis var. tritici (strain R3-111a-1)
Fusarium pseudograminearum (strain CS3096) (Wheat
Magnaporthe oryzae (strain P131) (Rice blast fungus)
Magnaporthe oryzae (strain Y34) (Rice blast fungus)
Magnaporthe oryzae (strain 70-15/ATCC MYA-4617/
Thielavia terrestris (strain ATCC 38088/NRRL 8126)
Gibberella fujikuroi (strain CBS 195.34/IMI 58289/
Pyronema omphalodes (strain CBS 100304) (Pyronema
confluens)
Gibberella moniliformis (strain M3125/FGSC 7600)
Magnaporthe oryzae (strain P131) (Rice blast fungus)
Magnaporthe oryzae (strain 70-15/ATCC MYA-4617/
Fusarium oxysporum f. sp. cubense (strain race 4)
Chaetomium thermophilum (strain DSM 1495/CBS
Botryotinia fuckeliana (strain BcDW1) (Noble rot fungus)
Verticillium alfalfae (strain VaMs.102/ATCC MYA-4576/
Arthroderma gypseum (strain ATCC MYA-4604/CBS
Uncinocarpus reesii (strain UAMH 1704)
Bipolaris oryzae ATCC 44560
Paracoccidioides brasiliensis (strain Pb03)
Paracoccidioides brasiliensis (strain Pb18)
Neosartorya fumigata (strain ATCC MYA-4609/Af293/
Neosartorya fumigata (strain CEA10/CBS 144.89/
Aspergillus niger (strain ATCC 1015/CBS 113.46/
Coniosporium apollinis (strain CBS 100218) (Rock-
Aspergillus niger (strain CBS 513.88/FGSC A1513)
Aspergillus niger (strain ATCC 1015/CBS 113.46/
Neosartorya fischeri (strain ATCC 1020/DSM 3700/
Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow
Aspergillus oryzae (strain 3.042) (Yellow koji mold)
Aspergillus oryzae (Yellow koji mold)
Magnaporthe poae (strain ATCC 64411/73-15)
Cladophialophora carrionii CBS 160.54
Podospora anserina (strain S/ATCC MYA-4624/DSM
Gibberella zeae (strain PH-1/ATCC MYA-4620/FGSC
Colletotrichum orbiculare (strain 104-T/ATCC 96160/
Neosartorya fischeri (strain ATCC 1020/DSM 3700/
Trichophyton verrucosum (strain HKI 0517)
Botryotinia fuckeliana (strain T4) (Noble rot fungus)
Trichophyton rubrum (strain ATCC MYA-4607/CBS
Botryotinia fuckeliana (strain T4) (Noble rot fungus)
Setosphaeria turcica (strain 28A) (Northern leaf blight
Bipolaris victoriae FI3
Bipolaris zeicola 26-R-13
Podospora anserina (strain S/ATCC MYA-4624/DSM
Sporothrix schenckii (strain ATCC 58251/de Perez
Exophiala dermatitidis (strain ATCC 34100/CBS 525.76/
Colletotrichum gloeosporioides (strain Cg-14)
Arthroderma benhamiae (strain ATCC MYA-4681/CBS
Macrophomina phaseolina (strain MS6) (Charcoal rot
Trichophyton tonsurans (strain CBS 112818) (Scalp
Trichophyton equinum (strain ATCC MYA-4606/CBS
Arthroderma benhamiae (strain ATCC MYA-4681/CBS
Arthroderma otae (strain ATCC MYA-4605/CBS
Aspergillus flavus (strain ATCC 200026/FGSC A1120/
Mycosphaerella graminicola (strain CBS 115943/
Penicillium chrysogenum (strain ATCC 28089/DSM
Alternaria solani
Colletotrichum higginsianum (strain IMI 349063)
Thielavia heterothallica (strain ATCC 42464/BCRC
Togninia minima (strain UCR-PA7) (Esca disease fungus)
Ophiostoma piceae (strain UAMH 11346) (Sap stain
Cladophialophora carrionii CBS 160.54
Botryotinia fuckeliana (strain BcDW1) (Noble rot fungus)
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/DSM 13023/
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/DSM 13023/
Caulobacter crescentus (strain NA1000/CB15N)
Caulobacter crescentus (strain ATCC 19089/CB15)
Parvibaculum lavamentivorans (strain DS-1/DSM 13023/
Caulobacter segnis (strain ATCC 21756/DSM 7131/
Novosphingobium sp. PP1Y
Parvibaculum sp. S18-4
Bradyrhizobium sp. STM 3843
Bradyrhizobium sp. (strain ORS278)
Bradyrhizobium sp. (strain BTAi1/ATCC BAA-1182)
Caulobacter crescentus OR37
Afipia broomeae ATCC 49717
Afipia clevelandensis ATCC 49720
Bradyrhizobiaceae bacterium SG-6C
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 10833/IAM
Bradyrhizobium oligotrophicum S58
Erythrobacter litoralis (strain HTCC2594)
Erythrobacter sp. SD-21
Bradyrhizobium sp. DFCI-1
Bradyrhizobium sp. DFCI-1
Bradyrhizobium diazoefficiens (strain JCM 10833/IAM
Rhodopseudomonas palustris (strain TIE-1)
Bradyrhizobium sp. CCGE-LA001
Parvibaculum lavamentivorans (strain DS-1/DSM 13023/
Rhodopseudomonas palustris (strain ATCC BAA-98/
Bradyrhizobium sp. S23321
Bradyrhizobium sp. ORS 285
Bradyrhizobium sp. ORS 375
Bradyrhizobium sp. (strain BTAi1/ATCC BAA-1182)
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 BisBS)
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 10833/IAM
Bradyrhizobium japonicum
Pseudomonas sp. 19-rlim
Bradyrhizobium sp. WSM1253
Bradyrhizobium sp. WSM471
Afipia sp. P52-10
Glaciecola psychrophila 170
Marinobacter lipolyticus SM19
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/CGMCC
Alcanivorax hongdengensis A-11-3
Alcanivorax dieselolei
Alcanivorax pacificus W11-5
Marinobacter sp. ES-1
Limnobacter sp. MED105
Marinobacter aquaeolei (strain ATCC 700491/DSM
Marinobacter sp. EVN1
Marinobacter sp. EN3
Marinobacter manganoxydans MnI7-9
Marinobacter hydrocarbonoclasticus ATCC 49840
Marinobacter hydrocarbonoclasticus (Pseudomonas
nautica)
Patulibacter medicamentivorans
Acinetobacter baumannii WC-141
Saccharomonospora marina XMU15
Mycobacterium marinum (strain ATCC BAA-535/M)
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-154-0310
Mycobacterium abscessus 6G-0728-S
Mycobacterium abscessus 3A-0810-R
Mycobacterium abscessus 3A-0122-S
Mycobacterium abscessus 3A-0122-R
Mycobacterium abscessus 6G-0212
Mycobacterium abscessus subsp. bolletii 1S-153-0915
Mycobacterium abscessus subsp. bolletii 1S-152-0914
Mycobacterium abscessus subsp. bolletii 1S-151-0930
Mycobacterium abscessus 6G-1108
Mycobacterium abscessus 6G-0125-S
Mycobacterium abscessus 6G-0125-R
Mycobacterium abscessus subsp. bolletii 2B-0307
Mycobacterium abscessus subsp. bolletii 2B-0107
Mycobacterium abscessus subsp. bolletii 2B-1231
Mycobacterium abscessus subsp. bolletii 2B-0912-S
Mycobacterium abscessus subsp. bolletii 2B-0912-R
Mycobacterium abscessus subsp. bolletii 2B-0626
Parvibaculum lavamentivorans (strain DS-1/DSM 13023/
Alcanivorax hongdengensis
Alcanivorax sp. DG881
Marinobacter sp. C1S70
Alcanivorax sp. P2S70
Marinobacter goseongensis
Hirschia baltica (strain ATCC 49814/DSM 5838/IFAM
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 = NBRC 102413 =
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 100887)
Rhodococcus erythropolis DN1
Alcanivorax dieselolei
Alcanivorax borkumensis
Alcanivorax sp. 97CO-5
Alcanivorax borkumensis (strain SK2/ATCC 700651/
Alcanivorax borkumensis
Amycolicicoccus subflavus (strain DSM 45089/DQS3-9A1)
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 100887)
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 06
Mycobacterium abscessus (strain ATCC 19977/DSM
Mycobacterium abscessus V06705
Mycobacterium abscessus M94
Mycobacterium avium subsp. hominissuis 10-4249
Mycobacterium parascrofulaceum ATCC BAA-614
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/DQS3-9A1)
Candidatus Microthrix parvicella RN1
Gordonia paraffinivorans NBRC 108238
Nocardioides sp. CF8
Mycobacterium chubuense (strain NBB4)
Gordonia polyisoprenivorans (strain DSM 44266/VH2)
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 700651/
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/LMG 18877/
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
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/DSM 13023/
Novosphingobium pentaromativorans US6-1
In some embodiments, the invention 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 invention 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 3, Table 4, Table 5, 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 invention 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 invention 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 invention 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 invention 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 invention 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 invention 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 invention. 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 invention 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 invention, 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 invention 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%.
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 chlorocrhomate (PCC) (Scheme 20).
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 21).
The present invention 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/CBS 7435/
Komagataella pastoris (strain GS115/ATCC 20864)
Komagataella pastoris (strain ATCC 76273/CBS 7435/
Komagataella pastoris (strain GS115/ATCC 20864)
Candida boidinii (Yeast)
Pichia angusta (Yeast) (Hansenula polymorpha)
Thanatephorus cucumeris (strain AG1-IB/isolate
Thanatephorus cucumeris (strain AG1-IB/isolate
Thanatephorus cucumeris (strain AG1-IB/isolate
Thanatephorus cucumeris (strain AG1-IB/isolate
Thanatephorus cucumeris (strain AG1-IB/isolate
Thanatephorus cucumeris (strain AG1-IB/isolate
Thanatephorus cucumeris (strain AG1-IB/isolate
Thanatephorus cucumeris (strain AG1-IB/isolate
Thanatephorus cucumeris (strain AG1-IB/isolate
Thanatephorus cucumeris (strain AG1-IB/isolate
Ogataea henricii
Candida methanosorbosa
Candida methanolovescens
Candida succiphila
Aspergillus niger (strain CBS 513.88/FGSC A1513)
Aspergillus niger (strain CBS 513.88/FGSC A1513)
Moniliophthora perniciosa (Witches'-broom disease
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/CBS
stipitatum)
Talaromyces stipitatus (strain ATCC 10500/CBS
stipitatum)
Talaromyces stipitatus (strain ATCC 10500/CBS
stipitatum)
Talaromyces stipitatus (strain ATCC 10500/CBS
stipitatum)
Ogataea glucozyma
Ogataea parapolymorpha (strain DL-1/ATCC 26012/
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 tick)
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 Lamole)
Ajellomyces dermatitidis ATCC 26199
Gibberella zeae (strain PH-1/ATCC MYA-4620/
Pichia sorbitophila (strain ATCC MYA-4447/
Emericella nidulans (strain FGSC A4/ATCC
Pyrenophora tritici-repentis (strain Pt-1C-BFP)
Paracoccidioides lutzii (strain ATCC MYA-826/
Candida parapsilosis (strain CDC 317/ATCC
Pseudozyma brasiliensis (strain GHG001) (Yeast)
Candida parapsilosis (strain CDC 317/ATCC
Sclerotinia borealis F-4157
Sordaria macrospora (strain ATCC MYA-333/
Sordaria macrospora (strain ATCC MYA-333/
Meyerozyma guilliermondii (strain ATCC 6260/
Trichophyton rubrum CBS 202.88
Arthrobotrys oligospora (strain ATCC 24927/CBS
Scheffersomyces stipitis (strain ATCC 58785/CBS
Scheffersomyces stipitis (strain ATCC 58785/CBS
Aspergillus oryzae (strain 3.042) (Yellow koji mold)
Fusarium oxysporum (strain Fo5176) (Fusarium
Rhizopus delemar (strain RA 99-880/ATCC MYA-4621/
Rhizopus delemar (strain RA 99-880/ATCC MYA-4621/
Fusarium oxysporum (strain Fo5176) (Fusarium
Penicillium roqueforti
Aspergillus clavatus (strain ATCC 1007/CBS
Arthroderma otae (strain ATCC MYA-4605/CBS
Trichophyton tonsurans (strain CBS 112818) (Scalp
Colletotrichum higginsianum (strain IMI 349063)
Ajellomyces capsulatus (strain H143) (Darling's
Trichophyton rubrum (strain ATCC MYA-4607/
Cochliobolus heterostrophus (strain C5/ATCC
Candida orthopsilosis (strain 90-125) (Yeast)
Candida orthopsilosis (strain 90-125) (Yeast)
Candida orthopsilosis (strain 90-125) (Yeast)
Pseudozyma aphidis DSM 70725
Coccidioides posadasii (strain C735) (Valley fever
Magnaporthe oryzae (strain P131) (Rice blast
Neurospora tetrasperma (strain FGSC 2508/ATCC
Hypocrea virens (strain Gv29-8/FGSC 10586)
Hypocrea virens (strain Gv29-8/FGSC 10586)
Aspergillus niger (strain CBS 513.88/FGSC
Verticillium dahliae (strain VdLs.17/ATCC MYA-4575/
Ustilago maydis (strain 521/FGSC 9021) (Corn
Fusarium oxysporum f. sp. lycopersici MN25
Fusarium oxysporum f. sp. lycopersici MN25
Candida tropicalis (Yeast)
Magnaporthe oryzae (strain 70-15/ATCC MYA-4617/
oryzae)
Candida tropicalis (Yeast)
Candida tropicalis (Yeast)
Phaeosphaeria nodorum (strain SN15/ATCC
Candida tropicalis (Yeast)
Pestalotiopsis fici W106-1
Magnaporthe oryzae (strain Y34) (Rice blast
Pseudogymnoascus destructans (strain ATCC
Pseudogymnoascus destructans (strain ATCC
Mycosphaerella fijiensis (strain CIRAD86) (Black
fijiensis)
Bipolaris oryzae ATCC 44560
Cladophialophora psammophila CBS 110553
Fusarium oxysporum f. sp. melonis 26406
Fusarium oxysporum f. sp. melonis 26406
Cyphellophora europaea CBS 101466
Aspergillus kawachii (strain NBRC 4308) (White
Aspergillus terreus (strain NIH 2624/FGSC
Coccidioides immitis (strain RS) (Valley fever
Ajellomyces dermatitidis (strain ER-3/ATCC
Fusarium oxysporum f. sp. cubense (strain race 1)
Rhodotorula glutinis (strain ATCC 204091/IIP 30/
Aspergillus niger (strain ATCC 1015/CBS 113.46/
Candida cloacae
Candida cloacae
Fusarium oxysporum f. sp. cubense (strain race 1)
Candida albicans (strain SC5314/ATCC MYA-2876)
Candida albicans (strain SC5314/ATCC MYA-2876)
Chaetomium thermophilum (strain DSM 1495/
Mucor circinelloides f. circinelloides (strain
circinelloides)
Mucor circinelloides f. circinelloides (strain
circinelloides)
Mucor circinelloides f. circinelloides (strain
circinelloides)
Botryotinia fuckeliana (strain BcDW1) (Noble rot
Podospora anserina (strain S/ATCC MYA-4624/
Neosartorya fumigata (strain ATCC MYA-4609/
fumigatus)
Fusarium oxysporum f. sp. vasinfectum 25433
Fusarium oxysporum f. sp. vasinfectum 25433
Trichophyton interdigitale H6
Beauveria bassiana (strain ARSEF 2860) (White
Fusarium oxysporum f. sp. radicis-lycopersici 26381
Fusarium oxysporum f. sp. radicis-lycopersici 26381
Neurospora tetrasperma (strain FGSC 2509/P0656)
Pseudozyma hubeiensis (strain SY62) (Yeast)
Lodderomyces elongisporus (strain ATCC 11503/
Malassezia globosa (strain ATCC MYA-4612/CBS
Byssochlamys spectabilis (strain No. 5/NBRC
Ajellomyces capsulatus (strain H88) (Darling's
Trichosporon asahii var. asahii (strain ATCC 90039/
Penicillium oxalicum (strain 114-2/CGMCC 5302)
Fusarium oxysporum f. sp. conglutinans race 2
Fusarium oxysporum f. sp. conglutinans race 2
Fusarium oxysporum f. sp. raphani 54005
Fusarium oxysporum f. sp. raphani 54005
Metarhizium acridum (strain CQMa 102)
Arthroderma benhamiae (strain ATCC MYA-4681/
Fusarium oxysporum f. sp. cubense tropical race 4
Fusarium oxysporum f. sp. cubense tropical race 4
Cochliobolus heterostrophus (strain C4/ATCC
Trichosporon asahii var. asahii (strain CBS 8904)
Mycosphaerella graminicola (strain CBS 115943/
tritici)
Botryotinia fuckeliana (strain T4) (Noble rot fungus)
Metarhizium anisopliae (strain ARSEF 23/ATCC
Cladophialophora carrionii CBS 160.54
Coccidioides posadasii (strain RMSCC 757/
Rhodosporidium toruloides (strain NP11) (Yeast)
Puccinia graminis f. sp. tritici (strain CRL
Trichophyton rubrum CBS 288.86
Colletotrichum fioriniae PJ7
Trichophyton rubrum CBS 289.86
Cladophialophora yegresii CBS 114405
Colletotrichum orbiculare (strain 104-T/ATCC
lagenarium)
Drechslerella stenobrocha 248
Neosartorya fumigata (strain CEA10/CBS 144.89/
Thielavia terrestris (strain ATCC 38088/NRRL
Gibberella fujikuroi (strain CBS 195.34/IMI 58289/
Gibberella fujikuroi (strain CBS 195.34/IMI 58289/
Aspergillus flavus (strain ATCC 200026/FGSC
Togninia minima (strain UCR-PA7) (Esca disease
Ajellomyces dermatitidis (strain ATCC 18188/
Macrophomina phaseolina (strain MS6) (Charcoal
Neurospora crassa (strain ATCC 24698/74-OR23-1A/
Neosartorya fischeri (strain ATCC 1020/DSM
fischerianus)
Fusarium pseudograminearum (strain CS3096)
Spathaspora passalidarum (strain NRRL Y-27907/
Spathaspora passalidarum (strain NRRL Y-27907/
Trichophyton verrucosum (strain HKI 0517)
Arthroderma gypseum (strain ATCC MYA-4604/
Hypocrea jecorina (strain QM6a) (Trichoderma
reesei)
Trichophyton rubrum MR1448
Aspergillus ruber CBS 135680
Glarea lozoyensis (strain ATCC 20868/MF5171)
Setosphaeria turcica (strain 28A) (Northern leaf
Paracoccidioides brasiliensis (strain Pb18)
Fusarium oxysporum Fo47
Fusarium oxysporum Fo47
Trichophyton rubrum MR1459
Penicillium marneffei (strain ATCC 18224/CBS
Sphaerulina musiva (strain SO2202) (Poplar stem
Gibberella moniliformis (strain M3125/FGSC
verticillioides)
Gibberella moniliformis (strain M3125/FGSC
verticillioides)
Pseudozyma antarctica (strain T-34) (Yeast)
Paracoccidioides brasiliensis (strain Pb03)
Rhizophagus irregularis (strain DAOM 181602/
Penicillium chrysogenum (strain ATCC 28089/
notatum)
Baudoinia compniacensis (strain UAMH 10762)
Hypocrea atroviridis (strain ATCC 20476/IMI
Colletotrichum gloeosporioides (strain Cg-14)
Cordyceps militaris (strain CM01) (Caterpillar
Pyronema omphalodes (strain CBS 100304)
Colletotrichum graminicola (strain M1.001/M2/
Glarea lozoyensis (strain ATCC 74030/MF5533)
Fusarium oxysporum f. sp. cubense (strain race 4)
Fusarium oxysporum f. sp. cubense (strain race 4)
Cochliobolus sativus (strain ND90Pr/ATCC
Mixia osmundae (strain CBS 9802/IAM 14324/
Mycosphaerella pini (strain NZE10/CBS 128990)
septosporum)
Grosmannia clavigera (strain kw1407/UAMH
clavigera)
Fusarium oxysporum FOSC 3-a
Fusarium oxysporum FOSC 3-a
Fusarium oxysporum FOSC 3-a
Nectria haematococca (strain 77-13-4/ATCC
Nectria haematococca (strain 77-13-4/ATCC
Tuber melanosporum (strain Mel28) (Perigord black
Ajellomyces dermatitidis (strain SLH14081)
Chaetomium globosum (strain ATCC 6205/CBS
Candida tenuis (strain ATCC 10573/BCRC 21748/
Trichophyton rubrum CBS 100081
Pyrenophora teres f. teres (strain 0-1) (Barley net
Colletotrichum gloeosporioides (strain Nara gc5)
Gibberella zeae (Wheat head blight fungus)
Trichophyton soudanense CBS 452.61
Sclerotinia sclerotiorum (strain ATCC 18683/1980/
Fusarium oxysporum f. sp. pisi HDV247
Fusarium oxysporum f. sp. pisi HDV247
Ustilago hordei (strain Uh4875-4) (Barley covered
Sporisorium reilianum (strain SRZ2) (Maize head
Bipolaris zeicola 26-R-13
Melampsora larici-populina (strain 98AG31/
Fusarium oxysporum f. sp. lycopersici (strain 4287/
Fusarium oxysporum f. sp. lycopersici (strain 4287/
Bipolaris victoriae FI3
Debaryomyces hansenii (strain ATCC 36239/CBS
Clavispora lusitaniae (strain ATCC 42720) (Yeast)
Candida albicans (strain WO-1) (Yeast)
Trichophyton rubrum MR850
Candida dubliniensis (strain CD36/ATCC MYA-646/
Starmerella bombicola
Thielavia heterothallica (strain ATCC 42464/
thermophila)
Claviceps purpurea (strain 20.1) (Ergot fungus)
Aspergillus oryzae (strain ATCC 42149/RIB 40)
Dictyostelium discoideum (Slime mold)
Triticum urartu (Red wild einkorn) (Crithodium
urartu)
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) (Medicago
tribuloides)
Arabidopsis thaliana (Mouse-ear cress)
Medicago truncatula (Barrel medic) (Medicago
tribuloides)
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 CCAP 1055/1)
Hordeum vulgare var. distichum (Two-rowed barley)
Hordeum vulgare var. distichum (Two-rowed barley)
Hordeum vulgare var. distichum (Two-rowed barley)
Hordeum vulgare var. distichum (Two-rowed barley)
Hordeum vulgare var. distichum (Two-rowed barley)
Ricinus communis (Castor bean)
Brassica rapa subsp. pekinensis (Chinese cabbage)
Ricinus communis (Castor bean)
Brassica rapa subsp. pekinensis (Chinese cabbage)
Brassica rapa subsp. pekinensis (Chinese cabbage)
Brassica rapa subsp. pekinensis (Chinese cabbage)
Brassica rapa subsp. pekinensis (Chinese cabbage)
Ricinus communis (Castor bean)
Zea mays (Maize)
Oryza glaberrima (African rice)
Zea mays (Maize)
Zea mays (Maize)
Aegilops tauschii (Tausch's goatgrass) (Aegilops
squarrosa)
Solanum habrochaites (Wild tomato) (Lycopersicon
Physcomitrella patens subsp. patens (Moss)
Physcomitrella patens subsp. patens (Moss)
Physcomitrella patens subsp. patens (Moss)
Solanum pennellii (Tomato) (Lycopersicon
pennellii)
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 tomato)
Phaseolus vulgaris (Kidney bean) (French bean)
Phaseolus vulgaris (Kidney bean) (French bean)
Phaseolus vulgaris (Kidney bean) (French bean)
Solanum tuberosum (Potato)
Solanum tuberosum (Potato)
Solanum tuberosum (Potato)
Glycine max (Soybean) (Glycine hispida)
Glycine max (Soybean) (Glycine hispida)
Populus trichocarpa (Western balsam poplar)
Picea sitchensis (Sitka spruce) (Pinus sitchensis)
Populus trichocarpa (Western balsam poplar)
Populus trichocarpa (Western balsam poplar)
Glycine max (Soybean) (Glycine hispida)
Glycine max (Soybean) (Glycine hispida)
Setaria italica (Foxtail millet) (Panicum italicum)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Setaria italica (Foxtail millet) (Panicum italicum)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Setaria italica (Foxtail millet) (Panicum italicum)
Setaria italica (Foxtail millet) (Panicum italicum)
Mimulus guttatus (Spotted monkey flower) (Yellow
Mimulus guttatus (Spotted monkey flower) (Yellow
Mimulus guttatus (Spotted monkey flower) (Yellow
Mimulus guttatus (Spotted monkey flower) (Yellow
Mimulus guttatus (Spotted monkey flower) (Yellow
Musa acuminata subsp. malaccensis (Wild banana)
Musa acuminata subsp. malaccensis (Wild banana)
Musa acuminata subsp. malaccensis (Wild banana)
Saprolegnia diclina VS20
Brachypodium distachyon (Purple false brome)
Brachypodium distachyon (Purple false brome)
Brachypodium distachyon (Purple false brome)
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-leaved rock-
Arabidopsis lyrata subsp. lyrata (Lyre-leaved rock-
Arabidopsis lyrata subsp. lyrata (Lyre-leaved rock-
Arabidopsis lyrata subsp. lyrata (Lyre-leaved rock-
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 C15 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) (Ralstonia
eutropha)
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) (Scaptodrosophila
lebanonensis)
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) (Pyres 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 fly)
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/DSM 428/
Mycobacterium tuberculosis (strain CDC 1551/Oshkosh)
Staphylococcus aureus (strain MW2)
Mycobacterium tuberculosis (strain ATCC 25618/H37Rv)
Staphylococcus aureus (strain N315)
Staphylococcus aureus (strain bovine RF122/ET3-1)
Sulfolobus acidocaldarius (strain ATCC 33909/DSM 639/
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 700699)
Staphylococcus epidermidis (strain ATCC 12228)
Staphylococcus epidermidis (strain ATCC 35984/RP62A)
Sulfolobus solfataricus (strain ATCC 35092/DSM 1617/
Sulfolobus tokodaii (strain DSM 16993/JCM 10545/NBRC
Anas platyrhynchos (Domestic duck) (Anas boschas)
Apteryx australis (Brown kiwi)
Ceratitis capitata (Mediterranean fruit fly) (Tephritis capitata)
Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra)
Gallus gallus (Chicken)
Columba livia (Domestic pigeon)
Coturnix coturnix japonica (Japanese quail) (Coturnix
japonica)
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 attwateri)
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/S288c)
Arabidopsis thaliana (Mouse-ear cress)
Schizosaccharomyces pombe (strain 972/ATCC 24843)
Drosophila lacicola (Fruit fly)
Mus musculus (Mouse)
Peromyscus maniculatus (North American deer mouse)
Rattus norvegicus (Rat)
Drosophila virilis (Fruit fly)
Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/
Aspergillus flavus (strain ATCC 200026/FGSC A1120/
Neurospora crassa (strain ATCC 24698/74-OR23-1A/CBS
Candida albicans (Yeast)
Oryza sativa subsp. japonica (Rice)
Drosophila mojavensis (Fruit fly)
Kluyveromyces lactis (strain ATCC 8585/CBS 2359/DSM
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) (Tephritis capitata)
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/CBS 6054/
Arabidopsis thaliana (Mouse-ear cress)
Saccharomyces cerevisiae (strain ATCC 204508/S288c)
Candida albicans (strain SC5314/ATCC MYA-2876)
Oryza sativa subsp. japonica (Rice)
Drosophila mojavensis (Fruit fly)
Kluyveromyces lactis (strain ATCC 8585/CBS 2359/DSM
Oryctolagus cuniculus (Rabbit)
Oryctolagus cuniculus (Rabbit)
Hordeum vulgare (Barley)
Solanum tuberosum (Potato)
Kluyveromyces lactis (strain ATCC 8585/CBS 2359/DSM
Saccharomyces cerevisiae (strain ATCC 204508/S288c)
Homo sapiens (Human)
Mus musculus (Mouse)
Rattus norvegicus (Rat)
Struthio camelus (Ostrich)
Kluyveromyces lactis (strain ATCC 8585/CBS 2359/DSM
Schizosaccharomyces pombe (strain 972/ATCC 24843)
Saccharomyces cerevisiae (strain YJM789) (Baker's yeast)
Saccharomyces cerevisiae (strain ATCC 204508/S288c)
Saccharomyces pastorianus (Lager yeast) (Saccharomyces
cerevisiae x Saccharomyces 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/S288c)
Homo sapiens (Human)
Peromyscus maniculatus (North American deer mouse)
Pongo abelii (Sumatran orangutan) (Pongo pygmaeus abelii)
Rattus norvegicus (Rat)
Homo sapiens (Human)
Rattus norvegicus (Rat)
Mus musculus (Mouse)
Mycobacterium tuberculosis (strain CDC 1551/Oshkosh)
Rhizobium meliloti (strain 1021) (Ensifer meliloti)
Mycobacterium tuberculosis (strain ATCC 25618/H37Rv)
Zymomonas mobilis subsp. mobilis (strain ATCC 31821/
Mycobacterium bovis (strain ATCC BAA-935/AF2122/97)
Mycobacterium tuberculosis (strain CDC 1551/Oshkosh)
Mycobacterium tuberculosis (strain ATCC 25618/H37Rv)
Zymomonas mobilis subsp. mobilis (strain ATCC 31821/
Zymomonas mobilis subsp. mobilis (strain ATCC 10988/
Mycobacterium tuberculosis (strain CDC 1551/Oshkosh)
Mycobacterium tuberculosis (strain ATCC 25618/H37Rv)
Clostridium acetobutylicum (strain ATCC 824/DSM 792/
Escherichia coli (strain K12)
Escherichia coli O157:H7
Rhodobacter sphaeroides (strain ATCC 17023/2.4.1/NCIB
Oryza sativa subsp. indica (Rice)
Escherichia coli (strain K12)
Geobacillus stearothermophilus (Bacillus stearothermophilus)
Emericella nidulans (strain FGSC A4/ATCC 38163/CBS
Emericella nidulans (strain FGSC A4/ATCC 38163/CBS
Emericella nidulans (strain FGSC A4/ATCC 38163/CBS
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/H37Rv)
Caenorhabditis elegans
Caenorhabditis elegans
Pseudomonas sp.
Escherichia coli (strain K12)
Moraxella sp. (strain TAE123)
Alligator mississippiensis (American alligator)
Catharanthus roseus (Madagascar periwinkle) (Vinca rosea)
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) (Uromastyx
hardwickii)
Saara hardwickii (Indian spiny-tailed lizard) (Uromastyx
hardwickii)
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) (Uromastyx
hardwickii)
Scyliorhinus canicula (Small-spotted catshark) (Squalus
canicula)
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 (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Homo sapiens (Human)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Arabidopsis lyrata subsp. lyrata (Lyre-leaved
Ectocarpus siliculosus (Brown alga)
Nicotiana attenuata (Coyote tobacco)
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 invention 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 gcdw 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−1h−1). In general, in vivo hydroxylation reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g·gcdw−1h−1, wherein gcdw 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 invention 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 gcdw 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−1h−1). In general, in vivo hydroxylation reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g·gcdw−1h−1, wherein gcdw 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−1h−1, or from about 0.1 to about 0.5 g·gcdw−1h−1, or greater than 0.5 g·gcdw−1h−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−1h−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 invention. 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 invention 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), 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 invention 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 invention. 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 invention 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 invention. 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 invention. 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 cosolvent 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 invention, 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 invention. 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.
Pheromone Compositions and Uses Thereof
As described above, many of the olefinic alcohol products made via the methods described herein are pheromones. Pheromones prepared according to the methods of the invention can be formulated for use as insect control compositions. The pheromone compositions can include a carrier, and/or be contained in a dispenser. 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, silica and China clay. 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; and mixtures thereof. Baits or feeding stimulants can also be added to the carrier.
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.
Pheromone compositions can contain other pheromones or attractants provided that the other compounds do not substantially interfere with the activity of the composition. The pheromone compositions can also include insecticides. Examples of suitable insecticides include, but are not limited to, buprofezin, pyriproxyfen, flonicamid, acetamiprid, dinotefuran, clothianidin, acephate, malathion, quinolphos, chloropyriphos, profenophos, bendiocarb, bifenthrin, chlorpyrifos, cyfluthrin, diazinon, pyrethrum, fenpropathrin, kinoprene, insecticidal soap or oil, and mixtures thereof.
Pheromone compositions can be used in conjuction 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 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.
A variety of pheromones, including those set forth in Table 1 can be prepared according to the methods of the invention and formulated as described above. For example, the methods of the invention can be used to prepare peach twig borer (PTB) sex pheromone, which is a mixture of (E)-dec-5-en-1-ol (17%) and (E)-dec-5-en-1-yl acetate (83%). The PTB sex pheromone can be used in conjunction with a sustained pheromone release device having a polymer container containing a mixture of the PTB 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 ampul, or a bag made of a polyolefin or an olefin component-containing copolymer. Sex pheromones of other pest insects such the cotton bollworm (Helicoverpa armigera), fall army worm (Spodoptera frugiperda), oriental fruit moth (Grapholita molesta) and leaf roller (Tortricidae) can be used in this type of sustained pheromone release device. The sex pheromones typically include one or more 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) and/or one or more aliphatic aldehyde compounds having from 10 to 16 carbon atoms (e.g., 7-hexadecenal, 11-hexadecenal, 13-octadecenal, and the like).
Pheromones prepared according to the methods of the invention, as well as compositions containing the pheromones, can be used to control the behavior and/or growth of insects in various environments. The pheromones can be used, for example, to attract or repel male or female insects to or from a particular target area. The pheromones can be used to attract insects away from vulnerable crop areas. The pheromones can also be used example to attract insects as part of a strategy for insect monitoring, mass trapping, lure/attract-and-kill or mating disruption.
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 the insecticide.
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 invention can be colored brightly, to provide additional attraction for the insects.
The trap is positioned in an area infested (or potentially infested) with insects. Generally, the trap is placed on or close to a tree or large plant. The aroma of the pheromone attracts the insects to the trap. The insects can then be caught, immobilised and/or killed within the trap, for example, by the killing agent present in the trap.
Pheromones prepared according to the methods of the invention can also be used to disrupt mating. 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.
Insect populations can be surveyed or monitored by counting the number of insects in a target area (e.g., the number of insects caught in a trap). Inspection by a horticulturist can provide information about the life stage of a population. Knowing where insects are, how many of them there are, and their life stage enables informed decisions to be made as to where and when insecticides or other treatments are warranted. 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.
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 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 invention 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 invention 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.
General Methods.
E. coli heat shock transformation: Heat shock transformation of plasmids/Gibson assembly products/ligation products were performed with competent BL21 (DE3) cells from NEB (C2527H). Set a water bath to 42° C. and remove the SOC media from storage at 4° C. and incubate at 37° C. Remove chemically competent cells from the −80° C. freezer and thaw on ice for 5-10 minutes. Add the DNA solution to be transformed to the cells and tap the tube gently to mix, keep the mixture on ice for 5 more minutes. Submerge the tube containing the cell/DNA mixture in the 42° C. water bath for 10 seconds and quickly place the tubes back on ice for 2 minutes. After the two minute incubation on ice, add 500 μl of SOC recovery media and incubate at 37° C. with 210 rpm shaking for 60 minutes. After incubation, 50-200 μl of the recovered cells are plated on LB/agar plates supplemented with the appropriate antibiotics.
E. coli plasmid isolation: Plasmid isolation of 2.5 ml overnight culture were performed with the SpinSmart™ Plasmid Miniprep DNA Purification kit (Danville Scientific). The overnight culture is collected by centrifugation at 14,000 rpm for 10 minutes. Resuspend the pellet in 250 μl of P1 buffer and transfer the mixture to a 1.5 ml microcentrifuge tube. Add 250 μl of P2 buffer and mix by inverting the tube. Add 300 μl of P3 buffer and mix by inverting the tube. Centrifuge the mixture at 14,000 rpm for 5 minutes and transfer the supernatant containing DNA onto a fresh plasmid binding spin column. Bind DNA onto the column by centrifugation at 14,000 rpm for 30 seconds, followed by washes with 500 μl of P4 wash buffer and 650 μl of P5 wash buffer. Dry the column by centrifugation at 14,000 rpm for 2 minutes and then discard the collection tube. Place the plasmid binding spind column in a 1.5 ml microcentrifuge tube. Add 50 μl of warm DI water (50° C.) and centrifuge at 14000 rpm to elute the DNA.
E. coli DNA isolation: DNA fragment isolation from agarose gel was performed with the Zymoclean Gel DNA Recovery Kits (Zymo research). Excise the desired gel fragment using a razor blade and transfer it to a 1.5 ml microcentrifuge tube. Add 250 μl of ADB to the tube and incubate at 50° C. for 5 to 10 minutes until the gel slice is dissolved. Transfer the melted agarose solution to a Zymo-spin™ column in a collection tube, centrifuge for 30-60 seconds and discard the flow through. Wash the column with 200 μl of DNA wash buffer and centrifuge for 30 seconds. Repeat the wash step. Dry the column by centrifugation at 14,000 rpm for 2 minutes. Place the Zymo-spin™ column in a 1.5 ml microcentrifuge tube. Add 12 μl of warm DI water (50° C.) and centrifuge at 14000 rpm to elute DNA.
PCR: PCR reactions were performed using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB: M0531S). In a typical 25 μl PCR reaction, 0.5 μl of the template DNA (plasmid, PCR product, synthesized DNA, etc) is mixed with 1.25 μl of each of the forward and reverse primers (10 mM) along with 0.75 μl of DMSO, 12.5 μl of the Phusion master mix and 8.75 μl of DI water. A typical thermocycler program consists of the follow steps:
SOE-PCR: A typical 50 μl reaction contains (1) 0.5 to 3 μl of each isolated DNA fragment being spliced (2) 2.5 μl of each the forward and reverse primers (10 mM), (3) 1.5 μl of DMSO, (4) 25 μl of Phusion master mix and (5) enough DI water to reach a final total volume of 50 μl. The thermocycler program is modified from that of table # by extending the time of step 3 to 20 seconds.
E. coli biotransformations: Each E. coli strain expressing a terminal hydroxylase as well as the necessary redox partners was cultured in shake flasks to produce cell mass and induce protein expression. At the end of the protein expression phase, the cells were collected by centrifugation and washed with the bioconversion buffer. The washed cells were resuspended in bioconversion buffer to yield a cell density of ˜100 g cell wet weight (g cww)/L. A volume of 1 ml of this cell mixture was transferred to a 20 ml amber vial. The bioconversion was initiated by the addition of 200 μl of a 50/50 (v/v) mixture of substrate and isopropyl alcohol. For E/Z-5-decene and 1-bromodecane, the reactions were quenched after 1 hour with the addition of 100 μl 3 M HCl and analyzed for product formation with GC/FID. For 1-dodecene, 1-dodecyne, and hexadecane, the reactions were quenched after 4 hours with HCl and analyzed with GC/FID.
The PTB pheromone is a mixture of (E)-dec-5-en-1-ol (17%) and (E)-dec-5-en-1-yl acetate (83%). Using a terminal hydroxylase described in Table 3, 4, or 5, (E)-dec-5-ene can be readily converted into (E)-dec-5-en-1-ol, which can be acetylated to yield (E)-dec-5-en-1-yl acetate, as shown in Scheme 22. A suitable terminal hydroxylase enables this original synthesis route and reduces the synthesis cost of this pheromone.
Asymmetric substrates shown in Tables 11, 12, and 13, including (Z)-hexadec-11-ene and diolefin substrates such as (E,E)-8,10-dodecadiene, are terminally hydroxylated via the enzymes presented in Tables 3, 4, and 5.
(Z)-non-3-ene
(Z)-non-3-en-1-ol
(E)-dec-5-ene
(E)-dec-5-en-1-ol
(Z)-dodec-8-ene
(Z)-dodec-8-en-12-ol
(E,E)-8,10-Dodecadiene
(E,E)-8,10-Dodecadien-1-ol
(Z)-hexadec-11-ene
(Z)-hexadec-11-en-1-yl acetate
Pandemis leafroller sex pheromone
(Z)-tetradec-9-ene
(Z)-hexadec-11-enal
(Z)-tetradec-9-en-1-yl acetate
(Z)-hexadec-11-ene
(Z)-hexadec-9-enal
(Z)-hexadec-9-ene
(Z)-hexadec-11-enal
(Z)-tetradec-11-ene
(Z)-tetradec-11-en-1-yl acetate
(Z)-3-nonene
(Z)-3-nonenol
Z-5-decene
Z-5-decen-1-ol
(E)-8-dodecene
(E)-8-dodecenol
(Z)-8-dodecene
(Z)-8-dodecenol
Z-7-dodecene
Z-7-dodecen-1-ol
Z-9-tetradecene
Z-9-tetradecen-1-ol
Z-11-tetradecene
Z-11-tetradecen-1-ol
E-11-tetradecene
E-11-tetradecen-1-ol
(Z,E)-9,11,13-Tetradecatriene
(Z,E)-9,11,13-Tetradecatrienol
Z-7-hexadecene
Z-7-hexadecen-1-ol
Z-9-hexadecene
Z-9-hexadecenol
Z-11-hexadecene
Z-11-hexadecenol
(Z,E)-9,11-Hexadecadiene
(Z,E)-9,11-Hexadecadien-1-ol
(Z,Z)-11,13-Hexadecadiene
(Z,Z)-11,13-Hexadecadien-1-o1
(Z,E)-11,13-Hexadecadiene
(Z,E)-11,13-Hexadecadien-1-ol
Z-13-octadecene
Z-13-octadecen-1-ol
The purpose of this example is to illustrate the construction and/or identity of the various biocatalysts described in the present disclosure.
Strains, plasmids, and oligonucleotides disclosed herein are listed in Tables 14-16.
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
All strains listed in Table 14 were obtained by transforming their corresponding plasmid into E. coli BL21(DE3) cells using standard heat shock protocol. Briefly, aliquots of competent E. coli BL21(DE3) were thawed on ice and mixed with 1 ng of isolated DNA plasmid. The mixture was immersed in a 42° C. water bath for 10 seconds, followed by incubation at 4° C. for 2 minutes and 37° C. for 30 minutes before plating on Luria-Bertani agar plates with the appropriate antibiotics.
Plasmid pPV001 was obtained directly from EMD Millipore. Plasmids pPV003, pPV004, pPV007, and pPV008 were directly obtained from the Institut fur Technische Biochemie (Stuttgart, Germany). Plasmid pPV002 was obtained from the California Institute of Technology (Pasadena, USA). Plasmids pPV001, pPV003 and pPV002 served as the vector backbone for pET28a(+), pColaDuet-1, and pCom10 based plasmids listed in Table 15. Gene inserts of pPV009, pPV010, pPV011, pPV012, pPV013, pPV014, pPV015, pPV016, pPV017, pPV018, pPV019, pPV020, pPV021, pPV022, pPV023, pPV024, pPV025, pPV026, pPV0028, pPV0029, and pPV030 designed for the expression of terminal hydroxylases and their redox partners were synthesized (Gen9, Inc. Cambridge, Mass.). The inserts of pPV009, pPV010, pPV011, pPV012, pPV013, pPV0014, pPV0015, pPV0016, pPV0017, pPV018, pPV019, pPV024, pPV025, pPV026, pPV0028 and pPV0029 were cloned into the vector by ligation of vector and insert DNA fragments generated by restriction digest at flanking sites indicated in Table 15. Plasmid pPV0020 was produced by ligation of DNA fragments obtained by restriction digest of pPV0018 (9195 bp fragment) and pPV0019 (2399 bp fragment) with XhoI and XbaI. Plasmid pPV021 was produced by Gibson assembly of DNA fragments obtained by (1) restriction digest of pPV0019 (9089 bp fragment) with EcoRI and SalI and (2) PCR product of 1406_provivi 023 with primers oPV001 and oPV002. The pPV022, pPV023 and pPV030 plasmids were produced by Gibson assembly of DNA fragments obtained by (1) restriction digest of pPV0019 (8550 bp fragment) with EcoRI and SfiI and (2) a splicing by overhang extension (SOE)-PCR product. For pPV022, the SOE-PCR was performed using primers oPV001 and oPV005 along with the PCR products from reactions containing (1) 1406_provivi024 with primers oPV001 and oPV004 and (2) pPV021 with primers oPV003 and oPV005. For pPV023, the SOE-PCR was similarly performed using primers oPV001 and oPV005 along with the PCR products from reactions containing (1) 1406_provivi026 with primers oPV001 and oPV004 and (2) pPV021 with primers oPV003 and oPV005. For pPV030, the SOE-PCR was similarly performed using primers oPV001 and oPV005 along with the PCR products from reactions containing (1) 1406_provivi025 with primers oPV001 and oPV004 and (2) pPV0021 with primers oPV003 and oPV005.
Enzymes with terminal hydroxylation activity reported in the literature have generally been characterized for their substrate specificity with linear alkanes and/or fatty acids of various lengths. In certain aspects, the present invention relies upon the observation that the presence of the alkene bonds does not affect the regioselectivity and substrate specificity of the hydroxylases. A subset of known terminal hydroxylases selected from Tables 3, 4, and 5 with preference for C8-C18 substrates is determined. These sequences are evaluated for terminal hydroxylation according to Scheme 23 with substrates shown in Tables 11, 12, and 13, including (E)-dec-5-ene, (Z)-hexadec-11-ene, and (E,E)-8,10-dodecadiene:
Strains containing these types of enzymes that have been previously used for whole-cell terminal hydroxylation of alkanes or fatty acids are shown in Table 6.
To evaluate the activity of candidate enzymes not already present in an expression strain, the appropriate DNA expression vectors are constructed and transformed in the desired hosts. The identified genes are synthesized as gene products or multiple DNA fragments and inserted into vectors that have been previously used for expression, e.g., pET28a(+) for CYP153 and pGEc47 for AlkB, using standard molecular biology techniques. The correct expression vectors are confirmed by DNA sequencing and then introduced into the desired host via transformation to obtain the desired expression strains.
The constructed expression strains are used to determine the in vivo terminal hydroxylation activity of each candidate enzyme for substrates shown in Tables 11, 12, and 13, including (E)-dec-5-ene, (Z)-hexadec-11-ene, and (E,E)-8,10-dodecadiene. Typically, each strain is cultured in a suitable medium, such as Terrific Broth, until a sufficient cell density to induce enzyme expression with Isopropyl β-D-1-thiogalactopyranoside or dicyclopropylketone. After a predetermined expression period, the cells are pelleted by centrifugation to remove the growth media. The biotransformation reaction is performed in nitrogen-free medium such as M9 or phosphate buffer to ensure the cells are in a resting state to maximize the supply of redox cofactors for the hydroxylation reaction. The alkene is present in the reaction as a neat organic overlay or part of the organic phase in a two-phase reaction. To evaluate strains with sufficient replicates, the initial reaction is performed on a 5-25 mL scale in a vial or flask. A carbon source such as glucose or glycerol can also be added to the reaction to support the regeneration of redox cofactors by the cells. The addition of a co-solvent such as bis(2-ethylhexyl) phthalate to improve the extraction of the product to the organic phase can be explored. In addition, reaction parameters such as reaction time, temperature, volume, ratio of organic and aqueous phases, aeration and cell density can be optimized.
The analysis of the whole-cell hydroxylation of substrates shown in Tables 11, 12, and 13, including (E)-dec-5-ene, (Z)-hexadec-11-ene, and (E,E)-8,10-dodecadiene reactions, requires the development of appropriate gas chromatography (GC) methods. The GC analysis of alkane and fatty acid terminal hydroxylation reactions has been performed with both mass spectrometry and flame ionization detectors. Authentic standards for (E)-dec-5-ene, (E)-dec-5-en-1-ol, and potential side-products, (E)-dec-5-ene-1,10-diol, (E)-dec-5-en-1-al, (E)-dec-5-enoic acid and 2,3-dibutyloxirane are obtained for method development and product quantification. Likewise, (Z)-hexadec-11-ene and (E,E)-8,10-dodecadiene and an assortment of likely hydroxylation or epoxidation products of these two molecules are used for method development and product quantification. A procedure for terminating the hydroxylation reaction and extracting the organic phase into a suitable GC solvent is used.
Demonstration of gram-scale production of (E)-dec-5-en-1-ol using biocatalytic terminal hydroxylation is performed in biotransformation reactions on a liter scale. To efficiently perform whole-cell hydroxylation on a liter scale, the reaction pH and aeration are controlled.
Evaluation of the most promising candidates from the 5-25 mL screening reactions in fermenters enables the identification and alleviation of reaction bottle-necks and inhibition effects of the product and by-products. Enzyme expression can also be further optimized in fermenters with enhanced control over culturing conditions.
The purpose of this example is to illustrate the biocatalytic hydroxylation of (E)-5-decene and (Z)-5-decene to (E)-5-decen-1-ol and (Z)-5-decen-1-ol, respectively, and the selectivity of the various enzymes towards either (E)-5-decene or (Z)-5-decene.
The 25 strains detailed in Example 3 were characterized for their ability to convert a mixture of (E)-5-decene and (Z)-5-decene to (E)-5-decen-1-ol and (Z)-5-decen-1-ol in whole cell bioconversion reactions.
Overnight cultures of these 25 strains were inoclulated from single colonies grown on LB agar plates containing 30 μg/ml of kanamycin into 2.5 mL of LB medium containing 30 μg/ml of kanamycin and incubated for 24 hours at 37° C. and 210 rpm. The overnight culture was used to inoculate 50 or 100 ml of Terrific Broth with a starting OD600 of 0.1. After incubation for approximately 2.5 hours at 37° C. and 210 rpm the cultures reached an OD600 of approximately 1.0-1.5, at which point they were induced with 0.5 mM IPTG and supplemented with 0.5 mM 5-aminolevulinic acid, 50 mg/L thiamine, 1.2 mM MgSO4, and 25 mL of a solution of trace elements (190 mg CaCl2)*2H2O, 90 mg ZnSO4*7H2O, 90 mg CoCl2*6H2O, 75 mg CuSO4*5H2O, 50 mg MnSO4*H2O, 11.1 mg Na2-EDTA*2H2O and 8.35 mg FeCl3*6H2O in 500 ml of ddH2O). The culture was further incubated for 20 hours at 20° C. and 180 rpm. The cultures were then pelleted via centrifugation at 3900×g at 4° C. for 10 min, washed once with bioconversion buffer (100 mM phosphate buffer (pH7.2), 1% glycerol/0.4% glucose, 100 μg/ml FeSO4*7H2O, and 30 μg/ml kanamycin), and pelleted again. Next, bioconversion buffer was added to the cell pellets targeting a cell wet weight of 100 g/L and the cell pellets were resuspended.
To carry out biotransformations, 1 mL of this mixture was transferred into sterile 40 mL amber screw cap vials and 1004 of an 84:16 mixture of (E)-5-decene:(Z)-5-decene was added along with 100 μL of isopropyl alcohol. This reaction mixture was incubated at 20° C. and 180 rpm for 1 hour before the reaction was quenched via addition of 100 μL of 3M HCL.
To extract the biotransformation products, 5 mL of n-hexane was added. The mixture was thoroughly mixed via shaking in an orbital shaker (20 min @ 250 rpm) and then allowed to settle undisturbed for 20 minutes. An aliquot (1 mL) of the organic layer was analyzed via gas chromatography (GC) using a J&W DB-23 column (30 m×25 mm×25 μm) coupled to an FID detector using the following temperature profile: 45° C. for 0.5 min; ramp 5° C./min to 50° C.; hold 0.5 min; ramp 30° C./min to 220° C.; hold 3.3 min. Retention for substrates and products were verified using authentic standards and were as follows: (E)-5-decene at 2.84 min, (Z)-5-decene at 2.91 min, (E)-5-decen-1-ol at 6.73 min, and (Z)-5-decen-1-ol at 6.79 min.
Results are shown in Table 17. These results demonstrate the capability of members of the CYP153 and AlkB family of enzymes to catalyze the hydroxylation of (E)-5-decene and (Z)-5-decene. Since the (E/Z)-5-decene substrate used in these bioconversion was an 84:16 mixture of the E:Z enantiomers, a product mixture with the same 84:16 ratio of (E)-5-decen-1-ol to (Z)-5-decen-1-ol would be obtained if the terminal hydroxylase exhibits no selectivity for either enantiomer. Our results demonstrate that the terminal hydroxylase does have a preference for one of the two substrate isomers, and isomeric enrichment for a particular alcohol product can be achieved by biohydroxylation.
The purpose of this example is to illustrate the biocatalytic hydroxylation of 1-dodecene to 11-dodecen-1-ol, and the selectivity of the various enzymes towards the production of either 11-dodecen-1-ol or 1,2-epoxydodecane.
The 25 strains detailed in Example 3 were characterized for their ability to convert 1-dodecene to 11-dodecen-1-ol and 1,2-epoxydodecane in whole cell bioconversion reactions.
Overnight cultures of these 25 strains were inoclulated from single colonies grown on LB agar plates containing 30 μg/ml of kanamycin into 2.5 mL of LB medium containing 30 μg/ml of kanamycin and incubated for 24 hours at 37° C. and 210 rpm. The overnight culture was used to inoculate 50 or 100 ml of Terrific Broth with a starting OD600 of 0.1. After incubation for approximately 2.5 hours at 37° C. and 210 rpm the cultures reached an OD600 of approximately 1.0-1.5, at which point they were induced with 0.5 mM IPTG and supplemented with 0.5 mM 5-aminolevulinic acid, 50 mg/L thiamine, 1.2 mM MgSO4, and 25 mL of a solution of trace elements (190 mg CaCl2*2H2O, 90 mg ZnSO4*7H2O, 90 mg CoCl2*6H2O, 75 mg CuSO4*5H2O, 50 mg MnSO4*H2O, 11.1 mg Na2-EDTA*2H2O and 8.35 mg FeCl3*6H2O in 500 ml of ddH2O). The culture was further incubated for 20 hours at 20° C. and 180 rpm. The cultures were then pelleted via centrifugation at 3900×g at 4° C. for 10 min, washed once with bioconversion buffer (100 mM phosphate buffer (pH7.2), 1% glycerol/0.4% glucose, 100 μg/ml FeSO4*7H2O, and 30 μg/ml kanamycin), and pelleted again. Next, bioconversion buffer was added to the cell pellets targeting a cell wet weight of 100 g/L and the cell pellets were resuspended.
To carry out biotransformations, 1 mL of this mixture was transferred into sterile 40 mL amber screw cap vials and 100 μL of 1-dodecene was added along with 100 μL of isopropyl alcohol. This reaction mixture was incubated at 20° C. and 180 rpm for 4 hour before the reaction was quenched via addition of 100 μL of 3M HCL.
To extract the biotransformation products, 5 mL of n-hexane was added. The mixture was thoroughly mixed via shaking in an orbital shaker (20 min @ 250 rpm) and then allowed to settle undisturbed for 20 minutes. An aliquot (1 mL) of the organic layer was analyzed via gas chromatography (GC) using a J&W DB-23 column (30 m×25 mm×25 μm) coupled to an FID detector using the following temperature profile: 45° C. for 0.5 min; ramp 5° C./min to 50° C.; hold 0.5 min; ramp 30° C./min to 220° C.; hold 3.3 min. Retention for substrates and products were verified using authentic standards and were as follows: 1-dodecene at 4.35 min, 11-dodecen-1-ol at 8.91 min, and 1,2-epoxydodecane at 7.86 min.
Results are shown in Table 18. These results demonstrate the capability of members of the CYP153 and AlkB family of enzymes to catalyze the hydroxylation of 1-dodecene to produce 11-dodecen-1-ol. Furthermore, some of these enzyme are able to selectively form 11-dodecen-1-ol over the energetically favored 1,2-epoxydodecane product.
The purpose of this example is to illustrate the biocatalytic hydroxylation of (Z)-5-hexadecene by members of the CYP153 family.
The following strains are constructed by (1) restriction digest of a synthesized DNA fragment containing the target gene insert and the desired expression plasmid followed by (2) ligation of the digested fragment and (3) transformation of the ligation mixture into the desired E. coli strain (Table 19).
E. coli strain
E. coli BL21 (DE3)
E. coli BL21 (DE3)
E. coli BL21 (DE3)
E. coli BL21 (DE3)
Cultures of the strains listed in Table 19 are inoculated, cultured, and subjected to a biotransformation procedure as described in Example 5 with the exception that (Z)-5-hexadecene was used as the substrate instead of the 84:16 mixture of (E)-5-decene:(Z)-5-decene. The biotransformation products are extracted and then analyzed via gas chromatography as described in Example 5 to identify products including (Z)-11-hexadecene-1-ol and (Z)-5-hexadecene-1-ol.
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 20 and 21.
P. pastoris CBS7435
P. pastoris CBS7435
Gene sequences for C. tropicalis CYP52A13 (Accession No. AA073953.1 (SEQ ID NO: 23)), C. tropicalis CPR (Accession No. P37201.1), C. maltosa CYP52A3 (Accession No. P24458.1 (SEQ ID NO: 24)), 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:13) and CYP52A13/CPR (SEQ ID NO:14) 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 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 22 and
The purpose of this example is to illustrate the biocatalytic hydroxylation of 1-dodecyne to 11-dodecyn-1-ol.
The 25 strains detailed in Example 3 were characterized for their ability to convert 1-dodecyne to 11-dodecyn-1-ol in whole cell bioconversion reactions.
Overnight cultures of these 25 strains were inoclulated from single colonies grown on LB agar plates containing 30 μg/ml of kanamycin into 2.5 mL of LB medium containing 30 μg/ml of kanamycin and incubated for 24 hours at 37° C. and 210 rpm. The overnight culture was used to inoculate 50 or 100 ml of Terrific Broth with a starting OD600 of 0.1. After incubation for approximately 2.5 hours at 37° C. and 210 rpm the cultures reached an OD600 of approximately 1.0-1.5, at which point they were induced with 0.5 mM IPTG and supplemented with 0.5 mM 5-aminolevulinic acid, 50 mg/L thiamine, 1.2 mM MgSO4, and 25 mL of a solution of trace elements (190 mg CaCl2*2H2O, 90 mg ZnSO4*7H2O, 90 mg CoCl2*6H2O, 75 mg CuSO4*5H2O, 50 mg MnSO4*H2O, 11.1 mg Na2-EDTA*2H2O and 8.35 mg FeCl3*6H2O in 500 ml of ddH2O). The culture was further incubated for 20 hours at 20° C. and 180 rpm. The cultures were then pelleted via centrifugation at 3900×g at 4° C. for 10 min, washed once with bioconversion buffer (100 mM phosphate buffer (pH7.2), 1% glycerol/0.4% glucose, 100 μg/ml FeSO4*7H2O, and 30 μg/ml kanamycin), and pelleted again. Next, bioconversion buffer was added to the cell pellets targeting a cell wet weight of 100 g/L and the cell pellets were resuspended.
To carry out biotransformations, 1 mL of this mixture was transferred into sterile 40 mL amber screw cap vials and 100 μL of 1-dodecyne was added along with 100 μL of isopropyl alcohol. This reaction mixture was incubated at 20° C. and 180 rpm for 4 hour before the reaction was quenched via addition of 100 μL of 3M HCL.
To extract the biotransformation products, 5 mL of n-hexane was added. The mixture was thoroughly mixed via shaking in an orbital shaker (20 min @ 250 rpm) and then allowed to settle undisturbed for 20 minutes. An aliquot (1 mL) of the organic layer was analyzed via gas chromatography (GC) using a J&W DB-23 column (30 m×25 mm×25 um) coupled to an FID detector using the following temperature profile: 45° C. for 0.5 min; ramp 5° C./min to 50° C.; hold 0.5 min; ramp 30° C./min to 220° C.; hold 3.3 min. Retention for substrates and products were verified using authentic standards and were as follows: 1-dodecyne at 6.34 min and 11-dodecyn-1-ol at 9.71 min.
Results are shown in Table 23. These results demonstrate the capability of members of the CYP153 and AlkB family of enzymes to catalyze the hydroxylation of 1-dodecyne to produce 11-dodecyn-1-ol.
The purpose of this example is to illustrate the biocatalytic hydroxylation of 1-bromodecane to 10-bromodecan-1-ol, and the selectivity of the various enzymes towards 10-bromodecan-1-ol formation over 1-decanal formation as the result of dehalogenation.
The 25 strains detailed in Example 3 were characterized for their ability to convert 1-bromodecane to 10-bromodecan-1-ol in whole cell bioconversion reactions.
Overnight cultures of these 25 strains were inoclulated from single colonies grown on LB agar plates containing 30 μg/ml of kanamycin into 2.5 mL of LB medium containing 30 μg/ml of kanamycin and incubated for 24 hours at 37° C. and 210 rpm. The overnight culture was used to inoculate 50 or 100 ml of Terrific Broth with a starting OD600 of 0.1. After incubation for approximately 2.5 hours at 37° C. and 210 rpm the cultures reached an OD600 of approximately 1.0-1.5, at which point they were induced with 0.5 mM IPTG and supplemented with 0.5 mM 5-aminolevulinic acid, 50 mg/L thiamine, 1.2 mM MgSO4, and 25 mL of a solution of trace elements (190 mg CaCl2*2H2O, 90 mg ZnSO4*7H2O, 90 mg CoCl2*6H2O, 75 mg CuSO4*5H2O, 50 mg MnSO4*H2O, 11.1 mg Na2-EDTA*2H2O and 8.35 mg FeCl3*6H2O in 500 ml of ddH2O). The culture was further incubated for 20 hours at 20° C. and 180 rpm. The cultures were then pelleted via centrifugation at 3900×g at 4° C. for 10 min, washed once with bioconversion buffer (100 mM phosphate buffer (pH7.2), 1% glycerol/0.4% glucose, 100 μg/ml FeSO4*7H2O, and 30 μg/ml kanamycin), and pelleted again. Next, bioconversion buffer was added to the cell pellets targeting a cell wet weight of 100 g/L and the cell pellets were resuspended.
To carry out biotransformations, 1 mL of this mixture was transferred into sterile 40 mL amber screw cap vials and 100 μL, of 1-bromodecane was added along with 100 μL, of isopropyl alcohol. This reaction mixture was incubated at 20° C. and 180 rpm for 1 hour before the reaction was quenched via addition of 100 μL of 3M HCL.
To extract the biotransformation products, 5 mL of n-hexane was added. The mixture was thoroughly mixed via shaking in an orbital shaker (20 min @ 250 rpm) and then allowed to settle undisturbed for 20 minutes. An aliquot (1 mL) of the organic layer was analyzed via gas chromatography (GC) using a J&W DB-23 column (30 m×25 mm×25 μm) coupled to an FID detector using the following temperature profile: 45° C. for 0.5 min; ramp 5° C./min to 50° C.; hold 0.5 min; ramp 30° C./min to 220° C.; hold 3.3 min. Retention for substrates and products were verified using authentic standards and were as follows: 1-bromodecane at 7.42 min, 10-bromodecan-1-ol at 11.6 min and 1-decanal at 7.24 min.
Results are shown in Table 24. These results demonstrate the capability of members of the CYP153 and AlkB family of enzymes to catalyze the hydroxylation of 1-bromodecane to 10-bromodecan-1-ol. Furthermore, these results demonstrate selective hydroxylation to from 10-bromodecan-1-ol over the dehalogenated product 1-decanal.
Synthesis of (E)-5-decen-1-ol is carried out according to Scheme 24.
E-5-Decene:
The synthesis of E-5-decene is carried out according to the Example 6 disclosed in the US Patent Application No. 2013/0023665 A1. Briefly, into a 25 mL Schlenk flask, the catalyst (Richard Pederson et al. Adv. Synth. Catal. 2002, 344, 728) (0.0032 mmol) and then 0.4 mL of 1-hexene is added under argon atmosphere. The mixture is stirred at 40° C. for 2 hours. The reaction is quenched by filtration through a plug of silica gel (˜2 cm) packed in a Pasteur pipette using pentane as eluent. The reaction solvent is removed under reduced pressured. The desired product, E-5-decene, is purified by distillation and characterized by GC and NMR.
E-5-decen-1-ol:
E-5-Decene is then subjected to biohydroxylation according to the process disclosed in Example 6 to generate E-5-decen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.
Synthesis of (Z)-3-hexen-1-ol is carried out according to Scheme 25.
Z-3-Hexene:
A 5-L jacketed 3-necked flask is equipped with a magnetic stir bar, gas feeding tube and a packed bed column containing a dry ice condenser. Z-selective catalyst (see, Scheme 19a) 2.97 g, 0.0035 mol, 0.023 mol % based on 1-butene added) and toluene (240 g) are added. The flask is sparged with argon for 15 min, while being cooled to 15° C. 1-Butene (841 g, 15.0 mol) is added by bubbling into the toluene solution over 10.5 hours. The rate of addition is such that the reaction temperature remains above 10° C. After 10.5 hours, the packed bed column and the dry ice condenser are replaced with a Friedrich condenser. The Friedrich condenser is circulated with 0° C. coolant. The reaction flask is cooled to 10° C. An argon purge with a flow rate of 1 L/minute is maintained for 12 hours. The metathesis catalyst is removed by the in-situ generation of catalyst-tris(hydroxymethyl)phosphine (THP) complex. Tetrakis(hydroxymethyl)phosphonium chloride (TKC) (80% purity, 20.80 g, 25 equivalent to catalyst) and NaHCO3 (7.35 g, 25 equivalent to catalyst) are added to the solution. The chiller/heater controlling the jacketed flask is set to 40° C. and stirred for 18 hours. The reaction is cooled to 10° C. and washed with water (500 mL) and brine (500 mL) and dried over anhydrous Na2SO4. Z-3-Hexene is isolated by distillation.
Z-3-Hexen-1-ol:
Z-3-Decene is subjected to biohydroxylation according to the process disclosed in Example 6 to generate Z-3-hexen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.
Synthesis of (Z)-11-hexadecenol is carried out according to Scheme 26.
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 (based on dodec-1-ene added; see Scheme 19a) 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 6 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.
Synthesis of (Z)-11-hexadecenol is carried out according to Scheme 27.
1-Dodecyne:
The synthesis of 1-dodecyne 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-dodecyne (5 mmol) in THF (20 mL), 2.5 M 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 subjected to biohydroxylation according to the process disclosed in Example 6 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.
Synthesis of (Z)-11-hexadecenol is carried out according to Scheme 28.
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), 2.5 M 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 6 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.
Synthesis of (Z)-11-hexadecenol is carried out according to Scheme 29.
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 6 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.
Synthesis of (Z)-11-hexadecenol is carried out according to Scheme 30.
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 dropwise 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 mobile phase. Z-5-hexadecene 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 6 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.
Synthesis of (Z)-11-hexadecenol is carried out according to Scheme 31.
1,12-Dodecanediol:
Dodecane is subjected to biohydroxylation according to the process disclosed in Example 6 to generate dodecane-1,12-diol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.
11-Dodecen-1-ol:
Monodehydration of 1,12-dodecanediol to 11-dodecen-1-ol is carried out according to a vapor-phase catalytic process as described in M. Segawa et al. Journal of Molecular Catalysis A: Chemical 2009, 310, 166. The catalytic reactions are performed in a fixed-bed down-flow reactor with the inside diameter of 17 mm. Prior to the reactions, an In2O3 sample (weight, W=0.50 g) is preheated in the reactor in N2 flow at 500° C. for 1 h. The temperature of catalyst bed set at a prescribed temperature between 300 and 375° C., and 1,12-dodecanediol is then fed through the reactor top at a liquid feed rate of 2.67 mL per hour together with N2 flow of 30 mL per min. The effluent is collected at −77° C., analyzed by GC. The 11-dodecen-1-ol product is purified by distillation.
Z-11-Hexadecen-1-ol:
The cross metathesis reactions of 1-hexene and 11-dodecen-1-ol is carried out in a 250 mL three-necked round-bottomed flask fitted with a condenser, thermometer and septum. The 11-dodecen-1-ol (20 mL) is transferred to the reaction flask along with 3 mole equivalents 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 (based on 11-dodecen-1-ol added) 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 the limiting starting material, 11-dodecen-1-ol, 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-11-hexadecen-1-ol is obtained by distillation.
Synthesis of (Z)-11-hexadecenol is carried out according to Scheme 32.
11-Dodecen-1-ol:
1-Dodecene is subjected to biohydroxylation according to the process disclosed in Example 6 to generate 11-dodecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.
Z-11-Hexadecen-1-ol:
The synthesis of Z-11-hexadecen-1-ol is carried out the same way as described in Example 13.
Synthesis of (Z)-11-hexadecenal via hydroxylation of 1-halodecane is carried out according to Scheme 33. In particular, Scheme 33 illustrates the synthesis of an important pheromone, (Z)-11-hexadecenal, utilizing biohydroxylation of 1-halodecane. In this process, biohydroxylation of 1-halodecane provides the desired α,ω-halodecanol. Upon protection of the hydroxyl moiety, the molecule can be alkylated with 1-hexyne to provide the C16 internal alkyne, which can be reduced to the cis-alkene via the use of the Lindlar's hydrogenation catalyst. Deprotection of the alcohol moiety, followed by oxidation of the free alcohol to aldehyde provides the desired product, (Z)-11-hexadecenal.
10-halodecanol:
1-Halodecane is subjected to biohydroxylation according to the process disclosed in Example 6 to generate 11-halodecanol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.
THP-Protected 10-halodecanol:
Into an oven-dried 50 mL round-bottom flask, 10-halodecan-1-ol (1 mmol) and p-toluenesulfonic acid monohydrate (catalytic amount) were dissolved in 20 mL of dichloromethane. The mixture was allowed to mix at 0° C. for 10 min. With stirring, 3,4-Dihydro-2H-pyran (5 mmol) dissolved in 10 mL of dichloromethane was then dropwise added to the reaction mixture at 0° C. The progress of the reaction was monitored by TLC and the reaction was observed to be completed within 3 hours. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by silica gel chromatography. Fractions contain the desired product were pulled and the solvent was removed under reduced pressure to afford the desired product.
THP-Protected 11-hexadecyn-1-ol:
To a −78° C. solution of 1-hexyne (5 mmol) in THF (20 mL), 2.5 M n-BuLi (5 mmol) in hexane is added dropwise via a syringe. A solution of THP-protected 10-halodecanol (5 mmol) and n-Bu4NI (TBAI) (0.16 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 to obtain the desired product, THP-protected 11-hexadecyn-1-ol, as pure component.
THP-Protected Z-11-Hexadecenol:
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 THP-protected 11-hexadecyn-1-ol (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-11-Hexadecenol:
THP-protected Z-11-hexadecenol (1 mmol) is dissolved in methanol (10 mL) with a catalytic amount of monohydrate p-TsOH. The mixture is heated to 70° C. for 30 min. The reaction solvent is removed under reduced pressure and the resulting residue is re-suspended in hexane and purified by silica gel flash chromatography using 9:1/hexane:ethyl acetate as mobile phase. Pure fractions containing the desired product is pulled and concentrated to dryness to provide the desired product, Z-11-hexadecenol.
Z-11-Hexadecenal:
To a 25 mL RBF, Pyridinium chlorochromate (PCC) (90 mg, 0.41 mmol) was dissolved in dichloromethane (15 mL) and the mixture was allowed to stir at ambient temperature for 10 min. A solution of Z-11-Hexadecen-1-ol (48 mg) in dichloromethane (15 ml) was then dropwise added to the reaction mixture. The progress of the oxidation was monitored by TLC and deemed to be finished within 2 hrs. The reaction mixture was filtered through a bed of silica and the filtrate was concentrated under reduced pressure. The resulting residue was purify by silica gel flash chromatography to provide 7 mg of Z-11-hexadecen-1-al, a yield of 14%.
Synthesis of Codling Moth pheromone via hydroxylation of 1-bromohexane is carried out according to Scheme 34. Illustrated in Scheme 34 below are possible approaches to the synthesis of Codling Moth pheromone utilizing biohydroxylation of 1-bromohexane and (E,E)-2,4-hexadiene to generate 6-bromo-hexan-1-ol and (E,E)-2,4-hexadien-1-ol, respectively, as key and novel steps. Alternatively, (E,E)-2,4-hexadien-1-ol can be synthesized by reduction of sorbic acid with lithium aluminum hydride. In a similar fashion, 1-bromohexane was subjected to biohydroxylation to generate 1-bromohexanol. With appropriate chemical manipulation, the coupling of (E,E)-2,4-hexadien-1-ol derivative to 1-bromohexanol derivative can be carried out to obtain the desired Codling Moth pheromone as shown in Scheme 34 below.
(E,E)-2,4-Hexadien-1-ol:
(E,E)-2,4-hexadiene is subjected to biohydroxylation according to the process disclosed in Example 6 to generate (E,E)-2,4-hexadienol. The product is isolated by extraction of the fermentation broth with ethyl acetate and purified by distillation.
Alternatively, (E,E)-2,4-hexadienol can also be made by reduction of sorbic acid with one equivalent of lithium aluminium hydride in diethyl ether solvent according to known literature procedures.
6-Bromo-1-hexanol:
1-Bromohexane is subjected to biohydroxylation according to the process disclosed in Example 6 to generate 6-bromo-1-hexanol. The product is isolated by extraction of the fermentation broth with ethyl acetate and purified by distillation.
THP-protected 6-bromo-1-hexanol:
Into an oven-dried 50 mL round-bottom flask, 6-Bromo-1-hexanol (1 mmol) and p-toluenesulfonic acid monohydrate (catalytic amount) are dissolved in 20 mL of dichloromethane. The mixture was allowed to mix at 0° C. for 10 min. With stirring, 3,4-Dihydro-2H-pyran (3 mmol) dissolved in 10 mL of dichloromethane was then dropwise added to the reaction mixture at 0° C. The progress of the reaction was monitored by TLC and the reaction was observed to be completed within 3 hours. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by silica gel chromatography. Fractions contain the desired product were pulled and the solvent was removed under reduced pressure to afford the desired product.
1-Bromo-2,3-Hexadiene:
Into an oven-dried 50 mL round-bottom flask, CaH2 (1 mmol) is suspended in anhydrous THF (10 mL) at −78° C. and to this a solution of 2,4-hexadien-1-ol in 10 mL of THF is then dropwise added. The reaction solution is allowed to stir at −78° C. for 30 min. Phosphorus tribromide (1 mmol) dissolves in THF (10 mL) is then dropwise added to the reaction mixture via an addition funnel. The reaction mixture is allowed to warm to ambient temperature and allows to react until complete consumption of the starting material is achieved that can be monitored by TLC. The reaction mixture is quenched with 5 mL of 1 M NH4Cl and extracted three times with ethyl acetate. The organic fractions are combined, dry with anhydrous MgSO4, and concentrate to dryness. The resulting residue is purified by silica gel chromatography to obtain 1-bromo-2,3-hexadiene as pure compound.
THP-protected (8E,10E)-dodecadien-1-ol:
Into an oven-dried 50 mL round-bottom flask, freshly prepared magnesium strip (0.5 g) is added along with a few crystals of iodide and 10 mL of anhydrous THF. A solution of 1-bromo-2,3-hexadiene in THF (10 mL) is then dropwise added to the reaction vessel and a gentle reflux of the reaction mixture is maintained via a heating oil bath. A solution of THP-protected 6-bromo-1-hexanol (1 mmol) in THF (10 mL) is then dropwise added to the reaction mixture via an additional funnel. The reaction mixture is maintained at reflux and the progress of the reaction is monitored by TLC. Once the starting material, 1-bromo-2,3-hexadiene, is observed to have been consumed, the reaction is quenched with 10 mL of water. The reaction mixture is extracted with ethyl acetate (3×) and the organic fractions are combined, dry with anhydrous MgSO4 and concentrate. THP-protected (8E,10E)-dodecadien-1-ol is further purified by silica gel chromatography.
(8E,10E)-dodecadien-1-ol:
THP-protected (8E,10E)-dodecadien-1-ol (1 mmol) is dissolved in methanol (10 mL) with a catalytic amount of monohydrate p-toluenesulphonic acid. The mixture is heated to 70° C. for 30 min. The reaction solvent is removed under reduced pressure and the resulting residue is re-suspended in hexane and purified by silica gel flash chromatography using 9:1/hexane:ethyl acetate as mobile phase. Pure fractions containing the desired product are combined and concentrated to dryness to provide the desired product, (8E,10E)-dodecadien-1-ol.
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 is a continuation of PCT/US2015/031219, filed May 15, 2015, which application claims priority to U.S. Provisional Application No. 61/994,662, filed May 16, 2014; U.S. Provisional Application No. 62/060,469, filed Oct. 6, 2014; U.S. Provisional Application No. 62/062,758, filed Oct. 10, 2014; U.S. Provisional Application No. 62/082,555, filed Nov. 20, 2014; U.S. Provisional Application No. 62/096,417, filed Dec. 23, 2014; and U.S. Provisional Application No. 62/096,429, filed Dec. 23, 2014; which applications are incorporated herein by reference in their entirety for all purposes.
Certain aspects of this invention were made with government support under award number IIP-1448692, awarded by the National Science Foundation. The government may have certain rights in the invention.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4455373 | Higgins | Jun 1984 | A |
| 4473643 | Higgins | Sep 1984 | A |
| 5104504 | Tanaka et al. | Apr 1992 | A |
| 5593872 | Gabelman et al. | Jan 1997 | A |
| 5783429 | Gabelman et al. | Jul 1998 | A |
| 6034028 | Hayashi et al. | Mar 2000 | A |
| 6215019 | Pederson et al. | Apr 2001 | B1 |
| 6331420 | Wilson et al. | Dec 2001 | B1 |
| 6540991 | Klassen et al. | Apr 2003 | B2 |
| 7405063 | Eirich et al. | Jul 2008 | B2 |
| 7524664 | Arnold et al. | Apr 2009 | B2 |
| 8143034 | Gross et al. | Mar 2012 | B2 |
| 8158391 | Gross et al. | Apr 2012 | B2 |
| 8309333 | Koch et al. | Nov 2012 | B1 |
| 8361769 | Koch et al. | Jan 2013 | B1 |
| 8597923 | Ness et al. | Dec 2013 | B2 |
| 20020022741 | Pederson et al. | Feb 2002 | A1 |
| 20020146387 | Klassen et al. | Oct 2002 | A1 |
| 20030022947 | McAtee et al. | Jan 2003 | A1 |
| 20070142680 | Ayoub et al. | Jun 2007 | A1 |
| 20080057577 | Arnold et al. | Mar 2008 | A1 |
| 20090054610 | Gross et al. | Feb 2009 | A1 |
| 20090137850 | Yanagawa et al. | May 2009 | A1 |
| 20120271085 | Nesterenko et al. | Oct 2012 | A1 |
| 20130085288 | Snead et al. | Apr 2013 | A1 |
| 20130274529 | Del Cardayre | Oct 2013 | A1 |
| 20140004598 | Picataggio et al. | Jan 2014 | A1 |
| 20140186905 | Schaffer et al. | Jul 2014 | A1 |
| 20150010968 | Engel et al. | Jan 2015 | A1 |
| Number | Date | Country |
|---|---|---|
| 2 617 703 | Jul 2013 | EP |
| 00002837 | Jan 2000 | WO |
| 2011008232 | Jan 2011 | WO |
| 2014079683 | May 2014 | WO |
| 2014201474 | Dec 2014 | WO |
| 2015014644 | Feb 2015 | WO |
| Entry |
|---|
| Devos et al., (Proteins: Structure, Function and Genetics, 2000, vol. 41: 98-107). |
| Witkowski et al., (Biochemistry 38:11643-11650, 1999. |
| Kisselev L., (Structure, 2002, vol. 10: 8-9. |
| Whisstock et al., (Quarterly Reviews of Biophysics 2003, vol. 36 (3): 307-340. |
| Catterjee etal. JACS 2003, 125, p. 11360-11370. |
| Abghari, A. and Chen, S., “Yarrowia lipolytica as an oleaginous cell factory platform for the production of fatty acid-based biofuel and bioproducts,” Front. Energy Res. 2(2):1-21, 2014. |
| Abramovitch, R. et al., “1,2-Benzisothiazole 1,1-dioxide: a convenient synthesis. The question of the possible aromaticity of 1,2-benzothiazepine 1,1-dioxides,” J. Chem. Soc., Chem. Commun., 520-521, 1983. |
| Albrecht, M. et al., “Expression of a ketolase gene mediates the synthesis of canthaxanthin in Synechococcus leading to tolerance against photoinhibition, pigment degradation and UV-B sensitivity of photosynthesis,” Photochem Photobiol, 73(5):551-555, 2001. |
| Ando, T. et al., “Insecticidal activity of new fluorinated pyrethroids and their stability toward chemical oxidation and photoreaction,” Pestic. Sci., 40:307-312, 1994. |
| Andrus, M. and Zhou, Z., “Highly Enantioselective Copper-Bisoxazoline-Catalyzed Allylic Oxidation of Cyclic Olefins with tert-Butyl p-nitroperbenzoate,” J. Am. Chem. Soc., 124:8806-8807, 2002. |
| Arterburn, J., “Selective oxidation of secondary alcohols,” Tetrahedron, 57(49), 9765-9788, 2001. |
| Bae, J. et al., “Development of a recombinant Escherichia coli-based biocatalyst to enable high styrene epoxidation activity with high product yield on energy source,” Process Biochem., 45:147-152, 2010. |
| Balasubramanian, R. et al., “Oxidation of methane by a biological dicopper centre,” Nature, 465:115-119, 2010. |
| Baldwin, C. and Woodley, J., “On oxygen limitation in a whole cell biocatalytic Baeyer-Villiger oxidation process,” Biotechnol. Bioeng., 95:362-369, 2006. |
| Banthorpe, D., “Purification and properties of alcohol oxidase from Tanacetum vulgare,” Phytochemistry, 15:391-394, 1976. |
| Beier, A. et al., “Metabolism of alkenes and ketones by Candida maltosa and related yeasts,” AMB Express, 4:75, 2014. |
| Bell, S. et al., “Butane and propane oxidation by engineered cytochrome P450cam,” Chem. Commun., 490-491, 2002. |
| Boddupalli, S. et al., “Fatty acid monooxygenation by cytochrome P-450BM-3,” J. Biol. Chem., 265:4233-4239, 1990. |
| Boetius, A. et al., “A marine microbial consortium apparently mediating anaerobic oxidation of methane,” Nature, 407:623-626, 2000. |
| Bogdan, A. and McQuade, D., “A biphasic oxidation of alcohols to aldehydes and ketones using a simplified packed-bed microreactor,” Beilstein Journal of Organic Chemistry, 5:17, 2009. |
| Boll, M. et al., “Anaerobic oxidation of aromatic compounds and hydrocarbons,” Curr. Opin. Chem. Biol., 6:604-611, 2002. |
| Bordeaux, M. et al., “A Regioselective Biocatalyst for Alkane Activation under Mild Conditions,” Angew. Chemie-International Ed, 50:2075-2079, 2011. |
| Bordeaux, M. et al., “Catalytic, mild, and selective oxyfunctionalization of linear alkanes: current challenges,” Angew Chem Int Ed Engl, 51:10712-10723, 2012. |
| Bosetti, A. et al., “Production of primary aliphatic alcohols with a recombinant Pseudomonas strain, encoding the alkane hydroxylase enzyme system,” Enzym. Microb. Technol., 14:702-708, 1992. |
| Braun, A. et al., “Steroid biotransformation in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes,” Microb. Cell Fact., 11:106, 2012. |
| Bronner, S. et al., “Ru-based Z-selective metathesis catalysts with modified cyclometalated carbene ligands,” Chem Sci, 5:4091-4098, 2014. |
| Buck, M. and Chong, J., “Alkylation of 1-alkynes in THF,” Tetrahedron Lett, 42:5825-5827, 2001. |
| Buhler, B. and Schmid, A., “Process implementation aspects for biocatalytic hydrocarbon oxyfunctionalization,” J. Biotechnol., 113:183-210, 2004. |
| Cannon, J. and Grubbs, R., “Alkene Chemoselectivity in Ruthenium-Catalyzed Z-Selective Olefin Metathesis,” Angew Chemie Int Ed, 52:9001-9004, 2013. |
| Cao, Z. et al., “Engineering the acetyl-CoA transportation system of candida tropicalis enhances the production of dicarboxylic acid,” Biotechnol. J., 1:68-74, 2006. |
| Cappaert, L. and Larroche, C., “Oxidation of a mixture of 2-(R) and 2-(S)-heptanol to 2-heptanone by Saccharomyces cerevisiae in a biphasic system,” Biocatal. Biotransformation, 22:291-296, 2004. |
| Cappelletti, M. et al., “Analyses of both the alkB Gene Transcriptional Start Site and alkB Promoter-Inducing Properties of Rhodococcus sp Strain BCP1 Grown on n-Alkanes,” Appl. Environ. Microbiol., 77(5):1619-1627, 2011. |
| Cardemil, E., “Alcohol-oxidizing enzymes from various organisms,” Comp Biochem Physiol B, 60B:1-7, 1978. |
| Caron, S. et al., “Large-Scale Oxidations in the Pharmaceutical Industry,” Chemical Reviews, 106(7):2943-2989, 2006. |
| Chatterjee, A. et al., “A general model for selectivity in olefin cross metathesis,” Journal of the American Chemical Society, 125(37):11360-11370, 2003. |
| Chen, M. and White, M., “A Predictably Selective Aliphatic C—H Oxidation Reaction for Complex Molecule Synthesis,” Science, 318:783-787, 2007. |
| Chen, M. et al., “Utilizing Terminal Oxidants to Achieve P450-Catalyzed Oxidation of Methane,” Adv. Synth. Catal., 354:964-968, 2012. |
| Cheng, Q. et al., “Candida yeast long chain fatty alcohol oxidase is a c-type haemoprotein and plays an important role in long chain fatty acid metabolism,” Biochim et Biophysica Acta, 1735:192-203, 2005. |
| Cheng, Q. et al., “Functional identification of AtFao3, a membrane bound long chain alcohol oxidase in Arabidopsis thaliana,” FEBS Lett, 574:62-68, 2004. |
| Chrobok, A. et al., “Supported ionic liquid phase catalysis for aerobic oxidation of primary alcohols,” Applied Catalysis, A: General, 389(1-2):179-185, 2010. |
| Ciriminna, R. and Pagliaro, M., “Industrial Oxidations with Organocatalyst TEMPO and Its Derivatives,” Organic Process Research & Development, 14(1):245-251, 2010. |
| Cornelissen, S. et al., “Cell physiology rather than enzyme kinetics can determine the efficiency of cytochrome P450-catalyzed C—H-oxyfunctionalization,” J. Ind. Microbiol. Biotechnol., 38:1359-1370, 2011. |
| Cornelissen, S. et al., “Whole-cell-based CYP153A6-catalyzed (S)-limonene hydroxylation efficiency depends on host background and profits from monoterpene uptake via AlkL,” Biotechnology and Bioengineering, 110:1282-1292, 2013. |
| Craft, D. et al., “Identification and Characterization of the CYP52 Family of Candida tropicalis ATCC 20336, Important for the Conversion of Fatty Acids and Alkanes to α,ω-Dicarboxylic Acids,” Appl. Environ. Microbiol., 69(10):5983-5991, 2003. |
| Crow, J., “Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species,” Nitric Oxide: Biology and Chemistry, 1(2):145-157, 1997. |
| D'Acunzo, F. et al., “A mechanistic survey of the oxidation of alcohols and ethers with the enzyme laccase and its mediation by TEMPO,” European Journal of Organic Chemistry, 24:4195-4201, 2002. |
| Daff, S. et al., “Redox control of the catalytic cycle of flavocytochrome P-450 BM3,” Biochemistry, 36:13816-13823, 1997. |
| De Smidt, O. et al., “The alcohol dehydrogenases of Saccharomyces cerevisiae: a comprehensive review,” FEMS Yeast Res, 8:967-978, 2008. |
| Dellomonaco, C. et al., “Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals,” Nature, 476:355-359, 2011. |
| Dickinson, F. and Dack, S., “The activity of yeast ADH I and ADH II with long-chain alcohols and diols,” Chem. Biol. Interact., 130-132:417-423, 2001. |
| Dickinson, F. and Wadforth, C., “Purification and some properties of alcohol oxidase from alkane-grown candida-tropicalis,” Biochem. J., 282:325-331, 1992. |
| Dong, Y. et al., “Engineering of LadA for enhanced hexadecane oxidation using random- and site-directed mutagenesis,” Appl. Microbiol. Biotechnol., 94:1019-1029, 2012. |
| Duff, S. and Murray W., “Production and application of methylotrophic yeast Pichia-pastoris,” Biotechnol Bioeng, 31:44-49, 1988. |
| Eirich, L. et al., “Cloning and characterization of three fatty alcohol oxidase genes from Candida tropicalis strain ATCC 20336,” Appl Environ Microbiol, 70(8):4872-4879, 2004. |
| Endo, K. and Grubbs, R., “Chelated ruthenium catalysts for Z-selective olefin metathesis,” J Am Chem Soc, 133(22):8525-8527, 2011. |
| Ernst, M. et al., “Enantioselective reduction of carbonyl compounds by whole-cell biotransformation, combining a formate dehydrogenase and a (R)-specific alcohol dehydrogenase,” Appl Microbiol Biotechnol, 66:629-634, 2005. |
| Eschenfeldt, W. et al., “Transformation of fatty acids catalyzed by cytochrome P450 monooxygenase enzymes of Candida tropicalis,” Appl. Environ. Microbiol. 69(10):5992-5999, 2003. |
| Farinas, E. et al., “Alkene epoxidation catalyzed by cytochrome P450 BM-3 139-3,” Tetrahedron, 60:525-528, 2004. |
| Farinas, E. et al., “Directed evolution of a cytochrome P450 monooxygenase for alkane oxidation,” Advanced Synth. Catal., 343:601-606, 2001. |
| Fasan, R., “Tuning P450 Enzymes as Oxidation Catalysts,” ACS Catal., 2:647-666, 2012. |
| Favre-Bulle, O. and Witholt, B., “Biooxidation of n-octane by a recombinant Escherichia coli in a two-liquid-phase system: Effect of medium components on cell growth and alkane oxidation activity,” Enzym. Microb. Technol., 14:931-937, 1992. |
| Feng, L. et al., “Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir,” Proc. Natl. Acad. Sci. U. S. A., 104(13):5602-5607, 2007. |
| Fickers, P. et al., “Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications,” FEMS Yeast Res., 5:527-543, 2005. |
| Fishman, A. et al., “Controlling the Regiospecific Oxidation of Aromatics via Active Site Engineering of Toluene para-Monooxygenase of Ralstonia pickettii PKO1,” J. Biol. Chem., 280(1):506-514, 2005. |
| Flook, M. et al., “Z-selective olefin metathesis processes catalyzed by a molybdenum hexaisopropylterphenoxide monopyrrolide complex,” Journal of the American Chemical Society, 131(23):7962-3, 2009. |
| Fontan, N. et al., “Synthesis of C40-Symmetrical Fully-Conjugated Carotenoids by Olefin Metathesis,” Eur. J. Org. Chem., 2011(33):6704-6712, 2011. |
| Friedrich, H., “The oxidation of alcohols to aldehydes or ketones high oxidation state ruthenium compounds as catalysts,” Platinum Metals Review, 43(3):94-102, 1999. |
| Fujii, T. et al., “Biotransformation of various alkanes using the Escherichia coli expressing an alkane hydroxylase system from Gordonia sp TF6,” Biosci. Biotechnol. Biochem., 68(10):2171-2177, 2004. |
| Fujii, T. et al., “Production of α,ω-alkanediols using Escherichia coli expressing a cytochrome p450 from Acinetobacter sp OC4,” Biosci. Biotechnol. Biochem., 70(6):1379-1385, 2006. |
| Fujita, N. et al., “Comparison of Two Vectors for Functional Expression of a Bacterial Cytochrome P450 Gene in Escherichia coli Using CYP153 Genes,” Biosci. Biotechnol. Biochem., 73(8):1825-1830, 2009. |
| Funhoff, E. et al., “CYP153A6, a soluble P450 oxygenase catalyzing terminal-alkane hydroxylation,” J. Bacteriol., 188(14):5220-5227, 2006. |
| Funhoff, E. et al., “Hydroxylation and epoxidation reactions catalyzed by CYP153 enzymes,” Enzyme and Microbial Technology, 40:806-812, 2007. |
| Garikipati, S. et al., “Whole-Cell Biocatalysis for 1-Naphthol Production in Liquid-Liquid Biphasic Systems,” Appl. Environ. Microbiol., 75(20):6545-6552, 2009. |
| Girhard, M. et al., “Characterization of the versatile monooxygenase CYP109B1 from Bacillus subtilis,” Appl Microbiol Biotechnol, 87:595-607, 2010. |
| Goswami, P. et al., “An overview on alcohol oxidases and their potential applications,” Applied Microbiology and Biotechnology, 97:4259-4275, 2013. |
| Grant, C. et al., “Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes,” Sci. Rep. 4:5844, 9 pages, 2014. |
| Grant, C. et al., “Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P-putida GPo1 expressed in E-coil,” Enzyme Microb. Technol., 48:480-486, 2011. |
| Griffin, B. et al., “Radical mechanism of aminopyrine oxidation by cumene hydroperoxide catalyzed by purified liver microsomal cytochrome P-450,” Arch. Biochem. Biophys., 205(2):543-553, 1980. |
| Griffin, K. and Matthey, J., “Selective oxidation of alcohols to carbonyls,” Innovations in Pharmaceutical Technology, 02(10), 110,112,114, 2002. |
| Groothaert, M. et al., “Selective Oxidation of Methane by the Bis(μ-oxo)dicopper Core Stabilized on ZSM-5 and Mordenite Zeolites,” J. Am. Chem. Soc., 127:1394-1395, 2005. |
| Gross, R. et al., “Engineered catalytic biofilms for continuous large scale production of n-octanol and (S)-styrene oxide,” Biotechnol. Bioeng., 110(2):424-436, 2013. |
| Groves, J. and Roman, J., “Nitrous Oxide Activation by a Ruthenium Porphyrin,” J. Am. Chem. Soc., 117:5594-5595, 1995. |
| Groves, J., “High-valent iron in chemical and biological oxidations,” J. Inorg. Biochem., 100:434-447, 2006. |
| Gudiminchi, R. et al., “Screening for cytochrome P450 expression in Pichia pastoris whole cells by P450-carbon monoxide complex determination,” Biotechnol. J., 8:146-152, 2013. |
| Gudiminchi, R. et al., “Whole-cell hydroxylation of n-octane by Escherichia coli strains expressing the CYP153A6 operon,” Appl. Microbiol. Biotechnol., 96:1507-1516, 2012. |
| Guengerich, F. et al., “Evidence for a 1-electron oxidation mechanism in N-dealkylation of N,N-dialkylanilines by cytochrome P450 2B1,” J. Biol. Chem., 271(44):27321-27329, 1996. |
| Guengerich, F., “Oxidation-reduction properties of rat liver cytochromes P-450 and NADPH-cytochrome P-450 reductase related to catalysis in reconstituted systems,” Biochemistry, 22:2811-2820, 1983. |
| Hagstrom, A. et al., “A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory,” Microb. Cell Fact., 12:125, 11 pages, 2013. |
| Hagstrom, A. et al., “Semi-Selective Fatty Acyl Reductases from Four Heliothine Moths Influence the Specific Pheromone Composition,” PLoS One, 7(5):e37230, 11 pages, 2012. |
| Hamberg, M. et al., “α-Dioxygenases,” Biochem. Biophys. Res. Commun., 338:169-174, 2005. |
| Hara, A. et al., “Cloning and functional analysis of alkB genes in Alcanivorax borkumensis SK2,” Environ. Microbiol., 6(3):191-197, 2004. |
| Hartmans, S. et al., “Microbial metabolism of short-chain unsaturated hydrocarbons,” FEMS Microbiol Rev, 5:235-264, 1989. |
| Hartung, J. and Grubbs, R., “Highly Z-selective and enantioselective ring-opening/cross-metathesis catalyzed by a resolved stereogenic-at-Ru complex,” J Am Chem Soc, 135(28):10183-10185, 2013. |
| Hartung, J. et al., “Enantioselective Olefin Metathesis with Cyclometalated Ruthenium Complexes,” Journal of the American Chemical Society, 136(37):13029-13037, 2014. |
| Herbert, M. et al., “Concise syntheses of insect pheromones using Z-selective cross metathesis,” Angew Chem Int Ed Engl, 52:310-314, 2013. |
| Hommel, R. and Ratledge, C., “Evidence for two fatty alcohol oxidases in the biosurfactant-producing yeast Candida (Torulopsis) bombicola,” FEMS Microbiol Lett, 58:183-186, 1990. |
| Hommel, R. et al., “The inducible microsomal fatty alcohol oxidase of Candida (Torulopsis) apicola,” Appl Microbiol Biotechnol, 40:729-734, 1994. |
| Hoover, J. et al., “Copper(I)/TEMPO-catalyzed aerobic oxidation of primary alcohols to aldehydes with ambient air,” Nature Protocols, 7(6):1161-1166, S1161/1-S1161/5, 2012. |
| Hou, C. et al., “Thermostable NAD-linked secondary alcohol-dehydrogenase from propane-grown pseudomonas-fluorescens NRRL-B-1244,” Appl Environ Microbiol, 46(1):98-105, 1983. |
| Hua, F. et al., “Trans-membrane transport of n-octadecane by Pseudomonas sp DG17,” J. Microbiol., 51(6):791-799, 2013. |
| Huang, F. et al., “Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola,” Appl. Environ. Microbiol., 80:766-776, 2014. |
| Iida, T. et al., “The cytochrome P450ALK multigene family of an n-alkane-assimilating yeast, Yarrowia lipolytica: cloning and characterization of genes coding for new CYP52 family members,” Yeast, 16:1077-1087, 2000. |
| Ji, Y. et al., “Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases,” Front. Microbiol., 4(58):1-13, 2013. |
| Jiang, A. et al., “Highly Z-Selective Metathesis Homocoupling of Terminal Olefins,” Journal of the American Chemical Society, 131(46):16630-16631, 2009. |
| Johnson Matthey PLC, The Catalyst Technical Handbook, West Deptford, NJ, USA, 94 pages, 2005. |
| Johnson Matthey PLC, The Catalyst Technical Handbook, Platinum Metals Review, 45(3), 110, 2001. |
| Jones, D. and Howe, R., “Microbiological oxidation of long-chain aliphatic compounds. I. Alkanes and 1-alkenes,” J. Chem. Soc. C, 2801-2808, 1968. |
| Jones, N. et al., “Engineering the selectivity of aliphatic C—H bond oxidation catalyzed by cytochrome P450cam,” Chem. Commun., 2413-2414, 1996. |
| Jordann, M. et al., “Experimental and DFT investigation of the 1-octene metathesis reaction mechanism with the Grubbs 1 precatalyst,” J. Mol. Catal. A: Chem., 254:145-154, 2006. |
| Julsing, M. et al., “Outer Membrane Protein AlkL Boosts Biocatalytic Oxyfunctionalization of Hydrophobic Substrates in Escherichia coli,” Appl. Environ. Microbiol., 78(16):5724-5733, 2012. |
| Kaehne, F. et al., “A recombinant α-dioxygenase from rice to produce fatty aldehydes using E. coli,” Appl Microbiol Biotechnol, 90:989-995, 2011. |
| Kajikawa, T. et al., “Olefin metathesis in carotenoid synthesis,” Org. Biomol. Chem., 7(22):4586-4589, 2009. |
| Karimi, B. et al., “Green, transition-metal-free aerobic oxidation of alcohols using a highly durable supported organocatalyst,” Angewandte Chemie, International Edition, 46(38):7210-7213, 2007. |
| Karra-Chaabouni M. et al., “Biooxidation of n-Hexanol by Alcohol Oxidase and Catalase in Biphasic and Micellar Systems Without Solvent,” Biotechnol Bioeng, 81:27-32, 2003. |
| Katopodis, A. et al., “Mechanistic studies on non-heme iron monooxygenase catalysis: epoxidation, aldehyde formation and demethylation by the ω-hydroxylation system of Pseudomonas oleovorans,” J. Am. Chem. Soc., 106:7928-7935, 1984. |
| Kedziora, K. et al., “Laccase/TEMPO-mediated system for the thermodynamically disfavored oxidation of 2,2-dihalo-l-phenylethanol derivatives,” Green Chemistry, 16(5):2448-2453, 2014. |
| Keitz, B. et al., “Cis-selective ring-opening metathesis polymerization with ruthenium catalysts,” J Am Chem Soc, 134(4):2040-2043, 2012. |
| Keitz, B. et al., “Improved ruthenium catalysts for Z-selective olefin metathesis,” J Am Chem Soc, 134(1):693-699, 2012. |
| Kemp, G. et al., “Activity and substrate-specificity of the fatty alcohol oxidase of candida-tropicalis in organic-solvents,” Appl Microbiol Biotechnol, 34:441-445, 1991. |
| Kemp, G. et al., “Inducible long-chain alcohol oxidase from alkane-grown candida-tropicalis,” Appl Microbiol Biotechnol, 29:370-374, 1988. |
| Kemp, G. et al., “Light sensitivity of the n-alkane-induced fatty alcohol oxidase from Candida tropicalis and Yarrowia lipolytica,” Appl Microbiol Biotechnol, 32:461-464, 1990. |
| Khan, R. et al., “Readily accessible and easily modifiable Ru-based catalysts for efficient and Z-selective ring-opening metathesis polymerization and ring-opening/cross-metathesis,” J Am Chem Soc, 135:10258-10261, 2013. |
| Kille, S. et al., “Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution,” Nat Chem, 3:738-743, 2011. |
| Kim, D. et al., “Functional expression and characterization of cytochrome P450 52A21 from Candida albicans,” Arch. Biochem. Biophys., 464:213-220, 2007. |
| Kirmair, L. and Skerra, A., “Biochemical Analysis of Recombinant AlkJ from Pseudomonas putida Reveals a Membrane-Associated, Flavin Adenine Dinucleotide-Dependent Dehydrogenase Suitable for the Biosynthetic Production of Aliphatic Aldehydes,” Appl. Environ. Microbiol., 80(8):2468-2477, 2014. |
| Koch, D. et al., “In vivo evolution of butane oxidation by terminal alkane hydroxylases AlkB and CYP153A6,” Appl. Environ. Microbiol., 75(2):337-344, 2009. |
| Kotani, H. et al., “Photocatalytic Generation of a Non-Heme Oxoiron(IV) Complex with Water as an Oxygen Source,” J. Am. Chem. Soc., 133:3249-3251, 2011. |
| Krebs, C. et al., “Non-Heme Fe(IV)-Oxo Intermediates,” Acc. Chem. Res., 40:484-492, 2007. |
| Kubo, T. et al., “Enantioselective epoxidation of terminal alkenes to (R)- and (S)-epoxides by engineered cytochromes P450 BM-3,” Chem. Eur. J., 12:1216-1220, 2006. |
| Kubota, M. et al., “Isolation and functional analysis of cytochrome P450 CYP153A genes from various environments,” Biosci Biotechnol Biochem, 69(12):2421-2430, 2005. |
| Kumar A. and Goswami, P., “Functional characterization of alcohol oxidases from Aspergillus terreus MTCC 6324,” Appl Microbiol Biotechnol, 72:906-911, 2006. |
| Labinger, J., “Selective alkane oxidation: hot and cold approaches to a hot problem,” J. Mol. Catal. A Chem., 220:27-35, 2004. |
| Lewis, J. and Arnold, F., “Catalysts on demand. selective oxidations by laboratory-evolved cytochrome P450 BM3,” Chimia (Aarau), 63(6):309-312, 2009. |
| Lewis, J. et al., “Combinatorial Alanine Substitution Enables Rapid Optimization of Cytochrome P450BM3 for Selective Hydroxylation of Large Substrates,” Chembiochem, 11:2502-2505, 2010. |
| Li, L. et al., “Crystal Structure of Long-Chain Alkane Monooxygenase (LadA) in Complex with Coenzyme FMN: Unveiling the Long-Chain Alkane Hydroxylase,” J. Mol. Biol., 376:453-465, 2008. |
| Lipscomb, J. et al., “Methane monooxygenase and compound Q: lessons in oxygen activation,” Int. Congr. Ser., 1233:205-212, 2002. |
| Liu, S. et al., “Optimal pH control strategy for high-level production of long-chain α,ω-dicarboxylic acid by Candida tropicalis,” Enzyme Microb. Technol., 34:73-77, 2004. |
| Liu, X. et al., “Two novel metal-independent long-chain alkyl alcohol dehydrogenases from Geobacillus thermodenitrificans NG80-2,” Microbiology, 155:2078-2085, 2009. |
| Liu, Y. et al., “Isolation of an alkane-degrading Alcanivorax sp strain 2B5 and cloning of the alkB gene,” Bioresour. Technol., 101:310-316, 2010. |
| Lo Piccolo, L. et al., “Involvement of an Alkane Hydroxylase System of Gordonia sp Strain SoCg in Degradation of Solid n-Alkanes,” Appl. Environ. Microbiol., 77(4):1204-1213, 2011. |
| Lu, W. et al., “Biosynthesis of Monomers for Plastics from Renewable Oils,” J. Am. Chem. Soc., 132:15451-15455, 2010. |
| Lucio Anelli, P. et al., “Fast and selective oxidation of primary alcohols to aldehydes or to carboxylic acids and of secondary alcohols to ketones mediated by oxoammonium salts under two-phase conditions,” Journal of Organic Chemistry, 52(12):2559-2562, 1987. |
| Malca, S. et al., “Bacterial CYP153A monooxygenases for the synthesis of omega-hydroxylated fatty acids,” Chemical Communications, 48:5115-5117, 2012. |
| Malca, S., “Substrate characterization and protein engineering of bacterial cytochrome P450 monooxygenases for the bio-based synthesis of omega-hydroxylated aliphatic compounds,” Institute of Technical Biochemistry at the University of Stuttgart (University of Stuttgart, 2013). |
| Marinescu, S. et al., “Isolation of pure disubstituted E olefins through Mo-catalyzed Z-selective ethenolysis of stereoisomeric mixtures,” Journal of the American Chemical Society, 133(30):11512-4, 2011. |
| Martinez, C. and Rupashinghe, S., “Cytochrome P450 bioreactors in the pharmaceutical industry: challenges and opportunities,” Curr Top Med Chem, 13:1470-1490, 2013. |
| Marx, V. et al., “Stereoselective access to Z and E macrocycles by ruthenium-catalyzed Z-selective ring-closing metathesis and ethenolysis,” J Am Chem Soc, 135(1): 94-97, 2013. |
| Marzorati, M. et al., “Selective laccase-mediated oxidation of sugars derivatives,” Green Chemistry, 7(5):310-315, 2005. |
| Masuda, H. et al., “Characterization of three propane-inducible oxygenases in Mycobacterium sp. strain ENV421,” Lett. Appl. Microbiol., 55:175-181, 2012. |
| Mathys, R. et al., “Integrated two-liquid phase bioconversion and product-recovery processes for the oxidation of alkanes: process design and economic evaluation,” Biotechnol Bioeng, 64:459-477, 1999. |
| Matsuyama, H. et al., “A new n-alkane oxidation system from Pseudomonas aeruginosa S7B1,” Agric. Biol. Chem., 45(1):9-14, 1981. |
| Mauersberger, S. et al., “Substrate-specificity and stereo selectivity of fatty alcohol oxidase from the yeast candida-maltosa,” Appl. Microbiol. Biotechnol., 37:66-73, 1992. |
| Maurer, S. et al., “Catalytic hydroxylation in biphasic systems using CYP102A1 mutants,” Adv. Synth. Catal., 347:1090-1098, 2005. |
| May, S. and Schwartz, R., “Stereoselective epoxidation of octadiene catalyzed by an enzyme system of Pseudomonas oleovorans,” J. Am. Chem. Soc., 96:4031-4032, 1974. |
| Mayoral, J. et al., “NADP(+)-dependent farnesol dehydrogenase, a corpora allata enzyme involved in juvenile hormone synthesis,” Proc. Natl. Acad. Sci. U. S. A., 106:21091-21096, 2009. |
| Meinhold, P. et al., “Engineering cytochrome P450 BM3 for terminal alkane hydroxylation,” Adv. Synth. Catal., 348:763-772, 2006. |
| Moreau, R. and Huang, A., “Oxidation of fatty alcohol in the cotyledons of jojoba seedlings,” Arch Biochem Biophys, 194(2):422-430, 1979. |
| Mori, K., “Synthesis of all the six components of the female-produced contact sex pheromone of the German cockroach, Blattella germanica (L.),” Tetrahedron, 64:4060-4071, 2008. |
| Munzer, D. et al., “Stereoselective hydroxylation of an achiral cyclopentanecarboxylic acid derivative using engineered P450s BM-3,” Chem. Commun., 2597-2599, 2005. |
| Murray, W. and Duff, S., “Biooxidation of aliphatic and aromatic high-molecular-weight alcohols by pichia-pastoris alcohol oxidase,” Appl. Microbiol. Biotechnol., 33:202-205, 1990. |
| Nguyen, H. et al., “Metabolic Engineering of Seeds Can Achieve Levels of ω-7 Fatty Acids Comparable with the Highest Levels Found in Natural Plant Sources,” Plant Physiology, 154(4):1897-1904, 2010. |
| Nie, Y. et al., “Diverse alkane hydroxylase genes in microorganisms and environments,” Sci. Rep., 4(4968) :1-11, 2014. |
| Nodate, M. et al., “Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp NCIMB 9784,” Appl. Microbiol.Biotechnol., 71:455-462, 2006. |
| Olaofe, O. et al., “The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6,” Microb. Cell Fact., 12:8, 12 pages, 2013. |
| Oprean, I. et al., “Synthesis of cis-7,8-epoxyoctadecane, species-specific component of the sex pheromone of nun moth Lymantria monacha (Lepidoptera, Limantriidae),” Stud Univ Babes-Bolyai, Chem, 51:33-38, 2006. |
| Ozimek, P. et al., “Alcohol oxidase: A complex peroxisomal, oligomeric flavoprotein,” FEMS Yeast Res, 5:975-983, 2005. |
| Pagliaro, M. et al., “Ru-based oxidation catalysis,” Chemical Society Reviews, 34(10):837-845, 2005. |
| Pederson, R. et al., “Applications of olefin cross metathesis to commercial products,” Advanced Synthesis & Catalysis, 344(6-7):728-735,2002. |
| Peryshkov, D. et al., “Z-Selective olefin metathesis reactions promoted by tungsten oxo alkylidene complexes,” Journal of the American Chemical Society, 133(51):20754-7, 2011. |
| Peter, S. et al., “Selective hydroxylation of alkanes by an extracellular fungal peroxygenase,” FEBS J., 278(19):3667-3675, 2011. |
| Peters, M. et al., “Regio- and Enantioselective Alkane Hydroxylation with Engineered Cytochromes P450 BM-3,” J. Am. Chem. Soc., 125:13442-13450, 2003. |
| Pflug, S. et al., “Development of a fed-batch process for the production of the cytochrome P450 monooxygenase CYP102A1 from Bacillus megaterium in E. coli,” J Biotechnol, 129:481-488, 2007. |
| Pham, S. et al., “Evolving P450pyr hydroxylase for highly enantioselective hydroxylation at non-activated carbon atom,” Chem. Commun., 48:4618-4620, 2012. |
| Presecki, A. et al., “Coenzyme Regeneration in Hexanol Oxidation Catalyzed by Alcohol Dehydrogenase,” Appl. Biochem. Biotechnol., 167:595-611, 2012. |
| Pribisko, M. et al., “Z-Selective ruthenium metathesis catalysts: Comparison of nitrate and nitrite X-type ligands,” Polyhedron Ahead of Print. doi: 10.1016/j.poly.2014.06.055, Published in final edited form as: Polyhedron. Dec. 14, 2014; 84: 144-149. |
| Qian, W. et al., “Clean and selective oxidation of alcohols catalyzed by ion-supported TEMPO in water,” Tetrahedron, 62(4):556-562,2006. |
| Quigley, B. and Grubbs, R., “Ruthenium-catalysed Z-selective cross metathesis of allylic-substituted olefins,” Chemical Science, 5(2):501-506, 2014. |
| Rickert, A. et al., “Enzymatic allylic oxidations with a lyophilisate of the edible fungus Pleurotus sapidus,” Green Chem., 14:639-644, 2012. |
| Roiban, G. et al., “Stereo- and regioselectivity in the P450-catalyzed oxidative tandem difunctionalization of 1-methylcyclohexene,” Tetrahedron, 69:5306-5311, 2013. |
| Rosebrugh, L. et al., “Highly active ruthenium metathesis catalysts exhibiting unprecedented activity and Z-selectivity,” J Am Chem Soc, 135(4):1276-1279, 2013. |
| Rui, Z. et al., “Microbial biosynthesis of medium-chain 1-alkenes by a nonheme iron oxidase,” Proc. Natl. Acad. Sci. U. S. A., 111(51):18237-18242, 2014. |
| Ryland, B. and Stahl, S., “Practical Aerobic Oxidations of Alcohols and AMINES with Homogeneous Copper/TEMPO and Related Catalyst Systems,” Angew Chemie Int Ed, 53:8824-8838, 2014. |
| Sarmah, P. et al., “Copper(II) catalysed oxidation of alcohols in aqueous medium,” Indian Journal of Chemistry, Section A: Inorganic, Bio-Inorganic, Physical, Theoretical & Analytical Chemistry, 48A(5):637-644, 2009. |
| Sato, S. et al., “Selective Dehydration of Alkanediols into Unsaturated Alcohols over Rare Earth Oxide Catalysts,” ACS Catal, 3:721-734, 2013. |
| Savitha, J. and Ratledge, C., “Alcohol oxidase of Aspergillus flavipes grown on hexadecanol,” FEMS Microbiol Lett, 80:221-224, 1991. |
| Scheller, U. et al., “Characterization of the n-alkane and fatty acid hydroxylating cytochrome P450 forms 52A3 and 52A4,” Arch. Biochem. Biophys., 328(2):245-254, 1996. |
| Scheller, U. et al., “Generation of the soluble and functional cytosolic domain of microsomal cytochrome P450 52A3,” J. Biol. Chem., 269(17):12779-12783, 1994. |
| Scheller, U. et al., “Oxygenation cascade in conversion of n-alkanes to α,ω-dioic acids catalyzed by cytochrome p450 52A3,” J. Biol. Chem., 273(49):32528-32534, 1998. |
| Scheps, D. et al., “Regioselective ω-hydroxylation of medium-chain n-alkanes and primary alcohols by CYP153 enzymes from Mycobacterium marinum and Polaromonas sp strain J5666,” Org. Biomol. Chem., 9:6727-6733, 2011. |
| Scheps, D. et al., “Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct,” Microb. Biotechnol., 6(6):694-707, 2013. |
| Scheps, D., “Cytochrome P450 monooxygenases: a study of the synthesis of industrial relevant aliphatic ω-hydroxy products,” Institute of Technical Biochemistry at the University of Stuttgart, 130 pages, 2013. |
| Schrewe, M. et al., “Kinetic Analysis of Terminal and Unactivated C—H Bond Oxyfunctionalization in Fatty Acid Methyl Esters by Monooxygenase-Based Whole-Cell Biocatalysis,” Advanced Synthesis & Catalysis, 353:3485-3495, 2011. |
| Schrewe, M. et al., “Whole-cell biocatalysis for selective and productive C—O functional group introduction and modification,” Chem. Soc. Rev., 42:6346-6377, 2013. |
| Schroer, K. et al., “Continuous asymmetric ketone reduction processes with recombinant Escherichia coli,” J. Biotechnol., 132:438-444, 2007. |
| Seghezzi, W. et al., “Characterization of a second alkane-inducible cytochrome P450-encoding gene, CYP52A2, from Candida tropicalis,” Gene, 106:51-60, 1991. |
| Seghezzi, W. et al., “Identification and characterization of additional members of the cytochrome-p450 multigene family CYP52 of Candida-tropicalis,” DNA Cell Biol., 11(10):767-780, 1992. |
| Shahane, S. et al., “Z Selectivity: Recent Advances in one of the Current Major Challenges of Olefin Metathesis,” ChemCatChem, 5:3436-3459, 2013. |
| Smith, A. et al., “Evolution of a Gram-Scale Synthesis of (+)-Discodermolide,” J Am Chem Soc, 122:8654-8664, 2000. |
| Smits, T. et al., “Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria,” J. Bacteriol., 184(6):1733-42, 2002. |
| Smits, T. et al., “Functional characterization of genes involved in alkane oxidation by Pseudomonas aeruginosa,” Antonie Van Leeuwenhoek, 84:193-200, 2003. |
| Smits, T. et al., “New alkane-responsive expression vectors for Escherichia coli and Pseudomonas,” Plasmid, 46:16-24, 2001. |
| Sugimoto, K. et al “Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode of plant odor reception and defense,” Proc Natl Acad Sci, 111(19):7144-7149, 2014. |
| Takai, H. et al., “Construction and characterization of a Yarrowia lipolytica mutant lacking genes encoding cytochromes P450 subfamily 52,” Fungal Genet. Biol., 49:58-64, 2012. |
| Tani, A. et al., “Thermostable NADP(+)-dependent medium-chain alcohol dehydrogenase from Acinetobacter sp strain M-1: Purification and characterization and gene expression in Escherichia coli,” Appl Environ Microbiol, 66(12):5231-5235, 2000. |
| Thevenieau, F. et al., “Characterization of Yarrowia lipolytica mutants affected in hydrophobic substrate utilization,” Fungal Genet. Biol., 44:531-542, 2007. |
| Townsend, E. et al., “Z-selective metathesis homocoupling of 1,3-dienes by molybdenum and tungsten monoaryloxide pyrrolide (MAP) complexes,” Journal of the American Chemical Society, 134(28):11334-7, 2012. |
| Uchio, R. and Shiio, I., “Tetradecane-1,14-dicarboxylic acid production from n-hexadecane by Candida cloacae” Agric. Biol. Chem., 36(8):1389-1397, 1972. |
| Urlacher, V. et al., “Biotransformation of β-ionone by engineered cytochrome P450 BM-3,” Appl Microbiol Biotechnol, 70:53-59, 2006. |
| Urlacher, V. et al., “Microbial P450 enzymes in biotechnology,” Appl. Microbiol. Biotechnol., 64:317-325, 2004. |
| Van Beilen, J. and Funhoff, E., “Alkane hydroxylases involved in microbial alkane degradation,” Appl. Microbiol. Biotechnol., 74:13-21, 2007. |
| Van Beilen, J. and Funhoff, E., “Expanding the alkane oxygenase toolbox: new enzymes and applications,” Curr. Opin. Biotechnol., 16:308-314, 2005. |
| Van Beilen, J. et al., “Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis,” Environ. Microbiol., 6(3):264-273, 2004. |
| Van Beilen, J. et al., “Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases,” Appl. Environ. Microbiol., 72(1):59-65, 2006. |
| Van Beilen, J. et al., “Diversity of alkane hydroxylase systems in the environment,” Oil Gas Sci. Technol, 58(4):427-440, 2003. |
| Van Beilen, J. et al., “Identification of an amino acid position that determines the substrate range of integral membrane alkane hydroxylases,” J. Bacteriol., 187(1):85-91, 2005. |
| Van Beilen, J. et al., “Practical issues in the application of oxygenases,” Trends Biotechnol., 21(4):170-177, 2003. |
| Van Beilen, J. et al., “Substrate-specificity of the alkane hydroxylase system of Pseudomonas-oleovorans GPO1,” Enzyme Microb. Technol., 16:904-911, 1994. |
| Van Der Gryp, P. et al., “Experimental, DFT and kinetic study of 1-octene metathesis with Hoveyda-Grubbs second generation precatalyst,” Journal of Molecular Catalysis A: Chemical, 355:85-95, 2012. |
| Van Der Klei, I. et al., “Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeasts: a review,” Yeast, 7:195-209, 1991. |
| Vangnai, A. and Arp, D., “An inducible 1-butanol dehydrogenase, a quinohaemoprotein, is involved in the oxidation of butane by ‘Pseudomonas butanovora,’” Microbiology, 147:745-756, 2001. |
| Vanhanen, S. et al., “A consensus sequence for long-chain fatty-acid alcohol oxidases from Candida identifies a family of genes involved in lipid ω-oxidation in yeast with homologues in plants and bacteria,” J Biol Chem, 275(6):4445-4452, 2000. |
| Wang, C. et al., “Mo-Based Complexes with Two Aryloxides and a Pentafluoroimido Ligand Catalysts for Efficient Z-Selective Synthesis of a Macrocyclic Trisubstituted Alkene by Ring-Closing Metathesis,” Angewandte Chemie International Edition, 52(7):1939-1943, 2013. |
| Wang, L. et al., “Gene diversity of CYP153A and AlkB alkane hydroxylases in oil-degrading bacteria isolated from the Atlantic Ocean,” Env. Microbiol, 12(5):1230-1242, 2010. |
| Weissbart, D. et al., “Regioselectivity of a plant lauric acid omega hydroxylase. Omega hydroxylation of cis and trans unsaturated lauric acid analogs and epoxygenation of the terminal olefin by plant cytochrome P-450,” Biochimica et Biophysica Acta, 1124:135-142, 1992. |
| Wentzel, A. et al., “Bacterial metabolism of long-chain n-alkanes,” Appl. Microbiol. Biotechnol., 76:1209-1221, 2007. |
| Whitehouse, C. et al., “P450BM3 (CYP102A1): connecting the dots,” Chem. Soc. Rev., 41:1218-1260, 2012. |
| Youngquist, J. et al., “Production of medium chain length fatty alcohols from glucose in Escherichia coli,” Metab. Eng., 20:177-186, 2013. |
| Yu, A. et al., “Production of Fatty Acid-Derived Valuable Chemicals in Synthetic Microbes,” Front. Bioeng. Biotechnol., 2(78):1-12, 2014. |
| Zampolli, J. et al., “Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism,” AMB Express, 4:73, 9 pages, 2014. |
| Zehentgruber, D. et al., “Studies on the enantioselective oxidation of β-ionone with a whole E. coli system expressing cytochrome P450 monooxygenase BM3,” J. Mol. Catal. B Enzym., 84:62-64, 2012. |
| Zhang, H. et al., “Preparation of Macrocyclic Z Enoates and (E,Z)- or (Z,E) Dienoates through Catalytic Stereoselective Ring-Closing Metathesis,” Journal of the American Chemical Society, 136(47):16493-16496, 2014. |
| Zhao, S. et al., “Cloning and characterization of long-chain fatty alcohol oxidase LjFAO1 in Lotus japonicas,” Biotechnol. Prog., 24:773-779, 2008. |
| Zimmer, T. et al., “The CYP52 multigene family of Candida maltosa encodes functionally diverse n-alkane-inducible cytochromes P450,” Biochem. Biophys. Res. Commun., 224:784-789, 1996. |
| Zotova, N. et al., “Catalysis in flow: the practical and selective aerobic oxidation of alcohols to aldehydes and ketones,” Green Chemistry, 12(12):2157-2163, 2010. |
| Number | Date | Country | |
|---|---|---|---|
| 20160108436 A1 | Apr 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61994662 | May 2014 | US | |
| 62060469 | Oct 2014 | US | |
| 62062758 | Oct 2014 | US | |
| 62082555 | Nov 2014 | US | |
| 62096417 | Dec 2014 | US | |
| 62096429 | Dec 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2015/031219 | May 2015 | US |
| Child | 14845212 | US |
