Patent Application
20220170026
Tropane alkaloids (TAs) are a class of anticholinergic secondary metabolites produced by plants of the nightshade family (Solanaceae). Several TAs, including atropine, hyoscyamine, and scopolamine, are classified as essential medicines by the World Health Organization for the treatment of diverse neurological disorders such as organophosphate and nerve agent poisoning, gastrointestinal spasms, and cardiac arrhythmia, as well as to control symptoms of Parkinson's disease. As such, an adequate and consistent supply of these TA molecules so that they are available to researchers and physicians is of interest. Current supply chains for medicinal TAs rely on extraction from unsustainable and geographically restricted plant monocultures, in which TAs accumulate to only 0.2-4% dry weight, and which are susceptible to pests, changes in land use, and climate. No total chemical syntheses for TAs from simple feedstocks have yet proven sufficiently economical for industrial use due to difficulties arising from TA stereochemistry. Moreover, poor economies of scale and long generation times have thus far rendered the engineering of transgenic plants or plant cultures with improved TA production an unviable strategy for sourcing these compounds. As such, methods for preparing TAs are of interest.
This invention includes non-plant organisms engineered for the production of diverse tropane alkaloids (TAs) from precursors and sugar. For example, included in this invention are engineered microbial strains for the production of medicinal TAs, which are hereby defined as naturally occurring TAs with established uses in current medical practice, including hyoscyamine, atropine, anisodamine, and scopolamine, and precursors and derivatives thereof. Also included in this invention are engineered microbial strains for the production of non-medicinal TAs, which are hereby defined as naturally occurring TAs without established uses in current medical practice but which may possess bioactivities of medicinal interest, including calystegines, cocaine, and precursors and derivatives thereof. This invention further includes engineered microbial strains for the production of non-natural TAs, which are hereby defined as TAs not produced by unmodified organisms, such as TAs produced via esterification of acyl donor and acyl acceptor compounds which are not esterified in naturally-occurring organisms, including derivatives of medicinal TAs and derivatives of non-medicinal TAs. An example of the schemes included in this invention are detailed in
The invention encompasses methods of producing pseudotropine and alkaloids derived from pseudotropine, for example calystegines, using microorganisms engineered to express at least one heterologous enzyme as microbial catalysts. This invention further includes methods of producing diverse compounds which can be used as acyl donors for the biosynthesis of TA scaffolds using microorganisms engineered to express at least one heterologous enzyme as microbial catalysts. This invention also includes methods of esterifying acyl donors and acceptors for the production of TA scaffolds using microorganisms engineered to express at least one heterologous enzyme as microbial catalysts. The invention further includes methods of modifying and culturing engineered microbial strains for the production of medicinal TAs such as hyoscyamine and scopolamine, non-medicinal TAs such as calystegines, and non-natural TAs such as those derived from esterification of tropine with acyl donor compounds other than 3-phenyllactic acid (PLA).
Host cells that are engineered to produce tropane alkaloids (TAs) that are of interest, such as hyoscyamine and scopolamine, are provided. TAs of interest may include TA precursors, TAs, and modifications of TAs, including derivatives of TAs. The host cells may have one or more modifications selected from: a feedback inhibition alleviating mutation in an enzyme gene; a transcriptional modulation modification of a biosynthetic enzyme gene; an inactivating mutation in an enzyme; and a heterologous coding sequence. Also provided are methods of producing a TA of interest using the host cells and compositions, e.g., kits, systems etc., that find use in methods of the invention.
An aspect of the invention provides a method for forming a product stream having a tropane alkaloid (TA) product. The method comprises providing engineered non-plant cells and a feedstock including nutrients and water to a batch reactor, which engineered non-plant cells have at least one modification selected from the group consisting of: a feedback inhibition alleviating mutation in a biosynthetic enzyme gene native to the cell; a transcriptional modulation modification of a biosynthetic enzyme gene native to the cell; and an inactivating mutation in an enzyme native to the cell. Additionally, the method comprises, in the batch reactor, subjecting the engineered non-plant cells to fermentation by incubating the engineered non-plant cells for a time period of at least about 5 minutes to produce a solution comprising the TA product and cellular material. The method also comprises using at least one separation unit to separate the TA product from the cellular material to provide said product stream comprising the TA product.
In another aspect, the invention provides a method for forming a product stream having a TA product. The method comprises providing engineered non-plant cells and a feedstock including nutrients and water to a reactor. The method also comprises, in the reactor, subjecting the engineered non-plant cells to fermentation by incubating the engineered yeast cells for a time period of at least about 5 minutes (e.g., 5 minutes or longer) to produce a solution comprising cellular material and the TA product. Additionally, the method comprises using at least one separation unit to separate the TA product from the cellular material to provide the product stream comprising the TA product.
Another aspect of the invention provides an engineered non-plant cell that produces a tropane alkaloid (TA) product, the engineered non-plant cell having at least one modification selected from the group consisting of: a feedback inhibition alleviating mutation in a biosynthetic enzyme gene native to the cell; a transcriptional modulation modification of a biosynthetic enzyme gene native to the cell; and an inactivating mutation in an enzyme native to the cell. The engineered non-plant cell comprises at least one heterologous coding sequence encoding at least one enzyme that is selected from the group of arginine decarboxylase, agmatine ureohydrolase, agmatinase, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidine ketide synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylpyruvate reductase, 3-phenyllactic acid UDP-glucosyltransferase 84A27, littorine synthase, littorine mutase, hyoscyamine dehydrogenase, hyoscyamine 6β-hydroxylase/dioxygenase, and cocaine synthase. In some examples, the engineered non-plant cell comprises a plurality of heterologous coding sequences encoding an enzyme that is selected from the group of arginine decarboxylase, agmatine ureohydrolase, agmatinase, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidine ketide synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylpyruvate reductase, 3-phenyllactic acid UDP-glucosyltransferase 84A27, littorine synthase, littorine mutase, hyoscyamine dehydrogenase, hyoscyamine 6β-hydroxylase/dioxygenase, and cocaine synthase. In some examples, the heterologous coding sequences may be operably connected. Heterologous coding sequences that are operably connected may be within the same pathway of producing a particular tropane alkaloid product. In some examples, the engineered non-plant cell comprises one or more modifications to intracellular compartmentalization that is selected from the group including, but not limited to, modified intracellular trafficking of enzymes, modified intracellular localization of enzymes, and modified intracellular transport of metabolites.
In another aspect of the invention, a therapeutic agent is provided. The therapeutic agent comprises a tropane alkaloid product.
The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.
Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the description.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference.
It is noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. For example, the term “a primer” refers to one or more primers, i.e., a single primer and multiple primers. It is further noted that the claims are drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
As used herein, the term “polypeptide” refers to a polymeric form of amino acids of any length, including peptides that range from 2-50 amino acids in length and polypeptides that are greater than 50 amino acids in length. The terms “polypeptide” and “protein” are used interchangeably herein. The term “polypeptide” includes polymers of coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones in which the conventional backbone has been replaced with non-naturally occurring or synthetic backbones. A polypeptide may be of any convenient length, e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, such as up to 500 or 1000 or more amino acids. “Peptides” may be 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, such as up to 50 amino acids. In some embodiments, peptides are between 5 and 30 amino acids in length.
As used herein the term “isolated,” refers to an moiety of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the moiety is associated with prior to purification.
As used herein, the term “encoded by” refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of 3 or more amino acids, such as 5 or more, 8 or more, 10 or more, 15 or more, or 20 or more amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed by the term are polypeptide sequences that are immunologically identifiable with a polypeptide encoded by the sequence.
A “vector” is capable of transferring gene sequences to target cells. As used herein, the terms, “vector construct,” “expression vector,” and “gene transfer vector,” are used interchangeably to mean any nucleic acid construct capable of directing the expression of a gene of interest and which may transfer gene sequences to target cells, which is accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors.
An “expression cassette” includes any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Such cassette is constructed into a “vector,” “vector construct,” “expression vector,” or “gene transfer vector,” in order to transfer the expression cassette into target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.
A “plurality” contains at least 2 members. In certain cases, a plurality may have 10 or more, such as 100 or more, 1000 or more, 10,000 or more, 100,000 or more, 106 or more, 107 or more, 108 or more, or 109 or more members. In any embodiments, a plurality can have 2-20 members.
The term “tropane alkaloid product” is intended to refer to any molecule whose skeleton contains an 8-azabicyclo[3.2.1]octane core group comprising a cycloheptane ring and a nitrogen bridge connecting carbon atoms 1 and 5, wherein the 8-azabicyclo[3.2.1]octanyl group is covalently bonded to an acyl group by means of an ester linkage at the 3 position, and/or wherein the 8-azabicyclo[3.2.1]octanyl group is functionalized with a hydroxyl group at the 3 position and one or more hydroxyl groups at the 2, 4, 5, 6, and/or 7 positions. Tropane alkaloid products include, but are not limited to, littorine, hyoscyamine, atropine, anisodamine, scopolamine, cocaine, and any other similar tropine/pseudotropine+acyl group natural or non-natural tropane alkaloids (e.g., calystegines).
The term “precursor of a tropane alkaloid product” is intended to refer to any molecule that can be biosynthesized by an organism from a carbon source and a nitrogen source and which can be converted to a tropane alkaloid product in one or more (e.g., one or two) biosynthetic steps; wherein the carbon source is a carbohydrate, a non-carbohydrate sugar, a sugar alcohol, a lipid, a fatty acid, or a substrate which is converted to one or more of the above carbon sources through a metabolic pathway; and wherein the nitrogen source is ammonia, urea, nitrate, nitrite, any amino acid excluding glutamic acid, arginine, ornithine, and citrulline, a peptide, a protein, or any substrate which is converted to one or more of the above nitrogen sources through a metabolic pathway.
The term “derivative of a tropane alkaloid product” is intended to refer to any molecule not naturally produced by an unmodified organism, wherein the skeleton of the molecule comprises a tropane alkaloid product and which differs from said tropane alkaloid product by the attachment of functional groups without modification of the skeleton itself. As used herein, attachment of functional groups includes, but is not limited to, hydroxylation, alkylation and N-alkylation, acetylation and N-acetylation, acylation and N-acylation, and halogenation.
Numeric ranges are inclusive of the numbers defining the range.
The methods described herein include multiple steps. Each step may be performed after a predetermined amount of time has elapsed between steps, as desired. As such, the time between performing each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, and including 5 hours or more. In certain embodiments, each subsequent step is performed immediately after completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting time after completion of the previous step, e.g., a few minutes to an overnight waiting time.
Other definitions of terms may appear throughout the specification.
Host cells that are engineered to produce tropane alkaloids (TAs) that are of interest, such as hyoscyamine and scopolamine, are provided. The host cells may have one or more engineered modifications selected from: a feedback inhibition alleviating mutation in an enzyme gene; a transcriptional modulation modification of a biosynthetic enzyme gene; an inactivating mutation in an enzyme; and a heterologous coding sequence. Also provided are methods of producing a TA of interest using the host cells and compositions, e.g., kits, systems etc., that find use in methods of the invention.
Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method is carried out in the order of events recited or in any other order which is logically possible.
In further describing the subject invention, TA precursors of interest, TAs, and modifications of TAs, including derivatives of TAs, are described first in greater detail, followed by host cells for producing the same. Next, methods of interest in which the host cells find use are reviewed. Kits that may be used in practicing methods of the invention are also described.
As summarized above, host cells which produce tropane alkaloid precursors (TA precursors) are provided. The TA precursor may be any intermediate or precursor compound in a synthetic pathway (e.g., as described herein) that leads to the production of a TA of interest (e.g., as described herein). In some cases, the TA precursor has a structure that may be characterized as a TA or a derivative thereof. In certain cases, the TA precursor has a structure that may be characterized as a fragment of a TA. In some cases, the TA precursor is an early TA. As used herein, by “early TA” is meant an early intermediate in the synthesis of a TA of interest in a cell, where the early TA is produced by a host cell from a host cell feedstock or simple starting compound. In some cases, the early TA is a TA intermediate that is produced by the subject host cell solely from a host cell feedstock (e.g., a carbon and nutrient source) without the need for addition of a starting compound to the cells. The term early TA may refer to a precursor of a TA end product of interest whether or not the early TA may itself be characterized as a tropane alkaloid.
In some cases, the TA precursor is an early TA, such as a pre-tropine tropane alkaloid or a pre-littorine tropane alkaloid. As such, host cells which produce pre-tropine tropane alkaloids (pre-tropine TAs) and pre-littorine tropane alkaloids (pre-littorine TAs) are provided. Tropine is a major branch point intermediate of interest in the synthesis of downstream TAs via cell engineering efforts to produce end products such as medicinal TA products derived from littorine (
As used herein, the terms “pre-esterification tropane alkaloid”, “pre-esterification TA”, and “pre-esterification TA precursor” are used interchangeably and refer to a biosynthetic precursor of littorine, cinnamoyltropine, or other product of acyl donor and acyl acceptor esterification, whether or not the structure of the esterification precursor itself is characterized as a tropane alkaloid. The term pre-esterification TA is meant to include biosynthetic precursors, intermediates and metabolites thereof, of any convenient member of a host cell biosynthetic pathway that may lead to esterification products such as littorine. In some cases, the pre-esterification TA includes a tropane alkaloid fragment, such as a tropine fragment, a phenylpropanoid fragment or a precursor or derivative thereof. In certain instances, the pre-esterification TA has a structure that may be characterized as a tropane alkaloid or a derivative thereof.
TA precursors of interest include, but are not limited to, tropine and phenyllactic acid (PLA), as well as tropine and PLA precursors, such as arginine, ornithine, agmatine, N-carbamoylputrescine (NCP), putrescine, N-methylputrescine (NMP), 4-methylaminobutanal, N-methylpyrrolinium (NMPy), 4-(1-methyl-2-pyrrodinyl)-3-oxobutanoic acid (MPOB), tropinone, phenylalanine, prephenic acid, and phenylpyruvic acid (PPA). In some embodiments, the one or more TA precursors are tropine and PLA. In certain instances, the one or more TA precursors are tropine and a phenylpropanoid carboxylic acid other than PLA, such as cinnamic acid.
Synthetic pathways to a TA precursor may be generated in the host cells, and may start with any convenient starting compound(s) or materials.
As summarized above, host cells which produce tropane alkaloids (TAs) of interest are provided. In some embodiments, the engineered strains of the invention will provide a platform for producing tropane alkaloids of interest and modifications thereof across several classes including, but not limited to, medicinal TAs such as those derived from tropine and PLA; non-medicinal TAs such as those derived from tropinone, pseudotropine, or norpseudotropine; and non-natural TAs such as those derived from the esterification of TA precursors (e.g., acyl donor and acyl acceptor compounds) other than tropine and PLA. Each of these classes is meant to include biosynthetic precursors, intermediates, and metabolites thereof, of any convenient member of a host cell biosynthetic pathway that may lead to a member of the class. Non-limiting examples of compounds are given below for each of these classes. In some embodiments, the structure of a given example may or may not be characterized itself as a tropane alkaloid. The present chemical entities are meant to include all possible isomers, including single enantiomers, racemic mixtures, optically pure forms, mixtures of diastereomers and intermediate mixtures.
Medicinal TAs may include, but are not limited to, littorine, hyoscyamine, atropine, anisodamine, scopolamine, and derivatives thereof that are naturally produced by plants.
Non-medicinal TAs may include, but are not limited to, calystegines, cocaine, and derivatives thereof that are naturally produced by plants.
Non-natural TAs may include, but are not limited to, cinnamoyltropine, cinnamoyl-3β-tropine, coumaroyltropine, coumaroyl-3β-tropine, benzoyltropine, benzoyl-3β-tropine, caffeoyltropine, caffeoyl-3β-tropine, feruloyltropine, and feruloyl-3β-tropine.
As summarized above, host cells which produce modified derivatives of tropane alkaloids (TAs) of interest are provided. In some embodiments, the engineered strains of the invention will provide a platform for derivatizing TAs of interest, including derivatizing TA precursors, medicinal TAs, non-medicinal TAs, and non-natural TAs which are produced by engineered host cells or which are fed to engineered host cells in the growth media.
As used herein, the terms “derivatization”, “functionalization”, “modification by derivatization”, and “modification by functionalization” refer to the modification of TAs or of TA precursors via the attachment of functional groups without modification of the TA skeleton itself. As used herein, attachment of functional groups includes, but is not limited to, hydroxylation, alkylation and N-alkylation, acetylation and N-acetylation, acylation and N-acylation, and halogenation.
In some embodiments of the invention, derivatization of TAs of interest may be achieved enzymatically by feeding pre-functionalized TA precursors, for example halogenated or alkylated amino acids, to host cells engineered to uptake and then convert fed TA precursors into TAs of interest. In other embodiments of the invention, derivatization of TAs of interest may be achieved enzymatically by engineering host cells to express enzymes which possess the desired activity in attaching a functional group to a target TA, in addition to the enzymes and cellular modifications required to produce the unmodified TA. In other embodiments of the invention, derivatization of TAs of interest may be achieved enzymatically by treating unmodified TAs produced by engineered host cells with purified enzymes capable of attaching desired functional groups, or with crude lysate of host cells engineered to express enzymes that have the desired derivatizing activity. In other embodiments of the invention, derivatization of TAs of interest may be achieved non-enzymatically by treating unmodified TAs produced by engineered host cells with chemical agents with attach desired functional groups.
Modified derivatives of TAs include, but are not limited to, p-hydroxyatropine, p-hydroxyhyoscyamine, p-fluorohyoscyamine, p-chlorohyoscyamine, p-bromohyoscyamine, p-fluoroscopolamine, p-chloropscopolamine, p-bromoscopolamine, N-methylhyoscyamine, N-butylhyoscyamine, N-methylscopolamine, N-butylscopolamine, N-acetylhyoscyamine, and N-acetylscopolamine.
As summarized above, one aspect of the invention is a host cell that produces one or more TAs of interest. Any convenient cells may be utilized in the subject host cells and methods. In some cases, the host cells are non-plant cells. In some instances, the host cells may be characterized as microbial cells. In certain cases, the host cells are insect cells, mammalian cells, bacterial cells, or fungal cells. Any convenient type of host cell may be utilized in producing the subject TA-producing cells, see, e.g., US2008/0176754 now published as U.S. Pat. No. 8,975,063, US2014/0273109 and WO2014/143744); the disclosures of which are incorporated by reference in their entirety. Host cells of interest include, but are not limited to, bacterial cells, such as Bacillus subtilis, Escherichia coli, Streptomyces, Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria, Lactobacillus, Leucononstoc, Macrococcus, Methylomonas, Methylobacter, Methylocella, Methylococcus, Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus, Propionibacterium, Proteus, Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, Zymomonas, and Salmonella typhimuium cells, insect cells such as Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cells, and yeast cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Yarrowia lipolytica, Candida albicans, Aspergillus spp., Rhizopus spp., Penicillium spp., and Trichoderma reesei cells. In some embodiments, the host cells are yeast cells or E. coli cells. In some cases, the host cell is a yeast cell. In some instances the host cell is from a strain of yeast engineered to produce a TA of interest. Any of the host cells described in US2008/0176754 now published as U.S. Pat. No. 8,975,063, US2014/0273109 and WO2014/143744, may be adapted for use in the subject cells and methods. In certain embodiments, the yeast cells may be of the species Saccharomyces cerevisiae (S. cerevisiae). In certain embodiments, the yeast cells may be of the species Schizosaccharomyces pombe. In certain embodiments, the yeast cells may be of the species Pichia pastoris. Yeast is of interest as a host cell because cytochrome P450 proteins, which are involved in some biosynthetic pathways of interest, are able to fold properly into the endoplasmic reticulum membrane so that their activity is maintained.
Yeast strains of interest that find use in the invention include, but are not limited to, CEN.PK (Genotype: MA Ta/a ura3-52/ura3-52 trp1-289/trp1-289 leu2-3_112/leu2-3_112 his3 Δ1/his3 Δ1 MAL2-8C/MAL2-8C SUC2/SUC2), S288C, W303, D273-10B, X2180, A364A, Σ1278B, AB972, SK1, and FL100. In certain cases, the yeast strain is any of S288C (MATα; SUC2 mal mel gal2 CUP1 flo1 flo8-1 hap1), BY4741 (MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0), BY4742 (MATα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0), BY4743 (MATa/MATα; his3Δ1/his3Δ1; leu2Δ0/leu2Δ0; met15Δ0/MET15; LYS2/lys2Δ0; ura3Δ0/ura3Δ0), and WAT11 or W(R), derivatives of the W303-B strain (MATa; ade2-1; his3-11, -15; leu2-3, -112; ura3-1; canR; cyr+) which express the Arabidopsis thaliana NADPH-P450 reductase ATR1 and the yeast NADPH-P450 reductase CPR1, respectively. In another embodiment, the yeast cell is W303alpha (MATα; his3-11, 15 trp1-1 leu2-3 ura3-1 ade2-1). The identity and genotype of additional yeast strains of interest may be found at EUROSCARF (web.uni-frankfurt.de/fb15/mikro/euroscarf/col_index.html).
In some instances, the host cell is a fungal cell. In certain embodiments, the fungal cells may be of the Aspergillus species and strains include Aspergillus niger (ATCC 1015, ATCC 9029, CBS 513.88), Aspergillus oryzae (ATCC 56747, RIB40), Aspergillus terreus (NIH 2624, ATCC 20542) and Aspergillus nidulans (FGSC A4).
In certain embodiments, heterologous coding sequences may be codon optimized for expression in Aspergillus sp. and expressed from an appropriate promoter. In certain embodiments, the promoter may be selected from phosphoglycerate kinase promoter (PGK), MbfA promoter, cytochrome c oxidase subunit promoter (CoxA), SrpB promoter, TvdA promoter, malate dehydrogenase promoter (MdhA), beta-mannosidase promoter (ManB). In certain embodiments, a terminator may be selected from glucoamylase terminator (GlaA) or TrpC terminator. In certain embodiments, the expression cassette consisting of a promoter, heterologous coding sequence, and terminator may be expressed from a plasmid or integrated into the genome of the host. In certain embodiments, selection of cells maintaining the plasmid or integration cassette may be performed with antibiotic selection such as hygromycin or nitrogen source utilization, such as using acetamide as a sole nitrogen source. In certain embodiments, DNA constructs may be introduced into the host cells using established transformation methods such as protoplast transformation, lithium acetate, or electroporation. In certain embodiments, cells may be cultured in liquid ME or solid MEA (3% malt extract, 0.5% peptone, and ±1.5% agar) or in Vogel's minimal medium with or without selection.
In some instances, the host cell is a bacterial cell. The bacterial cell may be selected from any bacterial genus. Examples of genera from which the bacterial cell may come include Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria, Lactobacillus, Leucononstoc, Macrococcus, Methylomonas, Methylobacter, Methylocella, Methylococcus, Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus, Propionibacterium, Proteus, Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, and Zymomonas. Examples of bacterial species which may be used with the methods of this disclosure include Arthrobacter nicotianae, Acetobacter aceti, Arthrobacter arilaitensis, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus pumilus, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium adolescentis, Brachybacterium tyrofermentans, Brevibacterium linens, Carnobacterium divergens, Corynebacterium flavescens, Enterococcus faecium, Gluconacetobacter europaeus, Gluconacetobacter johannae, Gluconobacter oxydans, Hafnia alvei, Halomonas elongata, Kocuria rhizophila, Lactobacillus acidifarinae, Lactobacillus jensenii, Lactococcus lactis, Lactobacillus yamanashiensis, Leuconostoc citreum, Macrococcus caseolyticus, Microbacterium foliorum, Micrococcus lylae, Oenococcus oeni, Pediococcus acidilactici, Propionibacterium acidipropionici, Proteus vulgaris, Pseudomonas fluorescens, Psychrobacter celer, Staphylococcus condimenti, Streptococcus thermophilus, Streptomyces griseus, Tetragenococcus halophilus, Weissella cibaria, Weissella koreensis, Zymomonas mobilis, Corynebacterium glutamicum, Bifidobacterium bifidum/breve/longum, Streptomyces lividans, Streptomyces coelicolor, Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus casei, Pseudoalteromonas citrea, Pseudomonas putida, Clostridium ljungdahlii/aceticum/acetobutylicum/beijerinckii/butyricum, and Moorella themocellum/thermoacetica.
In certain embodiments, the bacterial cells may be of a strain of Escherichia coll. In certain embodiments, the strain of E. coli may be selected from BL21, DH5α, XL1-Blue, HB101, BL21, and K12, In certain embodiments, heterologous coding sequences may be codon optimized for expression in E. coli and expressed from an appropriate promoter. In certain embodiments, the promoter may be selected from T7 promoter, tac promoter, trc promoter, tetracycline-inducible promoter (tet), lac operon promoter (lac), lacO1 promoter. In certain embodiments, the expression cassette consisting of a promoter, heterologous coding sequence, and terminator may be expressed from a plasmid or integrated into the genome. In certain embodiments, the plasmid is selected from pUC19 or pBAD. In certain embodiments, selection of cells maintaining the plasmid or integration cassette may be performed with antibiotic selection such as kanamycin, chloramphenicol, streptomycin, spectinomycin, gentamycin, erythromycin or ampicillin. In certain embodiments, DNA constructs may be introduced into the host cells using established transformation methods such as conjugation, heat shock chemical transformation, or electroporation. In certain embodiments, cells may be cultured in liquid Luria-Bertani (LB) media at about 37° C. with or without antibiotics.
In certain embodiments, the bacterial cells may be a strain of Bacillus subtilis. In certain embodiments, the strain of B. subtilis may be selected from 1779, GP25, RO-NN-1, 168, BSn5, BEST195, 1A382, and 62178. In certain embodiments, heterologous coding sequences may be codon optimized for expression in Bacillus sp. and expressed from an appropriate promoter. In certain embodiments, the promoter may be selected from grac promoter, p43 promoter, or trnQ promoter. In certain embodiments, the expression cassette consisting of the promoter, heterologous coding sequence, and terminator may be expressed from a plasmid or integrated into the genome. In certain embodiments, the plasmid is selected from pHP13 pE194, pC194, pHT01, or pHT43. In certain embodiments, integrating vectors such as pDG364 or pDG1730 may be used to integrate the expression cassette into the genome. In certain embodiments, selection of cells maintaining the plasmid or integration cassette may be performed with antibiotic selection such as erythromycin, kanamycin, tetracycline, and spectinomycin. In certain embodiments, DNA constructs may be introduced into the host cells using established transformation methods such as natural competence, heat shock, or chemical transformation. In certain embodiments, cells may be cultured in liquid Luria-Bertani (LB) media at 37° C. or M9 medium plus glucose and tryptophan.
The host cells may be engineered to include one or more modifications (such as two or more, three or more, four or more, five or more, or even more modifications) that provide for the production of TAs of interest. In some cases, by modification is meant a genetic modification, such as a mutation, addition, or deletion of a gene or fragment thereof, or transcription regulation of a gene or fragment thereof. In some cases, the one or more (such as two or more, three or more, or four or more) modifications is selected from: a feedback inhibition alleviating mutation in a biosynthetic enzyme gene native to the cell; a transcriptional modulation modification of a biosynthetic enzyme gene native to the cell; an inactivating mutation in an enzyme native to the cell; a heterologous coding sequence that encodes an enzyme; and a heterologous coding sequence that encodes a protein which modifies the sub-cellular trafficking and/or localization of an enzyme or a metabolite. A cell that includes one or more modifications may be referred to as a modified cell.
A modified cell may overproduce one or more precursor TA, TA, or modified TA molecules. By overproduce is meant that the cell has an improved or increased production of a TA molecule of interest relative to a control cell (e.g., an unmodified cell). By improved or increased production is meant both the production of some amount of the TA of interest where the control has no TA precursor production, as well as an increase of about 10% or more, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, such as 2-fold or more, such as 5-fold or more, including 10-fold or more in situations where the control has some TA of interest production.
In some cases, the host cell is capable of producing an increased amount of putrescine relative to a control host cell that lacks the one or more modifications (e.g., as described herein). In certain instances, the increased amount of putrescine is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell.
In some cases, the host cell is capable of producing an increased amount of N-methylpyrrolinium relative to a control host cell that lacks the one or more modifications (e.g., as described herein). In certain instances, the increased amount of N-methylpyrrolinium is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell.
In some cases, the host cell is capable of producing an increased amount of tropine relative to a control host cell that lacks the one or more modifications (e.g., as described herein). In certain instances, the increased amount of tropine is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell.
In some cases, the host cell is capable of producing an increased amount of phenylpyruvic acid relative to a control host cell that lacks the one or more modifications (e.g., as described herein). In certain instances, the increased amount of phenylpyruvic acid is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell.
In some cases, the host cell is capable of producing an increased amount of phenyllactic acid relative to a control host cell that lacks the one or more modifications (e.g., as described herein). In certain instances, the increased amount of phenyllactic acid is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell.
In some cases, the host cell is capable of producing an increased amount of littorine relative to a control host cell that lacks the one or more modifications (e.g., as described herein). In certain instances, the increased amount of littorine is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell.
In some cases, the host cell is capable of producing an increased amount of hyoscyamine relative to a control host cell that lacks the one or more modifications (e.g., as described herein). In certain instances, the increased amount of hyoscyamine is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell.
In some cases, the host cell is capable of producing an increased amount of scopolamine relative to a control host cell that lacks the one or more modifications (e.g., as described herein). In certain instances, the increased amount of scopolamine is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell.
In some embodiments, the host cell is capable of producing a 10% or more yield of tropine from a starting compound such as arginine, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more yield of tropine from a starting compound.
In some embodiments, the host cell is capable of producing a 10% or more yield of phenyllactic acid from a starting compound such as phenylalanine, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more yield of phenyllactic acid from a starting compound.
In some embodiments, the host cell is capable of producing a 10% or more yield of hyoscyamine from a starting compound such as arginine or phenylalanine, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more yield of hyoscyamine from a starting compound.
In some embodiments, the host cell is capable of producing a 10% or more yield of scopolamine from a starting compound such as arginine or phenylalanine, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more yield of scopolamine from a starting compound.
In some embodiments, the host cell overproduces one or more TA of interest molecules selected from the group consisting of arginine, ornithine, agmatine, putrescine, N-methylputrescine, 4-methylaminobutanal, N-methylpyrrolinium, 4-(1-methyl-2-pyrrodinyl)-3-oxobutanoic acid, tropinone, tropine, phenylalanine, prephenic acid, phenylpyruvic acid, phenyllactic acid, glucose-1-O-phenyllactate, littorine, hyoscyamine aldehyde, hyoscyamine, anisodamine, and scopolamine.
Any convenient combinations of the one or more modifications may be included in the subject host cells. In some cases, two or more (such as two or more, three or more, or four or more) different types of modifications are included. In certain instances, two or more (such as three or more, four or more, five or more, or even more) distinct modifications of the same type of modification are included in the subject cells.
In some embodiments of the host cell, when the cell includes one or more heterologous coding sequences that encode one or more enzymes, it includes at least one additional modification selected from the group consisting of: a feedback inhibition alleviating mutations in a biosynthetic enzyme gene native to the cell; a transcriptional modulation modification of a biosynthetic enzyme gene native to the cell; and an inactivating mutation in an enzyme native to the cell. In certain embodiments of the host cell, when the cell includes one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell, it includes a least one additional modification selected from the group consisting of: a transcriptional modulation modification of a biosynthetic enzyme gene native to the cell; an inactivating mutation in an enzyme native to the cell; and a heterologous coding sequence that encode an enzyme. In some embodiments of the host cell, when the cell includes one or more transcriptional modulation modifications of one or more biosynthetic enzyme genes native to the cell, it includes at least one additional modification selected from the group consisting of: a feedback inhibition alleviating mutation in a biosynthetic enzyme gene native to the cell; an inactivating mutation in an enzyme native to the cell; a heterologous coding sequence that encodes an enzyme; and a heterologous coding sequence that encodes a protein which modifies the sub-cellular trafficking and/or localization of an enzyme or a metabolite. In certain instances of the host cell, when the cell includes one or more inactivating mutations in one or more enzymes native to the cell, it includes at least one additional modification selected from the group consisting of: a feedback inhibition alleviating mutation in a biosynthetic enzyme gene native to the cell; a transcriptional modulation modification of a biosynthetic enzyme gene native to the cell; a heterologous coding sequence that encodes an enzyme; and a heterologous coding sequence that encodes a protein which modifies the sub-cellular trafficking and/or localization of an enzyme or a metabolite.
In certain embodiments of the host cell, the cell includes one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell; and one or more transcriptional modulation modifications of one or more biosynthetic enzyme gene native to the cell. In certain embodiments of the host cell, the cell includes one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell; and one or more inactivating mutations in an enzyme native to the cell. In certain embodiments of the host cell, the cell includes one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell; and one or more heterologous coding sequences. In some embodiments, the host cell includes one or more modifications (e.g., as described herein) that include one or more of the genes of interest described in Table 1.
In some instances, the host cells are cells that include one or more feedback inhibition alleviating mutations (such as two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell. In some cases, the one or more biosynthetic enzyme genes are native to the cell (e.g., is present in an unmodified cell). As used herein, the term “feedback inhibition alleviating mutation” refers to a mutation that alleviates a feedback inhibition control mechanism of a host cell. Feedback inhibition is a control mechanism of the cell in which an enzyme in the synthetic pathway of a regulated compound is inhibited when that compound has accumulated to a certain level, thereby balancing the amount of the compound in the cell. In some instances, the one or more feedback inhibition alleviating mutations is in an enzyme described in a biosynthetic pathway of
A variety of feedback inhibition control mechanisms and biosynthetic enzymes native to the host cell that are directed to regulation of levels of TA precursors may be targeted for alleviation in the host cell. The host cell may include one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell. The mutation may be located in any convenient biosynthetic enzyme genes native to the host cell where the biosynthetic enzyme is subject to regulatory control. In some embodiments, the one or more biosynthetic enzyme genes encode one or more enzymes selected from an ornithine decarboxylase (ODC), an ornithine decarboxylase antizyme, and a putrescine N-methyltransferase. In some embodiments, the one or more biosynthetic enzyme genes encode an ornithine decarboxylase. In some instances, the one or more biosynthetic enzyme genes encode an ornithine decarboxylase antizyme. In some embodiments, the one or more biosynthetic enzyme genes encode a putrescine N-methyltransferase. In certain instances, the one or more feedback inhibition alleviating mutations are present in a biosynthetic enzyme gene selected from SPE1, OAZ1, and PMT. In certain instances, the one or more feedback inhibition alleviating mutations are present in a biosynthetic enzyme gene that is SPE1. In certain instances, the one or more feedback inhibition alleviating mutations are present in a biosynthetic enzyme gene that is OAZ1. In certain instances, the one or more feedback inhibition alleviating mutations are present in a biosynthetic enzyme gene that is PMT. In some embodiments, the host cell includes one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes such as one of those genes described in Table 1.
Any convenient numbers and types of mutations may be utilized to alleviate a feedback inhibition control mechanism. As used herein, the term “mutation” refers to a deletion, insertion, or substitution of an amino acid(s) residue or nucleotide(s) residue relative to a reference sequence or motif. The mutation may be incorporated as a directed mutation to the native gene at the original locus. In some cases, the mutation may be incorporated as an additional copy of the gene introduced as a genetic integration at a separate locus, or as an additional copy on an episomal vector such as a 2μ or centromeric plasmid. In certain instances, the feedback inhibited copy of the enzyme is under the native cell transcriptional regulation. In some instances, feedback inhibited copy of the enzyme is introduced with engineered constitutive or dynamic regulation of protein expression by placing it under the control of a synthetic promoter.
In certain embodiments, the host cells of the present invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more feedback inhibition alleviating mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the host cell.
The host cells may include one or more transcriptional modulation modifications (such as two or more, three or more, four or more, five or more, or even more modifications) of one or more biosynthetic enzyme genes of the cell. In some cases, the one or more biosynthetic enzyme genes are native to the cell. Any convenient biosynthetic enzyme genes of the cell may be targeted for transcription modulation. By transcription modulation is meant that the expression of a gene of interest in a modified cell is modulated, e.g., increased or decreased, enhanced or repressed, relative to a control cell (e.g., an unmodified cell). In some cases, transcriptional modulation of the gene of interest includes increasing or enhancing expression. By increasing or enhancing expression is meant that the expression level of the gene of interest is increased by 2-fold or more, such as by 5-fold or more and sometimes by 25-, 50-, or 100-fold or more and in certain embodiments 300-fold or more or higher, as compared to a control, i.e., expression in the same cell not modified (e.g., by using any convenient gene expression assay). Alternatively, in cases where expression of the gene of interest in a cell is so low that it is undetectable, the expression level of the gene of interest is considered to be increased if expression is increased to a level that is easily detectable. In certain instances, transcriptional modulation of the gene of interest includes decreasing or repressing expression. By decreasing or repressing expression is meant that the expression level of the gene of interest is decreased by 2-fold or more, such as by 5-fold or more and sometimes by 25-, 50-, or 100-fold or more and in certain embodiments 300-fold or more or higher, as compared to a control. In some cases, expression is decreased to a level that is undetectable. Modifications of host cell processes of interest that may be adapted for use in the subject host cells are described in U.S. Publication No. 20140273109 (Ser. No. 14/211,611) by Smolke et al., the disclosure of which is herein incorporated by reference in its entirety.
Any convenient biosynthetic enzyme genes may be transcriptionally modulated, and include but are not limited to, those biosynthetic enzymes described in
In some embodiments, the transcriptional modulation modification includes substitution of a strong promoter for a native promoter of the one or more biosynthetic enzyme genes or the expression of an additional copy(ies) of the gene or genes under the control of a strong promoter. The promoters driving expression of the genes of interest may be constitutive promoters or inducible promoters, provided that the promoters may be active in the host cells. The genes of interest may be expressed from their native promoters, or non-native promoters may be used. Although not a requirement, such promoters should be medium to high strength in the host in which they are used. Promoters may be regulated or constitutive. In some embodiments, promoters that are not glucose repressed, or repressed only mildly by the presence of glucose in the culture medium, are used. There are numerous suitable promoters, examples of which include promoters of glycolytic genes such as the promoter of the B. subtilis tsr gene (encoding fructose biphosphate aldolase) or GAPDH promoter from yeast S. cerevisiae (coding for glyceraldehyde-phosphate dehydrogenase) (Bitter G. A., Meth. Enzymol. 152:673 684 (1987)). Other strong promoters of interest include, but are not limited to, the ADHI promoter of baker's yeast (Ruohonen L., et al, J. Biotechnol. 39:193 203 (1995)), the phosphate-starvation induced promoters such as the PHOS promoter of yeast (Hinnen, A., et al, in Yeast Genetic Engineering, Barr, P. J., et al. eds, Butterworths (1989), the alkaline phosphatase promoter from B. licheniformis (Lee. J. W. K., et al., J. Gen. Microbiol. 137:1127 1133 (1991)), GPD1 and TEF1. Yeast promoters of interest include, but are not limited to, inducible promoters such as Gal1-10, Gal1, GaIL, GalS, repressible promoter Met25, tetO, and constitutive promoters such as glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation-elongation factor-1-alpha promoter (TEF), cytochrome c-oxidase promoter (CYC1), MRP7 promoter, phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI), etc. In some instances, the strong promoter is GPD1. In certain instances, the strong promoter is TEF1. Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones are also known and include, but are not limited to, the glucorticoid responsive element (GRE) and thyroid hormone responsive element (TRE), see e.g., those promoters described in U.S. Pat. No. 7,045,290. Vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of genes of interest. It is understood that any convenient promoters specific to the host cell may be selected, e.g., E. coli. In some cases, promoter selection may be used to optimize transcription, and hence, enzyme levels to maximize production while minimizing energy resources.
The host cells may include one or more inactivating mutations to an enzyme of the cell (such as two or more, three or more, four or more, five or more, or even more). The inclusion of one or more inactivating mutations may modify the flux of a synthetic pathway of a host cell to increase the levels of a TA of interest or a desirable enzyme or precursor leading to the same. In some cases, the one or more inactivating mutations are to an enzyme native to the cell.
In some embodiments, the cell includes an inactivating mutation in an enzyme native to the cell. Any convenient enzymes may be targeted for inactivation. Enzymes of interest include, but are not limited to, those enzymes described in
Some methods, processes, and systems provided herein describe the concerted reaction of one or more TA precursors comprising an acyl donor group with one or more TA precursors comprising an acyl acceptor group to produce one or more TAs within a non-plant cell (hereafter referred to as TA acyl transfer reactions). Some of these methods, processes, and systems may comprise an engineered host cell. In some examples, the TA acyl transfer reaction is a key step in the conversion of a substrate to a diverse range of alkaloids. In some examples, the TA acyl transfer reaction comprises a condensation reaction.
In some examples, the TA acyl transfer may involve at least one condensation reaction. In some cases, at least one of the condensation reactions is carried out in the presence of an enzyme. In some cases, at least one of the condensation reactions is catalyzed by an enzyme. In some cases, at least one enzyme is useful to catalyze the condensation reaction.
In some methods, processes and systems described herein, a condensation reaction may be performed in the presence of an enzyme. In some examples, the enzyme may be an acyltransferase. The acyltransferase may use a TA with an alcohol or carboxylate functional group as a substrate. The acyltransferase may use a TA containing a carboxylate group activated via a 1-O-β glycosidic linkage to a sugar (hereafter referred to as a glycoside) as a substrate. The acyltransferase may convert the TA alcohol and carboxylate/glycoside functional groups to a corresponding ester derivative. Non-limiting examples of enzymes suitable for condensation of TA precursors in this disclosure include serine carboxypeptidase-like acyltransferases (SCPL-ATs). For example, littorine synthase (EC 2.3.1.-) may condense tropine and other TA precursors containing alcohol functional groups with 1-O-β-phenyllactoyl-glucose and other TA glycoside precursors to littorine and other corresponding ester products. In some examples, a protein that comprises an SCPL-AT domain of any one of the preceding examples may perform the condensation. In some examples, the SCPL-AT may catalyze the condensation reaction within a host cell, such as an engineered host cell, as described herein. In yet other examples, the SCPL-AT may catalyze the condensation reaction within a sub-cellular compartment inside a host cell, such as an engineered host cell, as described herein.
In some embodiments of the invention, the amino acid sequence of an acyltransferase enzyme which is used to perform a TA acyl transfer reaction, such as an SCPL-AT enzyme, is subject to one or more modifications which alters the post-translational processing, trafficking, folding, oligomerization, and/or sub-cellular localization of the enzyme. As some acyltransferase enzymes, including SCPL-AT enzymes, have never been demonstrated to exhibit catalytic activity in living, non-plant cells, such modifications may prove useful, or may be necessary, for activity in non-plant host cells. Examples of such modifications include, but are not limited to: addition, removal, or replacement of N-terminal signal peptide sequences; addition, removal, or replacement of internal propeptide sequences; addition or removal of asparagine-linked N-glycosylation sites; addition or removal of serine-linked O-glycosylation sites; and fusion of protein domains to the N- and/or C-terminus of the acyltransferase domain.
In one embodiment of the invention, an SCPL-AT enzyme domain is modified at its N-terminus by fusion of a soluble protein domain. This soluble domain masks any internal signal sequences in the acyltransferase domain, thereby modifying the trafficking and/or sub-cellular localization of the fused SCPL-AT domain. In some examples, the N-terminally fused domain induces trafficking of the SCPL-AT domain to sub-cellular compartments including, but not limited to, the ER membrane, ER lumen, cis-Golgi, trans-Golgi, lysosome, vacuole membrane, and vacuole lumen. The N-terminally fused soluble domain can also modify the oligomerization state of the SCPL-AT domain from its native state (monomer) to any state including, but not limited to, homodimer, heterodimer, homotrimer, heterotrimer, homotetramer, heterotetramer, homohexamer, heterohexamer, homooctamer, heterooctamer, or greater degrees of oligomerization.
In one example, the N-terminally fused soluble protein domain is a fluorescent protein selected from the group including, but not limited to, fluorescent proteins derived from Aequoria sp. and fluorescent proteins derived from Discosoma sp. In one example, the N-terminally fused soluble protein domain is red fluorescent protein from Discosoma sp. (DsRed). In other examples, the N-terminally fused soluble protein domain is another enzyme in the TA biosynthetic pathway, including but not limited to, ornithine decarboxylase, putrescine N-methyltransferase, pyrrolidine ketide synthase, tropinone reductase, phenylpyruvate reductase, phenyllactate UDP-glucosyltransferase 84A27, and hyoscyamine dehydrogenase.
Examples of amino acid sequences of soluble protein domains which can be fused to the N-terminus of a SCPL-AT domain that can then be used to perform a TA acyl transfer reaction within a non-plant cell are provided in Table 3. An amino acid sequence for a SCPL-AT enzyme comprising a fused N-terminal domain and that is utilized in TA acyl transfer reactions in non-plant cells may be 50% or more identical to a given amino acid sequence as listed in Table 3. For example, an amino acid sequence for such an acyltransferase may comprise an amino acid sequence that is at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to an amino acid sequence as provided herein. Additionally, in certain embodiments, an “identical” amino acid sequence contains at least 80%-99% identity at the amino acid level to the specific amino acid sequence. In some cases an “identical” amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more in certain cases, at least 95%, 96%, 97%, 98% and 99% identity, at the amino acid level. In some cases, the amino acid sequence may be identical but the DNA sequence is altered such as to optimize codon usage for the host organism, for example.
An engineered non-plant host cell may be provided that produces an acyltransferase that catalyzes a TA acyl transfer reaction, wherein the acyltransferase comprises an amino acid sequence whose N-terminus is fused to the amino acid sequence of a soluble protein domain selected from the group consisting of those sequences in Table 3. The acyltransferase that is produced within the engineered host cell may be recovered and purified so as to form a biocatalyst. The one or more enzymes that are recovered from the engineered host cell that produces the acyltransferase may be used in a process for carrying out a TA acyl transfer reaction. The process may include contacting the TA precursors possessing an alcohol and/or a carboxylate/glycoside functional group with an acyltransferase in an amount sufficient to convert the alcohol and/or carboxylate/glycoside group to a corresponding ester group. In examples, the TA precursors possessing an alcohol and/or a carboxylate/glycoside functional group may be contacted with a sufficient amount of the one or more enzymes such that at least 5% of said TA precursors are converted to the corresponding ester. In further examples, the TA possessing an alcohol and/or a carboxylate/glycoside functional group may be contacted with a sufficient amount of the one or more enzymes such that at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or 100% of said TA precursors are converted to the corresponding ester.
The one or more enzymes that may be used to carry out a TA acyl transfer reaction may contact the TA precursors in vitro. Additionally, or alternatively, the one or more enzymes that may be used to carry out a TA acyl transfer reaction may contact the TA precursors in vivo. Additionally, the one or more enzymes that may be used to carry out a TA acyl transfer reaction may be provided to a cell having the TA precursors within, or may be produced within an engineered non-plant host cell.
In some examples, the methods provide for engineered non-plant host cells that produce an alkaloid product, wherein the TA acyl transfer reaction may comprise a key step in the production of an alkaloid product. In some examples, the alkaloid produced is a medicinal TA. In still other embodiments, the alkaloid produced is derived from a medicinal TA, including, for example, non-natural TAs. In still other embodiments, the alkaloid product is selected from the group consisting of medicinal TA, non-medicinal TA, and non-natural TA.
In some examples, the substrates are TA precursors selected from the group consisting of tropine, pseudotropine, ecgonine, methylecgonine, phenyllactic acid, cinnamic acid, ferulic acid, coumaric acid, and glycosides of the listed compounds.
In some examples, the methods provide for engineered non-plant host cells that produce alkaloid products from tropine and 1-O-β-phenyllactoylglucose. The condensation of tropine and 1-O-β-phenyllactoylglucose to littorine may comprise a key step in the production of diverse alkaloid products from a precursor. In some examples, the precursor is an L-amino acid or a sugar (e.g., glucose). The diverse alkaloid products can include, without limitation, medicinal TAs, non-medicinal TAs, and non-natural TAs.
Any suitable carbon source may be used as a precursor toward a TA acyl transfer reaction. Suitable precursors can include, without limitation, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or a combination thereof. In some examples, unpurified mixtures from renewable feedstocks can be used (e.g., cornsteep liquor, sugar beet molasses, barley malt, biomass hydrolysate). In still other embodiments, the carbon precursor can be a one-carbon compound (e.g., methanol, carbon dioxide) or a two-carbon compound (e.g., ethanol). In yet other embodiments, other carbon-containing compounds can be utilized, for example, methylamine, glucosamine, and amino acids (e.g., L-arginine and L-phenylalanine). In some examples, a TA or a precursor of a TA possessing an alcohol and/or a carboxylate/glycoside functional group may be added directly to an engineered host cell of the invention, including, for example, tropine, pseudotropine, ecgonine, methylecgonine, phenyllactic acid, cinnamic acid, ferulic acid, coumaric acid, and glycosides of the listed compounds.
In some embodiments, the substrate used to carry out the vacuolar TA acyl transfer reaction may comprise one or more alcohol and/or carboxylate/glycoside functional groups, wherein only one of said functional groups is condensed to the corresponding ester.
Some methods, processes, and systems provided herein describe the conversion of TAs with aldehyde functional groups to TAs with alcohol (hydroxyl) functional groups, and the conversion of TAs with alcohol functional groups to TAs with aldehyde functional groups (hereafter referred to as TA alcohol-aldehyde interconversions). Some of these methods, processes, and systems may comprise an engineered host cell. In some examples, the TA alcohol-aldehyde interconversion is a key step in the conversion of a substrate to a diverse range of alkaloids. In some examples, the conversion of a TA aldehyde group to a TA alcohol group comprises a reduction reaction. In some cases, reduction of a substrate TA aldehyde to an alcohol may be performed by reducing an aldehyde substrate to the corresponding tetrahedral oxyanion intermediate, then protonating this intermediate to a hydroxyl as provided in
In some examples, the TA alcohol-aldehyde interconversion may involve at least one oxidation reaction or at least one reduction reaction. In some cases, at least one of the oxidation or reduction reactions is carried out in the presence of an enzyme. In some cases, at least one of the oxidation or reduction reactions is catalyzed by an enzyme. In some cases, the oxidation and reduction reactions are both carried out in the presence of at least one enzyme. In some cases, at least one enzyme is useful to catalyze the oxidation and reduction reactions. The oxidation and reduction reactions may be catalyzed by the same enzyme.
In some methods, processes and systems described herein, an oxidation or reduction reaction may be performed in the presence of an enzyme. In some examples, the enzyme may be a dehydrogenase. The dehydrogenase may use a TA with an alcohol or aldehyde functional group as a substrate. The dehydrogenase may convert the TA alcohol or aldehyde functional group to a corresponding aldehyde or alcohol derivative. The dehydrogenase may be referred to as hyoscyamine dehydrogenase (HDH). Non-limiting examples of enzymes suitable for oxidation and/or reduction of TAs in this disclosure include a cytochrome P450 oxidase, a 2-oxoglutarate-dependent oxidase, a flavoprotein oxidase, a short-chain dehydrogenase-reductase (SDR), a medium-chain dehydrogenase-reductase (MDR), a cinnamyl alcohol dehydrogenase (CAD), and an aldo-keto reductase (AKR). For example, tropinone reductase 1 (EC 1.1.1.206) may oxidize tropinone and other TA precursors with ketone functional groups to tropine (3α-tropanol) and other corresponding alcohol products. In some examples, a protein that comprises a dehydrogenase domain of any one of the preceding examples may perform the oxidation or reduction. In some examples, the dehydrogenase may catalyze the oxidation and/or reduction reactions within a host cell, such as an engineered host cell, as described herein.
Examples of amino acid sequences of a dehydrogenase enzyme that may be used to perform a TA alcohol-aldehyde interconversion are provided in Table 2. An amino acid sequence for a dehydrogenase that is utilized in TA alcohol-aldehyde interconversions may be 50% or more identical to a given amino acid sequence as listed in Table 2. For example, an amino acid sequence for such a dehydrogenase may comprise an amino acid sequence that is at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to an amino acid sequence as provided herein. Additionally, in certain embodiments, an “identical” amino acid sequence contains at least 80%-99% identity at the amino acid level to the specific amino acid sequence. In some cases an “identical” amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more in certain cases, at least 95%, 96%, 97%, 98% and 99% identity, at the amino acid level. In some cases, the amino acid sequence may be identical but the DNA sequence is altered such as to optimize codon usage for the host organism, for example.
An engineered host cell may be provided that produces a dehydrogenase that catalyzes a TA alcohol-aldehyde interconversion, wherein the dehydrogenase comprises an amino acid sequence selected from the group consisting of those sequences in Table 2. The dehydrogenase that is produced within the engineered host cell may be recovered and purified so as to form a biocatalyst. The one or more enzymes that are recovered from the engineered host cell that produces the dehydrogenase may be used in a process for carrying out a TA alcohol-aldehyde interconversion. The process may include contacting the TA possessing an alcohol and/or an aldehyde functional group with a dehydrogenase in an amount sufficient to convert the alcohol and/or aldehyde group of the TA to a corresponding aldehyde and/or alcohol group. In examples, the TA possessing an alcohol and/or an aldehyde functional group may be contacted with a sufficient amount of the one or more enzymes such that at least 5% of said TA is converted to its corresponding aldehyde and/or alcohol group. In further examples, the TA possessing an alcohol and/or an aldehyde functional group may be contacted with a sufficient amount of the one or more enzymes such that at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or 100% of said TA is converted to its corresponding aldehyde and/or alcohol group.
The one or more enzymes that may be used to carry out a TA alcohol-aldehyde interconversion may contact the TA in vitro. Additionally, or alternatively, the one or more enzymes that may be used to carry out a TA alcohol-aldehyde interconversion may contact the TA in vivo. Additionally, the one or more enzymes that may be used to carry out a TA alcohol-aldehyde interconversion may be provided to a cell having the TA within, or may be produced within an engineered host cell.
In some examples, the methods provide for engineered host cells that produce an alkaloid product, wherein the TA alcohol-aldehyde interconversion may comprise a key step in the production of an alkaloid product. In some examples, the alkaloid produced is a medicinal TA. In still other embodiments, the alkaloid produced is derived from a medicinal TA, including, for example, non-natural TAs. In another embodiment, a TA possessing an alcohol and/or an aldehyde functional group is an intermediate toward the product of the engineered host cell. In still other embodiments, the alkaloid product is selected from the group consisting of medicinal TA, non-medicinal TA, and non-natural TA.
In some examples, the substrate is a TA or a precursor of a TA selected from the group consisting of littorine, hyoscyamine aldehyde, hyoscyamine, anisodamine, and scopolamine.
In some examples, the methods provide for engineered host cells that produce alkaloid products from hyoscyamine aldehyde. The reduction of hyoscyamine aldehyde to hyoscyamine may comprise a key step in the production of diverse alkaloid products from a precursor. In some examples, the precursor is an L-amino acid or a sugar (e.g., glucose). The diverse alkaloid products can include, without limitation, medicinal TAs, non-medicinal TAs, and non-natural TAs.
Any suitable carbon source may be used as a precursor toward a TA alcohol-aldehyde interconversion. Suitable precursors can include, without limitation, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or a combination thereof. In some examples, unpurified mixtures from renewable feedstocks can be used (e.g., cornsteep liquor, sugar beet molasses, barley malt, biomass hydrolysate). In still other embodiments, the carbon precursor can be a one-carbon compound (e.g., methanol, carbon dioxide) or a two-carbon compound (e.g., ethanol). In yet other embodiments, other carbon-containing compounds can be utilized, for example, methylamine, glucosamine, and amino acids (e.g., L-arginine and L-phenylalanine). In some examples, a TA or a precursor of a TA possessing an alcohol and/or an aldehyde functional group may be added directly to an engineered host cell of the invention, including, for example, tropine, pseudotropine, ecgonine, methylecgonine, littorine, hyoscyamine aldehyde, hyoscyamine, anisodamine, and scopolamine.
In some embodiments, the substrate used to carry out the TA alcohol-aldehyde interconversion may comprise one or more alcohol and/or aldehyde functional groups, wherein only one of said functional groups is oxidized or reduced to the corresponding aldehyde or alcohol group.
Some methods, processes, and systems provided herein describe the use of proteins (hereafter referred to as ‘transporters’) to translocate metabolites across lipid membranes (hereafter referred to as ‘transmembrane transport’). Some of these methods, processes, and systems may comprise an engineered host cell. In some examples, transmembrane transport is a key step in the conversion of a substrate to a diverse range of alkaloids.
In certain embodiments, the host cell includes one or more heterologous coding sequences for one or more transporters or active fragments thereof that localize to a lipid membrane and translocate a TA or a TA precursor across the same lipid membrane. In some examples, the lipid membrane is the vacuole membrane. In other examples, the lipid membrane is the ER membrane. In some examples, the lipid membrane is the peroxisome membrane. In other examples, the lipid membrane is the cellular plasma membrane.
In some examples, TAs and TA precursors transported in this manner include, but are not limited to, putrescine, N-methylputrescine, 4-methylaminobutanal, N-methylpyrrolinium, tropinone, tropine, phenyllactic acid, 1-O-β-phenyllactoylglucose, littorine, hyoscyamine, anisodamine, and scopolamine. The accumulation of such TAs or TA precursors in specific sub-cellular compartments can preclude access by operably linked biosynthetic enzymes in different compartments; therefore, the use of transporters which translocate TAs or TA precursors from one compartment to another can mitigate such transport limitations. In certain cases, the expression of heterologous coding sequences for one or more transporters within a host cell can increase production of a TA or a TA precursor.
In some embodiments, the transporter or active fragment thereof is a multidrug and toxin extrusion (MATE) transporter. Any convenient MATE transporters which transport one or more of the aforementioned TAs or TA precursors find use in the subject host cells. Transporter proteins of interest include, but are not limited to, enzymes such as Nicotiana tabacum jasmonate-inducible alkaloid transporter 1 (NtJAT1), N. tabacum MATE1, N. tabacum MATE2, or any others as described in Table 1 and Table 4.
In certain embodiments, the transporter or active fragment thereof is a nitrate/peptide family (NPF) transporter. Any convenient NPF transporters which transport one or more of the aforementioned TAs or TA precursors find use in the subject host cells. In other embodiments, the transporter or active fragment thereof is an ATP-binding cassette (ABC) transporter. Any convenient NPF transporters which transport one or more of the aforementioned TAs or TA precursors find use in the subject host cells. In some embodiments, the transporter or active fragment thereof is a pleiotropic drug resistance (PDR) transporter. Any convenient PDR transporters which transport one or more of the aforementioned TAs or TA precursors find use in the subject host cells.
In certain embodiments, the host cell includes a heterologous coding sequence for a transporter or an active fragment thereof. In some embodiments of the invention, the amino acid sequence of a transporter is subject to one or more modifications which alters the sub-cellular localization, the direction of substrate translocation, and/or the topological orientation of the enzyme. Examples of such modifications include, but are not limited to: addition, removal, or replacement of N-terminal, C-terminal, or internal signal sequences; addition, removal, replacement, or rearrangement of transmembrane helices; and fusion of protein domains to the N- and/or C-terminus of the transporter.
Examples of amino acid sequences of transporters which can be used to mitigate substrate transport limitations and/or to increase accumulation of TAs or TA precursors in specific cellular compartments are provided in Table 4. An amino acid sequence for a transporter that is utilized in this manner in non-plant cells may be 50% or more identical to a given amino acid sequence as listed in Table 4. For example, an amino acid sequence for such a transporter may comprise an amino acid sequence that is at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to an amino acid sequence as provided herein. Additionally, in certain embodiments, an “identical” amino acid sequence contains at least 80%-99% identity at the amino acid level to the specific amino acid sequence. In some cases an “identical” amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more in certain cases, at least 95%, 96%, 97%, 98% and 99% identity, at the amino acid level. In some cases, the amino acid sequence may be identical but the DNA sequence is altered such as to optimize codon usage for the host organism, for example.
An engineered non-plant host cell may be provided that produces a transporter which translocates one or more TAs or TA precursors from one cellular compartment to another, wherein the transporter comprises an amino acid sequence selected from the group consisting of those sequences in Table 4. In some examples, the methods provide for engineered non-plant host cells that produce an alkaloid product, wherein TA transmembrane transport may comprise a key step in the production of an alkaloid product. In some examples, the alkaloid produced is a medicinal TA. In still other embodiments, the alkaloid produced is derived from a medicinal TA, including, for example, non-natural TAs. In still other embodiments, the alkaloid product is selected from the group consisting of medicinal TA, non-medicinal TA, and non-natural TA.
In some instances, the host cells are cells that harbor one or more heterologous coding sequences (such as two or more, three or more, four or more, five or more, or even more) which encode activity(ies) that enable the host cells to produce desired TAs of interest, e.g., as described herein. As used herein, the term “heterologous coding sequence” is used to indicate any polynucleotide that codes for, or ultimately codes for, a peptide or protein or its equivalent amino acid sequence, e.g., an enzyme, that is not normally present in the host organism and may be expressed in the host cell under proper conditions. As such, “heterologous coding sequences” includes multiple copies of coding sequences that are normally present in the host cell, such that the cell is expressing additional copies of a coding sequence that are not normally present in the cells. The heterologous coding sequences may be RNA or any type thereof, e.g., mRNA, DNA or any type thereof, e.g., cDNA, or a hybrid of RNA/DNA. Coding sequences of interest include, but are not limited to, full-length transcription units that include such features as the coding sequence, introns, promoter regions, 3′-UTRs, and enhancer regions.
In examples, the engineered host cell comprises a plurality of heterologous coding sequences each encoding an enzyme. In some examples, the plurality of enzymes encoded by the plurality of heterologous coding sequences may be distinct from each other. In some examples, some of the plurality of enzymes encoded by the plurality of heterologous coding sequences may be distinct from each other and some of the plurality of enzymes encoded by the plurality of heterologous coding sequences may be duplicate copies.
In some examples, the heterologous coding sequences may be operably connected. Heterologous coding sequences that are operably connected may be within the same pathway of producing a particular tropane alkaloid product. In some examples, the operably connected heterologous coding sequences may be directly sequential along the pathway of producing a particular tropane alkaloid product. In some examples, the operably connected heterologous coding sequences may have one or more native enzymes between one or more of the enzymes encoded by the plurality of heterologous coding sequences. In some examples, the heterologous coding sequences may have one or more heterologous enzymes between one or more of the enzymes encoded by the plurality of heterologous coding sequences. In some examples, the heterologous coding sequences may have one or more non-native enzymes between one or more of the enzymes encoded by the plurality of heterologous coding sequences.
In some embodiments, the host cell includes putrescine N-methyltransferase (PMT) activity. Any convenient PMT enzymes find use in the subject host cells. PMT enzymes of interest include, but are not limited to, enzymes such as EC 2.1.1.53, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for a PMT or an active fragment thereof.
In some instances, the host cell includes one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert NMP to 4MAB. In certain cases, the one or more enzymes is selected from plant methylputrescine oxidases (MPOs) and eukaryotic MPOs (e.g., EC 1.4.3.22).
In certain embodiments, the cell includes one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert NMPy to MPOB. In certain cases, the one or more enzymes is a type III polyketide synthase (e.g., EC 2.3.1.-). The one or more heterologous coding sequences may be derived from any convenient species (e.g., as described herein). In some cases, the one or more heterologous coding sequences may be derived from a species described in Table 1. In some cases, the one or more heterologous coding sequences are present in a gene or enzyme selected from those described in Table 1.
In certain embodiments, the host cell includes tropinone synthase activity. Any convenient tropinone synthase enzymes (e.g., CYP82M3) find use in the subject host cells. Tropinone synthase enzymes of interest include, but are not limited to, enzymes such as EC 1.14.14.-, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for a tropinone synthase or an active fragment thereof.
In certain embodiments, the host cell includes tropinone reductase activity. Any convenient tropinone reductase enzymes find use in the subject host cells. Tropinone reductase enzymes of interest include, but are not limited to, enzymes such as EC 1.1.1.206, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for a tropinone reductase or an active fragment thereof.
In some instances, the host cell includes phenylpyruvate reductase (PPR) activity. Any convenient PPR enzymes find use in the subject host cells. Some PPR enzymes of interest include, but are not limited to, enzymes such as EC 1.1.1.237, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for a PPR or an active fragment thereof.
In certain embodiments, the host cell includes phenyllactate glycosyltransferase activity. Any convenient phenyllactate glycosyltransferase enzymes find use in the subject host cells. Glycosyltransferase enzymes include, but are not limited to, enzymes such as 2.4.1.-, which transfer a glucose moiety from UDP-glucose to phenyllactate by means of a glycosidic ester linkage, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for a phenyllactate glycosyltransferase or an active fragment thereof.
In certain embodiments, the cell includes one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert tropine and 1-O-β-phenyllactoylglucose to littorine. In some embodiments, the host cell includes littorine synthase activity. Any convenient littorine synthase enzymes or enzymes comprising littorine synthase active fragments find use in the subject host cells. Littorine synthase enzymes of interest include, but are not limited to, enzymes such as EC 2.3.1.-, as described in Table 1, and enzymes comprising littorine synthase enzymes whose N-termini are fused to soluble protein domains described in Table 3. In certain embodiments, the host cell includes a heterologous coding sequence for a littorine synthase or an active fragment thereof.
In certain instances, the host cell includes littorine mutase activity. Any convenient littorine mutase enzymes find use in the subject host cells. Littorine mutase enzymes of interest include, but are not limited to, enzymes such as EC 1.14.19.-, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for a littorine mutase or an active fragment thereof.
In some embodiments, the host cell includes hyoscyamine dehydrogenase (HDH) activity. Any convenient HDH enzymes find use in the subject host cells. Some HDH enzymes of interest include, but are not limited to, those sequences described in Table 2. In certain embodiments, the host cell includes a heterologous coding sequence for an HDH or an active fragment thereof.
In certain embodiments, the host cell includes hyoscyamine 6β-hydroxylase/dioxygenase (H6H) activity. Any convenient H6H enzymes find use in the subject host cells. Some H6H enzymes of interest include, but are not limited to, enzymes such as EC 1.14.11.11, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for an H6H or an active fragment thereof.
In certain examples, the engineered host cell comprises a plurality of heterologous coding sequences each encoding a transmembrane metabolite transporter. In some examples, the plurality of transporters encoded by the plurality of heterologous coding sequences may be distinct from each other. In some examples, some of the plurality of transporters encoded by the plurality of heterologous coding sequences may be distinct from each other and some of the plurality of transporters encoded by the plurality of heterologous coding sequences may be duplicate copies.
As used herein, the term “heterologous coding sequences” also includes the coding portion of the peptide or enzyme, i.e., the cDNA or mRNA sequence, of the peptide or enzyme, as well as the coding portion of the full-length transcriptional unit, i.e., the gene including introns and exons, as well as “codon optimized” sequences, truncated sequences or other forms of altered sequences that code for the enzyme or code for its equivalent amino acid sequence, provided that the equivalent amino acid sequence produces a functional protein. Such equivalent amino acid sequences may have a deletion of one or more amino acids, with the deletion being N-terminal, C-terminal, or internal. Truncated forms are envisioned as long as they have the catalytic capability indicated herein. Fusions of two or more enzymes are also envisioned to facilitate the transfer of metabolites in the pathway, provided that catalytic activities are maintained. Also included are fusions of one or more enzymes or catalytic protein domains with one or more non-catalytic protein domains in a manner by which the non-catalytic protein domain facilitates the solubilization, folding, maturation, and/or activity of the fused catalytic domain.
Operable fragments, mutants or truncated forms may be identified by modeling and/or screening. This is made possible by addition or deletion of, for example, N-terminal, C-terminal, or internal regions of the protein in a step-wise fashion, followed by analysis of the resulting derivative with regard to its activity for the desired reaction compared to the original sequence. If the derivative in question operates in this capacity, it is considered to constitute an equivalent derivative of the enzyme proper.
Aspects of the present invention also relate to heterologous coding sequences that code for amino acid sequences that are equivalent to the native amino acid sequences for the various enzymes. An amino acid sequence that is “equivalent” is defined as an amino acid sequence that is not identical to the specific amino acid sequence, but rather contains at least some amino acid changes (deletions, substitutions, inversions, insertions, etc.) that do not essentially affect the biological activity of the protein as compared to a similar activity of the specific amino acid sequence, when used for a desired purpose. The biological activity refers to, in the example of a decarboxylase, its catalytic activity. Equivalent sequences are also meant to include those which have been engineered and/or evolved to have properties different from the original amino acid sequence. Mutable properties of interest include catalytic activity, substrate specificity, selectivity, stability, solubility, localization, etc. In certain embodiments, an “equivalent” amino acid sequence contains at least 80%-99% identity at the amino acid level to the specific amino acid sequence, in some cases at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more in certain cases, at least 95%, 96%, 97%, 98% and 99% identity, at the amino acid level. In some cases, the amino acid sequence may be identical but the DNA sequence is altered such as to optimize codon usage for the host organism, for example.
The host cells may also be modified to possess one or more genetic alterations to accommodate the heterologous coding sequences. Alterations of the native host genome include, but are not limited to, modifying the genome to reduce or ablate expression of a specific protein that may interfere with the desired pathway. The presence of such native proteins may rapidly convert one of the intermediates or final products of the pathway into a metabolite or other compound that is not usable in the desired pathway. Thus, if the activity of the native enzyme were reduced or altogether absent, the produced intermediates would be more readily available for incorporation into the desired product.
In some instances, where ablation of expression of a protein may be of interest, the alteration is in proteins involved in the pleiotropic drug response, including, but not limited to, ATP-binding cassette (ABC) transporters, multidrug resistance (MDR) pumps, and associated transcription factors. These proteins are involved in the export of TA molecules and TA precursors into the culture medium, thus deletion controls the export of the compounds into the media, making them more available for incorporation into the desired product. In some embodiments, host cell gene deletions of interest include genes associated with the unfolded protein response and endoplasmic reticulum (ER) proliferation. Such gene deletions may lead to improved TA production. The expression of cytochrome P450s may induce the unfolded protein response and may cause the ER to proliferate. Deletion of genes associated with these stress responses may control or reduce overall burden on the host cell and improve pathway performance. Genetic alterations may also include modifying the promoters of endogenous genes to increase expression and/or introducing additional copies of endogenous genes. Examples of this include the construction/use of strains which overexpress the endogenous yeast NADPH-P450 reductase Ncp1p to increase activity of heterologous P450 enzymes. In addition, endogenous enzymes such as Spe1p, Fms1p, Car1p, Arg2p, Aro8p, Aro9p, Pha2p, Ugp1p, and Leu2p which are directly involved in the synthesis of intermediate metabolites, may also be overexpressed.
Heterologous coding sequences of interest include but are not limited to sequences that encode enzymes, either wild-type or equivalent sequences, that are normally responsible for the production of TAs and precursors in plants. In some cases, the enzymes for which the heterologous sequences code may be any of the enzymes in the TA pathway, and may be from any convenient source. The choice and number of enzymes encoded by the heterologous coding sequences for the particular synthetic pathway may be selected based upon the desired product. In certain embodiments, the host cells of the present invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more heterologous coding sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 heterologous coding sequences.
In some cases, polypeptide sequences encoded by the heterologous coding sequences are as reported in GENBANK. Enzymes of interest include, but are not limited to, those enzymes described herein and those shown in Table 1. The host cells may include any combination of the listed enzymes, from any source. Unless otherwise indicated, accession numbers in Table 1 refer to GenBank. Some accession numbers refer to the Saccharomyces genome database (SGD), which is available on the world-wide web at yeastgenome.org.
In some embodiments, the host cell (e.g., a yeast strain) is engineered for selective production of a TA of interest by localizing one or more enzymes to a compartment in the cell. In some cases, an enzyme may be located in the host cell such that the compound produced by this enzyme spontaneously rearranges, or is converted by another enzyme to a desirable metabolite before reaching a localized enzyme that may convert the compound into an undesirable metabolite. The spatial distance between two enzymes may be selected to prevent one of the enzymes from acting directly on a compound to make an undesirable metabolite, and restrict production of undesirable end products (e.g., an undesirable opioid by-product). In some other cases, an enzyme may be localized in the host cell such that the sub-cellular compartment in which it is located provides a more optimum pH, cofactor concentration, redox potential, substrate concentration, and/or other biochemical parameter for its activity than the compartment in which the enzyme is naturally found. In certain cases, an enzyme may be localized to a specific compartment within the host cell such that the intracellular trafficking pathway by which the enzyme is transported to said compartment provides the necessary post-translational modifications for the enzyme to exhibit activity. Such post-translational modifications include, but are not limited to, acetylation, acetylglycosylation, amidation, carboxylation, methylation, glutathionylation, hydroxylation, glycosylation, phosphorylation, sulfonation, disulfide bond formation, cleavage of signal sequences, and multi-enzyme complex formation. In certain embodiments, any of the enzymes described herein, either singularly or together with a second enzyme, may be localized to any convenient compartment in the host cell, including but not limited to, an organelle, endoplasmic reticulum, golgi, vacuole, nucleus, plasma membrane, mitochondrion, peroxisome, periplasm, the lumen of any of the aforementioned organelles, or the membrane enclosing or associated with any of the aforementioned organelles. In cases where one or more enzymes are localized to a membrane associated with any of the aforementioned organelles, the enzyme may be oriented such that the catalytic domain of the enzyme faces the cytosol, the lumen of the organelle, and/or any other intracellular space. In some embodiments, the host cell includes one or more of the enzymes that include a localization tag. Any convenient tags may be utilized. In some cases, the localization tag is a peptidic sequence that is attached at the N-terminus and/or C-terminus of the enzyme.
Any convenient methods may be utilized for attaching a tag to the enzyme. In some cases, the localization tag is derived from an endogenous yeast protein. Such tags may provide a route to a variety of yeast organelles including, but not limited to, the endoplasmic reticulum (ER), Golgi apparatus (GA), mitochondria (MT), plasma membrane (PM), peroxisome (PDX), and vacuole (V). In certain embodiments, the tag is an ER routing tag (e.g., ER1). In certain embodiments, the tag is a vacuole tag (e.g., V1). In certain embodiments, the tag is a plasma membrane tag (e.g., P1). In certain embodiments, the tag is a peroxisome-targeting sequence (e.g., PTS1). In certain instances, the tag includes or is derived from, a transmembrane domain from within the tail-anchored class of proteins. In some embodiments, the localization tag locates the enzyme on the outside of an organelle. In certain embodiments, the localization tag locates the enzyme on the inside of an organelle. In some embodiments, the localization tag locates the enzyme such that one or more portions of the enzyme are found both inside and outside of an organelle.
In some embodiments of the invention, the host cell is modified by expression of one or more coding sequences encoding one or more enzymes comprising a localization tag described above. In certain embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that one or more enzymes is expressed in the cytosol. Examples of such enzymes include, but are not limited to, arginine decarboxylases, putrescine N-methyltransferases, pyrrolidine ketide synthases, tropinone reductases, phenylpyruvate reductases, UDP-glucosyltransferases, and 2-oxoglutarate-dependent dioxygenases such as hyoscyamine 6β-hydroxylase/dioxygenase. In certain embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that one or more enzymes is expressed in the ER membrane. Examples of such enzymes include, but are not limited to, cytochromes P450 such as tropinone synthase (CYP82M3) and littorine mutase (CYP80F1), and NADP+-cytochrome P450 reductases. In certain embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that one or more enzymes is expressed in the mitochondria. Examples of such enzymes include, but are not limited to, N-acetylglutamate synthases. In other embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that one or more enzymes is expressed in the peroxisome. Examples of such enzymes include, but are not limited to, amine oxidases such as N-methylputrescine oxidase. In other embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that one or more enzymes is expressed in the vacuole lumen. Examples of such enzymes include, but are not limited to, serine carboxypeptidase-like acyltransferases such as littorine synthase, and engineered variants thereof. In other embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that one or more enzymes or proteins is expressed in the vacuole membrane. Examples of such proteins include, but are not limited to, multidrug and toxin extrusion transporters, nitrate/peptide family transporters, and ATP-binding cassette transporters. In other embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that one or more enzymes or proteins is expressed in the plasma membrane. Examples of such proteins include, but are not limited to, ATP-binding cassette transporters, pleiotropic drug resistance transporters, and multidrug resistance transporters.
In some instances, the expression of each type of enzyme is increased through additional gene copies (i.e., multiple copies), which increases intermediate accumulation and/or TA of interest production. Embodiments of the present invention include increased TA of interest production in a host cell through simultaneous expression of multiple species variants of a single or multiple enzymes. In some cases, additional gene copies of a single or multiple enzymes are included in the host cell. Any convenient methods may be utilized including multiple copies of a heterologous coding sequence for an enzyme in the host cell.
In some embodiments, the host cell includes multiple copies of a heterologous coding sequence for an enzyme, such as 2 or more, 3 or more, 4 or more, 5 or more, or even 10 or more copies. In certain embodiments, the host cell includes multiple copies of heterologous coding sequences for one or more enzymes, such as multiple copies of two or more, three or more, four or more, etc. In some cases, the multiple copies of the heterologous coding sequence for an enzyme are derived from two or more different source organisms as compared to the host cell. For example, the host cell may include multiple copies of one heterologous coding sequence, where each of the copies is derived from a different source organism. As such, each copy may include some variations in explicit sequences based on inter-species differences of the enzyme of interest that is encoded by the heterologous coding sequence.
In some embodiments of the host cell, the heterologous coding sequence is from a source organism selected from the group consisting of Escherichia coli, Bacillus coagulans, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus spp, Wickerhamia fluorescens, Aequoria spp, Discosoma spp, Arabidopsis thaliana, Avena sativa, Solanum lycopersicum, Solanum tuberosum, Nicotiana tabacum, Nicotiana benthamiana, Atropa belladonna, Hyoscyamus niger, Hyoscyamus muticus, Datura stramonium, Datura metel, Datura innoxia, Duboisia myoporoides, Anisodus luridus, Anisodus tanguticus, Anisodus acutangulus, Brugmansia arborea, Brugmansia x candida, Brugmansia sanguinea, Erythroxylum coca, Cochlearia officinalis, Solanum spp, Nicotiana spp, Atropa spp, Hyoscyamus spp, Datura spp, Duboisia spp, Anisodus spp, Brugmansia spp, Erythroxylum spp, or Cochlearia spp. In certain instances, the heterologous coding sequence is from a source organism selected from A. belladonna, H. niger, and D. stramonium. In some embodiments, the host cell includes a heterologous coding sequence from one or more of the source organisms described in Table 1.
The engineered host cell medium may be sampled and monitored for the production of TAs of interest. The TAs of interest may be observed and measured using any convenient methods. Methods of interest include, but are not limited to, LC-MS methods (e.g., as described herein) where a sample of interest is analyzed by comparison with a known amount of a standard compound. Identity may be confirmed, e.g., by m/z and MS/MS fragmentation patterns, and quantitation or measurement of the compound may be achieved via LC trace peaks of know retention time and/or EIC MS peak analysis by reference to corresponding LC-MS analysis of a known amount of a standard of the compound.
As summarized above, aspects of the invention include methods of preparing a tropane alkaloid (TA) of interest. As such, aspects of the invention include culturing a host cell under conditions in which the one or more host cell modifications (e.g., as described herein) are functionally expressed such that the cell converts starting compounds of interest into product TAs of interest or precursors thereof (e.g., pre-esterification TAs). Also provided are methods that include culturing a host cell under conditions suitable for protein production such that one or more heterologous coding sequences are functionally expressed and convert starting compounds of interest into product TAs of interest. In some instances, the method is a method of preparing a tropane alkaloid (TA), include culturing a host cell (e.g., as described herein); adding a starting compound to the cell culture; and recovering the TA from the cell culture. In some embodiments of the method, the starting compound, TA product and host cell are described by one of the entries of Table 1.
Fermentation media may contain suitable carbon substrates. The source of carbon suitable to perform the methods of this disclosure may encompass a wide variety of carbon containing substrates. Suitable substrates may include, without limitation, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or a combination thereof. In some cases, unpurified mixtures from renewable feedstocks may be used (e.g., cornsteep liquor, sugar beet molasses, barley malt). In some cases, the carbon substrate may be a one-carbon substrate (e.g., methanol, carbon dioxide) or a two-carbon substrate (e.g., ethanol). In other cases, other carbon containing compounds may be utilized, for example, methylamine, glucosamine, and amino acids.
Any convenient methods of culturing host cells may be employed for producing the TA precursors and downstream TAs of interest. The particular protocol that is employed may vary, e.g., depending on host cell, the heterologous coding sequences, the desired TA precursors and downstream TAs of interest, etc. The cells may be present in any convenient environment, such as an environment in which the cells are capable of expressing one or more functional heterologous enzymes. In vitro, as used herein, simply means outside of a living cell, regardless of the location of the cell. As used herein, the term in vivo indicates inside a living cell, regardless of the location of the cell. In some embodiments, the cells are cultured under conditions that are conducive to enzyme expression and with appropriate substrates available to allow production of TA precursors and downstream TAs of interest in vivo. In some embodiments, the functional enzymes are extracted from the host for production of TAs under in vitro conditions. In some instances, the host cells are placed back into a multicellular host organism. The host cells are in any phase of growth, including, but not limited to, stationary phase and log-growth phase, etc. In addition, the cultures themselves may be continuous cultures or they may be batch cultures.
Cells may be grown in an appropriate fermentation medium at a temperature between 20-40° C. Cells may be grown with shaking at any convenient speed (e.g., 200 rpm). Cells may be grown at a suitable pH. Suitable pH ranges for the fermentation may be between pH 5-9. Fermentations may be performed under aerobic, anaerobic, or microaerobic conditions. Any suitable growth medium may be used. Suitable growth media may include, without limitation, common commercially prepared media such as synthetic defined (SD) minimal media or yeast extract peptone dextrose (YEPD) rich media. Any other rich, defined, or synthetic growth media appropriate to the microorganism may be used.
Cells may be cultured in a vessel of essentially any size and shape. Examples of vessels suitable to perform the methods of this disclosure may include, without limitation, multi-well shake plates, test tubes, flasks (baffled and non-baffled), and bioreactors. The volume of the culture may range from 10 microliters to greater than 10,000 liters.
The addition of agents to the growth media that are known to modulate metabolism in a manner desirable for the production of alkaloids may be included. In a non-limiting example, cyclic adenosine 2′3′-monophosphate may be added to the growth media to modulate catabolite repression.
Any convenient cell culture conditions for a particular cell type may be utilized. In certain embodiments, the host cells that include one or more modifications are cultured under standard or readily optimized conditions, with standard cell culture media and supplements. As one example, standard growth media when selective pressure for plasmid maintenance is not required may contain 20 g/L yeast extract, 10 g/L peptone, and 20 g/L dextrose (YPD). Host cells containing plasmids are grown in synthetic complete (SC) media containing 1.7 g/L yeast nitrogen base, 5 g/L ammonium sulfate, and 20 g/L dextrose supplemented with the appropriate amino acids required for growth and selection. Alternative carbon sources which may be useful for inducible enzyme expression include, but are not limited to, sucrose, raffinose, and galactose. Cells are grown at any convenient temperature (e.g., 30° C.) with shaking at any convenient rate (e.g., 200 rpm) in a vessel, e.g., in test tubes or flasks in volumes ranging from 1-1000 mL, or larger, in the laboratory.
Culture volumes may be scaled up for growth in larger fermentation vessels, for example, as part of an industrial process. The industrial fermentation process may be carried out under closed-batch, fed-batch, or continuous chemostat conditions, or any suitable mode of fermentation. In some cases, the cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for alkaloid production.
A batch fermentation is a closed system, in which the composition of the medium is set at the beginning of the fermentation and not altered during the fermentation process. The desired organism(s) are inoculated into the medium at the beginning of the fermentation. In some instances, the batch fermentation is run with alterations made to the system to control factors such as pH and oxygen concentration (but not carbon). In this type of fermentation system, the biomass and metabolite compositions of the system change continuously over the course of the fermentation. Cells typically proceed through a lag phase, then to a log phase (high growth rate), then to a stationary phase (growth rate reduced or halted), and eventually to a death phase (if left untreated).
A fed-batch fermentation is similar to a batch fermentation, except that the substrate is added in intervals to the system over the course of the fermentation process. Fed-batch systems are used to reduce the impact of catabolite repression on the metabolism of the host cells and under other circumstances where it is desired to have limited amounts of substrate in the growth media.
A continuous fermentation is an open system, in which a defined fermentation medium is added continuously to the bioreactor and an equal amount of fermentation media is continuously removed from the vessel for processing. Continuous fermentation systems are generally operated to maintain steady state growth conditions, such that cell loss due to medium being removed must be balanced by the growth rate in the fermentation. Continuous fermentations are generally operated at conditions where cells are at a constant high cell density. Continuous fermentations allow for the modulation of one or more factors that affect target product concentration and/or cell growth.
The liquid medium may include, but is not limited to, a rich or synthetic defined medium having an additive component described above. Media components may be dissolved in water and sterilized by heat, pressure, filtration, radiation, chemicals, or any combination thereof. Several media components may be prepared separately and sterilized, and then combined in the fermentation vessel. The culture medium may be buffered to aid in maintaining a constant pH throughout the fermentation.
Process parameters including temperature, dissolved oxygen, pH, stirring, aeration rate, and cell density may be monitored or controlled over the course of the fermentation. For example, temperature of a fermentation process may be monitored by a temperature probe immersed in the culture medium. The culture temperature may be controlled at the set point by regulating the jacket temperature. Water may be cooled in an external chiller and then flowed into the bioreactor control tower and circulated to the jacket at the temperature required to maintain the set point temperature in the vessel.
Additionally, a gas flow parameter may be monitored in a fermentation process. For example, gases may be flowed into the medium through a sparger. Gases suitable for the methods of this disclosure may include compressed air, oxygen, and nitrogen. Gas flow may be at a fixed rate or regulated to maintain a dissolved oxygen set point.
The pH of a culture medium may also be monitored. In examples, the pH may be monitored by a pH probe that is immersed in the culture medium inside the vessel. If pH control is in effect, the pH may be adjusted by acid and base pumps which add each solution to the medium at the required rate. The acid solutions used to control pH may be sulfuric acid or hydrochloric acid. The base solutions used to control pH may be sodium hydroxide, potassium hydroxide, or ammonium hydroxide.
Further, dissolved oxygen may be monitored in a culture medium by a dissolved oxygen probe immersed in the culture medium. If dissolved oxygen regulation is in effect, the oxygen level may be adjusted by increasing or decreasing the stirring speed. The dissolved oxygen level may also be adjusted by increasing or decreasing the gas flow rate. The gas may be compressed air, oxygen, or nitrogen.
Stir speed may also be monitored in a fermentation process. In examples, the stirrer motor may drive an agitator. The stirrer speed may be set at a consistent rpm throughout the fermentation or may be regulated dynamically to maintain a set dissolved oxygen level.
Additionally, turbidity may be monitored in a fermentation process. In examples, cell density may be measured using a turbidity probe. Alternatively, cell density may be measured by taking samples from the bioreactor and analyzing them in a spectrophotometer. Further, samples may be removed from the bioreactor at time intervals through a sterile sampling apparatus. The samples may be analyzed for alkaloids produced by the host cells. The samples may also be analyzed for other metabolites and sugars, the depletion of culture medium components, or the density of cells.
In another example, a feed stock parameter may be monitored during a fermentation process. In particular, feed stocks including sugars and other carbon sources, nutrients, and cofactors that may be added into the fermentation using an external pump. Other components may also be added during the fermentation including, without limitation, anti-foam, salts, chelating agents, surfactants, and organic liquids.
Any convenient codon optimization techniques for optimizing the expression of heterologous polynucleotides in host cells may be adapted for use in the subject host cells and methods, see e.g., Gustafsson, C. et al. (2004) Trends Biotechnol, 22, 346-353, which is incorporated by reference in its entirety.
The subject method may also include adding a starting compound to the cell culture. Any convenient methods of addition may be adapted for use in the subject methods. The cell culture may be supplemented with a sufficient amount of the starting materials of interest (e.g., as described herein), e.g., a mM to μM amount such as between about 1-5 mM of a starting compound. It is understood that the amount of starting material added, the timing and rate of addition, the form of material added, etc., may vary according to a variety of factors. The starting material may be added neat or pre-dissolved in a suitable solvent (e.g., cell culture media, water, or an organic solvent). The starting material may be added in concentrated form (e.g., 10× over desired concentration) to minimize dilution of the cell culture medium upon addition. The starting material may be added in one or more batches, or by continuous addition over an extended period of time (e.g., hours or days).
Methods for Isolating Products from the Fermentation Medium
The subject methods may also include recovering the TA of interest from the cell culture. Any convenient methods of separation and isolation (e.g., chromatography methods or precipitation methods) may be adapted for use in the subject methods to recover the TA of interest from the cell culture. Filtration methods may be used to separate soluble from insoluble fractions of the cell culture. In some cases, liquid chromatography methods (e.g., reverse phase HPLC, size exclusion, normal phase chromatography) may be used to separate the TA of interest from other soluble components of the cell culture. In some cases, extraction methods (e.g., liquid extraction, pH based purification, etc.) may be used to separate the TA of interest from other components of the cell culture.
The produced alkaloids may be isolated from the fermentation medium using methods known in the art. A number of recovery steps may be performed immediately after (or in some instances, during) the fermentation for initial recovery of the desired product. Through these steps, the alkaloids (e.g., TAs) may be separated from the cells, cellular debris and waste, and other nutrients, sugars, and organic molecules may remain in the spent culture medium. This process may be used to yield a TA-enriched product.
In an example, a product stream having a tropane alkaloid (TA) product is formed by providing engineered yeast cells and a feedstock including nutrients and water to a batch reactor. The engineered yeast cells may have at least one modification selected from the group consisting of: a feedback inhibition alleviating mutation in a biosynthetic enzyme gene native to the cell; a transcriptional modulation modification of a biosynthetic enzyme gene native to the cell; and an inactivating mutation in an enzyme native to the cell. When the engineered yeast cells are within the batch reactor, the engineered yeast cells may be subjected to fermentation. In particular, the engineered yeast cells may be subjected to fermentation by incubating the engineered yeast cells for a time period of at least about 5 minutes to produce a solution comprising the TA product and cellular material. Once the engineered yeast cells have been subjected to fermentation, at least one separation unit may be used to separate the TA product from the cellular material to provide the product stream comprising the TA product. In particular, the product stream may include the TA product as well as additional components, such as a clarified yeast culture medium. Additionally, a TA product may comprise one or more TAs of interest, such as one or more TA compounds.
Different methods may be used to remove cells from a bioreactor medium that include a TA of interest. In examples, cells may be removed by sedimentation over time. This process of sedimentation may be accelerated by chilling or by the addition of fining agents such as silica. The spent culture medium may then be siphoned from the top of the reactor or the cells may be decanted from the base of the reactor. Alternatively, cells may be removed by filtration through a filter, a membrane, or other porous material. Cells may also be removed by centrifugation, for example, by continuous flow centrifugation or by using a continuous extractor.
If some valuable TAs of interest are present inside the cells, the cells may be permeabilized or lysed and the cell debris may be removed by any of the methods described above. Agents used to permeabilize the cells may include, without limitation, organic solvents (e.g., DMSO) or salts (e.g., lithium acetate). Methods to lyse the cells may include the addition of surfactants such as sodium dodecyl sulfate, or mechanical disruption by bead milling or sonication.
TAs of interest may be extracted from the clarified spent culture medium through liquid-liquid extraction by the addition of an organic liquid that is immiscible with the aqueous culture medium. Examples of suitable organic liquids include, but are not limited to, isopropyl myristate, ethyl acetate, chloroform, butyl acetate, methylisobutyl ketone, methyl oleate, toluene, oleyl alcohol, ethyl butyrate. The organic liquid may be added to as little as 10% or as much as 100% of the volume of aqueous medium.
In some cases, the organic liquid may be added at the start of the fermentation or at any time during the fermentation. This process of extractive fermentation may increase the yield of TAs of interest from the host cells by continuously removing TA precursors or TAs to the organic phase.
Agitation may cause the organic phase to form an emulsion with the aqueous culture medium. Methods to encourage the separation of the two phases into distinct layers may include, without limitation, the addition of a demulsifier or a nucleating agent, or an adjustment of the pH. The emulsion may also be centrifuged to separate the two phases, for example, by continuous conical plate centrifugation.
Alternatively, the organic phase may be isolated from the aqueous culture medium so that it may be physically removed after extraction. For example, the solvent may be encapsulated in a membrane.
In examples, TAs of interest may be extracted from a fermentation medium using adsorption methods. In particular, TAs of interest may be extracted from clarified spent culture medium by the addition of a resin such as Amberlite® XAD4 or another agent that removes TAs by adsorption. The TAs of interest may then be released from the resin using an organic solvent. Examples of suitable organic solvents include, but are not limited to, methanol, ethanol, ethyl acetate, or acetone.
TAs of interest may also be extracted from a fermentation medium using filtration. At high pH, the TAs of interest may form a crystalline-like precipitate in the bioreactor. This precipitate may be removed directly by filtration through a filter, membrane, or other porous material. The precipitate may also be collected by centrifugation and/or decantation.
The extraction methods described above may be carried out either in situ (in the bioreactor) or ex situ (e.g., in an external loop through which media flows out of the bioreactor and contacts the extraction agent, then is recirculated back into the vessel). Alternatively, the extraction methods may be performed after the fermentation is terminated using the clarified medium removed from the bioreactor vessel.
Methods for Purifying Products from Alkaloid-Enriched Solutions
Subsequent purification steps may involve treating the post-fermentation TA precursor- or TA-enriched product using methods known in the art to recover individual product species of interest to high purity.
In one example, TA precursors or TAs extracted in an organic phase may be transferred to an aqueous solution. In some cases, the organic solvent may be evaporated by heat and/or vacuum, and the resulting powder may be dissolved in an aqueous solution of suitable pH. In a further example, the TA precursors or TAs may be extracted from the organic phase by addition of an aqueous solution at a suitable pH that promotes extraction of the TA precursors or TAs into the aqueous phase. The aqueous phase may then be removed by decantation, centrifugation, or another method.
The TA precursor- or TA-containing solution may be further treated to remove metals, for example, by treating with a suitable chelating agent. The TA precursor- or TA-containing solution may be further treated to remove other impurities, such as proteins and DNA, by precipitation. In one example, the TA precursor- or TA-containing solution is treated with an appropriate precipitation agent such as ethanol, methanol, acetone, or isopropanol. In an alternative example, DNA and protein may be removed by dialysis or by other methods of size exclusion that separate the smaller alkaloids from contaminating biological macromolecules.
In further examples, the TA precursor-, TA-, or modified TA-containing solution may be extracted to high purity by continuous cross-flow filtration using methods known in the art.
If the solution contains a mixture of TA precursors or TAs, it may be subjected to acid-base treatment to yield individual TA of interest species using methods known in the art. In this process, the pH of the aqueous solution is adjusted to precipitate individual TA precursors or TAs at their respective pKas.
For high purity, small-scale preparations, the TA precursors or TAs may be purified in a single step by liquid chromatography.
The clarified yeast culture medium (CYCM) may contain a plurality of impurities. The clarified yeast culture medium may be dehydrated by vacuum and/or heat to yield an alkaloid-rich powder. This product is analogous to the concentrate of nightshade leaves (CNL), which is used by active pharmaceutical ingredient (API) manufacturers for extraction of tropane alkaloids to be subjected to further chemical processing and purification. For the purposes of this invention, CNL is a representative example of any type of purified plant extract from which the desired alkaloids product(s) may ultimately be further purified. Table 5 highlights the impurities in these two products that may be specific to either CYCM or CNL or may be present in both. By analyzing a product of unknown origin for a subset of these impurities, a person of skill in the art could determine whether the product originated from a yeast or plant production host.
API-grade pharmaceutical ingredients are highly purified molecules. As such, impurities that could indicate the plant- or yeast-origin of an API (such as those listed in Tables 2 and 3) may not be present at that API stage of the product. Indeed, many of the API products derived from yeast strains of the present invention may be largely indistinguishable from the traditional plant-derived APIs. In some cases, however, conventional alkaloid compounds may be subjected to chemical modification using chemical synthesis approaches which may show up as chemical impurities in plant-based products that require such chemical modifications. For example, chemical derivatization may often result in a set of impurities related to the chemical synthesis processes. In certain situations, these modifications may be performed biologically in the yeast production platform, thereby avoiding some of the impurities associated with chemical derivation from being present in the yeast-derived product. In particular, these impurities from the chemical derivation product may be present in an API product that is produced using chemical synthesis processes but may be absent from an API product that is produced using a yeast-derived product. Alternatively, if a yeast-derived product is mixed with a chemically derived product, the resulting impurities may be present but in a lesser amount than would be expected in an API that only or primarily contains chemically derived products. In this example, by analyzing the API product for a subset of these impurities, a person of skill in the art could determine whether the product originated from a yeast production host or the traditional chemical derivatization route.
Non-limiting examples of impurities that may be present in chemically-derivatized tropane alkaloid APIs but not in biosynthesized APIs include hydrogen halides such as hydrogen chloride, hydrogen iodide, and hydrogen bromide formed by chemical N-alkylation, such as N-methylation and N-butylation of hyoscyamine and scopolamine.
However, in the case where the yeast-derived compound and the plant-derived compound are both subjected to chemical modification through chemical synthesis approaches, the same impurities associated with the chemical synthesis process may be expected in the products. In such a situation, the starting material (e.g., CYCM or CNL) may be analyzed as described above.
Methods of Engineering Host Cells
Also included are methods of engineering host cells for the purpose of producing TAs of interest or precursors thereof. Inserting DNA into host cells may be achieved using any convenient methods. The methods are used to insert the heterologous coding sequences into the host cells such that the host cells functionally express the enzymes and convert starting compounds of interest into product TAs of interest.
Any convenient promoters may be utilized in the subject host cells and methods.
The promoters driving expression of the heterologous coding sequences may be constitutive promoters or inducible promoters, provided that the promoters are active in the host cells. The heterologous coding sequences may be expressed from their native promoters, or non-native promoters may be used. Such promoters may be low to high strength in the host in which they are used. Promoters may be regulated or constitutive. In certain embodiments, promoters that are not glucose repressed, or repressed only mildly by the presence of glucose in the culture medium, are used. Promoters of interest include but are not limited to, promoters of glycolytic genes such as the promoter of the B. subtilis tsr gene (encoding the promoter region of the fructose bisphosphate aldolase gene) or the promoter from yeast S. cerevisiae gene coding for glyceraldehyde 3-phosphate dehydrogenase (GPD, GAPDH, or TDH3), the ADH1 promoter of baker's yeast, the phosphate-starvation induced promoters such as the PHOS promoter of yeast, the alkaline phosphatase promoter from B. licheniformis, yeast inducible promoters such as Gal1-10, Gal1, GalL, GalS, repressible promoter Met25, tetO, and constitutive promoters such as glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation-elongation factor-1-α promoter (TEF), cytochrome c-oxidase promoter (CYC1), MRP7 promoter, phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI), etc. Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones may also be used and include, but are not limited to, the glucorticoid responsive element (GRE) and thyroid hormone responsive element (TRE). These and other examples are described U.S. Pat. No. 7,045,290, which is incorporated by reference, including the references cited therein. Additional vectors containing constitutive or inducible promoters such as a factor, alcohol oxidase, and PGH may be used. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of genes. Any convenient appropriate promoters may be selected for the host cell, e.g., E. coli. One may also use promoter selection to optimize transcript, and hence, enzyme levels to maximize production while minimizing energy resources.
Any convenient vectors may be utilized in the subject host cells and methods. Vectors of interest include vectors for use in yeast and other cells. The types of yeast vectors may be broken up into 4 general categories: integrative vectors (YIp), autonomously replicating high copy-number vectors (YEp or 2p plasmids), autonomously replicating low copy-number vectors (YCp or centromeric plasmids) and vectors for cloning large fragments (YACs). Vector DNA is introduced into prokaryotic or eukaryotic cells via any convenient transformation or transfection techniques.
The host cells and methods of the invention, e.g., as described above, find use in a variety of applications. Applications of interest include, but are not limited to: research applications and therapeutic applications. Methods of the invention find use in a variety of different applications including any convenient application where the production of TAs is of interest.
The subject host cells and methods find use in a variety of therapeutic applications. Therapeutic applications of interest include those applications in which the preparation of pharmaceutical products that include TAs is of interest. The host cells described herein produce tropane alkaloid precursors (TA precursors) and TAs of interest. Tropinone and tropine are major branch point intermediates of interest in the synthesis of TAs including engineering efforts to produce end products such as medicinal TA products. The subject host cells may be utilized to produce TA precursors from simple and inexpensive starting materials that may find use in the production of TAs of interest, including tropinone, tropine, and TA end products. As such, the subject host cells find use in the supply of therapeutically active TAs or precursors thereof.
In some instances, the host cells and methods find use in the production of commercial scale amounts of TAs or precursors thereof where chemical synthesis of these compounds is low yielding and not a viable means for large-scale production. In certain cases, the host cells and methods are utilized in a fermentation facility that would include bioreactors (fermenters) of e.g., 5,000-200,000 liter capacity allowing for rapid production of TAs of interest or precursors thereof for therapeutic products. Such applications may include the industrial-scale production of TAs of interest from fermentable carbon sources such as cellulose, starch, and free sugars.
The subject host cells and methods find use in a variety of research applications. The subject host cells and methods may be used to analyze the effects of a variety of enzymes on the biosynthetic pathways of a variety of TAs of interest or precursors thereof. In addition, the host cells may be engineered to produce TAs or precursors thereof that find use in testing for bioactivity of interest in as yet unproven therapeutic functions. In some cases, the engineering of host cells to include a variety of heterologous coding sequences that encode for a variety of enzymes elucidates the high yielding biosynthetic pathways towards TAs of interest or precursors thereof. In certain cases, research applications include the production of precursors for therapeutic molecules of interest that may then be further chemically modified or derivatized to desired products or for screening for increased therapeutic activities of interest. In some instances, host cell strains are used to screen for enzyme activities that are of interest in such pathways, which may lead to enzyme discovery via conversion of TA metabolites produced in these strains.
The subject host cells and methods may be used as a production platform for plant specialized metabolites. The subject host cells and methods may be used as a platform for drug library development as well as plant enzyme discovery. For example, the subject host cells and methods may find use in the development of natural product based drug libraries by taking yeast strains producing interesting scaffold molecules, such as hyoscyamine and scopolamine, and further functionalizing the compound structure through combinatorial biosynthesis or by chemical means. By producing drug libraries in this way, any potential drug hits are already associated with a production host that is amenable to large-scale culture and production. As another example, these subject host cells and methods may find use in plant enzyme discovery. The subject host cells provide a clean background of defined metabolites to express plant expressed sequence tag (EST) libraries to identify new enzyme activities. The subject host cells and methods provide expression methods and culture conditions for the functional expression and increased activity of plant enzymes in yeast.
Aspects of the invention further include kits and systems, where the kits and systems may include one or more components employed in methods of the invention, e.g., host cells, starting compounds, heterologous coding sequences, vectors, culture medium, etc., as described herein. In some embodiments, the subject kit includes a host cell (e.g., as described herein), and one or more components selected from the following: starting compounds, a heterologous coding sequence and/or a vector including the same, vectors, growth feedstock, components suitable for use in expression systems (e.g., cells, cloning vectors, multiple cloning sites (MCS), bi-directional promoters, an internal ribosome entry site (IRES), etc.), and a culture medium.
Any of the components described herein may be provided in the kits, e.g., host cells including one or more modifications, starting compounds, culture medium, etc. A variety of components suitable for use in making and using heterologous coding sequences, cloning vectors and expression systems may find use in the subject kits. Kits may also include tubes, buffers, etc., and instructions for use. The various reagent components of the kits may be present in separate containers, or some or all of them may be pre-combined into a reagent mixture in a single container, as desired.
Also provided are systems for producing a TA of interest, where the systems may include engineered host cells including one or more modifications (e.g., as described herein), starting compounds, culture medium, a fermenter and fermentation equipment, e.g., an apparatus suitable for maintaining growth conditions for the host cells, sampling and monitoring equipment and components, and the like. A variety of components suitable for use in large scale fermentation of yeast cells may find use in the subject systems.
In some cases, the system includes components for the large scale fermentation of engineered host cells, and the monitoring and purification of TA compounds produced by the fermented host cells. In certain embodiments, one or more starting compounds (e.g., as described herein) are added to the system, under conditions by which the engineered host cells in the fermenter produce one or more desired TA products or precursors thereof. In some instances, the host cells produce a TA of interest (e.g., as described herein). In certain cases, the TA products of interest are medicinal TA products, such as hyoscyamine, N-methylhyoscyamine, anisodamine, scopolamine, N-methylscopolamine, and N-butylscopolamine.
In some cases, the system includes means for monitoring and or analyzing one or more TA compounds or precursors thereof produced by the subject host cells. For example, a LC-MS analysis system as described herein, a chromatography system, or any convenient system where the sample may be analyzed and compared to a standard, e.g., as described herein. The fermentation medium may be monitored at any convenient times before and during fermentation by sampling and analysis. When the conversion of starting compounds to TA products or precursors of interest is complete, the fermentation may be halted and purification of the TA products may be done. As such, in some cases, the subject system includes a purification component suitable for purifying the TA products or precursors of interest from the host cell medium into which it is produced. The purification component may include any convenient means that may be used to purify the TA products or precursors of fermentation, including but not limited to, silica chromatography, reverse-phase chromatography, ion exchange chromatography, HIC chromatography, size exclusion chromatography, liquid extraction, and pH extraction methods. In some cases, the subject system provides for the production and isolation of TA fermentation products of interest following the input of one or more starting compounds to the system.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
The following section provides examples of methods and procedures which can be used to construct, culture, and test microbial strains, such as yeast strains, for the production of TA precursors and TAs, as well as to conduct fermentations of such strains to produce TA precursors and TAs. Also included are examples of methods, procedures, and materials which can be used to generate the DNA sequences required for modification of microbial hosts, and to introduce desired DNA sequences into microbial hosts.
Chemical compounds and standards. Chemical standards of TA precursors and TAs for verifying the identity of and quantifying metabolites produced by engineered host cells may be purchased from commercial vendors. For example, putrescine dihydrochloride, N-methylputrescine, hygrine, tropinone, and tropine may be purchased from Santa Cruz Biotechnology (Dallas, Tex.). 4-(Methylamino)butyric acid hydrochloride may be purchased from Sigma (St. Louis, Mo.). γ-Methylaminobutyraldehyde (4MAB) diethyl acetal and littorine may be purchased from Toronto Research Chemicals (Toronto, ON). A chemical standard for NMPy can be synthesized by deprotecting one volume of the diethyl acetal with five volumes of 2 M HCl at 60° C. for 30 min as described previously (see Feth, F., Wray, V. & Wagner, K. G. Determination of methylputrescine oxidase by high performance liquid chromatography. Phytochemistry 24, 1653-1655 (1985)), incubating overnight at room temperature, and then washing the resulting concentrate twice with three volumes of diethyl ether to remove residual organic impurities.
Plasmid construction. Oligonucleotides used for generation of novel DNA sequences by polymerase chain reaction (PCR) and for DNA sequencing can be obtained from a DNA synthesis company, such as IDT DNA, Twist Bioscience, or the Stanford Protein and Nucleic Acid Facility (Stanford, Calif.). Native yeast genes can be amplified from S. cerevisiae genomic DNA via colony PCR (see Kwiatkowski, T. J., Zoghbi, H. Y., Ledbetter, S. A., Ellison, K. A. & Chinault, A. C. Rapid identification of yeast artificial chromosome clones by matrix pooling and crude iysate PCR. Nucleic Acids Res. 18, 7191 (1990)). Gene sequences for heterologous enzymes may be codon-optimized to improve expression in S. cerevisiae using suitable codon optimization software, such as the GeneArt GeneOptimizer software (Thermo Fisher Scientific). Heterologous gene sequences can then be synthesized as linear, double-stranded DNA fragments by a commercial DNA synthesis company. Two types of plasmids can be used for gene expression in yeast: direct expression (DE) plasmids for testing biosynthetic genes of interest and yeast integration (YI) holding plasmids to provide a template for genomic integration of selected promoter-gene-terminator cassettes.
DE plasmids comprise a gene of interest flanked by a constitutive promoter and terminator, a low-copy CEN6/ARS4 yeast origin of replication, and an auxotrophic selection marker. DE plasmids may be constructed by PCR-amplifying genes of interest to append 5′ and 3′ restriction sites using primer overhangs, digesting PCR products or synthesized gene fragments with appropriate pairs of restriction enzymes (for example, SpeI, BamHI, EcoRI, PstI, or XhoI), and then ligating gene fragments into similarly digested vectors with suitable yeast promoters, terminators, and replication sequences, such as plasmids pAG414GPD-ccdB, pAG415GPD-ccdB, or pAG416GPD-ccdB (see Alberti, S., Gitler, A. D. & Lindquist, S. A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast 24, 913-9 (2007)) using T4 DNA ligase.
YI plasmids comprise a gene of interest flanked by a constitutive promoter and terminator but lack a yeast origin of replication or auxotrophic selection marker. YI plasmids may be constructed by linearizing empty holding vectors with suitable promoters and terminators using ‘around-the-horn’ PCR with primers designed to bind at the 3′ and 5′ ends of the promoter and terminator, respectively. Genes of interest can also be PCR-amplified to append 5′ and 3′ overhangs with 35-40 bp of homology to the termini of the linearized vector backbones. Assembly of genes into YI vectors may then be performed using Gibson assembly. DE plasmids expressing GFP fusions of biosynthetic enzymes may be prepared by first assembling PCR-amplified DNA fragments separately encoding GFP, the target enzyme, and a YI vector backbone using Gibson assembly, and subsequently subcloning the fusion constructs from YI plasmids into DE vectors using restriction enzymes and ligation cloning as described.
PCR amplification may be performed using any high-fidelity recombinant DNA polymerase available from commercial suppliers and linear DNA may be purified using a suitable DNA column purification kit. Assembled plasmids can be propagated in any chemically competent E. coli strain using heat-shock transformation and selection in Luria-Bertani (LB) broth or on LB-agar plates with carbenicillin (100 μg/mL), kanamycin (50 μg/mL), or another antibiotic selection. Plasmid DNA can be isolated by alkaline lysis from overnight E. coli cultures grown at 37° C. and 250 rpm in selective LB media using plasmid purification columns according to the manufacturer's protocol. Plasmid sequences should be verified by Sanger sequencing.
Yeast strain construction. Any suitable laboratory strain of yeast may be used as a host organism. Yeast strains described in the examples of the Experimental section are derived from the parental strain CEN.PK2-1 D (see Entian, K. D. & Kötter, P. 25 Yeast Genetic Strain and Plasmid Collections. Methods Microbiol. 36, 629-666 (2007)), referred to as CEN.PK2. Strains can be grown non-selectively in yeast-peptone media supplemented with 2% w/v dextrose (YPD media), yeast nitrogen base (YNB) defined media supplemented with synthetic complete amino acid mixture (YNB-SC) and 2% w/v dextrose, or on agar plates of the aforementioned media. Strains transformed with plasmids bearing auxotrophic selection markers (URA3, TRP1, HIS3, and/or LEU2) may be grown selectively in YNB media supplemented with 2% w/v dextrose and the appropriate dropout solution (YNB-DO) or on YNB-DO agar plates. Yeast strains which are deficient in acetate metabolism can be grown on the aforementioned media supplemented with 0.1% w/v potassium acetate (i.e., YPAD or YNBA).
Yeast genomic modifications may be performed using the CRISPRm method (see Ryan, O. W. et al. Selection of chromosomal DNA libraries using a multiplex CRISPR system. Elife 3, 1-15 (2014)). CRISPRm plasmids express Streptococcus pyogenes Cas9 and a single guide RNA (sgRNA) targeting a locus of interest in the yeast genome, and may be constructed by assembly PCR and Gibson assembly of DNA fragments encoding SpCas9, tRNA promoter and HDV ribozyme, a 20-nt guide RNA sequence, and tracrRNA and terminator. For gene insertions, integration fragments comprising one or more genes of interest flanked by unique promoters and terminators may be constructed using PCR amplification and cloned into holding vectors by Gibson assembly. Integration fragments are PCR amplified using a suitable high-fidelity DNA polymerase with flanking 40 bp microhomology regions to adjacent fragments and/or to the yeast genome at the integration site. For gene disruptions, integration fragments comprise 6-8 stop codons in all three reading frames flanked by 40 bp of microhomology to the disruption site, which is located within the first half of the open reading frame. For complete gene deletions, integration fragments comprise an auxotrophic marker gene flanked by 40 bp of microhomology to the deletion site. Each integration fragment is co-transformed with the CRISPRm plasmid targeting the desired genomic site. Positive integrants may be identified by yeast colony PCR, Sanger sequencing, and/or functional screening by liquid chromatography and tandem mass spectrometry (LC-MS/MS).
Yeast transformations. Yeast strains may be transformed using any suitable method, including heat-shock, electroporation, and chemical transformation. For example, yeast strains described in the examples of the Experimental section were chemically transformed using the Frozen-EZ Yeast Transformation II Kit (Zymo Research). Individual yeast colonies are inoculated into YP(A)D media and grown overnight at 30° C. and 250 rpm. Saturated cultures are back-diluted between 1:10 and 1:50 in YP(A)D media and grown for an additional 5-7 hours to reach exponential phase. Cultures are pelleted by centrifugation at 500×g for 4 min and then washed twice by resuspending the pellet in 50 mM Tris-HCl buffer, pH 8.5. Washed pellets are resuspended in 20 μL of EZ2 solution per transformation and then mixed with 100-600 ng of total DNA and 200 μL of EZ3 solution. The yeast suspensions are incubated at 30° C. with gentle rotation for one hour. For plasmid transformations, the transformed yeast are directly plated onto YNB(A)-DO agar plates. For Cas9-mediated chromosomal modifications, yeast suspensions are instead mixed with 1 mL YP(A)D media, pelleted by centrifugation at 500×g for 4 min, and then resuspended in 250 μL of fresh YP(A)D media. The suspensions are then incubated at 30° C. with gentle rotation for an additional two hours to enable production of G418 resistance proteins and then spread onto YP(A)D plates containing 400 mg/L G418 (geneticin) sulfate. Plates are then incubated at 30° C. for 48-60 hours to allow colony formation.
Spot dilution assays. Strains are inoculated into YNB(A)-DO media and grown overnight at 30° C. and 250 rpm. Saturated overnight cultures are pelleted by centrifugation at 500×g for 4 min and resuspended in sterile Tris-HCl buffer, pH 8.0 to a concentration of 107 cells/mL based on OD600. Ten-fold serial dilutions of each strain are then prepared in Tris-HCl buffer and 10 μL of each dilution is spotted on pre-warmed YNB(A)-DO plates. Plates are incubated at 30° C. and imaged after 48 hours.
Growth conditions for metabolite assays. Small-scale metabolite production tests may be conducted in YNB(A)-SC or YNB(A)-DO media. Yeast colonies may be inoculated into 300-500 μL of media and grown in 2 mL deep-well 96-well plates covered with a gas-permeable film for 48-72 hours at 30° C., 460 rpm, and 80% relative humidity in a shaker.
Analysis of metabolite production. Metabolite profiles and titers may be analyzed using liquid chromatography and tandem mass spectrometry (LC-MS/MS). To separate cells from media for analysis, fermentation cultures may be pelleted by centrifugation at 3,500×g for 5 min at 12° C. and 100-200 μL aliquots of the supernatant can then be removed for direct analysis. Metabolite production may be analyzed by LC-MS/MS using any suitable HPLC device paired with a triple quadrupole mass spectrometer, such as the Agilent 1260 Infinity Binary HPLC and Agilent 6420 Triple Quadrupole mass spectrometer. Chromatography may be performed using a C18 reverse phase column, such as a Zorbax EclipsePlus C18 column (2.1×50 mm, 1.8 μm; Agilent Technologies), with 0.1% v/v formic acid in water as mobile phase solvent A and 0.1% v/v formic acid in acetonitrile as solvent B. The column is operated with a constant flow rate of 0.4 mL/min at 40° C. and a sample injection volume of 5 μL. Compound separation may be performed using the following gradient: 0.00-0.75 min, 1% B; 0.75-1.33 min, 1-25% B; 1.33-2.70 min, 25-40% B; 2.70-3.70 min, 40-60% B; 3.70-3.71 min, 60-95% B; 3.71-4.33 min, 95% B; 4.33-4.34 min, 95-1% B; 4.34-5.00 min, equilibration with 1% B. The LC eluent is directed to the MS from 0.01-5 min operating with electrospray ionization (ESI) in positive mode, source gas temperature 350° C., gas flow rate 11 L/min, and nebulizer pressure 40 psi. Metabolites can be quantified by integrated peak area based on multiple reaction monitoring (MRM) parameters and standard curves.
Fluorescence microscopy. Individual colonies of yeast strains transformed with plasmids encoding biosynthetic enzymes fused to fluorescent protein reporters are inoculated into 1 mL YNB-DO media and grown overnight at 30° C. and 250 rpm. Overnight cultures are pelleted by centrifugation at 500×g for 4 min and resuspended in 2 mL YNB-DO media with 2% w/v dextrose and then grown at 30° C. and 250 rpm for an additional 4-6 hours to reach exponential phase and allow expressed fluorescent proteins to fold completely. Approximately 5-10 μL of culture is then spotted onto a glass microscope slide and covered with a glass coverslip and then imaged using a suitable inverted fluorescence microscope with a 60× oil immersion objective. Fluorescence excitation may be performed using a xenon arc lamp and the following filter settings: GFP, ET470/40X excitation filter and ET525/50 emission filter; mCherry, ET572/35X excitation filter and ET632/60 emission filter. Emitted light is captured with a CCD camera, and subsequent image analysis may performed in any suitable scientific image analysis software, such as ImageJ (NIH).
Identification of novel gene variants from transcriptome databases. Novel genes and variants thereof may be identified using sequence alignment-based searches of transcriptome and genome databases. For example, orthologs of N. tabacum N-methylputrescine oxidase (NtMPO1) were identified using a tBLASTn search of the transcriptomes of D. metel and A. belladonna in the 1000 Plants Project database (see Matasci, N. et al. Data access for the 1,000 Plants (1KP) project. Gigascience 3, 17 (2014)). Coding sequences for putative genes identified using these search strategies can then be optimized for yeast expression and then cloned into expression vectors as described previously.
Enzyme structural analysis. Heterologous enzymes may be analyzed for structural features that may prove problematic during expression in yeast, such as large unstructured regions, by examining homology models constructed using any suitable homology modeling or de novo structure prediction software, such as RaptorX or Rosetta. Resultant protein models can be visualized using any three-dimensional molecular viewing software, such as PyMOL (Schrodinger) or UCSF Chimera. Enzyme affinity for specific substrates may be analyzed using any suitable ligand docking simulation software, such as AutoDock, SwissDock, GOLD, or Glide.
Analysis of protein expression in yeast by Western blot. For immunoblot analysis of yeast-expressed proteins, a suitable strain is transformed with an expression vector harboring an epitope-tagged protein of interest. Three days post-transformation, transformed colonies are inoculated into 2 mL YNB-DO media and grown overnight (˜16-20 h) to stationary phase at 30° C. and 460 rpm. Cells are pelleted by centrifugation at 3,000×g for 5 min, resuspended in 200 μL H2O, mixed with 200 μL of 0.2 M NaOH, and incubated at room temperature for 5 min to allow hydrolysis of cell wall glycoproteins. Cells are re-pelleted at 3,000×g for 5 min, resuspended in 75 μL H2O, mixed with 25 μL of 4× NuPAGE LDS sample buffer (Thermo Fisher), and then boiled at 95° C. for 3 min to lyse cells. Suspensions are pelleted by centrifugation at 16,000×g for 5 min to remove insoluble debris and supernatants are transferred to pre-chilled tubes. For analysis under reducing conditions, protein lysates are mixed with β-mercaptoethanol (final concentration 10%) and incubated at 70° C. for 10 min. Approximately 20-40 μg of total protein is loaded onto NuPAGE Bis-Tris 4-12% acrylamide gels (Thermo Fisher) with Precision Plus Dual Color protein molecular weight marker (BioRad). Electrophoresis is conducted in 1× NuPAGE MOPS SDS running buffer at 150 V for 90 min. Transfer of protein to a nitrocellulose membranes is performed at 15 V for 15 min using a Trans-Blot Semi-Dry apparatus (BioRad) and NuPAGE transfer buffer (Thermo Fisher) per manufacturer instructions. For reducing conditions, NuPAGE antioxidant (Thermo Fisher) is added to a final concentration of 1× to both the running buffer and transfer buffer. Membranes with transferred protein are washed for 5 min in Tris-buffered saline with Tween (TBS-T; 137 mM NaCl, 2.7 mM KCl, 19 mM Tris base, 0.1% Tween20, pH 7.4) and then blocked with 5% skim milk in TBS-T for 1 h at room temperature. Membranes are incubated overnight at 4° C. with a suitable dilution of an HRP-conjugated antibody in TBS-T with 5% milk, washed three times for 5 min each with TBS-T, and then visualized using Western Pico PLUS HRP substrate (Thermo Fisher) and a suitable imager.
A series of specific genetic modifications provide a biosynthetic process in Saccharomyces cerevisiae for the production of TAs from simple, inexpensive feedstocks or precursor molecules. Methods for constructing novel strains capable of producing the early TA molecules putrescine, N-methylputrescine, 4-methylaminobutanal, N-methylpyrrolinium (NMPy), tropinone, tropine, phenyllactic acid (PLA), and 1-O-β-phenyllactoylglucose (PLA glucoside) from non-TA precursors or simple feedstocks are described. NMPy is the natural precursor to all known TA molecules. Methods for manipulating the regulation of yeast biosynthetic pathways and for optimizing the production of amino acid-derived TA precursors are also described. Methods for constructing novel strains capable of producing non-medicinal TAs such as pseudotropine alkaloids and calystegines from simple feedstocks are described. Additionally, methods for constructing novel strains capable of producing medicinal TAs such as hyoscyamine, anisodamine, and scopolamine from non-TA precursors or simple feedstocks are described. Furthermore, methods for constructing novel strains capable of producing non-natural TAs such as cinnamoyltropine from non-TA precursors or simple feedstocks are described.
The tropine moiety of TAs is derived from the amino acid arginine via the polyamine molecule putrescine. Strains of S. cerevisiae are developed with improved flux through the arginine and polyamine biosynthesis pathways for the purposes of increasing intracellular concentrations of TA precursor molecules including putrescine, NMP, 4MAB, and NMPy. These strains combine genetic modifications for the purpose of increasing carbon and nitrogen flux from central metabolism towards arginine and polyamine biosynthesis in general, and include the introduction of key heterologous enzymes for additional production of the TA precursor putrescine. Genetic modifications are employed including the introduction of feedback inhibition alleviating mutations to genes encoding native biosynthetic enzymes and regulatory proteins, tuning of transcriptional regulation of native biosynthetic enzymes, deletion or disruption of genes encoding enzymes that divert precursor molecules away from the intended pathway, and introduction of heterologous enzymes for the conversion of endogenous molecules into TA precursor molecules.
1.1) The biosynthetic pathway in the engineered strain incorporates overexpression of native yeast genes involved in arginine metabolism and polyamine biosynthesis (
1.1.1) Examples of overexpressed native genes in yeast include, but are not limited to: glutamate N-acetyltransferase (Arg2p), which catalyzes the first step in arginine biosynthesis from glutamate; arginase (Car1p), which removes the guanidinium group of arginine to produce ornithine in the mitochondrial matrix; a mitochondrial membrane transporter (Ort1p), which exports ornithine from the mitochondrial matrix to the cytosol; ornithine decarboxylase (Spe1p), which decarboxylates cytosolic ornithine to putrescine; and a polyamine oxidase (Fms1p), which dealkylates spermine and spermidine to putrescine.
1.1.2) The impact of overexpression of these native enzymes on putrescine production was examined by co-transforming a yeast strain with different combinations of three low-copy plasmids, each expressing one of SPE1, ORT1, CAR1, ARG2, FMS1, or blue fluorescent protein (BFP) as a negative control. The titer of putrescine accumulated in the extracellular medium of co-transformed cells following 48 hours of growth in selective media was quantified by LC-MS/MS (
1.2) The biosynthetic pathway in the engineered strain incorporates expression of heterologous enzymes from polyamine production pathways found in organisms other than yeast to further increase putrescine production (
1.2.1) In addition to the ornithine-dependent pathway found in most plants, animals, and fungi, whereby putrescine is synthesized via deguanidination of arginine followed by decarboxylation of ornithine, many bacteria and plants also express an alternate route through which arginine is first decarboxylated by arginine decarboxylase (ADC) to yield agmatine. In plants, the guanidine group of agmatine is then converted to a urea by an iminohydrolase (AIH) to produce N-carbamoylputrescine (NCP), from which the amide group is then removed by an amidase (CPA) to yield putrescine (see Patel, J. et al. Dual functioning of plant arginases provides a third route for putrescine synthesis. Plant Sci. 262, 62-73 (2017)). Some bacteria have evolved an agmatine ureohydrolase (AUH) enzyme that enables direct removal of the guanidine group from agmatine to produce putrescine without an N-carbamoylated intermediate (see Klein, R. D. et al. Reconstitution of a bacterial/plant polyamine biosynthesis pathway in Saccharomyces cerevisiae. Microbiology 145 (Pt 2, 301-7 (1999)).
1.2.2) To reconstruct the heterologous putrescine biosynthetic pathways in yeast, the following enzymes may be used: ADC, AIH, CPA, and AUH. As an example of an engineered strain which possesses these enzymatic activities, an ADC from oat (Avena sativa; AsADC) with previously demonstrated activity in S. cerevisiae (see Klein, R. D. et al. Reconstitution of a bacterial/plant polyamine biosynthesis pathway in Saccharomyces cerevisiae. Microbiology 145 (Pt 2, 301-7 (1999)), an AIH from Arabidopsis thaliana (AtAIH), two CPA orthologs from tomato (Solanum lycopersicum; SICPA) and A. thaliana (AtCPA), and two AUHs from E. coli (speB) and A. thaliana (AtARGAH2) were selected for expression in yeast.
1.2.3) In order to establish the functionality of each heterologous enzyme in yeast, the three-step (arginine→agmatine→NCP→putrescine) or two-step (arginine→agmatine→putrescine) putrescine pathways were reconstituted in a stepwise fashion by co-transforming the wild-type yeast strain with low-copy plasmids expressing AsADC, AtAIH, and either S/CPA or AtCPA; or AsADC and either speB or AtARGAH2. To eliminate effects on cell growth and metabolite production arising from different levels of auxotrophy, all transformations were performed with three low-copy plasmids harboring different auxotrophic markers, using BFP as a negative control in place of a blank or absent plasmid. The relative accumulation of agmatine, NCP, and putrescine in the extracellular medium of transformed cells following 48 hours of growth in selective media were analyzed by LC-MS/MS, which indicated that all enzymes except for SICPA and AtARGAH2 retained activity in yeast (
1.3) The biosynthetic pathway in the engineered strain incorporates overexpression of native yeast genes involved in arginine and polyamine biosynthesis and expression of heterologous biosynthetic enzymes from polyamine production pathways found in organisms other than yeast to further increase putrescine production.
1.3.1) The top-performing triad of overexpressed native genes for putrescine biosynthesis (SPE1, ARG2, CAR1; 1.1.2) was combined with the top-performing heterologous putrescine pathway (AsADC, speB; 1.2.3) by co-transforming the wild-type yeast strain with a low-copy plasmid encoding SPE1, AsADC, and speB and low-copy plasmids encoding ARG2 and CAR1. Putrescine titers in the culture medium of transformed cells were measured by LC-MS/MS analysis after 48 hours. The resulting strain produced putrescine at titers of 47 mg/L, (
1.4) Polyamine biosynthesis in yeast is regulated by several mechanisms (
1.4.1) Native yeast genes involved in regulation of polyamine biosynthesis, and which may therefore be disrupted to improve intracellular putrescine accumulation, include but are not limited to the following examples (
1.4.2) Yeast single-gene disruption strains for each of MEU1, OAZ1, SPE4, SKY1, and AGP2 were constructed by inserting a series of tandem nonsense mutations within the first third of each open reading frame in wild-type yeast. To characterize the effects of each regulatory disruption in the context of the native and heterologous putrescine production pathways, either yeast ODC (SPE1) was overexpressed, or AsADC and speB were co-expressed from low-copy plasmids in each of the single-gene disruption strains. Putrescine titers in the extracellular medium were measured via LC-MS/MS after 72 hours of growth (
1.5) The biosynthetic pathway in the engineered strain combines the MEU1 and OAZ1 regulatory gene knockouts with overexpression of the native and heterologous putrescine biosynthetic genes in order to further increase putrescine production in the engineered strain. Additional copies of the native arginine and polyamine biosynthetic genes ARG2, CAR1, and FMS1 were integrated into the genome of a meu1/oaz1 double-disruption strain. This strain was transformed with a low-copy plasmid expressing SPE1, AsADC, and speB. LC-MS/MS analysis of the extracellular medium of this transformed strain indicated that putrescine titers reached 86 mg/L after 48 hours of growth in selective media (
Strains of S. cerevisiae are developed by modifying the putrescine-overproducing strain developed in Example 1 for the production of the TA precursor NMPy. These strains combine genetic modifications for the purpose of increasing carbon and nitrogen flux from putrescine towards NMPy biosynthesis, and include the introduction of key heterologous enzymes for production of the TA precursors NMP, 4MAB, and NMPy. Genetic modifications are employed including modification of the N- and/or C-terminal domains of enzymes of interest to improve activity in a heterologous host, and deletion or disruption of genes encoding enzymes that diver precursor molecules away from the intended pathway.
2.1) The biosynthetic pathway in the engineered strain enables production of NMPy from endogenous putrescine. Putrescine is first converted to N-methylputrescine (NMP) by a SAM-dependent N-methyltransferase (PMT), which is subsequently oxidized to 4-methylaminobutanal (4MAB) by a copper-dependent diamine oxidase (MPO). 4MAB, like many aldehyde compounds, is unstable in aqueous solution and spontaneously cyclizes via base-catalyzed nucleophilic attack to form NMPy (
2.1.1) The putrescine overproducing strain of Example 1.5, which harbors a low-copy plasmid expressing SPE1, AsADC, and speB for putrescine overproduction, was co-transformed with additional low-copy plasmids expressing a PMT from A. belladonna (AbPMT1) and a subsequent MPO enzyme from Nicotiana tabacum (NtMPO1). The accumulation of intermediates in the extracellular medium of transformed cells expressing each successive enzyme between putrescine and NMPy was compared via LC-MS/MS analysis after 48 hours of growth. The immediate product of NtMPO1 (4MAB) as well as its spontaneous cyclization product (NMPy) were produced with expression of AbPMT1 and NtMPO1 (
2.1.2) The accumulation of NMP was measured in the growth medium of putrescine-overproducing yeast strains with and without disruption of the MEU1 gene (described in Example 1.4.2) by LC-MS/MS analysis. This analysis indicated that the prior disruption of MEU1 in the putrescine-overproducing strain and its concomitant impact on SAM recycling did not inhibit putrescine N-methylation by AbPMT1 (
2.2) Enzymes may localize to different sub-cellular compartments when heterologously expressed than in their original host organism, resulting in reduced function. The biosynthetic pathway in the engineered strain may incorporate modifications to the polypeptide sequences of native and heterologous enzymes to induce localization of these modified enzymes to sub-cellular compartments other than those to which they localize naturally. For example, prior studies have shown that while NtPMT is expressed in the cytosol of tobacco cells, NtMPO1 localizes to the peroxisome lumen (see Naconsie, M., Kato, K., Shoji, T. & Hashimoto, T. Molecular evolution of n-methylputrescine oxidase in Tobacco. Plant Cell Physiol. 55, 436-444 (2014)).
2.2.1) The sub-cellular localization of NtMPO1 was examined by performing in silico prediction of enzyme subcellular localization using the SherLoc2 utility for signal peptide detection (see Briesemeister, S. et al. SherLoc2: A high-accuracy hybrid method for predicting subcellular localization of proteins. J. Proteome Res. 8, 5363-5366 (2009)). This analysis indicated that NtMPO1 harbors a strong yeast consensus peroxisome-targeting sequence (PTS) at its C-terminus (Ala-Lys-Leu, denoted PTS1), which suggests that NtMPO1 may localize to peroxisomes when expressed heterologously in yeast (
2.2.2) Fluorescence microscopy of wild-type yeast cells expressing either N- or C-terminal GFP-tagged AbPMT1 and NtMPO1 from low-copy plasmids indicated that while AbPMT1 is found primarily in the cytosol, localization of NtMPO1 to peroxisomes is contingent on an exposed C-terminal PTS (
2.2.3) Cytosolic expression of NtMPO1 achieved by masking the C-terminal PTS with a GFP fusion did not significantly impact extracellular 4MAB or NMPy levels (
2.3) The biosynthetic pathway in the engineered strain may incorporate orthologs of biosynthetic enzymes other than those listed in Table 1. Different orthologs of an enzyme may exhibit significant differences in activity when expressed in heterologous hosts. Therefore, orthologs of biosynthetic enzymes provided as examples herein and listed in Table 1 may also be used in engineered non-plant cells to perform the same biochemical conversions.
2.3.1) A tBLASTn search of the transcriptomes of A. belladonna and Datura metel in the 1000 Plants Project database (see Matasci, N. et al. Data access for the 1,000 Plants (1KP) project. Gigascience 3, 17 (2014)) was performed using the amino acid sequence of NtMPO1 as a query and an E-value threshold of 10−150. Two full-length ortholog sequences denoted AbMPO1 and DmMPO1 were identified, which each shared 91% sequence identity with NtMPO1 (
2.3.2) Yeast codon-optimized sequences for AbMPO1 and DmMPO1 were obtained and cloned into low-copy expression plasmids. To evaluate their activity, each of the three MPO variants was co-expressed with AbPMT1 from low-copy plasmids in the putrescine-overproducing strain of Example 1.5, and 4MAB and NMPy accumulation were measured in the extracellular medium by LC-MS/MS following 48 hours of growth in selective media. DmMPO1 showed comparable levels of 4MAB and NMPy production to the original NtMPO1 variant (
2.3.3) Differences in activity between orthologous enzymes can often be at least partially attributed to structural differences in their active sites. Template-based homology models of NtMPO1, AbMPO1, and DmMPO1 were constructed based on the crystal structure of a Pisum sativum copper-containing amino oxidase (PDB: 1KSI) using the RaptorX web server (see Källberg, M. et al. Template-based protein structure modeling using the RaptorX web server. Nat. Protoc. 7, 1511-22 (2012)). The homology models indicated that the orthologs possess long, unstructured N- and C-terminal tail regions (
2.3.4) Truncations of the two active orthologs, NtMPO1 and DmMPO1, were tested for activity in engineered yeast. N-terminal truncations removed the first 84 and 81 residues of the two orthologs, respectively. C-terminal truncations removed the last 21 residues. C-terminal truncations were also constructed wherein the unstructured tail was removed but the PTS was retained (denoted ΔC-PTS1). Each of the MPO truncations was coexpressed with AbPMT1 from low-copy plasmids in the putrescine-overproducing strain of Example 1.5, and 4MAB and NMPy accumulation in the media after 48 hours of growth were quantified by LC-MS/MS. No significant differences in activity were observed between the NtMPO1 truncations (
2.4) The biosynthetic pathway in the engineered strain incorporates one or more genetic modifications to reduce or eliminate the metabolic flux of undesirable side reactions. Biosynthetic enzymes expressed in heterologous hosts may participate in undesirable side reactions that draw metabolite flux away from the biosynthesis of desired compounds. For example, yeast aldehyde dehydrogenases may oxidize heterologous aldehyde molecules, such as 4MAB, to their cognate carboxylic acids. Based on LC-MS/MS analysis, accumulation of 4MAB acid was observed in the growth media of the putrescine-overproducing strain of Example 1.5 when AbPMT1 and DmMPO1ΔC-PTS1 were co-expressed from low-copy plasmids, but not in the absence of the MPO enzyme (
2.4.1) Six yeast genes (ALD2-ALD6 and HFD1) have been demonstrated in the literature to encode enzymes with aldehyde dehydrogenase activity (see Datta, S., Annapure, U. S. & Timson, D. J. Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii. Biosci. Rep. 37, BSR20160529 (2017); and also Nakahara, K. et al. The Sjögren-Larsson Syndrome Gene Encodes a Hexadecenal Dehydrogenase of the Sphingosine 1-Phosphate Degradation Pathway. Mol. Cell 46, 461-471 (2012)). The ALD2 and ALD3 genes encode a pair of nearly identical cytosolic dehydrogenases which catalyze the oxidation of 3-aminopropanal to β-alanine in the biosynthesis of pantothenic acid (see White, W. H., Skatrud, P. L., Xue, Z. & Toyn, J. H. Specialization of Function Among Aldehyde Dehydrogenases: Genetics 163, 69-77 (2003)). The ALD4, ALD5, and ALD6 genes respectively encode two mitochondrial and one cytosolic acetaldehyde dehydrogenase which, in addition to oxidizing acetaldehyde to acetate during fermentative growth on glucose and ethanol (see Saint-Prix, F., Bönquist, L. & Dequin, S. Functional analysis of the ALD gene family of Saccharomyces cerevisiae during anaerobic growth on glucose: The NADP+-dependent Ald6p and Ald5p isoforms play a major role in acetate formation. Microbiology 150, 2209-2220 (2004)), have been shown to oxidize an array of diverse aliphatic and aromatic aldehydes to carboxylic acids (see Datta, S., Annapure, U. S. & Timson, D. J. Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii. Biosci. Rep. 37, BSR20160529 (2017)). Individual knockouts strains for these four target genes were constructed by inserting a series of tandem nonsense mutations within the first third of their open reading frames in the putrescine-overproducing strain of Example 1.5. The contribution of each of the four dehydrogenases toward 4MAB oxidation was evaluated by co-expressing AbPMT1 and DmMPO1ΔC-PTS1 from low-copy plasmids in each single disruption strain and measuring 4MAB acid accumulation in the media by LC-MS/MS after 48 hours of growth. Marginal decreases in 4MAB acid levels were observed with the individual HFD1 and ALD4-6 disruptions (
2.4.2) Although ALD4-6 are considered essential genes due to their role in acetate and acetyl-CoA production, prior studies have demonstrated that the three genes are at least partially redundant and that the lethal phenotype of double and triple knockouts can be rescued by supplementing media with acetate (see Saint-Prix, F., Bönquist, L. & Dequin, S. Functional analysis of the ALD gene family of Saccharomyces cerevisiae during anaerobic growth on glucose: The NADP+-dependent Ald6p and Ald5p isoforms play a major role in acetate formation. Microbiology 150, 2209-2220 (2004); and also Luo, Z., Walkey, C. J., Madilao, L. L., Measday, V. & Van Vuuren, H. J. J. Functional improvement of Saccharomyces cerevisiae to reduce volatile acidity in wine. FEMS Yeast Res. 13, 485-494 (2013)). A quadruple knockout yeast strain was constructed with disruptions to the open reading frames of HFD1 and ALD4-6, and which expressed both AbPMT1 and DmMPO1ΔC-PTS1 from low-copy plasmids. This strain showed a 45% reduction in 4MAB acid levels (
2.4.3) An ALD-null strain was constructed by deleting the tandem ALD2-ALD3 genes from the genome of the quadruple knockout strain of example 2.4.2 and co-expressing AbPMT1 and DmMPO1ΔC-PTS1 from low-copy plasmids. Following 48 hours of growth, LC-MS/MS analysis indicated that deletion of ALD2 and ALD3 completely eliminated the 4MAB acid side product and increased 4MAB and NMPy production by 83% and 75%, respectively, compared to the strain with all six ALD genes intact (
2.4.4) An NMPy-producing yeast strain was constructed by integrating a previously plasmid-borne putrescine-overproduction gene cassette (SPE1, AsADC, speB) into the genome of the ALD-null strain of Example 2.4.3, and additionally integrating AbPMT1 and DmMPO1ΔC-PTS1 LC-MS/MS analysis confirmed that NMPy production in this strain after 48 hours of growth in non-selective media was comparable to that of the ALD-null strain of example 2.4.3 expressing the requisite putrescine production genes, AbPMT1 and DmMPO1ΔC-PTS1, from low-copy plasmids and cultured in selective media (
A type III polyketide synthase (PKS) and a cytochrome P450 enable conversion of NMPy to tropinone by way of the TA precursor MPOB. Tropinone can be reduced by a stereospecific reductase, denoted tropinone reductase 1 (TR1), to produce tropine (see Kim, N., Estrada, O., Chavez, B., Stewart, C. & D'Auria, J. C. Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis. Molecules 21, (2016)) (
3.1) The biosynthetic pathway in the engineered strain incorporates a pyrrolidine ketide synthase, a tropinone synthase CYP82M3, one or more cytochrome P450 reductases, and a tropinone reductase 1 to convert NMPy to tropine.
3.1.1) Yeast codon-optimized DNA sequences encoding A. belladonna pyrrolidine ketide synthase (AbPYKS), tropinone synthase (AbCYP82M3), and Datura stramonium tropinone reductase 1 (DsTR1) were obtained. Yeast codon-optimized sequences for a panel of four different CPRs, including three plant CPRs from A. thaliana, Eschscholzia californica (California poppy), and Papaver somniferum (opium poppy), and the native yeast CPR (NCP1), were also obtained for expression in yeast, since P450 enzymes require NADP-cytochrome P450 reductase (CPR) partners for continued electron exchange. A yeast strain was constructed by integrating DsTR1 into the genome of the NMPy-producing strain of Example 2.4.4, and expressing AbPYKS, AbCYP82M3, and each of the four CPRs from low-copy plasmids. To validate enzyme activity and identify potential bottlenecks, the accumulation of NMPy, MPOB, tropinone, and tropine were monitored by LC-MS/MS in the media of the transformed strains after 48 hours of growth (
3.2) The presence of metabolic bottlenecks, which are defined as biosynthetic enzymes or spontaneous steps whose low activity limits flux through a portion of a biosynthetic pathway, can result in sub-optimal production of desired TAs and precursors.
3.2.1) For example, analysis of the accumulation of TA intermediates in the media of the engineered strains of Example 3.1.1 indicated that although accumulation of tropinone, the product of AbCYP82M3, was minimal, a substantial portion of MPOB produced by AbPYKS remained unconsumed by AbCYP82M3 (
3.2.2) Integration of the tropine biosynthesis genes into the yeast genome can improve tropine production by enabling more stable AbCYP82M3 expression. A tropine-producing platform strain was constructed by integrating AtATR1 with AbPYKS and AbCYP82M3 into the genome of the NMPy-producing strain of Example 3.1.1. Tropine and hygrine accumulation for the integrated strain was compared to plasmid-based expression of the same genes via LC-MS/MS analysis after 48 hours (
3.3) The accumulation of side products in the biosynthetic pathway of the engineered strain can result in sub-optimal production of desired TAs and precursors.
3.3.1) For example, analysis of the accumulation of TA intermediates in the media of the engineered strains of Example 3.1.1 indicated substantial accumulation of hygrine, a derivative of NMPy, to titers almost four-fold greater than tropine (775-900 μg/L). In the relevant literature, hygrine has been observed to accumulate via spontaneous decarboxylation of MPOB (see Bedewitz, M. A., Jones, A. D., D'Auria, J. C. & Barry, C. S. Tropinone synthesis via an atypical polyketide synthase and P450-mediated cyclization. Nat. Commun. 9, 5281 (2018)) (
3.3.2) Modulation of growth temperature may be used to reduce the accumulation of side products in the biosynthetic pathway of the engineered strain to increase flux towards desired TAs and precursors. In one example, the impact of temperature on spontaneous hygrine production was evaluated by leveraging a kinetic principle that the rates of enzymatic and spontaneous reactions are decreased at lower temperatures. Since A. belladonna and other TA-producing Solanaceae are adapted for optimal growth at cooler climates, growth of yeast strains expressing Solanaceae genes at 25° C. may improve enzyme folding and/or activity, enabling comparable production of enzymatically-generated tropine to growth at 30° C. while reducing the rate of spontaneous hygrine production. Cultures of the tropine-producing strain of Example 3.2.2 were grown in non-selective defined media at 30° C. and 25° C. and the accumulation of tropine and hygrine was compared via LC-MS/MS analysis of the growth medium after 48 hours. Tropine titers were minimally impacted by the decrease in temperature. Hygrine accumulation was decreased by 42% at 25° C. compared to at 30° C., resulting in a 60% increase in the ratio of tropine to hygrine produced (
3.3.3) Reduction or elimination of undesirable side reactions can be used to improve metabolite flux towards desirable TAs and TA precursors in the biosynthetic pathway of the engineered strain. In one example, flux towards the TA precursor tropine may be improved by reducing hygrine production resulting from spontaneous decarboxylative condensation with acetate. The impact of removing fed acetate from the media of the NMPy-producing strain of Example 2.4.4 on hygrine and tropine production was evaluated. The effect of abolishing acetate auxotrophy in the engineered strain of Example 2.4.4 was evaluated by expressing functional copies of ALD4 and ALD6 on low-copy plasmids and then monitoring the accumulation of hygrine and 4MAB acid via LC-MS/MS analysis after 48 hours of growth. While reconstitution of ALD4 or ALD6 enabled growth on selective media in the absence of fed acetate (
3.3.4) A functional copy of the ALD6 gene was re-integrated into the tropine-producing strain of Example 3.2.2 at the previously disrupted ald6 locus. The impact of this integration on the accumulation of all metabolites between NMPy and tropine was measured via LC-MS/MS analysis after 48 hours of growth in non-selective media. Restoration of acetate metabolism via Ald6p resulted in a 2.7-fold increase in tropine titers, as well as a 1.6-fold increase in hygrine accumulation (
3.3.5) An additional copy of each biosynthetic enzyme gene between putrescine and tropine (i.e., AbPMT1, DmMPO1ΔC-PTS1, AbPYKS, and AbCYP82M3) was expressed from a low-copy plasmid in the engineered strain of Example 3.3.4 and production of TA intermediates was compared to that of the same strain expressing BFP by LC-MS/MS after 48 hours of growth in selective media. Expression of an additional copy of AbPYKS resulted in a 4.3-fold increase in NMP accumulation and a 1.3-fold increase in tropine production (
Yeast strains can be engineered for the production of non-medicinal TAs from early amino acid precursors such as L-arginine. As an example, the platform yeast strains described in Example 3 can be further engineered to produce pseudotropine alkaloids from L-arginine (
The platform yeast strain producing tropinone from L-arginine (see descriptions in Example 3) can be further engineered to incorporate a stereospecific reductase, for example tropinone reductase 2 (TR2; EC 1.1.1.236), to convert the biosynthesized tropinone to pseudotropine. An expression cassette harboring a strong constitutive promoter such as TDH3 and a coding sequence for a TR2 variant, for example TR2 from Datura stramonium (DsTR2), can be integrated into the genome of the tropinone-producing platform yeast strain. The resulting strain can be further engineered to produce hydroxylated derivatives of pseudotropine, for example calystegines, by integrating one or more expression cassettes harboring a strong constitutive promoter such as PGK1 and a hydroxylating enzyme such as a cytochrome P450 that acts on the pseudotropine scaffold. By incorporating multiple P450 enzymes, each acting on a different position of the pseudotropine skeleton, a variety of calystegines and derivatives thereof can be biosynthesized. The engineered strains can then be cultured in nonselective synthetic complete media at 30° C. or 25° C. for 48 to 96 hours, after which the accumulation of pseudotropine alkaloids in the culture media can be analyzed by LC-MS/MS.
Yeast strains can be engineered for the overproduction of phenylpyruvate, which represents the precursor of acyl donor molecules required for production of medicinal TAs (
In one example, a yeast strain can be engineered for increased phenylpyruvate production by incorporating additional copies of native genes which encode biosynthetic enzymes that produce phenylpyruvate from amino acids or other central metabolites. These additional copies can be controlled by strong constitutive promoters, such as GPD, TEF1, or PGK1. Examples of native gene targets include, but are not limited to, the aromatic acid aminotransferases ARO8 and ARO9, and the dehydratase PHA2. In one instance, one or more additional copies of ARO8 can be incorporated into the engineered strain under the control of a strong constitutive promoter. In one instance, one or more additional copies of ARO9 can be incorporated into the engineered strain under the control of a strong constitutive promoter. In another instance, one or more additional copies of PHA2 can be incorporated into the engineered strain under the control of a strong constitutive promoter. In one embodiment of the invention, one or more additional copies of one or more genes selected from the group including ARO8, ARO9, and PHA2 can be incorporated into the engineered strain under the control of unique, strong constitutive promoters.
Yeast strains can be engineered for the production of diverse phenylpropanoid acyl donor compounds from L-phenylalanine and L-tyrosine, including PLA, cinnamic acid, coumaric acid, ferulic acid, benzoic acid, and coenzyme A thioester and glycoside derivatives of these compounds, which can undergo esterification with tropine, pseudotropine, or derivatives thereof to biosynthesize medicinal TAs, non-medicinal TAs, and non-natural TAs (
6.1) As wild-type yeast produce only trace levels of PLA, production of this TA precursor must be increased to permit sufficient accumulation of downstream TAs. To improve PLA production, heterologous phenylpyruvate reductases (PPRs) may be expressed in the engineered host cells. PPR orthologs from E. coli, Lactobacillus, A. belladonna, and Wickerhamia fluorescens, as well as lactate dehydrogenases (LDHs) from Bacillus and Lactobacillus with reported activity on 3-phenylpyruvate (Table 1) were screened for activity in yeast by expressing each enzyme from a low-copy plasmid in CSY1251 and measuring PLA production by LC-MS/MS after 72 h of growth in selective media. All LDH candidates as well as the PPRs from L. plantarum, E. coli, and A. belladonna yielded modest (1.3- to 3.5-fold) improvements in PLA production relative to control, whereas expression of the PPR from W. fluorescens resulted in a nearly 80-fold increase in PLA production to ˜250 mg/L (
6.2) As another example, yeast strains can be engineered for the production of cinnamic acid and coumaric acid, which are phenylpropanoids that can be used as acyl donor compounds for esterification with tropine or pseudotropine to form non-natural TAs, from L-phenylalanine and L-tyrosine, respectively. Yeast can be engineered for production of cinnamic acid from L-phenylalanine by incorporating an ammonia-lyase such as a phenylalanine ammonia-lyase (PAL; EC 4.3.1.24). Similarly, yeast can be engineered for production of coumaric acid from L-tyrosine by incorporating an ammonia-lyase such as a tyrosine ammonia-lyase (TAL; EC 4.3.1.23). A yeast strain was engineered to produce cinnamic acid from L-phenylalanine by transforming it with a low-copy CEN/ARS plasmid with a TRP1 selective marker, TEF1 promoter, and a coding sequence for a PAL variant from Arabidopsis thaliana (AtPAL1). The resulting strain harboring the low-copy plasmid was grown in synthetic complete media with the appropriate amino acid dropout solution (-Ura) at 30° C. After 48 hours of growth, the media was analyzed for cinnamic acid content by LC-MS/MS analysis (
6.3) In A. belladonna, PLA is activated for acyl transfer to tropine via glucosylation by UDP-glucosyltransferase 84A27 (AbUGT) (see Qiu, F. et al., Functional genomics analysis reveals two novel genes required for littorine biosynthesis. New Phytol., nph.16317 (2019)). As plant UGTs participate in the biosynthesis of diverse phenylpropanoids and often exhibit broad substrate scope (see Ross, J., Li, Y., Lim, E.-K., D. J. Bowles, Higher plant glycosyltransferases. Genome Biol. 2, 3004.1-3004.6 (2001)), it is necessary to select a UGT with sufficiently high activity on a desired acyl donor.
6.3.1) As an example, the activity of AbUGT on different phenylpropanoid acyl donors, including the canonical substrate, PLA, was evaluated by expressing AbUGT from a low-copy plasmid in CSY1251 and measuring conversion of three phenylpropanoid acyl donors (PLA, cinnamic acid, ferulic acid) to their respective glucosides. While AbUGT glucosylated ˜60% and 90% of cinnamic acid and ferulic acid, respectively, glucosylation of PLA was the lowest of the tested substrates at <3% conversion (
6.3.2) Orthologs of AbUGT from other TA-producing Solanaceae may be evaluated for activity on PLA and other phenylpropanoids. In this example, transcripts encoding UGT84A27 from the transcriptomes of Brugmansia sanguinea (BsUGT) and D. metel (DmUGT) in the 1000 Plants Database using a tBLASTn search. Yeast codon-optimized sequences encoding these orthologous UGTs were screened for activity by expressing AbUGT, BsUGT, DmUGT, or a BFP negative control from low-copy plasmids in CSY1251. Glucoside production was measured in cultures of the transformed strains via LC-MS/MS after 72 h of growth in selective media supplemented with 500 μM PLA, cinnamic acid (CA), or ferulic acid (FA) as glucose acceptors. All three UGT orthologs exhibited substantial glucosylation of CA (34-65% conversion) and FA (85-90% conversion) and only trace activity on PLA (<3% conversion), with AbUGT showing the greatest conversion of PLA (2.7%) (
6.3.3) Given the disproportionate variation in activity of AbUGT on the structurally similar substrates cinnamate, ferulate, and PLA, a structure-guided rational mutagenesis approach may be implemented to engineer the active site of AbUGT for improved activity on PLA. In this example, a homology model of AbUGT bound to UDP-glucose was first constructed based on the crystal structure of Arabidopsis thaliana salicylate UDP-glucosyltransferase UGT74F2 (PDB: 5V2K) using the RaptorX web server (
6.3.4) Based on the results described in sections 6.1 and 6.3, strain CSY1288 was constructed by integrating yeast codon-optimized WfPPR and AbUGT into the genome of CSY1251, validated by verification of PLA production (66 mg/L) and minimal PLA glucoside accumulation (
6.4) As poor activity of AbUGT on PLA is likely to limit flux of TA precursors towards downstream TAs, flux of phenylalanine to PLA glucoside may be increased by incorporating genetic modifications which promote UDP-glucose accumulation and decrease glycoside degradation.
6.4.1) UDP-glucose is critical for the formation of storage polysaccharides, cell wall glucans, and glycoproteins, and thus its biosynthesis is tightly regulated (see Nishizawa, M., Tanabe, M., Yabuki, N., Kitada, K., Toh-e, A. Pho85 kinase, a yeast cyclin-dependent kinase, regulates the expression of UGP1 encoding UDP-glucose pyrophosphorylase. Yeast. 18, 239-249 (2001)). During growth on glucose, yeast direct glucose-6-phosphate along two major metabolic routes, glycolysis and starch biosynthesis. As citrate is an allosteric inhibitor of the glycolytic rate-limiting enzyme phosphofructokinase (see Li, Y. et al., Production of Rebaudioside A from Stevioside Catalyzed by the Engineered Saccharomyces cerevisiae. Appl. Biochem. Biotechnol. 178, 1586-1598 (2016)), partial suppression of glycolysis via citrate supplementation might increase UDP-glucose availability and glucoside production (
6.4.2) Overexpression of PGM2 and UGP1, whose gene products respectively catalyze the isomerization of glucose-6-phosphate to glucose-1-phosphate and conversion of glucose-1-phosphate to UDP-glucose, can be used to increase UDP-glucose supply.
6.4.2.1) Extra copies of PGM2 and UGP1 were expressed from low-copy plasmids in CSY1288 and PLA glucoside production was measured following 72 h of growth in selective media. While PGM2 overexpression yielded no improvement relative to control, overexpression of UGP1 resulted in a ˜1.8-fold increase in PLA glucoside production (
6.4.2.2) Native glucosidases may act on PLA and other TA precursor glucosides to reduce accumulation, as other heterologous glucosides have been shown to be hydrolyzed in this manner in yeast (see Schmidt, S., Rainieri, S., Witte, S., Matern, U., Martens, S., Identification of a Saccharomyces cerevisiae glucosidase that hydrolyzes flavonoid glucosides. Appl. Environ. Microbiol. 77, 1751-1757 (2011); see also Wang, H. et al., Engineering Saccharomyces cerevisiae with the deletion of endogenous glucosidases for the production of flavonoid glucosides. Microb. Cell Fact. 15, 1-12 (2016)). In this example, three native glucosidase genes—EXG1, SPR1, and EGH1—were disrupted in CSY1288 and PLA glucoside production was measured following 72 h of growth of disruption mutants in non-selective media. The disruption of EGH1 more than doubled PLA glucoside production (
Yeast strains can be engineered for the conversion of littorine to hyoscyamine aldehyde (
Yeast strains can be engineered for conversion of hyoscyamine to scopolamine (
To identify a dehydrogenase enzyme suitable for performing the TA alcohol-aldehyde interconversions of the methods disclosed herein, and in particular to reduce hyoscyamine aldehyde to hyoscyamine, a hyoscyamine dehydrogenase (HDH) open reading frame was identified from publically available plant RNA sequencing data. 9.1) Tissue-specific abundances (fragments per kilobase of contig per million mapped reads, FPKM) and putative protein structural and functional annotations for each of 43,861 unique transcripts identified from the A. belladonna transcriptome were obtained from the Michigan State University Medicinal Plant Genomics Resource. Transcripts encoding hyoscyamine dehydrogenase candidates were identified based on clustering of tissue-specific expression profiles with those of the bait genes CYP80F1 (littorine mutase) and H6H (hyoscyamine 6β-hydroxylase/dioxygenase), which respectively precede and follow the dehydrogenase step in the TA biosynthetic pathway, using the following computational filtering algorithm.
First, the complete list of 43,861 transcripts was filtered for those annotated with any of the following protein family (PFAM) IDs: PF00106, PF13561, PF08659, PF08240, PF00107, PF00248, PF00465, PF13685, PF13823, PF13602, PF16884, PF00248; or any of the following functional annotation keywords: alcohol dehydrogenase, aldehyde reductase, short chain, aldo/keto. Additionally, any transcripts with functional annotations containing the keywords putrescine, tropinone, and tropine were included in the filter as positive control TA-associated genes to validate clustering with bait genes. Next, mean tissue-specific expression profiles were generated for the CYP80F1 and H6H bait genes. For each of the two bait genes, linear regression models were constructed to express the bait gene expression profile (in FPKM) as a linear function of each candidate gene profile and correlation p-values were computed for each candidate. The candidates identified using each of the two bait genes were pooled and duplicates were removed. Combined p-values for each candidate were computed as the sum of the log 10 p-values of the correlations with each of the two bait genes. Transcripts matching known dehydrogenases in the TA biosynthetic pathway (i.e., tropinone reductases I and II) were removed, and the remaining candidates were ranked by combined p-value and by distance from bait genes via hierarchical clustering of tissue-specific expression profiles (
9.2) Nearly all candidates identified in Example 9.1 exhibited the same secondary root-specific expression pattern observed for known TA biosynthetic genes. A BLASTp search of the resulting ˜30 candidates against the UniPROT/SwissPROT database revealed that many transcripts were missing terminal or internal sequence regions. To address this, de novo transcriptome assembly was repeated from deposited raw RNAseq reads using the Trinity software package (see Haas, B. J. et al., De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 8, 1494-512 (2013)), and all missing sequence fragments for twelve of the HDH candidates were reconstituted by performing BLAST alignments of incomplete sequence regions against the newly assembled transcriptome (Table 2).
9.3) The missing HDH activity was identified by screening the candidates generated in Examples 9.1 and 9.2 in yeast.
9.3.1) Lack of an authentic commercial standard for hyoscyamine aldehyde and insufficient yield from chemical syntheses necessitated co-expression of HDH candidates with the upstream biosynthetic enzyme—the cytochrome P450 littorine mutase (CYP80F1)—for activity screening in vivo via fed littorine (see Example 7). As littorine exhibits similar chromatographic and mass spectrometric properties as the HDH product hyoscyamine, an HDH screening strain (CSY1292) was constructed by integrating yeast codon-optimized AbCYP80F1 and DsH6H (see Example 8) into the genome of CSY1251, enabling screening of HDH candidates via detection of scopolamine (m/z+ 304) produced from fed littorine (m/z+ 290) via a three-step biosynthetic pathway (
9.3.2) Yeast codon-optimized sequences encoding each of the twelve HDH candidates were expressed from a low-copy plasmid in strain CSY1292, and scopolamine production was measured following 72 h of growth in media supplemented with 1 mM littorine. One of the twelve candidates, HDH2 (referred to as AbHDH), exhibited a 35% decrease in hyoscyamine aldehyde levels and measurable accumulation of scopolamine (7.2 μg/L), indicating that it encoded the missing HDH activity (
9.4) Structural and phylogenetic analyses provided further insight into the catalytic mechanism and evolutionary history of HDH.
9.4.1) A homology model of AbHDH was constructed based on the crystal structure of Populus tremuloides sinapyl alcohol dehydrogenase (PtSAD; PDB: 1YQD) (
9.4.2) The catalytic mechanism of AbHDH was elucidated via molecular docking of the substrate, hyoscyamine aldehyde, into the active site using the Maestro/Glide software package (
9.5) To confirm whether orthologous oxidoreductases catalyze hyoscyamine biosynthesis in other TA-producing Solanaceae, variants of the AbHDH coding sequence were identified from transcriptomes of Datura innoxia and Datura stramonium using a tBLASTx search (
9.6) The medicinal TA biosynthetic branch comprising optimal enzyme variants and overexpression of a flux-limiting enzyme was integrated into a platform yeast strain. Strain CSY1294 was constructed by integrating yeast codon-optimized WfPPR and AbUGT, DsHDH, and a second copy of DsH6H into CSY1292. Scopolamine production from fed littorine was verified in CSY1294 (
Yeast strains can be engineered to express enzymes which catalyze the esterification of activated acyl donor compounds and acyl acceptor compounds to produce diverse TA scaffolds (
10.1) In plants, where SCPL-ATs are typically found to occur naturally, the coding sequence of SCPL-ATs include N-terminal signal peptides which direct the nascent polypeptide to the endoplasmic reticulum (ER). Once localized to the ER, the SCPL-AT polypeptide is transported by way of the secretory trafficking pathway through the Golgi to the vacuole lumen, where they are found to exhibit activity. During this ER-to-vacuole trafficking process, they undergo several post-translational modifications (
10.1.1) Signal peptide sequences can impact the processing and localization of SCPL-ATs in yeast.
10.1.1.1) The presence of a putative N-terminal signal peptide in AbLS suggests that it follows the expected SCPL ER-to-vacuole trafficking pathway in planta. AbLS localization in yeast was examined by expressing N- and C-terminal GFP fusions of AbLS from low-copy plasmids in CSY1294. Fluorescence microscopy revealed that the N-terminal fusion (GFP-AbLS) co-localized with the vacuolar membrane stain FM4-64 (
10.1.1.2) Vacuolar sequestration of SCPL-ATs in yeast might preclude access to cytosolic substrate pools, as yeast likely lack the requisite tonoplastic transporters present in plants for exchange of secondary metabolites with the cytosol. To determine whether forced localization of AbLS to other yeast compartments—presumably, with improved access to cytosolic metabolites—would enable activity, the wild-type N-terminal SP sequence was replaced with a panel of N-terminal signal sequences taken from yeast proteins targeted to the vacuole lumen (Prc1p and Pep4p), vacuole membrane facing the lumen (Dap2p), trans-Golgi network (Ochi p), ER membrane facing the lumen (Mns1p), and mitochondrial matrix (Citi p) (
10.1.2) Incorrect post-translational processing of SCPL-ATs in yeast might prevent expression of active enzyme.
10.1.2.1) Protein N-glycosylation patterns differ between yeast and plants, and previous reports have suggested that correct N-glycosylation of diverse plant enzymes is important for their folding, stability, and/or activity (see Kar, B., Verma, P., den Haan, R., Sharma, A. K., Effect of N-linked glycosylation on the activity and stability of a β-glucosidase from Putranjiva roxburghii. Int. J. Biol. Macromol. 112, 490-498 (2018); see also Podzimek, T. et al., N-glycosylation of tomato nuclease TBN1 produced in N. benthamiana and its effect on the enzyme activity. Plant Sci. 276, 152-161 (2018); see also Strasser, R., Plant protein glycosylation. Glycobiology. 26, 926-939 (2016)). In silico analysis of the AbLS polypeptide predicted four N-glycosylation sites (N152, N320, N376, N416) and no O-glycosylation of this protein was detected in N. benthamiana (
10.1.2.2) A subset of SCPL acyltransferases, including sinapoylglucose:choline sinapoyltransferase from Arabidopsis thaliana (AtSCT) and an avenacin synthase from Avena strigosa (AsSCPL1), have been shown to contain an internal propeptide linker which is proteolytically removed to produce an active heterodimer joined by disulfide bonds (see Shirley, A. M., Chapple, C., Biochemical characterization of sinapoylglucose:choline sinapoyltransferase, a serine carboxypeptidase-like protein that functions as an acyltransferase in plant secondary metabolism. J. Biol. Chem. 278, 19870-19877 (2003); see also Mugford, S. T. et al., A serine carboxypeptidase-like acyltransferase is required for synthesis of antimicrobial compounds and disease resistance in oats. Plant Cell. 21, 2473-2484 (2009)). Comparison of the AbLS amino acid sequence with those of previously characterized plant serine carboxypeptidases and SCPL acyltransferases revealed the presence of an internal 25- to 30-residue sequence which aligns with the highly variable propeptide of AtSCT, AsSCPL1, and wheat carboxypeptidase 2 (TaCBP2), suggesting that AbLS too undergoes endoproteolytic cleavage to form a heterodimer (
To address this failure mode, split AbLS controls were constructed in which the N- and C-terminal domains flanking the putative propeptide linker were expressed independently, with or without separate signal peptides. Additionally, AbLS variants in which the putative propeptide was replaced with either a flexible (GGGGS)n (SEQ ID NO: 26) linker, the internal propeptide from AtSCT previously demonstrated to be cleaved in yeast (see Shirley, A. M., Chapple, C., Biochemical characterization of sinapoylglucose:choline sinapoyltransferase, a serine carboxypeptidase-like protein that functions as an acyltransferase in plant secondary metabolism. J. Biol. Chem. 278, 19870-19877 (2003)), or a synthetic linker containing a poly-arginine site cleaved by the trans-Golgi protease Kex2p (see Chen, X., Zaro, J. L., Shen, W. C., Fusion protein linkers: Property, design and functionality. Adv. Drug Deliv. Rev. 65, 1357-1369 (2013); see also Redding, K., Seeger, M., Payne, G. S., Fuller, R. S., The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p. Mol. Biol. Cell. 7, 1667-1677 (1996)) were constructed (
To troubleshoot protein expression, each of the above C-terminal HA-tagged AbLS variants was expressed from low-copy plasmids in CSY1294 and apparent protein sizes were compared to split-AbLS controls by Western blot (
10.1.3) Functional expression of SCPL-ATs in yeast can be achieved by engineering N-terminal fusions that alter sorting from the TGN. Transport of soluble yeast proteins from the TGN to the vacuole requires recognition of a typically N-terminal signal sequence by vacuole protein sorting (Vps) cargo transport proteins, whereas integral membrane proteins which reach the yeast TGN appear to be sorted to the vacuole by default (see Stack, J. H., Receptor-Mediated Protein Sorting to the Vacuole in Yeast: Roles for Protein Kinase, Lipid Kinase and GTP-Binding Proteins. Annu. Rev. Cell Dev. Biol. 11, 1-33 (1995); see also Roberts, C. J., Nothwehr, S. F., Stevens, T. H., Membrane protein sorting in the yeast secretory pathway: Evidence that the vacuole may be the default compartment. J. Cell Biol. 119, 69-83 (1992)). Conversion of SCPL-ATs into transmembrane proteins by masking the SP with an N-terminally fused soluble domain can therefore resolve the obstruction in TGN sorting.
10.1.3.1) In one example, AbLS variants were constructed with a panel of N-terminally fused soluble domains, including fluorescent proteins from the Aequoria (GFP, BFP, mVenus) and Discosoma (mCherry, DsRed) families; small ubiquitin-related modifier (Smt3p) with a mutated protease cleavage site (SUMO*); and the upstream enzyme in the TA pathway, AbUGT. These variants and wild-type AbLS were expressed from low-copy plasmids in CSY1294 and screened for littorine synthase activity following 96 h of growth in selective media. All N-terminally fused AbLS variants exhibited measurable accumulation of hyoscyamine and scopolamine. Fusion of Aequoria GFP-derived fluorescent proteins to AbLS resulted in hyoscyamine and scopolamine production of ˜1 μg/L and ˜0.1 μg/L, respectively; whereas fusion of Discosoma-derived fluorescent proteins led to considerably higher TA production, with the greatest titers achieved via DsRed fusion (10.3 μg/L hyoscyamine, 0.87 μg/L scopolamine) (
10.2) To generate a strain capable of complete TA biosynthesis, a yeast codon-optimized DsRed-AbLS and a second copy of UGP1 were integrated into the genome of CSY1294 at the disrupted EGH1 site to generate CSY1296. CSY1296 exhibited de novo hyoscyamine and scopolamine production at titers of 10.2 μg/L and 1.0 μg/L, respectively.
As the enzymes which carry out TA biosynthesis are distributed across multiple sub-cellular compartments (cytosol, ER membrane, peroxisome, vacuole, mitochondria), and yeast are unlikely to possess the transporters found in plants which enable mobilization of TA biosynthetic intermediates between different compartments, intracellular metabolite transport is likely to restrict TA production.
11.1) Inter-compartment transport limitations may be addressed by functional expression of plant transporters in non-plant host cells. Vacuolar compartmentalization of DsRed-AbLS (
11.2) To evaluate the subcellular localization of these transporters, and determine likely mechanisms of action, fluorescence microscopy of CSY1296 expressing C-terminal GFP fusions of NtJAT1 or NtMATE2 from low-copy plasmids was performed. The analysis supports that NtJAT1 localizes almost exclusively to the vacuolar membrane (co-localizing with DsRed-AbLS), whereas NtMATE2 is partitioned between the vacuolar and plasma membranes (
In addition to being engineered for the production of medicinal and non-medicinal TAs which occur naturally in organisms, yeast can also be engineered for the production of non-natural TAs (
12.1) In one example, the platform tropine-producing yeast strain described in Example 3 can be further engineered to produce the acyl donor compound cinnamic acid (as described in Example 6) and to express cinnamate-activating enzymes and esterifying enzymes to produce non-natural TAs such as cinnamoyltropine.
12.1.1) Cinnamate can be produced from phenylalanine via a phenylalanine ammonia-lyase, for example PAL1 from A. thaliana (AtPAL1). Since EcCS requires a coenzyme A (CoA)-activated acyl donor, a 4-coumarate-CoA ligase with established activity on cinnamate, such as 4CL5 from A. thaliana (At4CL5) (see Eudes, A. et al. Exploiting members of the BAND acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast. Microbial Cell Factories, 15, (2016)), can be expressed to enable cinnamoyl-CoA biosynthesis in yeast. The platform tropine-producing yeast strain described in Example 3 was transformed with a low-copy plasmid enabling production of cinnamic acid as described in Example 6.
12.1.2) The engineered strain of Example 12.1.1 was further modified to produce cinnamoyltropine by transforming it with a high-copy 2p plasmid with a URA3 selective marker, HXT7 and PMA1 promoters, and coding sequences for a 4-coumarate-CoA ligase variant from A. thaliana (At4CL5) and a cocaine synthase from Erythroxylum coca (EcCS). The resulting strain harboring the low- and high-copy plasmids was grown in synthetic complete media with the appropriate amino acid dropout solution (-Ura -Trp) at 25° C. After 72 hours of growth, the culture medium was analyzed for cinnamoyltropine by LC-MS/MS analysis (
12.1.3) Based on the 272→124 LC-MS/MS transition for cinnamoyltropine described in Example 12.1.2, a multiple reaction monitoring (MRM) LC-MS/MS method was developed to measure de novo cinnamoyltropine production. Cinnamoyltropine accumulated to substantial levels in the extracellular medium of the engineered strain of example 12.1.2, but not in the absence of AtPAL1, At4CL5, and EcCS (
Production titers of TA precursors and TAs can be improved by modifying the culture media composition. For example, the media types can vary in the media base (e.g., yeast peptone, yeast nitrogen base), carbon source (e.g., glucose, maltodextrin), and nitrogen source (e.g., amino acids, ammonium sulfate, urea). Media types can also vary in the relative proportions of each component, such as the concentration of carbon source and the concentration of nitrogen source, or the concentration of each individual amino acid.
13.1) Tropine-producing yeast strains (as described in Example 3) were initially grown in defined media (i.e., YNB with ammonium sulfate and all amino acids) with varying carbon sources and tropine production assayed after 48 hours of growth at 25° C. The highest production of tropine was observed with 2% galactose (
13.2) Tropine-producing yeast strains (as described in Example 3) were cultured in defined media with 2% dextrose for growth and supplemented with 2% of an additional carbon source, and tropine production was assayed after 48 hours of growth at 25° C. The highest production of tropine was observed with 2% dextrose and 2% glycerol (
13.3) Improvements in de novo medicinal TA biosynthesis in engineered yeast can be achieved via alleviation of flux bottlenecks and transport limitations.
13.3.1) Improvements in TA production were achieved via overexpression of bottleneck enzymes and media optimization. As production of tropine in CSY1296 (˜mg/L) is unlikely to limit flux to scopolamine (˜μg/L), metabolic bottlenecks limiting scopolamine production were identified by expressing an additional copy of each heterologous enzyme between phenylpyruvate and scopolamine (
13.3.2) An improved scopolamine-producing strain was constructed by integrating NtJAT1 and a second copy of WfPPR and DsH6H into CSY1296. The resulting strain CSY1297 showed 2.4- and 7.1-fold respective increases in hyoscyamine and scopolamine accumulation relative to CSY1296 (
Escherichia
coli
Escherichia
coli
Bacillus
subtilis
Escherichia
coli
Lactobacillus sp.
Lactobacillus
plantarum
Bacillus
coagulans
Lactobacillus
casei
Lactobacillus
plantarum
A. belladonna
A. belladonna
A. belladonna
A. belladonna plant
A. belladonna plant
A. belladonna plant
A. belladonna plant
A. belladonna plant
A. belladonna plant
A. belladonna plant
A. belladonna plant
A. belladonna plant
A. belladonna plant
D. innoxia plant
D. stramonium plant
Atropa belladonna
Nicotiana tabacum
Nicotiana tabacum
Nicotiana tabacum
Notwithstanding the appended clauses, the disclosure may be defined by the following clauses:
Clause 1. An engineered non-plant cell that produces a tropane alkaloid product, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product.
Clause 2. The cell of clause 1, wherein the cell is a microbial cell.
Clause 3. The cell of clauses 1 or 2, wherein the engineered cell comprises a plurality of heterologous coding sequences for encoding a plurality of enzymes, wherein at least one of the enzymes is selected from the group consisting of arginine decarboxylase, agmatine ureohydrolase, agmatinase, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidine ketide synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylpyruvate reductase, 3-phenyllactic acid UDP-glucosyltransferase 84A27, littorine synthase, littorine mutase, hyoscyamine dehydrogenase, hyoscyamine 6β-hydroxylase/dioxygenase, and cocaine synthase.
Clause 4. The cell of any of clauses 1-3, wherein endogenous arginine metabolism is modified in the cell.
Clause 5. The cell of any of clauses 1-4, wherein endogenous phenylalanine and phenylpropanoid metabolism is modified clauses the cell.
Clause 6. The cell of any of claims 1-5, wherein endogenous polyamine regulatory mechanisms are disrupted in the cell.
Clause 7. The cell of any of the clauses 1-6, wherein endogenous acetate metabolism is modified in the cell.
Clause 8. The cell of any of the clauses 1-7, wherein endogenous glycoside metabolism is modified in the cell.
Clause 9. The cell of any of clauses 1-8, wherein the cell produces a tropane alkaloid product, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product selected from the group consisting of a hyoscyamine, atropine, anisodamine, scopolamine, calystegine, cocaine, or a non-natural tropane alkaloid.
Clause 10. The cell of any of the clauses 1-9, wherein the engineered cell comprises a plurality of heterologous coding sequences encoding for a plurality of enzymes which comprise one or more soluble protein domains fused to the N-terminus of a serine carboxypeptidase-like acyltransferase domain.
Clause 11. The cell of any of the clauses 1-10, wherein the transport of TAs, TA precursors, and/or TA derivatives across intracellular membranes or across the plasma membrane is modified in the cell.
Clause 12. The cell of any of the clauses 1-11, wherein the engineered cell comprises a plurality of heterologous coding sequences for encoding a plurality of transporters, wherein at least one of the transporters is selected from the group consisting of a multidrug and toxin extrusion transporter, a nitrate/peptide family transporter, an ATP-binding cassette transporter, and a pleiotropic drug resistance transporter.
Clause 13. A method for producing a tropane alkaloid, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product comprising
(a) culturing a cell of any of clauses 1-12 under conditions suitable for protein production;
(b) adding a starting compound to the cell culture; and
(c) recovering the tropane alkaloid or the precursor of a tropane alkaloid product from the culture.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.
This application claims the benefit of U.S. provisional application Ser. Nos. 62/815,709, filed on Mar. 8, 2019, 62/848,419, filed on May 15, 2019 and 62/891,771, filed on Aug. 26, 2019, which applications are incorporated by reference herein.
This invention was made with Government support under contracts GM110699 and AT007886 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/021577 | 3/6/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62891771 | Aug 2019 | US | |
| 62848419 | May 2019 | US | |
| 62815709 | Mar 2019 | US |
