Tropane alkaloid (TA)-producing non-plant host cells and methods of making and using same

Abstract

Among other things, provided herein are engineered non-plant cells that produce a tropane alkaloid product, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product. Methods for producing a tropane alkaloid, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product utilizing the cells are also described.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application Nos. 62 / 815,709, filed March 8, 2019, 62 / 848,419, filed May 15, 2019, and 62 / 891,771, filed August 26, 2019, which applications are incorporated herein by reference.

[0002] Government Rights 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 this invention. [Background technology]

[0003] preface Tropane alkaloids (TAs) are a class of anticholinergic secondary metabolites produced by plants in the Solanaceae family. Several TAs, including atropine, hyoscyamine, and scopolamine, have been classified by the World Health Organization as essential medicines for the treatment of diverse neurological disorders such as organophosphate and nerve gas poisoning, gastrointestinal spasms, and cardiac arrhythmias, as well as for suppressing the symptoms of Parkinson's disease. Therefore, it is important to ensure an adequate and consistent supply of these TA molecules and make them available to researchers and physicians. The current supply chain for medicinal TAs relies on extraction from unsustainable, geographically restricted plant monocultures, where TAs accumulate to only 0.2–4% of the dry weight and are vulnerable to pests, land-use change, and climate. Complete chemical synthesis of TAs from simple raw materials has not yet proven sufficiently economical for industrial use due to difficulties arising from TA stereochemistry. Furthermore, poor economies of scale and long production times have so far made engineering transgenic plants or plant cultures with improved TA production an unfeasible strategy for sourcing these compounds. Therefore, the method for preparing TA is important. Summary of the Invention

[0004] overview The present invention includes non-plant organisms engineered for the production of various tropane alkaloids (TAs) from precursors and sugars. For example, the present invention includes engineered microbial strains for the production of medicinal TAs, defined herein as naturally occurring TAs with established uses in current medical practice, including hyoscyamine, atropine, anisodamine, and scopolamine, as well as their precursors and derivatives. Also included are engineered microbial strains for the production of non-medicinal TAs, defined herein as naturally occurring TAs without established uses in current medical practice, but which may possess biological activities of medical interest, including calystegine, cocaine, and their precursors and derivatives. The present invention further includes engineered microbial strains for the production of non-naturally occurring TAs, defined herein as TAs not produced by unmodified organisms, such as TAs produced via esterification of acyl donor and acyl acceptor compounds that are not esterified in naturally occurring organisms, including derivatives of medicinal and non-medicinal TAs. Exemplary schemes included in the present invention are detailed in Figures 1-3.

[0005] The present invention encompasses methods for producing pseudotropines and pseudotropine-derived alkaloids, such as calystegine, using microorganisms engineered to express at least one heterologous enzyme as a microbial catalyst. The present invention further encompasses methods for producing a variety of compounds that can be used as acyl donors for the biosynthesis of TA scaffolds using microorganisms engineered to express at least one heterologous enzyme as a microbial catalyst. The present invention also encompasses methods for esterifying acyl donors and acceptors to produce TA scaffolds using microorganisms engineered to express at least one heterologous enzyme as a microbial catalyst. The present invention further encompasses methods for modifying and culturing engineered microbial strains for the production of medicinal TAs, such as hyoscyamine and scopolamine, non-medicinal TAs, such as calystegine, and unnatural TAs, such as those derived from the esterification of tropine with acyl donor compounds other than 3-phenyllactic acid (PLA).

[0006] Host cells engineered to produce a tropane alkaloid (TA), such as hyoscyamine and scopolamine, are provided. The TA can include a TA precursor, a TA, and a modified TA, including a derivative of a TA. The host cell can have one or more modifications selected from the following: feedback inhibition to alleviate mutations in the enzyme gene, transcriptional regulatory modifications of the biosynthetic enzyme gene, inactivating mutations in the enzyme, and a heterologous coding sequence. Methods for producing a TA of interest using the host cells and compositions, e.g., kits, systems, etc., that find use in the methods of the invention are also provided.

[0007] One aspect of the present invention provides a method for forming a product stream having a tropane alkaloid (TA) product. The method includes providing engineered non-plant cells and a feedstock comprising nutrients and water to a batch reactor, wherein the engineered non-plant cells have at least one modification selected from the group consisting of: feedback inhibition that alleviates a mutation in a cell's native biosynthetic enzyme gene, a transcriptional regulatory modification of the cell's native biosynthetic enzyme gene, and an inactivating mutation in the cell's native enzyme. The method further includes subjecting the engineered non-plant cells to fermentation in the batch reactor by incubating the engineered non-plant cells for at least about 5 minutes to produce a solution comprising the TA product and cellular material. The method also includes separating the TA product from the cellular material using at least one separation unit to provide a product stream comprising the TA product.

[0008] In another aspect, the present invention provides a method for forming a product stream having a TA product. The method includes providing a reactor with a feedstock comprising engineered non-plant cells and nutrients and water. The method also includes subjecting the engineered non-plant cells to fermentation in the reactor by incubating the engineered yeast cells for at least about 5 minutes (e.g., 5 minutes or more) to produce a solution comprising the cellular material and the TA product. The method further includes separating the TA product from the cellular material using at least one separation unit to provide a product stream comprising the TA product.

[0009] Another aspect of the present invention provides engineered non-plant cells that produce tropane alkaloid (TA) products, wherein the engineered non-plant cells have at least one modification selected from the group consisting of feedback inhibition that alleviates a mutation in a cell's native biosynthetic enzyme gene, a transcriptional regulatory modification of the cell's native biosynthetic enzyme gene, and an inactivating mutation in the cell's native enzyme. The engineered non-plant cell comprises at least one heterologous coding sequence encoding at least one enzyme 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-phenyllactate UDP-glucosyltransferase 84A27, littorine synthase, littorine mutase, hyoscyamine dehydrogenase, hyoscyamine 6β-hydroxylase / dioxygenase, and cocaine synthase. In some examples, the engineered non-plant cell contains multiple heterologous coding sequences encoding enzymes 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-phenyllactate 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. The operably connected heterologous coding sequences may be in the same pathway to produce a specific tropane alkaloid product. In some examples, the engineered non-plant cells comprise one or more modifications to intracellular compartmentalization selected from the group including, but not limited to, modified intracellular trafficking of enzymes, modified intracellular localization of enzymes, and modified intracellular trafficking of metabolites.

[0010] In another aspect of the present invention, a therapeutic agent is provided, the therapeutic agent comprising a tropane alkaloid product. [The present invention 1001] 1. An engineered non-plant cell that produces a precursor of a tropane alkaloid product, a tropane alkaloid product, or a derivative of a tropane alkaloid product, comprising: the engineered non-plant cell comprises a plurality of heterologous coding sequences encoding a plurality of enzymes in a pathway for producing a precursor of the tropane alkaloid product, the tropane alkaloid product, or a derivative of the tropane alkaloid product; The cells are engineered non-plant cells that contain one or more alterations to one or more endogenous metabolic pathways or regulatory mechanisms selected from the group consisting of endogenous arginine metabolism, endogenous phenylalanine and phenylpropanoid metabolism, endogenous polyamine regulatory mechanisms and metabolism, endogenous acetate metabolism, and endogenous glycoside metabolism. [The present invention 1002] The cell of the present invention 1001, wherein the cell comprises one or more alterations to one or more endogenous metabolic pathways or regulatory mechanisms selected from the group consisting of endogenous arginine metabolism, endogenous phenylalanine and phenylpropanoid metabolism, endogenous polyamine regulatory mechanisms and metabolism, and endogenous acetate metabolism. [The present invention 1003] 1001. The cell of claim 1001, wherein said cell comprises one or more alterations to endogenous glycoside metabolism. [The present invention 1004] The cell of any one of 1001 to 1003, wherein the cell is a microbial cell. [The present invention 1005] 1004. The cell of claim 10, wherein the cell is a fungal cell. [The present invention 1006] the engineered cell comprises one or more heterologous coding sequences for one or more enzymes; The cell of any of claims 1001 to 1005, 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, phenylalanine ammonia-lyase, tyrosine ammonia-lyase, phenylpyruvate reductase, 4-coumarate-CoA ligase, 3-phenyllactate UDP-glucosyltransferase 84A27, littorine synthase, littorine mutase, hyoscyamine dehydrogenase, hyoscyamine 6β-hydroxylase / dioxygenase, and cocaine synthase. [The present invention 1007] endogenous arginine metabolism is altered in the cell by modifications to one or more coding sequences for one or more endogenous enzymes; The cell of any one of claims 1001 to 1006, wherein at least one of the enzymes is selected from the group consisting of glutamate N-acetyltransferase, acetylglutamate kinase, N-acetyl-γ-glutamylphosphate reductase, acetylornithine aminotransferase, ornithine acetyltransferase, ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lyase, and arginase. [The present invention 1008] endogenous phenylalanine and phenylpropanoid metabolism is altered in said cell by modifications to one or more coding sequences for one or more endogenous enzymes; The cell of any of claims 1001 to 1007, wherein at least one of the enzymes is selected from the group consisting of a pentafunctional AROM polypeptide, chorismate synthase, chorismate mutase, prephenate dehydratase, aromatic aminotransferase, and phenylacrylic acid decarboxylase. [The present invention 1009] endogenous polyamine regulatory mechanisms are altered in said cell by modifications to one or more coding sequences of one or more endogenous proteins; The cell of any of claims 1001 to 1008, wherein at least one of the proteins is selected from the group consisting of methylthioadenosine phosphorylase, ornithine decarboxylase, ornithine decarboxylase antizyme, polyamine oxidase, spermidine synthase, spermine synthase, polyamine transporter, and polyamine permease. [The present invention 1010] endogenous acetate metabolism is altered in the cell by modifications to one or more coding sequences for one or more endogenous enzymes; The cell of any of claims 1001 to 1009, wherein at least one of the enzymes is selected from the group consisting of alcohol dehydrogenase and aldehyde dehydrogenase. [The present invention 1011] endogenous glycoside metabolism is altered in the cell by modifications to one or more coding sequences for one or more endogenous enzymes; The cell of any of claims 1001 to 1010, wherein at least one of the enzymes is selected from the group consisting of glucan 1,3-β-glucosidase and steryl-β-glucosidase. [The present invention 1012] The cell of any of claims 1006 to 1011, wherein the modification to one or more coding sequences is selected from the group consisting of feedback inhibition that alleviates a mutation in a biosynthetic enzyme or regulatory protein gene native to the cell, a transcriptional regulatory modification of a biosynthetic enzyme gene native to the cell, and an inactivating mutation in an enzyme or protein native to the cell. [The present invention 1013] the engineered cells are one or more heterologous coding sequences encoding one or more enzymes, including one or more soluble protein domains fused to the N-terminus of a serine carboxypeptidase-like acyltransferase domain to enable functional expression of the acyltransferase domain in an intracellular compartment of the engineered cell; Any of 1001 to 1012 cells of the present invention, comprising: [The present invention 1014] 10. The cell of any of claims 1001 to 1013, wherein the cell produces a precursor of a tropane alkaloid product selected from the group consisting of agmatine, N-carbamoylputrescine, N-methylputrescine, 4-methylaminobutanal, N-methylpyrrolinium, 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid, tropinone, tropine, pseudotropine, ecgonine, methylecgonine, coenzyme A covalently attached to phenyllactic acid by a thioester bond, or a sugar covalently attached to cinnamic acid, ferulic acid, coumaric acid, or phenyllactic acid by a glycosidic bond. [The present invention 1015] The cell of any of claims 1001 to 1014, wherein the cell produces a tropane alkaloid product selected from the group consisting of hyoscyamine, atropine, anisodamine, scopolamine, calystegine, cocaine, or an unnatural tropane alkaloid. [The present invention 1016] The cell of any of claims 1001 to 1015, wherein the cell produces a derivative of a tropane alkaloid product selected from the group consisting of p-hydroxyatropine, p-hydroxyhyoscyamine, p-fluorohyoscyamine, p-chlorohyoscyamine, p-bromohyoscyamine, p-fluoroscopolamine, p-chloroscopolamine, p-bromoscopolamine, N-methylhyoscyamine, N-butylhyoscyamine, N-methylscopolamine, N-butylscopolamine, N-acetylhyoscyamine, and N-acetylscopolamine. [The present invention 1017] 1015. The cell of claim 10, wherein said cell produces a tropane alkaloid product selected from the group consisting of hyoscyamine, atropine, or scopolamine. [The present invention 1018] The cell of any of claims 1001 to 1017, wherein transport of one or more TAs, one or more TA precursors, and / or one or more TA derivatives across intracellular membranes or across the plasma membrane is altered in said cell. [The present invention 1019] the altered transport is enabled by one or more heterologous coding sequences encoding one or more transporters; The cell of the present invention, wherein at least one of the transporters is selected from the group consisting of multidrug and toxin efflux transporters, nitrate / peptide family transporters, ATP-binding cassette transporters, and pleiotropic drug resistance transporters. [The present invention 1020] 1. An engineered non-plant cell that produces a tropane alkaloid product or a derivative of a tropane alkaloid product, comprising: The engineered non-plant cell comprises a plurality of heterologous coding sequences encoding a plurality of enzymes in a pathway for producing the tropane alkaloid product or a derivative of the tropane alkaloid product. [The present invention 1021] The cell of the present invention 1020, wherein the cell is a microbial cell. [The present invention 1022] The cell of claim 1021, wherein the cell is a fungal cell. [The present invention 1023] the engineered cell comprises one or more heterologous coding sequences for one or more enzymes; The cell of any of claims 1020 to 1022, 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, phenylalanine ammonia-lyase, tyrosine ammonia-lyase, phenylpyruvate reductase, 4-coumarate-CoA ligase, 3-phenyllactate UDP-glucosyltransferase 84A27, littorine synthase, littorine mutase, hyoscyamine dehydrogenase, hyoscyamine 6β-hydroxylase / dioxygenase, and cocaine synthase. [The present invention 1024] the engineered cells are one or more heterologous coding sequences encoding one or more enzymes, including one or more soluble protein domains fused to the N-terminus of a serine carboxypeptidase-like acyltransferase domain to enable functional expression of the acyltransferase domain in an intracellular compartment of the engineered cell; The cell of any one of 1020 to 1023 of the present invention, comprising: [The present invention 1025] The cell of any of claims 1020 to 1024, wherein the cell produces a tropane alkaloid product selected from the group consisting of hyoscyamine, atropine, anisodamine, scopolamine, calystegine, cocaine, or an unnatural tropane alkaloid. [The present invention 1026] The cell of any of claims 1020 to 1025, wherein the cell produces a derivative of a tropane alkaloid product selected from the group consisting of p-hydroxyatropine, p-hydroxyhyoscyamine, p-fluorohyoscyamine, p-chlorohyoscyamine, p-bromohyoscyamine, p-fluoroscopolamine, p-chloroscopolamine, p-bromoscopolamine, N-methylhyoscyamine, N-butylhyoscyamine, N-methylscopolamine, N-butylscopolamine, N-acetylhyoscyamine, and N-acetylscopolamine. [The present invention 1027] 1025. The cell of claim 1025, wherein said cell produces a tropane alkaloid product selected from the group consisting of hyoscyamine, atropine, or scopolamine. [The present invention 1028] The cell of any of claims 1020 to 1027, wherein transport of one or more TAs, one or more TA precursors, and / or one or more TA derivatives across intracellular membranes or across the plasma membrane is altered in said cell. [The present invention 1029] the altered transport is enabled by one or more heterologous coding sequences encoding one or more transporters; 1028. The cell of claim 1028, wherein at least one of the transporters is selected from the group consisting of multidrug and toxin efflux transporters, nitrate / peptide family transporters, ATP-binding cassette transporters, and pleiotropic drug resistance transporters. [The present invention 1030] 1. A method for producing a tropane alkaloid product, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product, comprising: (a) culturing any one of the cells of the present inventions 1001 to 1029 under conditions suitable for protein production; (b) adding a starting compound to the cell culture; (c) recovering the tropane alkaloid product, a precursor of the tropane alkaloid product, or a derivative of the tropane alkaloid product from the culture. [The present invention 1031] The method of claim 1030, wherein said cells are cultured in fed-batch or batch fermentation. [The present invention 1032] 1032. The process of claim 1030 or 1031, wherein the starting compound added to the cell culture is a sugar, or contains one or more sugars, or is a substrate that is converted into one or more sugars during microbial fermentation. [The present invention 1033] 1032. The method of claim 1030 or 1031, wherein the starting compound added to the cell culture is an amino acid or a mixture comprising one or more amino acids, or a substrate that is converted into one or more amino acids during microbial fermentation. [The present invention 1034] 1032. The process of any one of claims 1030 to 1031, wherein said starting compound added to said cell culture is a precursor of a tropane alkaloid product. [This invention 1035] Any of the methods of claims 1030 to 1034, wherein the precursor of the tropane alkaloid product, the tropane alkaloid product, or a derivative of the tropane alkaloid product is recovered via a process comprising liquid-liquid extraction, chromatographic separation, distillation, or recrystallization. [Brief explanation of the drawings]

[0011] 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. Rather, the dimensions of the various features are arbitrarily increased or reduced for clarity. The drawings include the following figures:

[0012] [Figure 1]An exemplary biosynthetic scheme for converting L-arginine to a non-medicinal TA is illustrated. ADC, arginine decarboxylase; ARG, arginase; AUH, agmatine ureohydrolase; ODC, ornithine decarboxylase; PAO, polyamine oxidase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; spontaneous (non-enzymatic) step; PYKS, pyrrolidine ketide synthase; CYP82M3, tropinone synthase; CPR, cytochrome P450-NADP+ reductase; TR2, tropinone reductase 2; P450, cytochrome P450. Arginine, ornithine, spermine, spermidine, and putrescine are naturally synthesized in yeast. All other metabolites shown are not naturally produced in yeast. The end products shown in the boxes are examples of non-medicinal TAs. [Figure 2]Illustrated are exemplary biosynthetic pathways that can convert amino acids into desired medicinal TA molecules and their precursor molecules. This example shows the conversion of L-arginine and L-phenylalanine into medicinal TAs. ADC, arginine decarboxylase; ARG, arginase; AUH, agmatine ureohydrolase; ODC, ornithine decarboxylase; PAO, polyamine oxidase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; spontaneous, spontaneous (non-enzymatic) step; PYKS, pyrrolidine ketide synthase; CYP82M3, tropinone synthase; CPR, cytochrome P450 -NADP+ reductase; TR1, tropinone reductase 1; ArAT, aromatic aminotransferase; PPR, phenylpyruvate reductase; UGT84A27, 3-phenyllactate UDP-glucosyltransferase; LS, littorine synthase; CYP80F1, littorine mutase; HDH, (S)-hyoscyamine dehydrogenase; H6H, (S)-hyoscyamine 6β-hydroxylase / dioxygenase. Arginine, ornithine, spermine, spermidine, putrescine, phenylalanine, 3-phenylpyruvate, and trace amounts of 3-phenyllactate are naturally synthesized in yeast. All other metabolites shown are not naturally produced in yeast. The end products shown in the boxes are examples of medicinal TAs. [Figure 3]Illustrated are exemplary biosynthetic pathways that can convert amino acids into unnatural TAs and their precursor molecules. In this example, L-arginine and L-phenylalanine are converted into unnatural TAs. ADC, arginine decarboxylase; ARG, arginase; AUH, agmatine ureohydrolase; ODC, ornithine decarboxylase; PAO, polyamine oxidase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; spontaneous (non-enzymatic) steps; PYKS, pyrrolidine ketide synthase; CYP82M3, tropinone synthase; CPR, cytochrome P450-NADP+ reductase; TR1, tropinone reductase 1; PAL, phenylalanine ammonia-lyase; 4CL, 4-coumarate-CoA ligase; CS, cocaine synthase. Arginine, ornithine, spermine, spermidine, putrescine, and phenylalanine are naturally synthesized in yeast. All other metabolites shown are not naturally produced in yeast. The end products shown in boxes are examples of non-natural TAs. [Figure 4]

[0023] Figure 1 illustrates an example biosynthetic pathway for the production of putrescine from amino acids and other polyamine molecules. The diagram shows how endogenous yeast and heterologous biosynthetic pathways can be used to produce putrescine from central metabolites. [Figure 5] We demonstrate that yeast strains engineered for overexpression of endogenous biosynthetic enzymes involved in arginine and polyamine metabolism can produce putrescine in liquid culture. Additional copies of the native genes were expressed from low-copy plasmids in wild-type yeast (CEN.PK2). Transformed strains were grown in selective medium containing 2% dextrose at 30°C for 48 hours before LC-MS / MS analysis. All data represent the mean of at least three biological replicates, and error bars indicate standard deviation. Student's two-tailed t-test: *P<0.05, **P<0.01, ***P<0.001. Unless otherwise stated, statistical significance is indicated compared to the corresponding control (i.e., CEN.PK2). [Figure 6]We demonstrate that yeast strains engineered for heterologous expression of biosynthetic enzymes from organisms other than yeast involved in arginine and polyamine metabolism can produce putrescine in liquid culture. In this example, yeast strains were engineered to express heterologous biosynthetic pathways from plants and bacteria. The heterologous enzymes were expressed from low-copy plasmids in wild-type yeast. Transformed strains were grown in selective medium containing 2% dextrose at 30 °C for 48 h before LC-MS / MS analysis. All data represent the mean of at least three biological replicates, and error bars indicate standard deviation. Student's two-tailed t-test: *P<0.05, **P<0.01, ***P<0.001. Unless otherwise stated, statistical significance is indicated compared to the corresponding control (i.e., CEN.PK2). [Figure 7] We demonstrate that yeast strains engineered for heterologous expression of biosynthetic enzymes involved in arginine and polyamine metabolism from organisms other than yeast can produce the TA precursor and intermediates agmatine, N-carbamoylputrescine, and putrescine in liquid culture. This figure demonstrates functional validation of the agmatine / putrescine biosynthetic pathway genes in yeast. The wild-type yeast strain CEN.PK2 was transformed with three low-copy plasmids to co-express zero (negative control) to three of the indicated biosynthetic genes. Plasmids expressing blue fluorescent protein (BFP) were used as negative controls for each of the three auxotrophic selection markers URA3, TRP1, and LEU2. Transformed strains were cultured in selective medium containing 2% dextrose at 30 °C for 48 h before LC-MS / MS analysis of metabolite production. All data show titers measured by LC-MS / MS peak area compared to the negative control (CEN.PK2). Data represent the average of three biological replicates, and error bars indicate standard deviation. [Figure 8] FIG. 1 illustrates the endogenous regulatory pathways that tightly control intracellular putrescine levels during normal yeast growth. [Figure 9]A heat map of putrescine production in yeast strains with disrupted endogenous polyamine biosynthetic regulatory mechanisms is shown. For overexpression of the native or heterologous putrescine pathway, the indicated genes were expressed from low-copy plasmids in wild-type yeast (WT) or each single-disruption strain. Strains were grown in selective medium containing 2% dextrose (YNB-DO) at 30 °C for 72 h before LC-MS / MS analysis. All data represent the average of at least three biological replicates. This figure demonstrates that yeast strains with single disruptions of polyamine metabolism genes and overexpression of the endogenous or heterologous putrescine biosynthetic pathway can produce putrescine in liquid culture. [Figure 10] An overview of the engineering efforts to increase putrescine production in yeast is shown. A "+" symbol indicates expression of at least one gene from the pathway, and a "-" symbol indicates no expression of a gene from the pathway. Strains were grown in selective medium containing 2% dextrose at 30°C for 48 h before LC-MS / MS analysis. All data represent the mean of at least three biological replicates, and error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. Unless otherwise stated, statistical significance is shown compared to the corresponding control (i.e., CEN.PK2). [Figure 11] Figure 1 shows LC-MS / MS chromatograms illustrating the stepwise conversion of putrescine to the TA intermediate NMPy and the by-product 4MAB acid in engineered yeast, via the intermediates NMP and 4MAB, according to an embodiment of the present invention. A proposed mechanism for the formation of the 4MAB acid by-product via the activity of an endogenous yeast enzyme (ALD) is shown. Extracted ion chromatogram MRM traces are shown for each metabolite along the pathway, along with reference standards using the highest precursor ion / product ion transition for each metabolite. The control represents strain CSY1235 (see Example 1.5) expressing SPE1, AsADC, and speB on a low-copy plasmid. Chromatogram traces are representative of three biological replicates. Enzyme symbols: PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; ALD, aldehyde dehydrogenase. [Figure 12] LC-MS / MS chromatograms illustrating the relative production of TA precursors (A) putrescine, (B) NMP, (C, E) 4MAB, and (D, F) NMPy in liquid cultures of engineered yeast expressing AbPMT1 and MPO enzymes according to embodiments of the present invention. (A) MRM chromatogram of putrescine (m / z +89→72) for CSY1235 harboring pCS4239 for putrescine overproduction. (B) MRM chromatogram of NMP (m / z +103→72) for CSY1235 harboring pCS4239 and expressing AbPMT1 from a low-copy plasmid. (C, D) MRM chromatograms of 4MAB (m / z +102 → 71) and NMPy (m / z +84 → 57), respectively, for CSY1235 harboring pCS4239 and expressing AbPMT1 and NtMPO1 from low-copy plasmids. (E, F) MRM chromatograms of 4MAB (m / z +102 → 71) and NMPy (m / z +84 → 57), respectively, for CSY1235 harboring pCS4239 and expressing AbPMT1 and DmMPO1ΔC-PTS1 from low-copy plasmids. The Y-axis of the traces is raw MRM ion counts. All chromatograms were generated by LC-MS / MS analysis of extracellular medium after 48 hours of growth at 30 °C in selective medium containing 2% dextrose. Traces are representative of at least three biological replicates. [Figure 13] The effect of MEU1 disruption on SAM-dependent putrescine N-methylation by AbPMT1 is shown. Wild-type strain CEN.PK2 or meu1-disrupted strain CSY1229 was cotransformed with low-copy plasmids expressing SPE1, AsADC, and speB, as well as AbPMT1. Data show the average NMP titer compared to the CEN.PK2 control, as quantified by LC-MS / MS peak area of ​​three biological replicates after 48 hours of growth at 30°C in selective medium containing 2% dextrose. Error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. [Figure 14]Figure 1 shows in silico predictions of the subcellular localization of NMPy biosynthetic genes in plant and yeast / fungal cells using the SherLoc2 web server. Values ​​and colors indicate the probability score (0–1) of localization to each compartment: CYT, cytosol; NUC, nucleus; VAC, vacuole; CHL, chloroplast; MIT, mitochondria; POX, peroxisome. [Figure 15] (A) Colocalization of N- and C-terminally GFP-tagged NtMPO1 with the PEX3 peroxisomal marker, and (B) the effect of N- and C-terminal GFP tagging of NtMPO1 on the production of the TA precursors 4MAB and NMPy in liquid cultures of engineered yeast, according to an embodiment of the present invention. This figure shows experimental validation of the subcellular localization of NtMPO1. (A) Fluorescence microscopy of N- and C-terminal GFP fusions of NtMPO1 coexpressed with the peroxisomal marker mCherry-PEX3 in wild-type yeast (CEN.PK2). White arrows indicate the colocalization of GFP-tagged NtMPO1 with peroxisomes. Scale bar, 10 μm. (B) The effect of enforcing cytosolic localization of NtMPO1 on 4MAB or NMPy production. Wild-type yeast (CEN.PK2) was co-transformed with low-copy plasmids expressing wild-type NtMPO1, or N-terminal GFP fusions or C-terminal GFP fusions, and low-copy plasmids expressing SPE1, AsADC, and speB, as well as AbPMT1. LC-MS / MS analysis was performed after 48 hours of growth at 30°C in selective medium containing 2% dextrose. Data represent the mean of three biological replicates. Error bars indicate standard deviation. The most likely subcellular compartment is indicated based on the microscopy data in (a). [Figure 16] Fluorescence microscopy data showing the subcellular localization of AbPMT1 and NtMPO1 when heterologously expressed in yeast are presented. Microscopy was performed on wild-type yeast expressing N- or C-terminally GFP-tagged AbPMT1 or NtMPO1 from low-copy plasmids. Scale bar, 10 μm. [Figure 17](A) Sequence alignment of NtMPO1 identified from plant transcriptome data with the putative MPO enzymes AbMPO1 and DmMPO1 (SEQ ID NOs: 27-29, from top to bottom). (B) Comparison of the production of the TA precursors 4MAB and NMPy in liquid culture of engineered yeast strains expressing NtMPO1, AbMPO1, or DmMPO1. (C) Comparison of the predicted three-dimensional structures of NtMPO1, AbMPO1, and DmMPO1 determined from homology modeling, according to embodiments of the present invention. (A) Alignment of the query NtMPO1 sequence against AbMPO1 and DmMPO1 candidates from the 1000 Plants Project database. Blue indicates conservation of amino acid structure, and red indicates mismatches. (B) Comparison of the relative activities of MPO orthologs. The putrescine-overproducing strain CSY1235 (see Example 1.5) was cotransformed with low-copy plasmids expressing SPE1, AsADC, and speB, AbPMT1, and one of three MPO variants. LC-MS / MS analysis was performed after 48 h of growth at 30 °C in selective medium. Data represent the average of three biological replicates. Error bars indicate standard deviation. (C) Homology model of the MPO enzyme (pink) constructed based on the crystal structure of pea (Pisum sativum) copper-containing aminooxidase (PDB: 1KSI, blue) using the RaptorX web server. Top: NtMPO1; middle: AbMPO1; bottom: DmMPO1. [Figure 18]Figure 1 illustrates 4MAB production in liquid culture of engineered yeast strains overproducing putrescine and expressing AbPMT1 and N- and C-terminal truncated forms of NtMPO1 and DmMPO1. This figure shows the effect of N- and C-terminal truncations of methylputrescine oxidase on 4MAB production in engineered yeast. The wild-type (WT) enzyme and the indicated truncated forms were expressed from low-copy plasmids in the putrescine-overproducing strain CSY1235 (see Example 1.5). Strains were grown in selective medium containing 2% dextrose at 30°C for 48 hours before LC-MS / MS analysis. All data represent the mean of at least three biological replicates, and error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. [Figure 19] Figure 1 shows the production of the TA precursors 4MAB and NMPy and the byproduct 4MAB acid in liquid culture of an engineered yeast strain harboring a single disruption in one of the four native aldehyde dehydrogenase genes. This figure illustrates the effect of disruption of individual aldehyde dehydrogenases on the accumulation of 4MAB acid. The putrescine-overproducing strain CSY1235 (control) or daughter strains carrying nonsense mutation disruptions in hfd1, ald4, ald5, or ald6 were transformed with low-copy plasmids expressing SPE1, AsADC, and speB, AbPMT1, and DmMPO1ΔC-PTS1. Bars indicate the relative 4MAB acid titer measured by LC-MS / MS peak area normalized to CSY1235 (no ALD disruption) after 48 h of growth at 30 °C in selective medium. Data represent the average of three biological replicates, and error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. [Figure 20]Figure 1 shows the production of (A) the 4MAB acid by-product and (B) the TA precursors 4MAB and NMPy in liquid cultures of engineered yeast strains harboring one or more disruptions to native aldehyde dehydrogenase. This figure illustrates the effect of aldehyde dehydrogenase gene disruptions on the production of (A) the 4MAB acid by-product and (B) 4MAB and NMPy in engineered yeast. "+" and "-" symbols indicate the presence or absence of functional enzyme, respectively. Strains were grown in selective medium containing 2% dextrose (YNB-DO) at 30 °C for 48 h before LC-MS / MS analysis. All data represent the mean of at least three biological replicates, and error bars indicate standard deviation. Student's two-tailed t-test: *P<0.05, **P<0.01, ***P<0.001. Unless otherwise stated, statistical significance is indicated compared to the corresponding control (CSY1235). [Figure 21] This figure illustrates a comparison of the production of the TA precursor NMPy in liquid culture of engineered yeast strains with either low-copy plasmid-based or genomic expression of the putrescine overproduction genes, AbPMT1, and truncated DmMPO1, according to an embodiment of the present invention. The figure shows a comparison of 4MAB and NMPy production with plasmid-based (CSY1241) and genomic (CSY1243) expression of the NMPy biosynthetic genes. Strain CSY1241 was transformed with low-copy plasmids expressing the putrescine overproduction genes (SPE1, AsADC, speB), AbPMT1, and DmMPO1ΔC-PTS1. Strain CSY1243 expressed all of the aforementioned genes from copies integrated into the genome. NMPy levels were quantified by LC-MS / MS after 48 hours of growth at 30°C in selective (CSY1241) or nonselective (CSY1243) medium. Data represent the mean of at least two biological replicates, and error bars indicate the standard deviation. [Figure 22] 1 shows a biosynthetic pathway for the production of the by-product hygulin from NMPy and MPOB according to an embodiment of the present invention. Putative major and minor side reactions in yeast are indicated by thick and dotted arrows, respectively. [Figure 23]This figure illustrates the comparison of the production of the TA precursors tropinone and tropine and the byproduct hyglin in liquid culture of engineered yeast strains expressing low-copy plasmid-based AbPYKS, AbCYP82M3, DsTR1, and one of four different CPRs. This figure shows the production of tropine and related intermediates with expression of AbPYKS, AbCYP82M3, and DsTR1 in engineered yeast. The indicated genes are expressed from low-copy plasmids in CSY1246, and "+" and "-" symbols indicate the presence or absence of the enzyme. Strains were grown at 30°C for 48 hours in selective medium containing 2% dextrose before LC-MS / MS analysis. Data represent the mean of three biological replicates, and error bars indicate the standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. [Figure 24] (A) LC-MS / MS chromatogram showing the characteristic triple peak of the TA precursor MPOB produced in liquid culture of an engineered yeast strain, and (B) illustrate the production of the TA precursors NMPy and MPOB in liquid culture of a yeast strain engineered to express AbPYKS, AbCYP82M3, and one of four CPRs from a plasmid. This figure shows the accumulation of NMPy and MPOB in the medium of an engineered strain expressing AbPYKS. (A) Representative LC-MS / MS multiple reaction monitoring (MRM) chromatogram for detecting MPOB in the extracellular medium of CSY1246, which expresses AbPYKS only from a low-copy plasmid. The three characteristic MPOB isoform peaks are labeled (I), (II), and (III). LC-MS / MS analysis was performed after 48 hours of growth at 30 °C in selective medium. (B) Relative abundance of NMPy and MPOB (all three peaks) in the extracellular medium of CSY1246 expressing AbPYKS, AbCYP82M3, and one of the four CPRs from a low-copy plasmid after 48 h of growth at 30 °C in selective medium. "+" and "-" symbols indicate the presence or absence of the gene. Data represent the mean of three biological replicates, and error bars indicate the standard deviation. [Figure 25]This figure illustrates the effect of growth temperature on the production of the TA precursor tropine and the by-product hyglin in liquid cultures of engineered yeast. This figure shows the effect of growth temperature on spontaneous hyglin production in a tropine-producing yeast strain (CSY1248). Relative selectivity represents the ratio of the relative titer of tropine to the relative titer of hyglin. Strains were grown in non-selective medium containing 2% dextrose at 30°C or 25°C for 48 hours before LC-MS / MS analysis. Data represent the mean of three biological replicates, and error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. [Figure 26] (A) Illustrates the effect of ALD4 and ALD6 reconstitution on the growth of an engineered yeast strain that produces tropine in medium with or without acetate supplementation, and (B) the effect of removing acetate auxotrophy on the production of the by-products 4MAB acid and hyglin in liquid cultures of an engineered yeast strain that produces tropine, according to embodiments of the invention. This figure shows the effect of removing acetate auxotrophy in an engineered tropine-producing yeast strain. (A) The effect of reconstituting functional ALD4 or ALD6 genes on the growth of an NMPy-producing yeast strain (CSY1246) with or without acetate supplementation. ALD4 and ALD6 were expressed from low-copy plasmids. "WT" indicates CSY1246 containing the control (BFP) plasmid. The adjacent columns indicate 10-fold dilutions. (B) Production of 4MAB acid and hyglin by-products with reconstituted acetate metabolism in engineered yeast. The "+" and "-" symbols indicate the presence or absence of the supplied metabolite (acetate) or the ALD4 and ALD6 genes expressed from low-copy plasmids. Strains were grown in selective medium containing 2% dextrose (YNB-DO) at 30°C for 48 h before LC-MS / MS analysis. Data represent the mean of three biological replicates, and error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. [Figure 27](A) The effect of acetate requirement on the accumulation of the TA precursor between NMPy and tropinone in liquid cultures of yeast strains engineered to produce tropine, and (B) representative LC-MS / MS chromatograms of the TA precursor MPOB produced in liquid cultures of yeast strains engineered to produce tropine with and without acetate requirement, according to embodiments of the invention. This figure shows the effect of reconstitution of ALD6 activity on metabolite flux through NMPy toward tropine in engineered yeast. (A) Production of the intermediate between NMPy and tropinone in engineered strains with and without functional Ald6p. Intermediate abundance was measured by LC-MS / MS MRM in the extracellular medium of an integrated tropin-producing strain (CSY1248) grown in nonselective medium supplemented with 0.1% w / v potassium acetate (gray) or a tropin-producing strain with reconstituted ALD6 (CSY1249) grown in nonselective medium without acetate supplementation (pink) at 25 °C for 48 h. Data represent the mean of three biological replicates, and error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. (B) Representative MRM chromatograms of MPOB production from CSY1248 (gray) and CSY1249 (red) cultured as described in (a). [Figure 28] This figure illustrates the progression of improved production of the TA precursor tropine and by-product hyglin in liquid cultures of engineered yeast strains. This figure presents an overview of strains engineered to increase tropine production in yeast. The "-" symbol indicates the absence of a gene, while "p" and "i" indicate gene expression from a low-copy plasmid or genomic integration, respectively. Strains were grown in selective or non-selective media containing 2% dextrose at 30°C or 25°C for 48 hours before LC-MS / MS analysis. Data represent the mean of three biological replicates, and error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. [Figure 29]This figure illustrates the effect of expressing additional copies of the heterologous biosynthetic enzymes PMT1, MPO1ΔC-PTS1, PYKS, and CYP82M3 on the production of each TA precursor between putrescine and tropine in liquid cultures of engineered yeast, according to an embodiment of the present invention. The figure identifies metabolic bottlenecks in an optimized tropine-producing strain (CSY1249). Strain CSY1249 was transformed with a control plasmid expressing BFP ("no overexpression") or low-copy plasmids expressing additional copies of AbPMT1, DmMPO1ΔC-PTS1, AbPYKS, or AbCYP82M3. Intermediate levels in the extracellular medium were quantified by LC-MS / MS after 48 hours of growth at 25°C in selective medium. Data represent the average of three biological replicates, and error bars indicate standard deviation. [Figure 30] Figure 1 shows the effect of additional copies of the bottleneck enzymes PMT and PYKS on tropine production in engineered yeast. This figure demonstrates the alleviation of metabolic bottlenecks by genomic integration of additional copies of the PMT and PYKS enzymes. Tropine-producing strains CSY1249 and CSY1251 were grown in non-selective medium at 25°C for 48 hours before LC-MS / MS analysis of the growth medium. Data represent the mean of three biological replicates, and error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. [Figure 31]Production of the TA precursor acyl donor compound PLA in liquid culture of an engineered yeast strain expressing heterologous lactate dehydrogenase and phenylpyruvate reductase enzymes. This figure shows LC-MS / MS analysis of a yeast strain engineered to convert L-phenylalanine to 3-phenyllactic acid. Yeast strains were engineered to contain low-copy CEN / ARS plasmids carrying the LEU2 selectable marker, the TDH3 promoter, and the coding sequence for BFP as a negative control, LDH variants from B. coagulans (BcLLDH), L. casei (LcLLDH), L. plantarum (LpLLDH), or PPR variants from A. belladonna (AbPPR), L. plantarum (LpPPR), Escherichia coli (hcxB), or W. fluorescens (WfPPR). Yeast were grown from freshly transformed colonies in 300 μL of selective medium (-Leu) in 96-well deep-well microtiter plates. After 72 hours of growth in a shaking incubator at 25°C and 460 rpm, yeast were pelleted and the culture supernatant was analyzed by LC-MS / MS. Data show the relative 3-phenyllactic acid titer based on extracted ion chromatograms (ammonium adduct, EIC m / z+ = 184), normalized to trace levels present in the negative control. Data represent the mean of three biological replicates, and error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. [Figure 32]LC-MS / MS chromatograms showing the production of cinnamic acid, a TA precursor acyl donor compound, in liquid culture of an engineered yeast strain expressing phenylalanine ammonia-lyase. This figure shows LC-MS / MS analysis of a yeast strain engineered to convert L-phenylalanine to cinnamic acid. The yeast strains were engineered to carry a TRP1 selectable marker, a TEF1 promoter, and a low-copy CEN / ARS plasmid carrying the coding sequence for either (i) BFP or (ii) A. thaliana phenylalanine ammonia-lyase (AtPAL1). Yeast were grown from freshly transformed colonies in 300 μL of selective medium (-Trp) in 96-well deep-well microtiter plates. After 48 h of growth in a shaking incubator at 30 °C and 460 rpm, the yeast were pelleted and the culture supernatant was analyzed by LC-MS / MS. Chromatogram traces show the cinnamic acids produced by these strains based on the most abundant multiple reaction monitoring (MRM) transition of cinnamic acid (m / z +149 → 131). Each trace represents three samples. [Figure 33] This figure illustrates the substrate specificity of the UDP-glucosyltransferase 84A27 (UGT84A27) ortholog from the TA-producing Solanaceae expressed in engineered yeast. This figure shows a comparison of the activity of UGT84A27 orthologs toward three different phenylpropanoid compounds expressed in engineered yeast. (A) Phenypropanoids tested as glucose (Glu) acceptors for UGT84A27 in engineered yeast. Top, (D)-3-phenyllactic acid (PLA); middle, trans-cinnamic acid (CA); bottom, trans-ferulic acid (FA). (B) Heat map of the conversion rates of fed phenylpropanoids to glucosides by yeast engineered to express UGT84A27. UGT84A27 orthologs or a BFP negative control were expressed from a low-copy plasmid in CSY1251. Transfected cells were cultured in selective medium supplemented with 500 μM PLA, CA, or FA for 72 h before LC-MS / MS analysis. Data represent the mean ± standard deviation of n = 3 biologically independent samples. [Figure 34]An example of chromatographic and mass spectrometric analysis of UGT84A27 activity is illustrated. This figure shows representative LC-MS / MS traces demonstrating the conversion of PLA, CA, and FA to their cognate glucosides by AbUGT in CSY1251 cultured for 120 h as in Figure 33B to allow for more complete glucosylation. For PLA, the acid (top trace in each panel) and glucosides (bottom trace in each panel) were distinguished by their different NH4 + adduct parent masses as well as different retention times. For CA and FA, rapid fragmentation was required to detect the glucosides based on the less-retained peaks generated by the phenylpropanoid fragments. [Figure 35] Active site engineering based on structural information from AbUGT to alter substrate specificity is shown. This figure shows structural analysis of the AbUGT3D structure to identify potential mutations that enhance PLA activity. (A) A homology model of AbUGT84A27 constructed based on the crystal structure of the UDP-bound Arabidopsis thaliana salicylic acid UDP-glucosyltransferase UGT74F2 (PDB: 5V2K). PLA (orange) is shown in a favorable binding pose with UDP-glucose (pink) based on docking simulations. (B) A close-up view of the AbUGT active site containing docked D-PLA and UDP-glucose. Potential mutations (F130Y, L205F, I292Q) identified to improve PLA selectivity are shown, and dashed lines indicate putative polar / hydrogen-bond interactions. [Figure 36] Substrate specificity of AbUGT84A27 active site mutants is shown. This figure shows a heat map of the conversion rates of supplied phenylpropanoids to glucosides by yeast engineered to express AbUGT mutants. AbUGT wild-type, active site mutants, or a BFP negative control were expressed from low-copy plasmids in CSY1251. Transformed cells were grown in selective medium supplemented with 500 μM PLA, CA, or FA for 72 hours before LC-MS / MS analysis. Data represent the mean ± standard deviation of n=3 biologically independent samples. [Figure 37]LC-MS / MS chromatograms verifying the stepwise biosynthesis of PLA-glucosides in yeast engineered for tropine production are shown. The figure shows multiple reaction monitoring (MRM) and extracted ion chromatogram (EIC) traces from the culture medium of a yeast strain engineered for stepwise reconstitution of PLA-glucosides. Strains were grown in non-selective medium for 72 h prior to LC-MS / MS analysis of the culture supernatant. Chromatogram traces are representative of three biological replicates. [Figure 38] Schematic diagram of the biosynthetic pathways for the two metabolic fates of glucose in yeast. This diagram shows the effect of citrate on glucoside production through inhibition of glycolysis. Abbreviations: HXK, hexokinase; GPI, glucose-6-phosphate isomerase; PFK, phosphofructokinase; PGM, phosphoglucomutase; UGP, UDP-glucose pyrophosphorylase. [Figure 39] Figure 1 shows the effect of citrate supplementation on heterologous glucosides production in engineered yeast. This figure shows the effect of 2% citrate supplementation on the conversion of phenylpropanoid acids to glucosides by yeast engineered for AbUGT expression. Strain CSY1288 was grown in nonselective medium with or without 2% citrate, with or without additional supplementation, or with 500 μM trans-cinnamic acid (CA) or trans-ferulic acid (FA) supplementation to assess glucosylation of endogenously produced PLA. Cultures were grown for 72 h before LC-MS / MS analysis. Data represent the mean of n = 3 biologically independent samples (open circles), and error bars indicate standard deviation. Two-tailed Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001. [Figure 40]Relative PLA-glucoside production in yeast strains engineered for overexpression of UDP-glucose biosynthetic enzymes is shown. This figure demonstrates the effect of overexpression of native enzymes involved in the biosynthesis of the glucoside precursor UDP-glucose on PLA-glucoside production in engineered yeast. The enzymes or negative control (BFP) were expressed from low-copy plasmids in strain CSY1288. Strains were grown in selective medium for 72 hours before LC-MS / MS analysis of metabolites in the culture supernatant. Data represent the mean of n = 3 biologically independent samples (open circles); error bars indicate standard deviation. Two-tailed Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance compared to corresponding controls is indicated. [Figure 41] Figure 1 shows relative PLA-glucoside production in CSY1288 with disruption of endogenous glucosidases. This figure demonstrates the effect of disrupting each of the three native glycosidase genes on PLA-glucoside accumulation in engineered yeast. Strains were grown in non-selective medium for 72 hours before LC-MS / MS analysis of culture supernatants. Data represent the mean of n=3 biologically independent samples (open circles); error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. Statistical significance compared to corresponding controls is indicated. [Figure 42]LC-MS / MS chromatograms show the production of the medicinal TA precursor hyoscyamine aldehyde from littorine in liquid cultures of engineered yeast cells expressing AbCYP80F1. This figure shows LC-MS / MS analysis of a yeast strain engineered to convert (R)-littorine to hyoscyamine aldehyde. The yeast strain was engineered to harbor a low-copy CEN / ARS plasmid carrying a LEU2 selectable marker, a TDH3 promoter, and the coding sequence for the littorine mutase CYP80F1 (AbCYP80F1) from A. belladonna. The strain also harbors a TRP1 selectable marker, a TDH3 promoter, and a second low-copy plasmid carrying the coding sequence for (i) BFP as a negative control, (ii) S. cerevisiae CPR (NCP1), or (iii) Arabidopsis thaliana CPR (AtATR1). Yeast were grown from freshly transformed colonies in 300 μL of selective medium (-Leu--Trp) supplemented with 1 mM littorine in 96-well deep-well microtiter plates. After 48 h of growth in a shaking incubator at 30°C and 460 rpm, yeast were pelleted and the culture supernatant was analyzed by LC-MS / MS. Chromatogram traces indicate the hyoscyamine aldehyde produced by these strains based on the most abundant MRM transition (m / z +288→124). The arrow indicates the putative hyoscyamine aldehyde peak. Each trace represents triplicate samples. [Figure 43]Production of the medicinal TA scopolamine from the medicinal TA hyoscyamine in liquid culture of engineered yeast cells expressing an ortholog of hyoscyamine 6β-hydroxylase / dioxygenase (H6H). This figure shows the conversion of (S)-hyoscyamine to (S)-scopolamine by engineered yeast strains expressing H6H orthologs. Yeast strains were engineered to carry a low-copy CEN / ARS plasmid carrying a LEU2 selectable marker, the TDH3 promoter, and the coding sequence for BFP as a negative control, or H6H variants from D. stramonium (DsH6H), A. acutangulus (AaH6H), B. arborea (BaH6H), or D. metel (DmH6H). Yeast were grown from freshly transformed colonies in 300 μL of selective medium (-Leu) supplemented with 1 mM hyoscyamine in 96-well deep-well microtiter plates. After 48 h of growth in a shaking incubator at 30°C and 460 rpm, yeast were pelleted and the culture supernatant was analyzed by LC-MS / MS. Data represent the average of three biological replicates and are normalized to the amount of scopolamine contaminant in the supplied hyoscyamine. Error bars represent standard deviation. Relative scopolamine titer was quantified based on the peak area of ​​the m / z +304→138 MRM transition. [Figure 44]This figure illustrates the effect of cofactor availability and media supplementation on the conversion of hyoscyamine to scopolamine in liquid cultures of engineered yeast cells expressing DsH6H. This figure shows the effect of cofactor supplementation on the conversion of (S)-hyoscyamine to (S)-scopolamine in engineered yeast. Yeast strains were engineered to carry a LEU2 selectable marker, a TDH3 promoter, and a low-copy CEN / ARS plasmid carrying the coding sequence for either (i) BFP as a negative control or (iii) hyoscyamine 6β-hydroxylase / dioxygenase from D. stramonium (DsH6H). Yeast were grown from freshly transformed colonies in 300 μL of selective medium (-Leu) supplemented with the indicated substrates and / or cofactors in 96-well deep-well microtiter plates. After 48 h of growth in a shaking incubator at 30 °C and 460 rpm, the yeast were pelleted and the media supernatant was analyzed by LC-MS / MS. (S)-Scopolamine relative potency was quantified based on the integrated peak area of ​​the m / z +304→138 MRM transition and normalized to a strain expressing DsH6H supplemented with all cofactors and substrates. Data represent the average of three biological replicates, and error bars indicate standard deviation. Hyo, (S)-hyoscyamine; 2-OG, 2-oxoglutarate; L-AA, L-ascorbic acid. [Figure 45] Hierarchical clustering heatmap of candidate hyoscyamine dehydrogenase genes identified from the A. belladonna transcriptome by analysis of tissue co-expression data. This figure shows the clustering of tissue-specific expression profiles of transcripts from the A. belladonna transcriptome that potentially encode enzymes with hyoscyamine dehydrogenase activity. Expression of each candidate transcript is scaled row-by-row using a normal distribution. The dendrogram shows the hierarchical clustering of candidates by their tissue-specific expression profiles. Known TA pathway genes are identified by name, and putative HDH candidates are indicated by locus ID. Black triangles indicate candidates that have been screened for activity, and double black triangles indicate candidates with experimentally validated HDH activity. [Figure 46] This figure illustrates the production of the medicinal TA scopolamine from littorine in liquid cultures of engineered yeast cells expressing candidate hyoscyamine dehydrogenase (HDH) genes. This figure shows experimental screening of the activity of HDH candidates identified from the A. belladonna transcriptome in engineered yeast. Yeast strains were engineered to express A. belladonna littorine mutase (AbCYP80F1) and D. stramonium hyoscyamine 6β-hydroxylase / dioxygenase (DsH6H) from constitutive promoters within expression cassettes integrated into the genome, as well as one of 13 HDH candidates each from a low-copy CEN / ARS plasmid carrying the TRP1 selectable marker and the TDH3 promoter. Yeast were grown from freshly transformed colonies in 300 μL of selective medium (-Trp) supplemented with 1 mM littorine in 96-well deep-well microtiter plates. After 72 hours of growth in a shaking incubator at 30°C and 460 rpm, yeast cells were pelleted and the culture supernatant was analyzed by LC-MS / MS. Hyoscyamine aldehyde relative titer was quantified based on the integrated peak area of ​​the m / z +288→124 MRM transition and normalized to the peak area of ​​an engineered strain expressing BFP instead of the HDH candidate. (S)-Scopolamine titer was quantified based on the integrated peak area of ​​the m / z +304→138 MRM transition and a calibration curve of authentic scopolamine standards. Data represent the average of three biological replicates, and error bars indicate standard deviation. [Figure 47] The three-dimensional structure of hyoscyamine dehydrogenase from A. belladonna is illustrated. The figure shows a cartoon representation of the structure of AbHDH as a homology model built based on the crystal structure of Populus tremuloides sinapil alcohol dehydrogenase (PtSAD; PDB:1YQD) as a template. NADPH and Zn2+ are shown in the active site. The inset box shows a close-up of the AbHDH active site, including NADPH and docked hyoscyamine aldehyde. Dashed lines indicate interactions important for catalysis. [Figure 48] Figure 1 shows a phylogenetic tree of the three identified HDH orthologs (AbHDH, DiHDH, and DsHDH) along with their closest protein hits in the UniProt / SwissProt database. The figure illustrates the clustering of the three identified HDH enzyme orthologs with closely related protein sequences based on a BLAST search of the UniProt / SwissProt database. The sequences shown include the top 50 BLASTp hits based on E-values ​​and 10 additional hits selected from the next 100 ranks. Phylogenetic relationships were derived via bootstrap neighbor-joining with n = 1000 runs in ClustalX2, and the resulting tree was visualized with FigTree software. Abbreviations: ADH, alcohol dehydrogenase; CADH, cinnamyl alcohol dehydrogenase; MTDH, mannitol dehydrogenase; DPAS, dehydroprecondylocarpine acetate synthase; 8HGDH, 8-hydroxygeraniol dehydrogenase; GDH, geraniol dehydrogenase; GS, geissoschizine synthase; REDX, unspecified redox protein. [Figure 49]This figure illustrates the production of the medicinal TA scopolamine from littorine in liquid cultures of engineered yeast cells expressing hyoscyamine dehydrogenase orthologs. This figure shows a comparison of activity between identified HDH enzyme orthologs expressed in the engineered yeast. The yeast strains were engineered to express A. belladonna littorine mutase (AbCYP80F1) and D. stramonium hyoscyamine 6β-hydroxylase / dioxygenase (DsH6H) from constitutive promoters within expression cassettes integrated into the genome, and to express one of three HDH orthologs (AbHDH, DiHDH, or DsHDH) from a low-copy CEN / ARS plasmid carrying a TRP1 selectable marker and the TDH3 promoter, and to express an additional copy of DsH6H from a low-copy CEN / ARS plasmid carrying a LEU2 selectable marker and the TDH3 promoter. Yeast were grown from freshly transformed colonies in 300 μL of selective medium (-Leu-Trp) supplemented with 1 mM littorine in 96-well deep-well microtiter plates. After 72 h of growth in a shaking incubator at 30°C and 460 rpm, yeast were pelleted and the culture supernatant was analyzed by LC-MS / MS. Hyoscyamine aldehyde relative titer was quantified based on the integrated peak area of ​​the m / z +288→124 MRM transition and normalized to the peak area of ​​an engineered strain expressing AbHDH and BFP instead of DsH6H. (S)-Scopolamine titer was quantified based on the integrated peak area of ​​the m / z +304→138 MRM transition and a calibration curve of authentic scopolamine standards. Data represent the average of three biological replicates, and error bars indicate standard deviation. [Figure 50]This figure illustrates experimental validation of the conversion of fed littorine to scopolamine by yeast engineered for expression of CYP80F1, HDH, and H6H. The figure shows multiple reaction monitoring (MRM) LC-MS / MS traces from the culture medium of a yeast strain engineered for the conversion of littorine to scopolamine. The strain was grown in non-selective medium supplemented with 1 mM littorine for 72 h prior to LC-MS / MS analysis of metabolites in the culture supernatant. The dark trace in the lower right panel (CSY1294, scopolamine) represents a 125 nM (38 μg / L) scopolamine standard. Chromatogram traces are representative of three biological replicates. [Figure 51] The canonical plant ER-to-vacuole transport and maturation pathway of SCPL acyltransferase (SCPL-AT). This diagram shows a schematic of a typical ER-to-vacuole protein transport pathway followed by a plant SCPL-AT, with A. belladonna littorine synthase (AbLS) shown as an example. Circled numbers indicate key steps in SCPL-AT expression and activity, including (1) ER lumen and (2) Golgi maturation, (3) vacuolar transport, (4) substrate import, and (5) product export. [Figure 52] Colocalization of wild-type littorine synthase from A. belladonna expressed in engineered yeast. This figure shows epifluorescence microscopy of yeast engineered for expression of N-terminally GFP-tagged AbLS (GFP-AbLS) and stained with the vacuolar membrane dye FM4-64. Microscopy was performed on CSY1294 expressing GFP-AbLS from a low-copy plasmid. Scale bar, 5 μm. [Figure 53]This figure illustrates a strategy for forcing littorine synthase to different yeast subcellular compartments via signal sequence replacement. This figure illustrates a protein engineering approach to modify the subcellular localization of AbLS to address potential limitations on substrate availability in different compartments. (A) Schematic of the yeast subcellular compartments targeted for AbLS localization via signal sequence swapping. The signal sequence source protein is indicated for each compartment. (B) Terminus and residues selected for AbLS signal sequence replacement. The residues comprising each signal sequence domain were selected based on structural annotations in the UniProt / SwissProt database. [Figure 54] Western blot of wild-type AbLS expressed in tobacco and treated with deglycosylase is shown. This figure demonstrates the identification of glycosylation-modified AbLS expressed in plants. C-terminal HA-tagged AbLS was transiently expressed in N. benthamiana leaves via agroinfiltration. Crude leaf extracts were either untreated (lane 1: '-') or treated with peptide N-glycosidase F (PNGase F; lane 2: 'N') or O-glycosidase (lane 3: 'O') to remove N- or O-linked glycosylation, respectively. Crude extracts were separated by electrophoresis on a NuPAGE 4-12% Bis-Tris gel and then transferred to a nitrocellulose membrane for immunodetection using a chimeric rabbit IgGκ anti-HA HRP-conjugated antibody. All electrophoresis and blotting steps were performed under disulfide-reducing conditions (see online methods). Lane "L", Bio-Rad Precision Plus DualColor protein ladder. [Figure 55]Western blots of AbLS glycosylation site mutants expressed in yeast and tobacco are shown. This figure compares the N-glycosylation patterns present in AbLS expressed in yeast and tobacco. C-terminal HA-tagged wild-type AbLS, single glycosylation site point mutants (N→Q), or quadruple mutants were transiently expressed via agroinfiltration in N. benthamiana ('Nb') (A) or from a low-copy plasmid in CSY1294 ('yeast') (B). Preparation of crude extracts from tobacco and yeast was performed under denaturing, disulfide-reducing conditions (see online methods). Crude extracts were separated by electrophoresis on NuPAGE 4-12% Bis-Tris gels and then transferred to nitrocellulose membranes for immunodetection using a chimeric rabbit IgGκ anti-HA HRP-conjugated antibody. All electrophoresis and blotting steps were performed under disulfide-reducing conditions (see online methods). For (A) and (B), corresponding yeast- and tobacco-expressed controls are included for comparison. Lane "L", Bio-Rad Precision Plus DualColor protein ladder. [Figure 56]Phylogenetic identification of putative endoproteolytic propeptide removal in littorine synthase. The figure shows a sequence alignment of AbLS with characterized serine carboxypeptidases and SCPL acyltransferases known to possess (AtSCT, AsSCPL1, TaCBP2) or lack (AtSMT, yPRC1) an internal propeptide linker (bold, gray) that is proteolytically removed. The putative N-terminal signal peptide is shown in bold (black), and disulfide bonds are shown as connecting lines. AtSCT, Arabidopsis thaliana sinapoylglucose:choline sinapoyltransferase; AtSMT, A. thaliana sinapoylglucose:malate sinapoyltransferase; AbLS, Atropa belladonna littorine synthase; AsSCPL1, Avena strigosa avenacin synthase; TaCBP2, Triticum aestivum carboxypeptidase 2; yPRC1, yeast carboxypeptidase Y. From top to bottom: SEQ ID NOs: 30-35. [Figure 57] Structural identification of putative endoproteolytic propeptide removal in littorine synthase. This figure shows a comparison of the three-dimensional structures of two SCPL-ATs, one of which is known to contain an internal propeptide sequence that is removed by proteolysis. Left: Crystal structure of TaCBP2 (PDB:1WHT) in (top) pictorial representation and (bottom) surface representation, showing the disulfide bond and internal propeptide removal site. Right: Homology model of AbLS based on the crystal structure of TaCBP2 in (top) pictorial representation and (bottom) surface representation, showing the N-terminal signal peptide, disulfide bond, and putative internal propeptide that appears to block access to the active site. [Figure 58]Analysis of the proteolytic cleavage patterns of AbLS split control and putative propeptide-exchange variants in yeast. This figure shows Western blot analysis of protein fragment sizes generated by AbLS split control and propeptide variants expressed in engineered yeast. C-terminally HA-tagged AbLS variants were expressed from a low-copy plasmid in CSY1294 (lanes 1-6), and HA-tagged wild-type AbLS expressed in Nicotiana benthamiana (Nb) is shown as an additional control (lane 7). Gel electrophoresis and blotting were performed under disulfide-reducing conditions, and detection was performed using an anti-HA antibody (see online methods). Lane symbols: L, protein molecular weight ladder; WT, wild-type AbLS; SPL, AbLS split at the putative propeptide, with signal peptides in both fragments; SPL-T, AbLS split at the putative propeptide, without signal peptides in either fragment; GS, AbLS variant in which the wild-type propeptide was replaced with a flexible Gly-Ser linker; SCT, AbLS variant in which the wild-type propeptide was replaced with the AtSCT propeptide sequence; CUT, AbLS variant with the wild-type propeptide replaced with a synthetic polyarginine site recognized and cleaved by the Kex2p protease. [Figure 59] This figure illustrates the de novo production of hyoscyamine and scopolamine in yeast strains engineered for expression of N-terminal fusions of AbLS. This figure shows a comparison of de novo production of hyoscyamine and scopolamine in yeast strains expressing AbLS with different soluble protein domains fused to the N-terminus. Wild-type (control) or AbLS fusions were expressed from low-copy plasmids in CSY1294. Transformants were grown in selective medium for 96 hours before LC-MS / MS analysis of metabolites in culture supernatants. Data represent the mean of n = 3 biologically independent samples (open circles); error bars indicate standard deviation. Two-tailed Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001. [Figure 60]Fluorescence microscopy of tobacco alkaloid transporters expressed in CSY1296 to alleviate vacuolar TA transport restriction. This figure shows fluorescence microscopy images of engineered yeast expressing tobacco alkaloid transporters fused to GFP at the C-terminus to allow for confirmation of subcellular localization. C-terminal GFP fusions of (A) NtJAT1 and (B) NtMATE2 were expressed from low-copy plasmids in CSY1296. Scale bar, 5 µm. [Figure 61] Production of tropine, hyoscyamine, and scopolamine is shown in CSY1296 engineered for expression of heterologous alkaloid transporters. This figure demonstrates the utility of various plant alkaloid transporters in alleviating intracellular substrate transport limitations in yeast engineered for TA production. Tobacco (Nicotiana tabacum) jasmonate-inducible alkaloid transporter 1 (NtJAT1), multidrug and toxin efflux (MATE) transporter 1 or 2, or a negative control (BFP) were expressed from low-copy plasmids in CSY1296. Transformants were grown in selective medium for 96 hours before LC-MS / MS analysis of metabolites in the culture supernatant. Data represent the mean of three biologically independent samples (open circles), and error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. [Figure 62](A) LC-MS / MS chromatograms in product ion mode and (B) multiple reaction monitoring mode are shown, demonstrating de novo production of the unnatural TA cinnamoyltropine in engineered yeast. This figure shows LC-MS / MS analysis of engineered yeast strains producing the unnatural TA cinnamoyltropine. (A) Tandem MS / MS spectra of the extracellular medium of (i) the tropine-producing strain CSY1251, (ii) CSY1251 expressing phenylalanine ammonia lyase (AtPAL1), 4-coumarate-CoA ligase 5 (At4CL5), and cocaine synthase (EcCS) (denoted as CSY1282), or (iii) an authentic cinnamoyltropine standard relative to the parent mass at m / z = 272. The blue diamond indicates the parent compound peak. (B) Verification of EcCS acyltransferase activity toward cinnamic acid and α-tropine via substrate feeding. Strains were transformed with a combination of plasmids expressing AtPAL1 (low-copy plasmid pCS4252) and / or At4CL5 and EcCS (high-copy plasmid pCS4207) and grown in media supplemented with different substrates as follows: (i) CEN.PK2 + At4CL5 + EcCS + 0.1 mM trans -cinnamic acid, (ii) CEN.PK2 + At4CL5 + EcCS + 0.5 mM α-tropine, (ii (i) CEN.PK2 + AtPAL1 + At4CL5 + EcCS; (iv) CEN.PK2 + AtPAL1 + At4CL5 + EcCS + 0.5 mM α-tropine; (v) CSY1251 + At4CL5 + EcCS; (vi) CSY1251 + At4CL5 + EcCS + 0.2 mM trans-cinnamic acid; (vii) CSY1251 + AtPAL1 + At4CL5 + EcCS; (viii) 25 nM cinnamoyltropine standard. For (A) and (B), yeast strains were grown in selective medium (YNB-DO + 2% dextrose + 5% glycerol) at 25 °C for 72 h before LC-MS / MS analysis. [Figure 63]This figure illustrates the effect of various carbon sources, (A) fed alone or (B) fed together with dextrose, on the production of tropine and related TA precursors in liquid cultures of engineered yeast. This figure demonstrates carbon source optimization to improve tropine production in engineered yeast. Overnight cultures of the tropine-producing strain CSY1249 (see Example 3.3.4) were grown in non-selective rich medium (YPD). The overnight cultures were pelleted and resuspended in non-selective defined medium (YNB-SC) containing all amino acids and (A) 2% of each carbon source or (B) 2% dextrose, plus an additional 2% of each carbon source containing dextrose. Cultures were grown at 25°C for 48 hours before analysis of the growth medium by LC-MS / MS. Data show the relative titer of each metabolite, normalized to (A) 2% dextrose or (B) 2% + 2% dextrose. Data represent the average of three biological replicates, and error bars indicate standard deviation. [Figure 64] Metabolic bottleneck analysis of the scopolamine-producing strain CSY1296 is illustrated. This figure shows the effect of expressing additional copies of flux restriction enzymes on the production of TA and TA precursors in engineered yeast. Additional copies of each biosynthetic enzyme between tropine and scopolamine were expressed from the following low-copy plasmids in the CSY1296 strain: (A) WfPPR, pCS4436; (B) AbUGT, pCS4440; (C) DsRed-AbLS, pCS4526; (D) AbCYP80F1, pCS4438; (E) DsHDH, pCS4478; (F) DsH6H, pCS4439; or BFP controls (pCS4208, pCS4212, or pCS4213) corresponding to the same auxotrophic marker as each biosynthetic gene plasmid. Transformants were grown in appropriate selective media at 25°C for 96 hours, after which metabolites in the growth medium were quantified by LC-MS / MS. Data represent the mean of three biologically independent samples (open circles), and error bars indicate standard deviation. Two-tailed Student's t-test: *P<0.05, **P<0.01, ***P<0.001. [Figure 65]The effect of alleviating flux and transport limitations on the production of hyoscyamine and scopolamine in engineered yeast is shown. This figure shows a comparison of de novo production of hyoscyamine and scopolamine in yeast strains CSY1296 and CSY1297, the latter of which harbors additional genomic copies of flux-restricting enzymes (WfPPR and DsH6H) and the tobacco vacuolar alkaloid importer (NtJAT1). Strains were grown in nonselective medium for 96 h before LC-MS / MS analysis of metabolites in culture supernatants. Data represent the mean of n = 3 biologically independent samples (open circles); error bars indicate standard deviation. Two-tailed Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION OF THE INVENTION

[0013] definition Before describing the exemplary embodiments in more detail, the following definitions are set forth to illustrate and define the meaning and scope of terms used in this description.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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, NY (1991) provide those skilled in the art with the general meaning of many of the terms used herein. Furthermore, for clarity and ease of reference, certain terms are defined below.

[0015] It should be 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 "primer" refers to one or more primers, i.e., a single primer and multiple primers. It should be further noted that the claims have been drafted to exclude any optional elements. Accordingly, this statement is intended to serve as a precedent for the use of exclusive terminology such as "solely," "only," and the like, or the use of "negative" limitations in connection with the recitation of claim elements.

[0016] As used herein, the terms "determining," "measuring," and "evaluating," and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

[0017] As used herein, the term "polypeptide" refers to a polymeric form of amino acids of any length, including peptides ranging from 2 to 50 amino acids in length and polypeptides greater than 50 amino acids in length. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" includes coded and non-coded amino acids, polymers of amino acids that have been chemically or biochemically modified or derivatized, and polypeptides with modified peptide backbones in which the conventional backbone is replaced with a non-natural or synthetic backbone. Polypeptides can be of any convenient length, e.g., 2 or more amino acids, e.g., 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, e.g., 500 or 1000 or more amino acids. A "peptide" can be 2 or more amino acids, e.g., 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, e.g., up to 50 amino acids. In some embodiments, peptides are 5 to 30 amino acids in length.

[0018] As used herein, the term "isolated" refers to a 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, or even at least 99% free from other components with which the moiety is associated prior to purification.

[0019] As used herein, the term "encoded by" refers to a nucleic acid sequence that encodes a polypeptide sequence, or a portion thereof, that includes an amino acid sequence of 3 or more amino acids, e.g., 5 or more, 8 or more, 10 or more, 15 or more, or 20 or more amino acids, from the polypeptide encoded by the nucleic acid sequence. The term also includes polypeptide sequences that are immunologically identifiable as the polypeptide encoded by that sequence.

[0020] A "vector" can introduce a gene sequence into a target cell. As used herein, the terms "vector construct," "expression vector," and "gene transfer vector" are typically used interchangeably to refer to any nucleic acid construct that can direct the expression of a gene of interest and can introduce a gene sequence into a target cell, which can be achieved by genomic integration of all or part of the vector, or by transient or genetic maintenance of the vector as an extrachromosomal element. Thus, this term includes cloning and expression vehicles, as well as integrating vectors.

[0021] An "expression cassette" includes any nucleic acid construct capable of directing the expression of a gene / coding sequence of interest operably linked to the promoter of the expression cassette. Such cassettes can be assembled into "vectors," "vector constructs," "expression vectors," or "gene transfer vectors" for introducing the expression cassette into target cells. Thus, the term includes cloning and expression vehicles as well as viral vectors.

[0022] A "plurality" includes at least two members. In certain cases, a plurality can have 10 or more, e.g., 100 or more, 1000 or more, 10,000 or more, 100,000 or more, 10 or more, 10 or more, 10 or more, 10 or more, or 10 or more members. In any embodiment, a plurality can have from 2 to 20 members.

[0023] The term "tropane alkaloid product" is intended to refer to any molecule whose backbone contains an 8-azabicyclo[3.2.1]octane core group including a cycloheptane ring and a nitrogen bridge connecting carbon atoms 1 and 5, wherein the 8-azabicyclo[3.2.1]octanyl group is covalently linked to an acyl group by an ester bond at the 3-position and / or 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 positions 2, 4, 5, 6, and / or 7. Tropane alkaloid products include, but are not limited to, littorine, hyoscyamine, atropine, anisodamine, scopolamine, cocaine, and other similar tropine / pseudotropine plus acyl group natural or unnatural tropane alkaloids (e.g., calystegine).

[0024] 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 converted into a tropane alkaloid product in one or more (e.g., one or two) biosynthetic steps, where the carbon source is a carbohydrate, non-carbohydrate sugar, sugar alcohol, lipid, fatty acid, or substrate that is converted via a metabolic pathway into one or more of the above carbon sources, and the nitrogen source is any amino acid, peptide, protein, except for ammonia, urea, nitrate, nitrite, glutamate, arginine, ornithine, and citrulline, or any substrate that is converted via a metabolic pathway into one or more of the above nitrogen sources.

[0025] The term "derivative of a tropane alkaloid product" is intended to refer to any molecule not naturally produced by an unmodified organism, whose molecular backbone comprises a tropane alkaloid product and which differs from said tropane alkaloid product by the attachment of a functional group without altering the backbone itself. As used herein, attachment of a functional group includes, but is not limited to, hydroxylation, alkylation and N-alkylation, acetylation and N-acetylation, acylation and N-acylation, and halogenation.

[0026] Numeric ranges are inclusive of the numbers defining the range.

[0027] The methods described herein include multiple steps. Each step can be performed after a predetermined time has elapsed between steps, if necessary. Thus, the time between performing each step can 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 5 hours or more. In certain embodiments, each subsequent step is performed immediately after the completion of the previous step. In other embodiments, a step can be performed after an incubation or waiting time after the completion of the previous step, for example, after a waiting time of several minutes to overnight.

[0028] Other definitions of terms may appear throughout this specification.

[0029] Detailed Description Host cells engineered to produce a desired tropane alkaloid (TA), such as hyoscyamine and scopolamine, are provided. The host cells may have one or more engineered modifications selected from the following: feedback inhibition to alleviate mutations in enzyme genes, transcriptional regulatory modifications in biosynthetic enzyme genes, enzyme inactivating mutations, and heterologous coding sequences. Methods of producing a desired TA using the host cells and compositions, e.g., kits, systems, etc., that find use in the methods of the invention are also provided.

[0030] Before describing the present invention in more detail, it is to be understood that the invention is not limited to particular embodiments described, 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.

[0031] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, 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. When the stated range includes one or both of the limits, excluding either or both of the included limits is also encompassed within the invention.

[0032] Certain ranges are presented herein with the term "about" preceding the numerical values. The term "about" is used herein to provide literal support for the exact number it precedes and for numbers that are near or approximately the number it precedes. In determining whether a number is near or approximately a particular recited number, the near or approximately unrecited number may be a number that is substantially equivalent to the specifically recited number in the context in which it is presented.

[0033] Unless otherwise defined, 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 can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0034] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated 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 publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0035] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements 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 invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0036] In further describing the present invention, the TA precursors, TAs, and modifications of TAs, including TA derivatives of interest, are first described in more detail, followed by a description of host cells for producing them. Methods of interest in which the host cells find use are then outlined. Kits that can be used in practicing the methods of the present invention are also described.

[0037] Tropane alkaloid (TA) precursor As summarized above, host cells that produce tropane alkaloid precursors (TA precursors) are provided. The TA precursor can 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 can be characterized as a TA or a derivative thereof. In some cases, the TA precursor has a structure that can be characterized as a fragment of a TA. In some cases, the TA precursor is an initial TA. As used herein, "initial TA" refers to an early intermediate in the synthesis of a TA of interest in a cell, where the early TA is produced by the host cell from host cell feedstock or a simple starting compound. In some cases, the initial TA is a TA intermediate that is produced by the subject host cell solely from host cell feedstock (e.g., carbon and nutrient sources) without the need to add starting compounds to the cell. The term initial TA can refer to a precursor of a TA end product of interest, regardless of whether the initial TA itself can be characterized as a tropane alkaloid.

[0038] In some cases, the TA precursor is an initial TA, such as a pretropine tropane alkaloid or a preritulin tropane alkaloid. Thus, host cells producing pretropine tropane alkaloids (pretropine TAs) and preritulin tropane alkaloids (preritulin TAs) are provided. Tropine is an important key branching point intermediate in the synthesis of downstream TAs through cell engineering efforts to produce end products, such as medicinal TA products derived from littorine (Figure 2). The subject host cells are capable of producing TA precursors from simple, inexpensive starting materials that can find use in the production of tropine, littorine, and downstream TA end products.

[0039] As used herein, the terms "pre-esterified tropane alkaloid," "pre-esterified TA," and "pre-esterified TA precursor" are used interchangeably and refer to biosynthetic precursors of littorine, cinnamoyltropine, or other products of acyl donor and acyl acceptor esterification, regardless of whether the structure of the esterified precursor itself is characterized as a tropane alkaloid. The term pre-esterified TA is intended to include biosynthetic precursors, intermediates, and metabolites thereof of any convenient member of a host cell biosynthetic pathway that can lead to an esterified product, such as littorine. In some cases, the pre-esterified 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-esterified TA has a structure that can be characterized as a tropane alkaloid or a derivative thereof.

[0040] 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-pyrrolidinyl)-3-oxobutanoic acid (MPOB), tropinone, phenylalanine, prephenic acid, and phenylpyruvic acid (PPA). In some embodiments, one or more TA precursors are tropine and PLA. In certain examples, one or more TA precursors are phenylpropanoid carboxylic acids other than PLA, such as tropine and cinnamic acid. Figures 1, 2, and 3 illustrate the biosynthesis of non-medicinal, medicinal, and unnatural TAs from various TA and non-TA precursor molecules, respectively.

[0041] The synthetic pathway to a TA precursor can occur in a host cell and can begin with any convenient starting compound(s) or material. Figures 1-4 illustrate synthetic pathways of interest to a TA precursor starting from an amino acid. The starting material may not be naturally occurring, or the starting material may be naturally present in the host cell. Based on the synthetic pathway present in the host cell, any convenient compound and material can be used as a starting material. The source of the starting material can be derived from the host cell itself, e.g., arginine or phenylalanine, or the starting material can be added or supplemented to the host cell from an external source. Thus, in some cases, a starting compound refers to a compound in a cell's synthetic pathway that is added to the host cell from an external source that is not part of the growth feedstock or cell growth medium. Starting compounds of interest include, but are not limited to, N-methylputrescine, 4-methylaminobutanal, tropinone, tropine, PLA, cinnamic acid, and any of the compounds shown in Figures 1-4. For example, when host cells are grown in liquid culture, the cell medium can be supplemented with a starting material that is delivered to the cells and converted by the cells into the desired product. Starting materials of interest include, but are not limited to, inexpensive feedstocks and simple precursor molecules. In some cases, host cells utilize feedstocks containing simple carbon sources as starting materials, which the host cells utilize to produce compounds in the cell's synthetic pathway. Host cell growth feedstocks can include one or more components, such as carbon sources such as cellulose, starch, and free sugars, and nitrogen sources such as ammonium salts or inexpensive amino acids. In some cases, the growth feedstock used as starting material can be obtained from sustainable sources, such as biomass grown on marginal arable land, such as switchgrass and algae, or biomass waste from other industrial or agricultural activities.

[0042] Tropane alkaloids (TA) As outlined above, host cells that produce tropane alkaloids (TAs) of interest are provided. In some embodiments, the engineered strains of the present invention 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; or unnatural TAs, such as those derived from the esterification of tropine and TA precursors other than PLA (e.g., acyl donor and acyl acceptor compounds). Each of these classes is intended to include biosynthetic precursors, intermediates, and their metabolites of any convenient member of a host cell biosynthetic pathway that may lead to a member of the class. Non-limiting examples of compounds for each of these classes are provided below. In some embodiments, the structures of a given example may or may not themselves be characterized as tropane alkaloids. The chemical entities are intended to include all possible isomers, including single enantiomers, racemic mixtures, optically pure forms, diastereomeric mixtures, and intermediate mixtures.

[0043] Medicinal TAs include, but are not limited to, littorine, hyoscyamine, atropine, anisodamine, scopolamine, and their derivatives, which are naturally produced by plants.

[0044] Non-medicinal TAs include, but are not limited to, calystegine, cocaine, and their derivatives, which are naturally produced by plants.

[0045] Non-natural TAs can 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.

[0046] Modification of TA, including derivatives As outlined above, host cells that produce modified derivatives of tropane alkaloids (TAs) of interest are provided. In some embodiments, the engineered strains of the invention provide a platform for deriving TAs of interest, including deriving TA precursors, medicinal TAs, non-medicinal TAs, and non-naturally occurring TAs produced by or provided to the engineered host cells in the growth medium.

[0047] As used herein, the terms "derivatized," "functionalized," "modified by derivatization," and "modified by functionalization" refer to the modification of a TA or TA precursor through the attachment of a functional group without modifying the TA backbone itself. As used herein, attachment of a functional group includes, but is not limited to, hydroxylation, alkylation and N-alkylation, acetylation and N-acetylation, acylation and N-acylation, and halogenation.

[0048] In some embodiments of the present invention, derivatization of a TA of interest can be achieved enzymatically by feeding a pre-functionalized TA precursor, e.g., a halogenated or alkylated amino acid, to a host cell engineered to incorporate the fed TA precursor and subsequently convert it to the TA of interest. In other embodiments of the present invention, derivatization of a TA of interest can be achieved enzymatically by engineering a host cell to express an enzyme with the desired activity for attaching a functional group to the target TA, in addition to the enzymes and cellular modifications necessary to produce the unmodified TA. In other embodiments of the present invention, derivatization of a TA of interest can be achieved enzymatically by treating the unmodified TA produced by the engineered host cell with a purified enzyme capable of attaching the desired functional group, or with a crude lysate of a host cell engineered to express an enzyme with the desired derivatization activity. In other embodiments of the present invention, derivatization of a TA of interest can be achieved non-enzymatically by treating the unmodified TA produced by the engineered host cell with a chemical agent to which the desired functional group is attached.

[0049] Modified derivatives of TA include, but are not limited to, p-hydroxyatropine, p-hydroxyhyoscyamine, p-fluorohyoscyamine, p-chlorohyoscyamine, p-bromohyoscyamine, p-fluoroscopolamine, p-chloroscopolamine, p-bromoscopolamine, N-methylhyoscyamine, N-butylhyoscyamine, N-methylscopolamine, N-butylscopolamine, N-acetylhyoscyamine, and N-acetylscopolamine.

[0050] host cell As outlined above, one aspect of the present invention is a host cell that produces one or more TAs of interest. Any convenient cell can be utilized in the subject host cells and methods. In some cases, the host cell is a non-plant cell. In some cases, the host cell can be characterized as a microbial cell. In some cases, the host cell is an insect cell, a mammalian cell, a bacterial cell, or a fungal cell. Any convenient type of host cell can be utilized in producing the subject TA-producing cells; see, e.g., U.S. Patent No. 8,975,063, U.S. Patent No. 2014 / 0273109, and U.S. Patent No. 2008 / 0176754, now published as 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, and the like. coli, Streptomyces, Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter bacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria, Lactobacillus, Leuconostoc, 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 Examples of suitable bacterial cells include Salmonella typhimurium 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 cell comprises:The host cell may be a yeast cell or an Escherichia coli (E. coli) cell. Optionally, the host cell is a yeast cell. Optionally, the host cell is derived from a yeast strain engineered to produce the TA of interest. Any of the host cells described in U.S. Patent No. 8,975,063, U.S. Patent No. 2014 / 0273109, and U.S. Patent No. 2008 / 0176754, now published as WO2014 / 143744, can be adapted for use in the subject cells and methods. In certain embodiments, the yeast cell may be of the species Saccharomyces cerevisiae (S. cerevisiae). In certain embodiments, the yeast cell may be of the species Schizosaccharomyces pombe. In certain embodiments, the yeast cell may be of the species Pichia pastoris. Yeast is important as a host cell because cytochrome P450 proteins involved in several biosynthetic pathways of interest are properly folded into the endoplasmic reticulum membrane to maintain their activity.

[0051] Yeast strains of interest that find use in the present invention include, but are not limited to, CEN.PK (genotype: MATa / α ura3-52 / ura3-52 trp1-289 / trp1-289 leu2-3_112 / leu2-3_112 his3Δ1 / his3Δ1 MAL2-8C / MAL2-8C SUC2 / SUC2), S288C, W303, D273-10B, X2180, A364A, Σ1278B, AB972, SK1, and FL100. In particular cases, the yeast strain is S288C (MATα; SUC2 mal mel gal2 CUP1 flo1 flo8-1). hap1), BY4741 (MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0), BY4742 (MATα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0), BY4743 (MATa / MATα; his3Δ1 / his3Δ1; leu2Δ0 / leu2Δ0; met15Δ0 / MET15; LYS2 / lys2Δ0; ura3Δ0 / ura3Δ0), and derivatives of strain W303-B, which are WAT11 or W(R) (MATa; ade2-1; his3-11, -15; leu2-3, -112; ura3-1; canR; cyr+) (Arabidopsis thaliana). thaliana NADPH-P450 reductase ATR1 and yeast NADPH-P450 reductase CPR1). In another embodiment, the yeast cell is W303alpha (MATα; his3-11,15 trp1-1 leu2-3 ura3-1 ade2-1). The identities and genotypes of additional yeast strains of interest can be found at EUROSCARF (web.uni-frankfurt.de / fb15 / mikro / euroscarf / col_index.html).

[0052] In some cases, the host cell is a fungal cell. In certain embodiments, the fungal cell may be of the Aspergillus species, including strains of 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).

[0053] In certain embodiments, the heterologous coding sequence may be codon-optimized for expression in Aspergillus species and expressed from an appropriate promoter. In certain embodiments, the promoter may be selected from the phosphoglycerate kinase promoter (PGK), MbfA promoter, cytochrome c oxidase subunit promoter (CoxA), SrpB promoter, TvdA promoter, malate dehydrogenase promoter (MdhA), and beta-mannosidase promoter (ManB). In certain embodiments, the terminator may be selected from the glucoamylase terminator (GlaA) or the TrpC terminator. 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 host genome. In certain embodiments, selection of cells maintaining the plasmid or integrated cassette may be performed by antibiotic selection, such as hygromycin, or by utilizing a nitrogen source, such as using acetamide as the sole nitrogen source. In certain embodiments, DNA constructs can be introduced into host cells using established transformation methods such as protoplast transformation, lithium acetate, or electroporation. In certain embodiments, cells can 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.

[0054] In some cases, the host cell is a bacterial cell. The bacterial cell can be selected from any bacterial genus. Examples of genera from which the bacterial cell originates 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, Psychrobac ter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, and Zymomonas. Examples of bacterial species that can be used in the methods of the present disclosure include Arthrobacter nicotianae, Acetobacter aceti, Arthrobacter arilaitensis, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus pumilus, Bacillus sphaericus, Bacillus spp. ...sphaericus, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium adolescentis, Brachybacterium tyrofermentans, Brevibacterium linens, Carnobacterium divergens, Corynebacterium flavescens, Enterococcus faecium, Gluconacetobacter europaeus, Gluconacetobacter johannae, Gluconobacter oxydans 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 oeni), Pediococcus acidilactici, Propionibacterium acidipropioniciAcidipropionici, Proteus vulgaris, Pseudomonas fluorescens, Psychrobacter celer, Staphylococcus condimenti, Streptococcus thermophilus, Streptomyces griseus, Tetragenococcus halophilus, Weissella cibaria, Weissella koreensis, Zymomonas mobilis, Corynebacterium glutamicum, Bifidobacterium 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 Contains Moorella themocellum / thermoacetica.

[0055] In certain embodiments, the bacterial cell may be Escherichia coli. In certain embodiments, the strain of E. coli may be selected from BL21, DH5α, XL1-Blue, HB101, BL21, and K12. In certain embodiments, the heterologous coding sequence may be codon-optimized for expression in E. coli and expressed from a suitable promoter. In certain embodiments, the promoter may be a T7 promoter, a tac promoter, a trc promoter, a tetracycline-inducible promoter (tet) 、 lac operon promoter (lac) 、 The lacO1 promoter may be selected from the lacO1 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 pUC19 or pBAD. In certain embodiments, selection of cells that maintain the plasmid or integrated cassette may be performed using antibiotic selection, such as kanamycin, chloramphenicol, streptomycin, spectinomycin, gentamicin, erythromycin, or ampicillin. In certain embodiments, the DNA construct may be introduced into the host cell using established transformation methods, such as conjugation, heat shock chemical transformation, or electroporation. In certain embodiments, the cells may be cultured in liquid Luria-Bertani (LB) medium at approximately 37°C, with or without antibiotics.

[0056] In certain embodiments, the bacterial cell may be a strain of Bacillus subtilis. In certain embodiments, the B. subtilis strain may be selected from 1779, GP25, RO-NN-1, 168, BSn5, BEST195, 1A382, and 62178. In certain embodiments, the heterologous coding sequence may be codon-optimized for expression in Bacillus species and expressed from an appropriate promoter. In certain embodiments, the promoter may be selected from the 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, the expression cassette may be integrated into the genome using an integration vector such as pDG364 or pDG1730. In certain embodiments, selection of cells that maintain the plasmid or integration cassette may be carried out using antibiotic selection, such as erythromycin, kanamycin, tetracycline, and spectinomycin. In certain embodiments, the DNA construct may be introduced into 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) medium or M9 medium supplemented with glucose and tryptophan at 37°C.

[0057] Genetic modification of host cells A host cell can be engineered to contain one or more modifications (e.g., two or more, three or more, four or more, five or more, or even more modifications) that provide for production of a desired TA. In some cases, modification refers to a genetic modification, such as a mutation, addition, or deletion of a gene or fragment thereof, or transcriptional regulation of a gene or fragment thereof. In some cases, the one or more (e.g., two or more, three or more, or four or more) modifications are selected from the following: feedback inhibition that alleviates a mutation in a cell's native biosynthetic enzyme gene, a transcriptional regulation modification of a cell's native biosynthetic enzyme gene, an inactivating mutation of a cell's native enzyme, a heterologous coding sequence encoding an enzyme, a heterologous coding sequence encoding a protein that alters intracellular trafficking and / or localization of an enzyme or metabolite. A cell containing one or more modifications may be referred to as a modified cell.

[0058] The modified cells can overproduce one or more precursor TAs, TAs, or modified TA molecules. Overproduction means that the cells have improved or increased production of the TA molecule of interest compared to control cells (e.g., unmodified cells). Improved or increased production refers to both the production of an amount of the TA of interest when the control has no production of the TA precursor, as well as an increase of about 10% or more, e.g., 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, e.g., 2-fold or more, e.g., 5-fold or more, including 10-fold or more, in situations where some TA of interest is produced in the control.

[0059] In some cases, the host cell can produce an increased amount of putrescine compared to a control host cell lacking one or more modifications (e.g., as described herein). In particular examples, the increase in putrescine is about 10% or more compared to the control host cell, e.g., 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 compared to the control host cell.

[0060] In some cases, the host cells can produce increased amounts of N-methylpyrrolinium compared to control host cells lacking one or more modifications (e.g., as described herein). In particular examples, the increase in N-methylpyrrolinium is about 10% or more compared to the control host cells, e.g., 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 compared to the control host cells.

[0061] In some cases, the host cells can produce increased amounts of tropine compared to control host cells lacking one or more modifications (e.g., as described herein). In particular examples, the increase in tropine is about 10% or more compared to the control host cells, e.g., 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 compared to the control host cells.

[0062] In some cases, the host cell can produce an increased amount of phenylpyruvic acid compared to a control host cell lacking one or more modifications (e.g., as described herein). In certain examples, the increase in phenylpyruvic acid is about 10% or more compared to the control host cell, e.g., 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 compared to the control host cell.

[0063] In some cases, the host cell can produce an increased amount of phenyllactic acid compared to a control host cell lacking one or more modifications (e.g., as described herein). In certain examples, the increase in phenyllactic acid is about 10% or more compared to the control host cell, for example, 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 compared to the control host cell.

[0064] In some cases, the host cell can produce an increased amount of littorin compared to a control host cell lacking one or more modifications (e.g., as described herein). In certain examples, the increase in littorin is about 10% or more compared 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 compared to the control host cell.

[0065] In some cases, the host cells can produce increased amounts of hyoscyamine compared to control host cells lacking one or more modifications (e.g., as described herein). In particular examples, the increase in hyoscyamine is about 10% or more compared to the control host cells, e.g., 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 compared to the control host cells.

[0066] In some cases, the host cells can produce increased amounts of scopolamine compared to control host cells lacking one or more modifications (e.g., as described herein). In certain examples, the increase in scopolamine is about 10% or more compared to the control host cells, e.g., 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 compared to the control host cells.

[0067] In some embodiments, the host cell is capable of producing tropine with a yield of 10% or greater from a starting compound such as arginine, e.g., tropine with a yield of 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or 90% or greater from the starting compound.

[0068] In some embodiments, the host cell is capable of producing phenyllactic acid in a yield of 10% or greater from a starting compound such as phenylalanine, for example, 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or even 90% or greater yield from the starting compound.

[0069] In some embodiments, the host cell is capable of producing hyoscyamine in a yield of 10% or greater from a starting compound such as arginine or phenylalanine, for example, in a yield of 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or even 90% or greater from the starting compound.

[0070] In some embodiments, the host cell is capable of producing scopolamine in a yield of 10% or greater from a starting compound such as arginine or phenylalanine, for example, in a yield of 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or even 90% or greater from the starting compound.

[0071] In some embodiments, the host cell overproduces one or more TA molecules of interest selected from the group consisting of arginine, ornithine, agmatine, putrescine, N-methylputrescine, 4-methylaminobutanal, N-methylpyrrolinium, 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid, tropinone, tropine, phenylalanine, prephenic acid, phenylpyruvic acid, phenyllactic acid, glucose-1-O-phenyllactate, littorine, hyoscyamine aldehyde, hyoscyamine, anisodamine, and scopolamine.

[0072] Any convenient combination of one or more modifications can be included in the subject host cell. 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 examples, two or more (for example, three or more, four or more, five or more, or more) separate modifications of the same type are included in the subject cell.

[0073] In some embodiments of the host cell, when the cell contains one or more heterologous coding sequences encoding one or more enzymes, it contains at least one additional modification selected from the group consisting of: feedback inhibition that mitigates mutations in the cell's native biosynthetic enzyme genes, transcriptional regulatory modifications of the cell's native biosynthetic enzyme genes, and inactivating mutations of the cell's native enzymes. In certain embodiments of the host cell, when the cell contains one or more feedback inhibition that mitigates mutations in one or more cell's native biosynthetic enzyme genes, it contains at least one additional modification selected from the group consisting of: transcriptional regulatory modifications of the cell's native biosynthetic enzyme genes, inactivating mutations of the cell's native enzymes, and heterologous coding sequences encoding enzymes. In some embodiments of the host cell, when the cell contains one or more transcriptional regulatory modifications of one or more cell's native biosynthetic enzyme genes, it contains at least one additional modification selected from the group consisting of: feedback inhibition that mitigates mutations in the cell's native biosynthetic enzyme genes, inactivating mutations of the cell's native enzymes, heterologous coding sequences encoding enzymes, and heterologous coding sequences encoding proteins that modify intracellular transport and / or localization of enzymes or metabolites. In particular examples of host cells, when the cell contains one or more inactivating mutations in one or more enzymes native to the cell, it contains at least one additional modification selected from the group consisting of: feedback inhibition that alleviates the mutation in the cell's native biosynthetic enzyme, a transcriptional regulatory modification of the cell's native biosynthetic enzyme gene, a heterologous coding sequence encoding an enzyme, and a heterologous coding sequence encoding a protein that modifies the intracellular transport and / or localization of the enzyme or metabolite.

[0074] In certain embodiments of the host cell, the cell comprises one or more feedback inhibitions that mitigate mutations in one or more biosynthetic enzyme genes native to the cell, and one or more transcriptional regulatory modifications of one or more biosynthetic enzyme genes native to the cell. In certain embodiments of the host cell, the cell comprises one or more feedback inhibitions that mitigate mutations in one or more biosynthetic enzyme genes native to the cell, and one or more inactivating mutations in enzymes native to the cell. In certain embodiments of the host cell, the cell comprises one or more feedback inhibitions that mitigate 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 comprises one or more modifications (e.g., as described herein) including one or more genes of interest listed in Table 1.

[0075] Feedback inhibition reduces mutations In some cases, the host cell is a cell that contains one or more feedback inhibitors (e.g., two or more, three or more, four or more, five or more, or more) that alleviate a mutation 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., present in an unmodified cell). As used herein, the term "feedback inhibition-relieving mutation" refers to a mutation that alleviates a feedback inhibition control mechanism of the host cell. Feedback inhibition is a cellular control mechanism in which an enzyme in a synthetic pathway of a regulated compound is inhibited when that compound accumulates to a certain level, thereby balancing the amount of the compound in the cell. In some cases, the one or more feedback inhibition-relieving mutations are in enzymes listed in the biosynthetic pathways of Figures 1-4 or the schematic diagram of Figure 8. The feedback inhibition-relieving mutation reduces inhibition of a regulatory enzyme in the cell of interest compared to a control cell, providing increased levels of the regulatory compound or its downstream biosynthetic product. In some cases, alleviating inhibition of a regulatory enzyme reduces the IC of the inhibition. 50By increased level is meant an increase of 2-fold or more, e.g., 3-fold or more, 5-fold or more, 10-fold or more, 30-fold or more, 100-fold or more, 300-fold or more, 1000-fold or more, or more. By increased level is meant an increase of 110% or more, e.g., 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200% or more, of the level of the regulated compound in a control cell or downstream product thereof, e.g., at least 3-fold or more, at least 5-fold or more, at least 10-fold or more, or more.

[0076] Various feedback inhibitory control mechanisms native to the host cell and biosynthetic enzymes that regulate the level of TA precursors can be targeted for alleviation in the host cell. The host cell can contain one or more feedback inhibitors that alleviate mutations in one or more biosynthetic enzyme genes native to the cell. The mutations can be located in any convenient biosynthetic enzyme gene 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 ornithine decarboxylase (ODC), ornithine decarboxylase antizyme, and putrescine N-methyltransferase. In some embodiments, the one or more biosynthetic enzyme genes encode ornithine decarboxylase. In some cases, the one or more biosynthetic enzyme genes encode ornithine decarboxylase antizyme. In some embodiments, the one or more biosynthetic enzyme genes encode putrescine N-methyltransferase. In particular examples, the one or more feedback inhibitors that alleviate mutations are present in a biosynthetic enzyme gene selected from SPE1, OAZ1, and PMT. In certain examples, the one or more feedback inhibitions that mitigate mutations are present in a biosynthetic enzyme gene that is SPE1. In certain examples, the one or more feedback inhibitions that mitigate mutations are present in a biosynthetic enzyme gene that is OAZ1. In certain examples, the one or more feedback inhibitions that mitigate mutations are present in a biosynthetic enzyme gene that is PMT. In some embodiments, the host cell comprises one or more feedback inhibitions that mitigate mutations in one or more biosynthetic enzyme genes, such as one of those genes listed in Table 1.

[0077] Any convenient number and type of mutations can be used to alleviate feedback inhibition control mechanisms. As used herein, the term "mutation" refers to the deletion, insertion, or substitution of amino acid or nucleotide residues relative to a reference sequence or motif. Mutations may be incorporated as directed mutations into the native gene at its original locus. In some cases, mutations may be incorporated as additional copies of the gene introduced by gene integration at another locus or as additional copies on an episomal vector, such as a 2μ or centromeric plasmid. In certain instances, feedback inhibition copies of the enzyme are under the transcriptional control of the native cell. In some cases, feedback inhibition copies of the enzyme are introduced by engineered constitutive or dynamic regulation of protein expression by placing them under the control of a synthetic promoter.

[0078] In certain embodiments, a host cell of the invention can comprise feedback inhibitions that mitigate one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more mutations, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 feedback inhibitions that mitigate mutations in one or more biosynthetic enzyme genes native to the host cell.

[0079] Altered transcriptional regulation A host cell can include one or more transcriptional regulatory modifications (e.g., 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, one or more biosynthetic enzyme genes are native to the cell. Any convenient biosynthetic enzyme gene of the cell can be targeted for transcriptional modulation. Transcriptional modulation means that the expression of a gene of interest in a modified cell is modulated, e.g., increased or decreased, enhanced or suppressed, compared to a control cell (e.g., an unmodified cell). In some cases, transcriptional modulation of a gene of interest includes increased or enhanced expression. Increasing or enhancing expression means that the expression level of the gene of interest is increased by 2-fold or more, for example, 5-fold or more, sometimes 25-fold, 50-fold, or 100-fold or more, and in certain embodiments, 300-fold or more, compared to expression in a control, i.e., the same unmodified cell (e.g., by using any convenient gene expression assay). Alternatively, if expression of a gene of interest in a cell is very low and undetectable, an increase in expression to a readily detectable level is considered to be an increased expression level of the gene of interest. In certain instances, transcriptional regulation of a gene of interest includes reducing or suppressing expression.Reducing or suppressing expression means that the expression level of the gene of interest is reduced by 2 times or more, for example, 5 times or more, sometimes 25 times, 50 times, or 100 times or more, and in certain embodiments, 300 times or more compared to a control.In some cases, expression is reduced to an undetectable level.The modification of a host cell process of interest that can be adapted for use in a host cell of interest is described in U.S. Patent Application Publication No. 2014 / 0273109 (14 / 211,611) by Smolke et al., the disclosure of which is incorporated herein by reference in its entirety.

[0080] Any convenient biosynthetic enzyme gene can be transcriptionally regulated, including, but not limited to, the biosynthetic enzymes set forth in Figures 1-3, such as ARG2, CAR1, SPE1, FMS1, PHA2, ARO8, ARO9, and UGP1. In some cases, the one or more biosynthetic enzyme genes are selected from ARG2, CAR1, SPE1, and FMS1. In some cases, the one or more biosynthetic enzyme genes are ARG2. In particular examples, the one or more biosynthetic enzyme genes are CAR1. In some embodiments, the one or more biosynthetic enzyme genes are SPE1. In some embodiments, the one or more biosynthetic enzyme genes are FMS1. In some embodiments, the host cell comprises one or more transcriptional regulatory modifications to one or more genes, such as one of these genes set forth in Table 1. In some embodiments, the host cell comprises one or more transcriptional regulatory modifications to one or more genes, such as one of the genes set forth in the biosynthetic pathway of one of Figures 1-4 or the schematic diagram of Figure 8.

[0081] In some embodiments, transcriptional regulatory modifications include replacing the native promoter of one or more biosynthetic enzyme genes with a strong promoter, or expressing additional copies of one or more genes under the control of a strong promoter. The promoter driving expression of the gene of interest can be constitutive or inducible, provided that the promoter can be active in the host cell. Genes of interest can be expressed from their native promoters, or non-native promoters can be used. Although not required, such promoters should be moderate to high in strength in the host in which they are used. In some embodiments, where the promoter can be regulated or constitutive, promoters that are not repressed by glucose or that are only moderately repressed by the presence of glucose in the culture medium are used. Many suitable promoters exist, examples of which include promoters of glycolytic genes, such as the promoter of the B. subtilis tsr gene (encoding fructose bisphosphate aldolase) or the promoter of the GAPDH gene (encoding glyceraldehyde-phosphate dehydrogenase) from the yeast S. cerevisiae (Bitter GA, Meth. Enzymol. 152:673-684 (1987)). Other strong promoters of interest include, but are not limited to, the ADHI promoter from baker's yeast (Ruohonen L., et al., J. Biotechnol. 39:193-203 (1995)), phosphate starvation-inducible promoters such as the PHO5 promoter from yeast (Hinnen, A., et al., in Yeast Genetic Engineering, Barr, PJ, 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, GalL, and GalS; repressible promoters such as Met25 and 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), and triosephosphate isomerase (TPI). In some cases, the strong promoter is GPD1. In particular examples, the strong promoter is TEF1. Autonomously replicating yeast expression vectors containing promoters that can be induced by hormones such as glucocorticoids, steroids, and thyroid hormones are also known, including, but not limited to, glucocorticoid response elements (GREs) and thyroid hormone response elements (TREs); see, for example, the promoters described in U.S. Patent No. 7,045,290. Vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, can be used. Furthermore, any promoter / enhancer combination (as defined by the Eukaryotic Promoter Database EPDB) can be used to drive expression of the gene of interest. It is understood that any convenient promoter specific to the host cell, e.g., E. coli, can be selected. In some cases, promoter selection can be used to optimize transcription, and thus enzyme levels, to maximize production while minimizing energy resources.

[0082] Inactivating mutations A host cell can include one or more inactivating mutations (e.g., two or more, three or more, four or more, five or more, or more) to a cellular enzyme. By including one or more inactivating mutations, the flow of a synthetic pathway in the host cell can be altered to increase the level of a TA of interest or a desired enzyme or precursor leading to the TA. In some cases, the one or more inactivating mutations are to an enzyme native to the cell. Figure 8 shows the natural regulatory mechanisms of yeast that affect the polyamine production pathway, and Figure 9 shows the effect of disrupting these natural regulatory systems on putrescine production. As used herein, "inactivating mutation" means one or more mutations to a gene or regulatory DNA sequence of the cell, where the mutation(s) inactivate the biological activity of a protein expressed by the gene of interest. In some cases, the gene is native to the cell. In some cases, the gene encodes an enzyme that is inactivated and is part of or connected to the synthetic pathway of the TA of interest to be produced by the host cell. In some cases, the inactivating mutation is located in a regulatory DNA sequence that controls the gene of interest. In some cases, the inactivating mutation is to the promoter of the gene. Any convenient mutation (e.g., as described herein) can be used to inactivate a gene or regulatory DNA sequence of interest. "Inactivated" or "inactivation" means that the biological activity of the protein expressed by the mutated gene is reduced by 10% or more, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more, compared to the control protein expressed by a non-mutated control gene. In some cases, the protein is an enzyme, and the inactivating mutation reduces the activity of the enzyme.

[0083] In some embodiments, the cells contain an inactivating mutation in a native enzyme of the cells. Any convenient enzyme can be targeted for inactivation. Enzymes of interest include, but are not limited to, those set forth in Figures 1-4, 8, 11, 22, and 41, whose action in a biosynthetic pathway of a host cell tends to reduce the level of a TA of interest. Optionally, the enzyme has methylthioadenosine phosphorylase activity. In certain embodiments, the enzyme containing an inactivating mutation is MEU1 (see, e.g., Figures 8, 9, and 13). Optionally, the enzyme has ornithine decarboxylase antizyme activity. In certain embodiments, the enzyme containing an inactivating mutation is OAZ1. Optionally, the enzyme has spermidine synthase activity. In certain embodiments, the enzyme containing an inactivating mutation is SPE3. Optionally, the enzyme has spermine synthase activity. In some embodiments, the enzyme containing an inactivating mutation is SPE4. Optionally, the enzyme is a membrane transporter with polyamine efflux activity. In certain embodiments, the enzyme or protein comprising an inactivating mutation is TPO5. Optionally, the enzyme has phenylacrylic acid decarboxylase activity. In certain embodiments, the enzyme comprising an inactivating mutation is PAD1. Optionally, the enzyme has alcohol dehydrogenase activity. In some embodiments, the enzyme comprising an inactivating mutation is selected from ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1. In certain embodiments, the enzyme comprising an inactivating mutation(s) is ADH2. In certain embodiments, the enzyme comprising an inactivating mutation(s) is ADH3. In certain embodiments, the enzyme comprising an inactivating mutation(s) is ADH4. In certain embodiments, the enzyme comprising an inactivating mutation(s) is ADH5. In certain embodiments, the enzyme comprising an inactivating mutation(s) is ADH6. In certain embodiments, the enzyme comprising an inactivating mutation(s) is ADH7. Optionally, the enzyme has aldehyde oxidoreductase activity. In certain embodiments, the enzyme comprising an inactivating mutation is selected from HFD1, ALD2, ALD3, ALD4, ALD5, and ALD6.In certain embodiments, the enzyme comprising the mutation(s) and the inactivating mutation(s) is HFD1. In certain embodiments, the enzyme comprising the inactivating mutation(s) is ALD2. In certain embodiments, the enzyme comprising the inactivating mutation(s) is ALD3. In certain embodiments, the enzyme comprising the inactivating mutation(s) is ALD4. In certain embodiments, the enzyme comprising the inactivating mutation(s) is ALD5. In certain embodiments, the enzyme comprising the inactivating mutation(s) is ALD6. In some embodiments, the enzyme has glucosidase activity. In certain embodiments, the enzyme comprising the inactivating mutation(s) is selected from EXG1, SPR1, and EGH1. In certain embodiments, the enzyme comprising the inactivating mutation(s) is EXG1. In certain embodiments, the enzyme comprising the inactivating mutation(s) is SPR1. In certain embodiments, the enzyme comprising the inactivating mutation(s) is EGH1. In some embodiments, the host cell comprises one or more inactivating mutations in one or more of the genes listed in Table 1.

[0084] Methods for performing TA acyl transfer reactions using functional expression of acyltransferases in non-plant hosts Several methods, processes, and systems provided herein describe the concerted reaction of one or more TA precursors containing an acyl donor group with one or more TA precursors containing an acyl acceptor group (hereinafter referred to as TA acyl transfer reactions) to produce one or more TAs in non-plant cells. Some of these methods, processes, and systems may include engineered host cells. In some examples, the TA acyl transfer reaction is a key step in converting substrates to a diverse range of alkaloids. In some examples, the TA acyl transfer reaction includes a condensation reaction.

[0085] In some instances, the TA acyl transfer can include at least one condensation reaction. In some instances, at least one of the condensation reactions is performed in the presence of an enzyme. In some instances, at least one of the condensation reactions is catalyzed by an enzyme. In some instances, at least one enzyme is useful for catalyzing the condensation reaction.

[0086] In some methods, processes, and systems described herein, condensation reactions can be carried out in the presence of an enzyme. In some examples, the enzyme can be an acyltransferase. The acyltransferase can use TAs with alcohol or carboxylate functional groups as substrates. The acyltransferase can use TAs containing carboxylate groups activated via 1-O-β glycosidic linkages to sugars (hereinafter referred to as glycosides) as substrates. The acyltransferase can convert the TA alcohol and carboxylate / glycoside functional groups to the corresponding ester derivatives. Non-limiting examples of enzymes suitable for condensing TA precursors in this disclosure include serine carboxypeptidase-like acyltransferases (SCPL-ATs). For example, littorine synthase (EC 2.3.1.-) can condense tropine and other TA precursors containing an alcohol functional group with 1-O-β-phenyllactoyl-glucose and other TA glycoside precursors to produce littorine and other corresponding ester products. In some examples, a protein comprising an SCPL-AT domain from any one of the foregoing examples can carry out condensation. In some examples, the SCPL-AT can catalyze a condensation reaction within a host cell, such as an engineered host cell, as described herein. In yet other examples, the SCPL-AT can catalyze a condensation reaction within an intracellular compartment within a host cell, such as an engineered host cell, as described herein.

[0087] In some embodiments of the present invention, the amino acid sequence of an acyltransferase enzyme, such as an SCPL-AT enzyme, used to perform a TA acyl transfer reaction is modified to alter post-translational processing, trafficking, folding, oligomerization, and / or subcellular localization of the enzyme. Because some acyltransferase enzymes, including the SCPL-AT enzyme, have not been demonstrated to exhibit catalytic activity in living non-plant cells, such modifications may be useful or necessary for activity in non-plant host cells. Examples of such modifications include, but are not limited to, the addition, removal, or substitution of an N-terminal signal peptide sequence; the addition, removal, or substitution of an internal propeptide sequence; the addition or removal of an asparagine-linked N-glycosylation site; the addition or removal of a serine-linked O-glycosylation site; and the fusion of a protein domain to the N-terminus and / or C-terminus of the acyltransferase domain.

[0088] In one embodiment of the present invention, the SCPL-AT enzymatic domain is modified at its N-terminus by fusion with a soluble protein domain. This soluble domain masks the internal signal sequence of the acyltransferase domain, thereby altering the trafficking and / or subcellular localization of the fused SCPL-AT domain. In some examples, the N-terminal fusion domain directs trafficking of the SCPL-AT domain to subcellular compartments, including, but not limited to, the ER membrane, ER lumen, cis-Golgi, trans-Golgi, lysosomes, vacuolar membrane, and vacuolar lumen. The N-terminal fused soluble domain can also alter 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 higher oligomerization.

[0089] 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 from Aequoria sp. and Discosoma sp. In one example, the N-terminally fused soluble protein domain is red fluorescent protein (DsRed) from Discosoma sp. In another example, 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.

[0090] Examples of amino acid sequences of soluble protein domains that can be fused to the N-terminus of an SCPL-AT domain that can be used to carry out a TA acyl transfer reaction in non-plant cells are provided in Table 3. The amino acid sequence of an SCPL-AT enzyme that includes a fused N-terminal domain and is utilized in a TA acyl transfer reaction in a non-plant cell can be 50% or more identical to a given amino acid sequence listed in Table 3. For example, the amino acid sequence of such an acyltransferase can include 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 the amino acid sequences provided herein. Furthermore, in certain embodiments, an "identical" amino acid sequence contains at least 80% to 99% identity at the amino acid level to a particular 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 in certain cases even at least 95%, 96%, 97%, 98%, or 99% identity at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence has been altered, for example, to optimize codon usage for the host organism.

[0091] Engineered non-plant host cells can be provided that produce an acyltransferase that catalyzes a TA acyl transfer reaction, where the acyltransferase comprises an amino acid sequence fused at its N-terminus to the amino acid sequence of a soluble protein domain selected from the group consisting of those sequences in Table 3. The acyltransferase produced in the engineered host cells can be recovered and purified to form a biocatalyst. One or more enzymes recovered from the engineered host cells that produce the acyltransferase can be used in a process for performing a TA acyl transfer reaction. The process can include contacting a TA precursor having alcohol and / or carboxylate / glycoside functional groups with a sufficient amount of the acyltransferase to convert the alcohol and / or carboxylate / glycoside groups to the corresponding ester groups. In an example, a TA precursor having alcohol and / or carboxylate / glycoside functional groups can be contacted with a sufficient amount of one or more enzymes such that at least 5% of the TA precursor is converted to the corresponding ester. In further examples, a TA having alcohol and / or carboxylate / glycoside functional groups can be contacted with a sufficient amount of 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 the TA precursor undergoes the corresponding esterification.

[0092] One or more enzymes that can be used to carry out a TA acyl transfer reaction can be contacted with a TA precursor in vitro. Additionally, or alternatively, one or more enzymes that can be used to carry out a TA acyl transfer reaction can be contacted with a TA precursor in vivo. Furthermore, one or more enzymes that can be used to carry out a TA acyl transfer reaction can be provided to a cell that harbors a TA precursor or can be produced within an engineered non-plant host cell.

[0093] In some examples, the method provides an engineered non-plant host cell that produces an alkaloid product, wherein TA acyl transfer reaction can comprise a key step in the production of the alkaloid product. In some examples, the alkaloid produced is a medicinal TA. In yet other embodiments, the alkaloid produced is derived from a medicinal TA, including, for example, a non-naturally occurring TA. In still other embodiments, the alkaloid product is selected from the group consisting of a medicinal TA, a non-medicinal TA, and a non-naturally occurring TA.

[0094] In some examples, the substrate is a TA precursor selected from the group consisting of tropine, pseudotropine, ecgonine, methylecgonine, phenyllactic acid, cinnamic acid, ferulic acid, coumaric acid, and glycosides of the described compounds.

[0095] In some examples, the method provides an engineered non-plant host cell that produces an alkaloid product from tropine and 1-O-β-phenyllactoylglucose. The condensation of tropine and 1-O-β-phenyllactoylglucose to form littorine can constitute a key step in the production of diverse alkaloid products from precursors. In some examples, the precursor is an L-amino acid or a sugar (e.g., glucose). The diverse alkaloid products can include, but are not limited to, medicinal TAs, non-medicinal TAs, and unnatural TAs.

[0096] Any suitable carbon source can be used as a precursor for the TA transacylation reaction. Suitable precursors can include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or combinations thereof. In some examples, unrefined mixtures from renewable sources can be used (e.g., corn steep liquor, sugar beet molasses, barley malt, biomass hydrolysates). 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 still other embodiments, other carbon-containing compounds, such as methylamine, glucosamine, and amino acids (e.g., L-arginine and L-phenylalanine), can be utilized. In some instances, TAs or precursors of TAs having alcohol and / or carboxylate / glycoside functional groups can be added directly to engineered host cells of the invention, including, for example, tropine, pseudotropine, ecgonine, methylecgonine, phenyllactic acid, cinnamic acid, ferulic acid, coumaric acid, and glycosides of the compounds described.

[0097] In some embodiments, the substrate used to perform the vacuolar TA acyl transfer reaction may contain one or more alcohol and / or carboxylate / glycoside functional groups, only one of which is condensed to the corresponding ester.

[0098] TA alcohol-aldehyde interconversion Several methods, processes, and systems provided herein describe the conversion of TAs bearing aldehyde functional groups to TAs bearing alcohol (hydroxyl) functional groups, and the conversion of TAs bearing alcohol functional groups to TAs bearing aldehyde functional groups (hereinafter referred to as TA alcohol-aldehyde interconversion). Some of these methods, processes, and systems may include engineered host cells. In some instances, TA alcohol-aldehyde interconversion is a key step in converting substrates to a diverse range of alkaloids. In some instances, the conversion of a TA aldehyde group to a TA alcohol group involves a reduction reaction. In some cases, the reduction of the substrate TA aldehyde to an alcohol can be carried out by reducing the aldehyde substrate to the corresponding tetrahedral oxyanion intermediate, and then protonating this intermediate to the hydroxyl, as presented in Figure 2 and generally shown in Scheme 1. As provided in Scheme 1, R 1 may be H, CH3, or a higher alkyl group, and R 2 and R 3 can be H, OH, or OCH3, and R 4 may be H, and R 5 may be H, OH, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 acyl, F, Cl, or Br. TIFF0007779737000001.tif35150

[0099] In some examples, the TA alcohol-aldehyde interconversion may include at least one oxidation reaction or at least one reduction reaction. In some cases, at least one of the oxidation or reduction reactions is performed 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, both the oxidation and reduction reactions are carried out in the presence of at least one enzyme. In some cases, at least one enzyme is useful for catalyzing both the oxidation and reduction reactions. The oxidation and reduction reactions may be catalyzed by the same enzyme.

[0100] In some methods, processes, and systems described herein, the oxidation or reduction reaction can be carried out in the presence of an enzyme. In some examples, the enzyme can be a dehydrogenase. The dehydrogenase can use a TA having an alcohol or aldehyde functional group as a substrate. The dehydrogenase can convert the TA alcohol or aldehyde functional group to the corresponding aldehyde or alcohol derivative. The dehydrogenase is sometimes referred to as hyoscyamine dehydrogenase (HDH). Non-limiting examples of enzymes suitable for the oxidation and / or reduction of TAs in the present disclosure include cytochrome P450 oxidase, 2-oxoglutarate-dependent oxidase, flavoprotein oxidase, short-chain dehydrogenase-reductase (SDR), medium-chain dehydrogenase-reductase (MDR), cinnamyl alcohol dehydrogenase (CAD), and aldo-keto reductase (AKR). For example, tropinone reductase 1 (EC 1.1.1.206) can oxidize tropinone and other TA precursors bearing a ketone functional group to tropine (3α-tropanol) and other corresponding alcohol products. In some instances, a protein comprising a dehydrogenase domain from any one of the foregoing examples can carry out the oxidation or reduction. In some instances, the dehydrogenase can catalyze oxidation and / or reduction reactions within a host cell, such as an engineered host cell, as described herein.

[0101] Examples of amino acid sequences of dehydrogenase enzymes that can be used to perform TA alcohol-aldehyde interconversions are provided in Table 2. The amino acid sequences of dehydrogenases utilized in TA alcohol-aldehyde interconversions can be 50% or more identical to a given amino acid sequence set forth in Table 2. For example, the amino acid sequences of such dehydrogenases can comprise amino acid sequences that are 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 the amino acid sequences provided herein. Furthermore, in certain embodiments, an "identical" amino acid sequence contains at least 80% to 99% identity at the amino acid level to a particular 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 in certain cases even at least 95%, 96%, 97%, 98%, or 99% identity at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence has been altered, for example, to optimize codon usage for the host organism.

[0102] Engineered host cells can be provided that produce a dehydrogenase that catalyzes TA alcohol-aldehyde interconversion, where the dehydrogenase comprises an amino acid sequence selected from the group consisting of those sequences in Table 2. The dehydrogenase produced in the engineered host cells can be recovered and purified to form a biocatalyst. One or more enzymes recovered from the engineered host cells that produce the dehydrogenase can be used in a process for performing TA alcohol-aldehyde interconversion. The process can include contacting a TA having an alcohol and / or aldehyde functional group with a sufficient amount of dehydrogenase to convert the alcohol and / or aldehyde group of the TA to the corresponding aldehyde and / or alcohol group. In an example, a TA having an alcohol and / or aldehyde functional group can be contacted with a sufficient amount of one or more enzymes such that at least 5% of the TA is converted to its corresponding aldehyde and / or alcohol group. In further examples, TAs having alcohol and / or aldehyde functional groups can be contacted with a sufficient amount of 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 the TAs are converted to their corresponding aldehyde and / or alcohol groups.

[0103] One or more enzymes that can be used to perform TA alcohol-aldehyde interconversion can be contacted with the TA in vitro. Additionally, or alternatively, one or more enzymes that can be used to perform TA alcohol-aldehyde interconversion can be contacted with the TA in vivo. Furthermore, one or more enzymes that can be used to perform TA alcohol-aldehyde interconversion can be provided to a cell that harbors the TA or can be produced within an engineered host cell.

[0104] In some examples, the method provides an engineered host cell that produces an alkaloid product, where TA alcohol-aldehyde interconversion can comprise a key step in the production of the alkaloid product. In some examples, the alkaloid produced is a medicinal TA. In yet other embodiments, the alkaloid produced is derived from a medicinal TA, including, for example, a non-naturally occurring TA. In another embodiment, a TA having an alcohol and / or aldehyde functional group is an intermediate to the product of the engineered host cell. In yet other embodiments, the alkaloid product is selected from the group consisting of a medicinal TA, a non-medicinal TA, and a non-naturally occurring TA.

[0105] 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.

[0106] In some instances, the method provides an engineered host cell that produces an alkaloid product from hyoscyamine aldehyde. The reduction of hyoscyamine aldehyde to hyoscyamine can comprise a key step in the production of various alkaloid products from precursors. In some instances, the precursor is an L-amino acid or a sugar (e.g., glucose). Various alkaloid products can include, but are not limited to, medicinal TAs, non-medicinal TAs, and unnatural TAs.

[0107] Any suitable carbon source can be used as a precursor for TA alcohol-aldehyde interconversion. Suitable precursors can include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or combinations thereof. In some examples, unrefined mixtures from renewable sources can be used (e.g., corn steep liquor, sugar beet molasses, barley malt, biomass hydrolysates). 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 still other embodiments, other carbon-containing compounds, such as methylamine, glucosamine, and amino acids (e.g., L-arginine and L-phenylalanine), can be utilized. In some instances, TAs or precursors of TAs having alcohol and / or aldehyde functional groups can be added directly to engineered host cells of the invention, including, for example, tropine, pseudotropine, ecgonine, methylecgonine, littorine, hyoscyamine aldehyde, hyoscyamine, anisodamine, and scopolamine.

[0108] In some embodiments, the substrate used to perform the TA alcohol-aldehyde interconversion may contain one or more alcohol and / or aldehyde functional groups, with only one of the functional groups being oxidized or reduced to the corresponding aldehyde or alcohol group.

[0109] Methods for increasing intracellular and extracellular metabolite transport Some methods, processes, and systems provided herein describe the use of proteins (hereinafter referred to as "transporters") to move metabolites across lipid membranes (hereinafter referred to as "transmembrane transport"). Some of these methods, processes, and systems may involve engineered host cells. In some instances, transmembrane transport is a key step in the conversion of substrates into a diverse range of alkaloids.

[0110] In certain embodiments, the host cell comprises one or more heterologous coding sequences for one or more transporters or active fragments thereof that are localized in a lipid membrane and translocate a TA or TA precursor across the same lipid membrane. In some examples, the lipid membrane is a vacuolar membrane. In other examples, the lipid membrane is an endoplasmic reticulum membrane. In some examples, the lipid membrane is a peroxisomal membrane. In other examples, the lipid membrane is a cell plasma membrane.

[0111] 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. Accumulation of such TAs or TA precursors in a particular intracellular compartment can prevent access by operably linked biosynthetic enzymes in a different compartment; therefore, the use of transporters that move TAs or TA precursors from one compartment to another can alleviate such transport limitations. In certain cases, expression of heterologous coding sequences for one or more transporters in a host cell can increase production of TAs or TA precursors.

[0112] In some embodiments, the transporter or active fragment thereof is a multidrug and toxin extrusion (MATE) transporter. Any convenient MATE transporter that transports one or more of the aforementioned TAs or TA precursors finds use in the host cell of interest. Transporter proteins of interest include, but are not limited to, Nicotiana tabacum jasmonate-inducible alkaloid transporter 1 (NtJAT1), N. tabacum MATE1, N. tabacum MATE2, or other enzymes listed in Tables 1 and 4.

[0113] In certain embodiments, the transporter or active fragment thereof is a nitrate / peptide family (NPF) transporter. Any convenient NPF transporter that transports one or more of the aforementioned TAs or TA precursors finds use in the host cells of the subject. In other embodiments, the transporter or active fragment thereof is an ATP-binding cassette (ABC) transporter. Any convenient NPF transporter that transports one or more of the aforementioned TAs or TA precursors finds use in the host cells of the subject. In some embodiments, the transporter or active fragment thereof is a pleiotropic drug resistance (PDR) transporter. Any convenient PDR transporter that transports one or more of the aforementioned TAs or TA precursors finds use in the host cells of the subject.

[0114] In certain embodiments, the host cell comprises a heterologous coding sequence for a transporter or an active fragment thereof. In some embodiments of the present invention, the amino acid sequence of the transporter is modified in one or more ways that alters the subcellular localization, the direction of substrate movement, and / or the topological orientation of the enzyme. Examples of such modifications include, but are not limited to, the addition, removal, or substitution of an N-terminal, C-terminal, or internal signal sequence; the addition, removal, replacement, or rearrangement of a transmembrane helix; and the fusion of a protein domain to the N-terminus and / or C-terminus of the transporter.

[0115] Examples of amino acid sequences of transporters that can be used to alleviate substrate transport limitations and / or increase accumulation of TAs or TA precursors in specific cellular compartments are provided in Table 4. The amino acid sequences of transporters so utilized in non-plant cells can be 50% or more identical to a given amino acid sequence set forth in Table 4. For example, the amino acid sequences of such transporters can 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, 99% or more identical to an amino acid sequence provided herein. Furthermore, in certain embodiments, an "identical" amino acid sequence contains at least 80% to 99% identity at the amino acid level to a particular 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 in certain cases even at least 95%, 96%, 97%, 98%, or 99% identity at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence has been altered, for example, to optimize codon usage for the host organism.

[0116] An engineered non-plant host cell can be provided that produces a transporter that moves 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 method provides an engineered non-plant host cell that produces an alkaloid product, wherein TA transmembrane transport can comprise a key step in the production of the alkaloid product. In some examples, the alkaloid produced is a medicinal TA. In yet other embodiments, the alkaloid produced is derived from a medicinal TA, including, for example, a non-naturally occurring TA. In still other embodiments, the alkaloid product is selected from the group consisting of a medicinal TA, a non-medicinal TA, and a non-naturally occurring TA.

[0117] Heterologous coding sequences In some cases, the host cell is a cell that harbors one or more heterologous coding sequences (e.g., two or more, three or more, four or more, five or more, or more) that encode an activity(ies) that enables the host cell to produce a desired TA of interest, e.g., as described herein. As used herein, the term "heterologous coding sequence" is used to refer to any polynucleotide that encodes, or ultimately encodes, a peptide or protein or its equivalent amino acid sequence, e.g., an enzyme, that is not normally present in the host organism and that can be expressed in the host cell under appropriate conditions. Thus, a "heterologous coding sequence" includes multiple copies of a coding sequence normally present in the host cell, thereby causing the cell to express additional copies of a coding sequence not normally present in the cell. A heterologous coding sequence can be RNA or any type thereof, e.g., mRNA, DNA or any type thereof, e.g., cDNA, or an RNA / DNA hybrid. Coding sequences of interest include, but are not limited to, full-length transcription units containing features such as coding sequences, introns, promoter regions, 3'-UTRs, and enhancer regions.

[0118] In examples, the engineered host cell comprises multiple heterologous coding sequences, each encoding an enzyme. In some examples, the multiple enzymes encoded by the multiple heterologous coding sequences may be different from each other. In some examples, some of the multiple enzymes encoded by the multiple heterologous coding sequences may be different from each other, and some of the multiple enzymes encoded by the multiple heterologous coding sequences may be duplicate copies.

[0119] In some instances, heterologous coding sequences may be operably connected. Operatively connected heterologous coding sequences may be in the same pathway that produces a particular tropane alkaloid product. In some instances, operably connected heterologous coding sequences may be directly contiguous along a pathway that produces a particular tropane alkaloid product. In some instances, operably connected heterologous coding sequences may have one or more native enzymes among one or more enzymes encoded by the multiple heterologous coding sequences. In some instances, a heterologous coding sequence may have one or more heterologous enzymes among one or more enzymes encoded by the multiple heterologous coding sequences. In some instances, a heterologous coding sequence may have one or more non-native enzymes among one or more enzymes encoded by the multiple heterologous coding sequences.

[0120] In some embodiments, the host cell comprises putrescine N-methyltransferase (PMT) activity. Convenient PMT enzymes find use in the host cell of interest. PMT enzymes of interest include, but are not limited to, enzymes such as EC 2.1.1.53, as set forth in Table 1. In certain embodiments, the host cell comprises a heterologous coding sequence for a PMT or an active fragment thereof.

[0121] In some cases, the host cell comprises one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert NMP to 4MAB. In particular cases, the one or more enzymes are selected from plant methylputrescine oxidase (MPO) and eukaryotic MPO (e.g., EC 1.4.3.22).

[0122] In certain embodiments, the cells contain 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 are type III polyketide synthases (e.g., EC 2.3.1.-). The one or more heterologous coding sequences can be derived from any convenient species (e.g., those described herein). In some cases, the one or more heterologous coding sequences can be derived from a species listed in Table 1. In some cases, the one or more heterologous coding sequences are present in a gene or enzyme selected from those listed in Table 1.

[0123] In certain embodiments, the host cell comprises a tropinone synthase activity. Any convenient tropinone synthase enzyme (e.g., CYP82M3) finds use in the host cell of interest. Tropinone synthase enzymes of interest include, but are not limited to, enzymes such as those listed in Table 1, EC 1.14.14.-. In certain embodiments, the host cell comprises a heterologous coding sequence for a tropinone synthase or an active fragment thereof.

[0124] In certain embodiments, the host cell comprises tropinone reductase activity. Convenient tropinone reductase enzymes find use in the host cell of interest. Tropinone reductase enzymes of interest include, but are not limited to, enzymes such as those listed in Table 1, EC 1.1.1.206. In certain embodiments, the host cell comprises a heterologous coding sequence for a tropinone reductase or an active fragment thereof.

[0125] In some cases, the host cell comprises phenylpyruvate reductase (PPR) activity. Convenient PPR enzymes find use in the host cell of interest. Some PPR enzymes of interest include, but are not limited to, enzymes such as EC 1.1.1.237, as listed in Table 1. In certain embodiments, the host cell comprises a heterologous coding sequence for a PPR or an active fragment thereof.

[0126] In certain embodiments, the host cell comprises a phenyllactic acid glycosyltransferase activity. Convenient phenyllactic acid glycosyltransferase enzymes find use in the host cell of interest. Glycosyltransferase enzymes include, but are not limited to, enzymes such as 2.4.1.-, which transfer a glucose moiety from UDP-glucose to phenyllactic acid via a glycosidic ester bond, as described in Table 1. In certain embodiments, the host cell comprises a heterologous coding sequence for a phenyllactic acid glycosyltransferase or an active fragment thereof.

[0127] In certain embodiments, the cells contain 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 cells contain littorine synthase activity. Any convenient littorine synthase enzyme or enzyme containing an active fragment of littorine synthase finds use in the host cells of interest. Littorine synthase enzymes of interest include, but are not limited to, enzymes such as those in EC 2.3.1.-, as described in Table 1, and enzymes containing a littorine synthase enzyme fused at the N-terminus to a soluble protein domain, as described in Table 3. In certain embodiments, the host cells contain a heterologous coding sequence for littorine synthase or an active fragment thereof.

[0128] In certain instances, the host cell comprises a littorine mutase activity. Convenient littorine mutase enzymes find use in the host cell of interest. Littorine mutase enzymes of interest include, but are not limited to, enzymes such as those listed in Table 1, EC 1.14.19.-, etc. In certain embodiments, the host cell comprises a heterologous coding sequence for a littorine mutase or an active fragment thereof.

[0129] In some embodiments, the host cell comprises hyoscyamine dehydrogenase (HDH) activity. Convenient HDH enzymes find use in the host cell of interest. Some HDH enzymes of interest include, but are not limited to, the sequences set forth in Table 2. In certain embodiments, the host cell comprises a heterologous coding sequence for HDH or an active fragment thereof.

[0130] In certain embodiments, the host cell comprises hyoscyamine 6β-hydroxylase / dioxygenase (H6H) activity. Convenient H6H enzymes find use in the host cell of interest. Some H6H enzymes of interest include, but are not limited to, enzymes such as those listed in Table 1 under EC 1.14.11.11. In certain embodiments, the host cell comprises a heterologous coding sequence for H6H or an active fragment thereof.

[0131] In certain instances, the engineered host cell comprises multiple heterologous coding sequences, each encoding a transmembrane metabolite transporter. In some instances, the multiple transporters encoded by the multiple heterologous coding sequences may be different from each other. In some instances, some of the multiple transporters encoded by the multiple heterologous coding sequences may be different from each other, and some of the multiple transporters encoded by the multiple heterologous coding sequences may be duplicate copies.

[0132] As used herein, the term "heterologous coding sequence" also includes the coding portion of a peptide or enzyme, i.e., the cDNA or mRNA sequence of the peptide or enzyme, as well as the coding portion of a full-length transcription unit, i.e., a gene containing introns and exons, as well as "codon-optimized" sequences, truncated sequences, or other forms of modified sequences that encode an enzyme or its equivalent amino acid sequence, provided that the equivalent amino acid sequence produces a functional protein. Such equivalent amino acid sequences may have one or more amino acid deletions, which may be N-terminal, C-terminal, or internal. Truncated forms are contemplated as long as they retain the catalytic ability described herein. Fusion of two or more enzymes to facilitate metabolite transfer within a pathway, provided catalytic activity is maintained, is also contemplated. Fusion of one or more enzymes or catalytic protein domains with one or more non-catalytic protein domains in such a way that the non-catalytic protein domain facilitates the solubilization, folding, maturation, and / or activity of the fused catalytic domain is also included.

[0133] Operable fragments, mutants, or truncations can be identified by modeling and / or screening. This can be done, for example, by incrementally adding or deleting N-terminal, C-terminal, or internal regions of the protein, followed by analyzing the resulting derivatives for their activity in the desired reaction compared to the original sequence. If the derivative in question functions in this capacity, it is considered to constitute an equivalent derivative of the appropriate enzyme.

[0134] Embodiments of the present invention also relate to heterologous coding sequences that encode amino acid sequences equivalent to the native amino acid sequences of various enzymes. An "equivalent" amino acid sequence is defined as an amino acid sequence that is not identical to a particular amino acid sequence and contains at least some amino acid changes (deletions, substitutions, inversions, insertions, etc.) that do not essentially affect the biological activity of the protein when used for the desired purpose, compared to the similar activity of the particular amino acid sequence. Biological activity, in the example of a decarboxylase, refers to its catalytic activity. Equivalent sequences are also meant to include those that have been engineered and / or evolved to have properties different from those of the original amino acid sequence. Variable properties of interest include catalytic activity, substrate specificity, selectivity, stability, solubility, localization, etc. In certain embodiments, an "equivalent" amino acid sequence comprises at least 80% to 99% identity at the amino acid level to a particular amino acid, and optionally at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, and even optionally at least 95%, 96%, 97%, 98%, 99% identity at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence has been altered, for example, to optimize codon usage for the host organism.

[0135] Host cells can also be modified to have one or more genetic changes to accommodate heterologous coding sequences. Alterations to the native host genome include, but are not limited to, modifying the genome to reduce or eliminate the expression of specific proteins that may interfere with the desired pathway. The presence of such native proteins can rapidly convert one of the pathway's intermediates or end products into metabolites or other compounds that cannot be used in the desired pathway. Thus, if the activity of the native enzyme is reduced or completely absent, the produced intermediate will be more readily available for incorporation into the desired product.

[0136] In some instances where elimination of protein expression may be important, the changes are in proteins involved in pleiotropic drug responses, including, but not limited to, ATP-binding cassette (ABC) transporters, multidrug resistance (MDR) pumps, and related transcription factors. These proteins are involved in the export of TA molecules and TA precursors into the medium, so their deletion controls the export of compounds into the medium and makes them available for incorporation into the desired product. In some embodiments, the host cell gene deletion of interest includes genes associated with the unfolded protein response and endoplasmic reticulum (ER) proliferation. Deletion of such genes may lead to improved TA production. Expression of cytochrome P450 can trigger the unfolded protein response and proliferate the ER. Deletion of genes associated with these stress responses can control or reduce the overall burden on the host cell and improve pathway performance. Genetic alterations may also include modifying the promoter of endogenous genes to increase expression and / or introducing additional copies of endogenous genes. Examples of this include the construction / use of strains that overexpress the endogenous yeast NADPH-P450 reductase Ncp1p to enhance the activity of heterologous P450 enzymes. Additionally, endogenous enzymes directly involved in the synthesis of intermediate metabolites, such as Spe1p, Fms1p, Car1p, Arg2p, Aro8p, Aro9p, Pha2p, Ugp1p, and Leu2p, may also be overexpressed.

[0137] Heterologous coding sequences of interest include sequences encoding enzymes typically involved in the production of TAs and precursors in plants, either wild-type or equivalent sequences. In some cases, the enzyme encoded by the heterologous sequence can be any of the enzymes in the TA pathway and can be from any convenient source. The choice and number of enzymes encoded by the heterologous coding sequence of a particular synthetic pathway can be selected based on the desired product. In certain embodiments, the host cell of the present invention can contain one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or even fifteen or more heterologous coding sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 heterologous coding sequences.

[0138] In some cases, the polypeptide sequence encoded by the heterologous coding sequence is as reported in GENBANK. Enzymes of interest include, but are not limited to, those described herein and those shown in Table 1. The host cell may contain any combination of the listed enzymes from any source. Unless otherwise specified, accession numbers in Table 1 refer to GenBank. Some accession numbers refer to the Saccharomyces Genome Database (SGD), available on the World Wide Web at yeastgenome.org.

[0139] In some embodiments, host cells (e.g., yeast strains) are engineered to selectively produce a desired TA by localizing one or more enzymes to a compartment within the cell. In some cases, enzymes can be positioned in the host cell so that the compound produced by the enzyme spontaneously rearranges before reaching a localized enzyme that may convert the compound to an undesired metabolite, or is converted to a desirable metabolite by another enzyme. The spatial distance between two enzymes can be selected to prevent one of the enzymes from acting directly on the compound to produce an undesired metabolite, limiting the production of undesired end products (e.g., undesired opioid by-products). In other cases, an enzyme can be localized in the host cell so that the subcellular compartment in which it is located provides a more optimal pH, cofactor concentration, redox potential, substrate concentration, and / or other biochemical parameters for its activity than the compartment in which the enzyme is naturally found. In certain cases, enzymes can be localized to specific compartments within host cells so that the intracellular transport pathways that transport the enzyme to those compartments provide the post-translational modifications necessary 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, signal sequence cleavage, and multi-enzyme complex formation. In certain embodiments, any of the enzymes described herein, alone or together with a second enzyme, can be localized in any convenient compartment of a host cell, including, but not limited to, an organelle, the endoplasmic reticulum, the Golgi, the vacuole, the nucleus, the plasma membrane, mitochondria, peroxisomes, the periplasm, the lumen of any of the aforementioned organelles, or a membrane surrounding or associated with any of the aforementioned organelles. If one or more enzymes are localized to a membrane associated with any of the aforementioned organelles, the enzymes can be oriented so 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 contains one or more enzymes that include a localization tag. Any convenient tag can be utilized.In some cases, the localization tag is a peptide sequence attached to the N-terminus and / or C-terminus of the enzyme.

[0140] Any convenient method can be utilized to attach a tag to an enzyme. In some cases, the localization tag is derived from an endogenous yeast protein. Such tags may provide a route to various yeast organelles, including, but not limited to, the endoplasmic reticulum (ER), Golgi apparatus (GA), mitochondria (MT), plasma membrane (PM), peroxisomes (POX), vacuoles (V), and the like. In certain embodiments, the tag is an ER routing tag (e.g., ER1). In certain embodiments, the tag is a vacuolar 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 comprises or is derived from a transmembrane domain from within a protein of the tail-anchored class. In some embodiments, the localization tag positions the enzyme outside of an organelle. In certain embodiments, the localization tag positions the enzyme inside of an organelle. In some embodiments, the localization tag positions the enzyme so that one or more portions of the enzyme are found both inside and outside the organelle.

[0141] In some embodiments of the present invention, host cells are modified by the expression of one or more coding sequences encoding one or more enzymes containing the above-described localization tags. In certain embodiments, host cells are modified by the expression of one or more heterologous coding sequences such that one or more enzymes are expressed in the cytosol. Examples of such enzymes include, but are not limited to, arginine decarboxylase, putrescine N-methyltransferase, pyrrolidine ketide synthase, tropinone reductase, phenylpyruvate reductase, UDP-glucosyltransferase, and 2-oxoglutarate-dependent dioxygenases such as hyoscyamine 6β-hydroxylase / dioxygenase. In certain embodiments, host cells are modified by the expression of one or more heterologous coding sequences such that one or more enzymes are expressed in the ER membrane. Examples of such enzymes include cytochrome P450s such as tropinone synthase (CYP82M3) and littorine mutase (CYP80F1), and NADP +In certain embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that one or more enzymes are expressed in the mitochondria. Examples of such enzymes include, but are not limited to, N-acetylglutamate synthase. In other embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that one or more enzymes are 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 are expressed in the vacuolar 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 are expressed in the vacuolar membrane. Examples of such proteins include, but are not limited to, multidrug and toxin efflux transporters, nitrate / peptide family transporters, and ATP-binding cassette transporters.In other embodiments, host cells are modified by expressing one or more heterologous coding sequences so that one or more enzymes or proteins are 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.

[0142] In some cases, expression of each type of enzyme is increased by additional gene copies (i.e., multiple copies), thereby increasing intermediate accumulation and / or production of the TA of interest. Embodiments of the present invention include increasing production of the TA of interest in a host cell through the co-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 method of including multiple copies of heterologous coding sequences for enzymes in the host cell can be utilized.

[0143] In some embodiments, the host cell contains multiple copies of the heterologous coding sequence for the enzyme, e.g., two or more, three or more, four or more, five or more, or ten or more copies. In certain embodiments, the host cell contains multiple copies of the heterologous coding sequence for one or more enzymes, such as two or more, three or more, four or more, etc. copies. In some cases, the multiple copies of the heterologous coding sequence for the enzyme are derived from two or more different source organisms compared to the host cell. For example, the host cell may contain multiple copies of a single heterologous coding sequence, each of the copies being derived from a different source organism. Thus, each copy may contain some variation in the explicit sequence based on interspecies differences in the enzyme of interest encoded by the heterologous coding sequence.

[0144] In some embodiments of the host cell, the heterologous coding sequence is 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 stramonium, and the like. metel, Datura innoxia, Duboisia myoporoides, Anisodus luridus, Anisodus tanguticus, Anisodus acutangulus, Brugmansia arborea, Brugmansia × candida, Brugmansia sanguinea, Erythroxylum coca, Cochlearia officinalis, Solanum spp., Nicotiana spp., Atropa spp. spp), Hyoscyamus spp, Datura spp, Duboisia spp, Anisodus spp, Brugmansia sppspp), Erythroxylum spp, or Cochlearia spp. In particular examples, the heterologous coding sequence is derived from a source organism selected from A. belladonna, H. niger, and D. stramonium. In some embodiments, the host cell comprises a heterologous coding sequence from one or more source organisms listed in Table 1.

[0145] The engineered host cell culture medium can be sampled and monitored for production of the TA of interest. The TA of interest can be observed and measured using any convenient method. Methods of interest include, but are not limited to, LC-MS methods (e.g., as described herein) in which a sample of interest is analyzed by comparison with a known amount of a standard compound. Identity can be confirmed, for example, by m / z and MS / MS fragmentation pattern, and quantitation or measurement of the compound can be achieved by EIC MS peak analysis referencing LC trace peaks of known retention times and / or LC-MS analysis of corresponding known amounts of standard compounds.

[0146] method Process Steps As summarized above, aspects of the present invention include methods for preparing a desired tropane alkaloid (TA). Accordingly, aspects of the present invention include culturing a host cell under conditions in which one or more host cell modifications (e.g., as described herein) are functionally expressed, such that the cell converts a desired starting compound into a desired product TA or its precursor (e.g., TA prior to esterification). 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 to convert a desired starting compound into a desired product TA. In some cases, the method is a method for preparing a tropane alkaloid (TA) and includes 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 this method, the starting compound, TA product, and host cell are described by one of the entries in Table 1.

[0147] The fermentation medium may contain a suitable carbon substrate. Suitable carbon sources for carrying out the methods of the present disclosure may include a wide variety of carbon-containing substrates. Suitable substrates may include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or combinations thereof. In some cases, unrefined mixtures from renewable sources can be used (e.g., corn steep 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, such as methylamine, glucosamine, and amino acids, may be utilized.

[0148] Any convenient method for culturing host cells can be used to produce the desired TA precursor and downstream TA. The specific protocol used can vary, for example, depending on the host cell, heterologous coding sequence, desired TA precursor, and downstream TA. The cells can be in any convenient environment, such as an environment in which the cells can express one or more functional heterologous enzymes. As used herein, in vitro simply refers to the outside of a living cell, regardless of the location of the cells. As used herein, the term in vivo refers to the inside of a living cell, regardless of the location of the cells. In some embodiments, cells are cultured under conditions conducive to enzyme expression with appropriate substrates available to enable in vivo production of the desired TA precursor and downstream TA. In some embodiments, functional enzymes are extracted from the host for production of the TA under in vitro conditions. Optionally, the host cells are returned to the multicellular host organism. The host cells can be in any phase of growth, including, but not limited to, stationary and logarithmic phases. Furthermore, the cultures themselves can be continuous cultures, or they can be batch cultures.

[0149] Cells can be grown in an appropriate fermentation medium at a temperature between 20 and 40°C. Cells can be grown with shaking at any convenient speed (e.g., 200 rpm). Cells can be grown at a suitable pH. A suitable pH range for fermentation can be between pH 5 and 9. Fermentation can be carried out under aerobic, anaerobic, or microaerobic conditions. Any suitable growth medium can be used. Suitable growth media can include, but are not limited to, common commercially prepared media such as synthetic defined (SD) minimal medium or yeast extract peptone dextrose (YEPD) rich medium. Other rich, defined, or synthetic growth media suitable for the microorganism can be used.

[0150] Cells can be cultured in vessels of essentially any size and shape. Examples of vessels suitable for carrying out the methods of the present disclosure include, but are not limited to, multi-well shaker plates, test tubes, flasks (baffled and non-baffled), and bioreactors. Culture volumes can range from 10 microliters to over 10,000 liters.

[0151] This may include adding to the growth medium agents known to regulate metabolism in a manner favorable for alkaloid production. In a non-limiting example, cyclic adenosine 2'3'-monophosphate can be added to the growth medium to regulate catabolite repression.

[0152] Any convenient cell culture conditions for a particular cell type can be utilized. In certain embodiments, host cells containing one or more modifications are cultured under standard or easily optimized conditions with standard cell culture media and supplements. As an example, a standard growth medium, 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). Plasmid-containing host cells are grown in synthetic complete (SC) medium containing 20 g / L dextrose, supplemented with 1.7 g / L yeast nitrogen base basal medium, 5 g / L ammonium sulfate, and the appropriate amino acids required for growth and selection. Alternative carbon sources that may be useful for inducible enzyme expression include, but are not limited to, sucrose, raffinose, and galactose. Cells are grown in the laboratory in vessels, e.g., test tubes or flasks, in volumes ranging from 1 to 1000 mL or more, with shaking at any convenient speed (e.g., 200 rpm), and at any convenient temperature (e.g., 30°C).

[0153] The culture volume can be scaled up for growth in larger fermentation vessels, for example, as part of an industrial process. Industrial fermentation processes can be carried out under closed-batch, fed-batch, or continuous chemostat conditions, or any suitable fermentation mode. In some cases, cells can be immobilized on a substrate as whole-cell catalysts and subjected to fermentation conditions for alkaloid production.

[0154] Batch fermentation is a closed system in which the composition of the medium is set at the beginning of the fermentation and does not change during the fermentation process. At the beginning of the fermentation, the medium is inoculated with the desired organism(s). In some cases, batch fermentation is performed with modifications 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 composition of the system change continuously over the course of the fermentation. Cells typically progress through a lag phase, a logarithmic phase (high growth rate), a stationary phase (a slowing or cessation of growth rate), and ultimately a death phase (if left untreated).

[0155] Fed-batch fermentation is similar to batch fermentation, except that substrate is added to the system at intervals during the fermentation process. Fed-batch systems are used to reduce the effects of catabolite repression on host cell metabolism and in other situations where it is desirable to limit the amount of substrate in the growth medium.

[0156] Continuous fermentation is an open system in which a defined fermentation medium is continuously added to a bioreactor and an equal amount of fermentation medium is continuously removed from the vessel for processing. Continuous fermentation systems are generally operated to maintain steady-state growth conditions, so that cell loss due to medium removal must be balanced by the growth rate in the fermentation. Continuous fermentation is generally operated under conditions in which cells are at a constant, high cell density. Continuous fermentation allows for the adjustment of one or more factors that affect the concentration of the target product and / or cell growth.

[0157] Liquid media can include, but are not limited to, rich or synthetic defined media with additive components as described above. Media components can be dissolved in water and sterilized by heat, pressure, filtration, radiation, chemicals, or any combination thereof. Some media components can be prepared and sterilized separately and then combined in the fermentation vessel. Culture media can be buffered to help maintain a constant pH throughout fermentation.

[0158] Process parameters such as temperature, dissolved oxygen, pH, agitation, aeration rate, and cell density can be monitored or controlled during fermentation. For example, the temperature of a fermentation process can be monitored by a temperature probe immersed in the culture medium. The culture temperature can be controlled at a set point by adjusting the jacket temperature. Water is cooled in an external chiller, then flows into the bioreactor control tower and circulated through the jacket at the temperature required to maintain the set temperature within the vessel.

[0159] Additionally, gas flow parameters can be monitored during the fermentation process. For example, gas can be introduced into the culture medium through a sparger. Suitable gases for the disclosed method can include compressed air, oxygen, and nitrogen. Gas flow can be fixed or adjusted to maintain a set point for dissolved oxygen.

[0160] The pH of the culture medium can also be monitored. In one example, the pH can be monitored by a pH probe immersed in the culture medium within the vessel. If pH control is enabled, the pH can be adjusted by acid and base pumps that add each solution to the medium at the required rate. Acidic solutions used to control pH can be sulfuric acid or hydrochloric acid. Basic solutions used to control pH can be sodium hydroxide, potassium hydroxide, or ammonium hydroxide.

[0161] Additionally, dissolved oxygen can be monitored in the culture medium by a dissolved oxygen probe immersed in the culture medium. If dissolved oxygen regulation is effective, the oxygen level can be adjusted by increasing or decreasing the agitation speed. The dissolved oxygen level can also be adjusted by increasing or decreasing the gas flow rate. The gas can be compressed air, oxygen, or nitrogen.

[0162] Agitation speed can also be monitored during the fermentation process. In an example, a stirrer motor can drive an agitator. The agitator speed can be set at a constant rpm throughout the fermentation or can be dynamically adjusted to maintain a set dissolved oxygen level.

[0163] Additionally, turbidity can be monitored in fermentation processes. In an example, cell density can be measured using a turbidity probe. Alternatively, cell density can be measured by taking samples from the bioreactor and analyzing them with a spectrophotometer. Additionally, samples can be removed from the bioreactor at timed intervals via a sterile sampling device. Samples can be analyzed for alkaloids produced by the host cells. Samples can also be analyzed for other metabolites and sugars, depletion of culture medium components, or cell density.

[0164] In another example, feed parameters can be monitored during the fermentation process. In particular, feedstocks containing sugars and other carbon sources, nutrients, and cofactors can be added to the fermentation using external pumps. Other ingredients can also be added during the fermentation, including, but not limited to, antifoam agents, salts, chelating agents, surfactants, and organic liquids.

[0165] Any convenient codon optimization technique for optimizing expression of heterologous polynucleotides in host cells can be adapted for use in the host cell and method of interest, see, e.g., Gustafsson, C. et al. (2004) Trends Biotechnol, 22, 346-353, which is incorporated by reference in its entirety.

[0166] The subject methods may also include adding a starting compound to the cell culture. Any convenient addition method can be adapted for use in the subject methods. The cell culture may be supplemented with a sufficient amount of the desired starting material (e.g., as described herein), e.g., in mM to μM amounts, such as about 1 to 5 mM starting compound. It is understood that the amount of starting material added, the timing and rate of addition, the form of the added material, etc., may vary depending on various factors. The starting material may be added neat or pre-dissolved in a suitable solvent (e.g., cell culture medium, water, or organic solvent). The starting material may be added in a concentrated form (e.g., 10x the 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., several hours or days).

[0167] Method for separating product from fermentation medium The subject methods can also include recovering the TA of interest from the cell culture. Any convenient method of separation and isolation (e.g., chromatography or precipitation) can be adapted for use in the subject methods for recovering the TA of interest from the cell culture. Filtration methods can be used to separate the soluble and insoluble fractions of the cell culture. In some cases, liquid chromatography methods (e.g., reverse-phase HPLC, size exclusion, normal-phase chromatography) can 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.) can be used to separate the TA of interest from other components of the cell culture.

[0168] The produced alkaloids can be isolated from the fermentation medium using methods known in the art. Several recovery steps can be performed immediately after (or possibly during) fermentation for initial recovery of the desired product. Through these steps, the alkaloids (e.g., TAs) can be separated from the cells, cell debris, and waste products, while other nutrients, sugars, and organic molecules may remain in the spent culture medium. This process can be used to produce TA-enriched products.

[0169] In one example, a product stream having a tropane alkaloid (TA) product is formed by providing engineered yeast cells and feedstocks containing nutrients and water to a batch reactor. The engineered yeast cells may have at least one modification selected from the group consisting of feedback inhibition that alleviates a mutation in a cell's native biosynthetic enzyme gene, a transcriptional regulatory modification of the cell's native biosynthetic enzyme gene, and an inactivating mutation in a cell's native enzyme. When the engineered yeast cells are in the batch reactor, the engineered yeast cells may be subjected to fermentation. Specifically, the engineered yeast cells may be subjected to fermentation by incubating the engineered yeast cells for at least about 5 minutes to produce a solution containing the TA product and cellular material. Once the engineered yeast cells are subjected to fermentation, at least one separation unit may be used to separate the TA product from the cellular material to provide a product stream containing the TA product. Specifically, the product stream may contain the TA product as well as additional components, such as clarified yeast medium. Furthermore, the TA product may contain one or more TAs of interest, such as one or more TA compounds.

[0170] Various methods can be used to remove cells from bioreactor media containing the TA of interest. For example, cells can be removed by settling over time. This settling process can be accelerated by cooling or the addition of a clarifying agent such as silica. The spent culture medium can then be siphoned from the top of the reactor, or the cells can be decanted from the bottom of the reactor. Alternatively, cells can be removed by filtration through a filter, membrane, or other porous material. Cells can also be removed by centrifugation, for example, by continuous-flow centrifugation, or by using a continuous extractor.

[0171] If some valuable TA of interest is present intracellularly, the cells can be permeabilized or lysed, and cellular debris removed by any of the methods described above. Agents used to permeabilize cells may include, but are not limited to, organic solvents (e.g., DMSO) or salts (e.g., lithium acetate). Methods for lysing cells may include the addition of detergents such as sodium dodecyl sulfate, or mechanical disruption by bead milling or sonication.

[0172] The TA of interest can be extracted from the clarified spent culture medium by liquid-liquid extraction by adding 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, methyl isobutyl ketone, methyl oleate, toluene, oleyl alcohol, and ethyl butyrate. The organic liquid can be added to a minimum of 10% or a maximum of 100% of the volume of the aqueous medium.

[0173] In some cases, the organic liquid can be added at the beginning of the fermentation or at any time during the fermentation. This process of extractive fermentation can increase the yield of the desired TA from the host cells by continuously removing the TA precursor or TA into the organic phase.

[0174] Agitation can cause the organic phase to form an emulsion with the aqueous medium. Methods to promote separation of the two phases into distinct layers can include, but are not limited to, adding a demulsifier or nucleating agent, or adjusting the pH. The emulsion can also be centrifuged to separate the two phases, for example, by continuous conical plate centrifugation.

[0175] Alternatively, the organic phase can be isolated from the aqueous culture medium and physically removed after extraction, for example, the solvent can be encapsulated in a membrane.

[0176] In one example, the TA of interest can be extracted from the fermentation medium using adsorption methods. In particular, the TA of interest can be extracted from the clarified spent culture medium by adding a resin such as Amberlite® XAD4 or another agent that removes the TA by adsorption. The TA of interest can 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.

[0177] The TA of interest can also be extracted from the fermentation medium using filtration. At high pH, ​​the TA of interest can form a crystal-like precipitate in the bioreactor. This precipitate can be directly removed by filtering through a filter, membrane, or other porous material. The precipitate can also be collected by centrifugation and / or decantation.

[0178] The extraction methods described above can be performed either in situ (within the bioreactor) or ex situ (e.g., in an external loop where the medium flows out of the bioreactor, contacts an extractant, and is then recycled back into the vessel). Alternatively, the extraction method can be carried out after fermentation has terminated using clarified medium removed from the bioreactor vessel.

[0179] Method for purifying products from alkaloid-rich solutions Subsequent purification steps may involve treating the TA precursor or TA-enriched product after fermentation using methods known in the art to recover the individual product species of interest in high purity.

[0180] In one example, the TA precursor or TA extracted into the organic phase can be transferred to an aqueous solution. In some cases, the organic solvent can be evaporated by heat and / or vacuum, and the resulting powder can be dissolved in an aqueous solution of a suitable pH. In a further example, the TA precursor or TA can be extracted from the organic phase by adding an aqueous solution of a suitable pH that promotes extraction of the TA precursor or TA into the aqueous phase. The aqueous phase can then be removed by decantation, centrifugation, or another method.

[0181] The TA precursor or TA-containing solution may be further processed to remove metals, for example, by treatment with a suitable chelating agent. The TA precursor or TA-containing solution may be further processed to remove other impurities, such as proteins and DNA, by precipitation. In one example, the TA precursor or TA-containing solution is treated with a suitable precipitating agent, such as ethanol, methanol, acetone, or isopropanol. In another example, DNA and proteins may be removed by dialysis or other methods of size exclusion that separate smaller alkaloids from contaminating biopolymers.

[0182] In a further example, a TA precursor, TA, or modified TA-containing solution can be extracted to high purity by continuous cross-flow filtration using methods known in the art.

[0183] If the solution contains a mixture of TA precursors or TAs, it can be subjected to acid-base treatment to produce individual TAs of the desired species using methods known in the art. In this process, the pH of the aqueous solution is adjusted to precipitate the individual TA precursors or TAs at their respective pKa.

[0184] For small-scale preparations of high purity, the TA precursor or TA can be purified in a single step by liquid chromatography.

[0185] Yeast-derived alkaloid APIs vs. plant-derived APIs Clarified yeast medium (CYCM) can contain multiple impurities. Clarified yeast culture medium can be dehydrated by vacuum and / or heat to produce an alkaloid-enriched powder. This product is similar to nightshade leaf concentrate (CNL), which is used by active pharmaceutical ingredient (API) manufacturers for the extraction of tropane alkaloids, which are then 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 alkaloid product(s) can ultimately be further purified. Table 5 highlights impurities in these two products that may be inherent to either CYCM or CNL, or that may be present in both. By analyzing a product of unknown origin for a subset of these impurities, one skilled in the art can determine whether the product is derived from the yeast or plant production host.

[0186] API-grade pharmaceutical ingredients are highly purified molecules. As such, impurities that may indicate the plant or yeast origin of the API (such as those listed in Tables 2 and 3) may not be present at the API stage of the product. In fact, many of the API products derived from the yeast strains of the present invention may be nearly indistinguishable from conventional plant-derived APIs. However, in some cases, conventional alkaloid compounds may undergo chemical modification using chemical synthesis approaches and may appear as chemical impurities in plant-based products that require such chemical modification. For example, chemical derivatization often results in a set of impurities associated with chemical synthesis processes. In certain situations, these modifications can be performed biologically in a yeast production platform, thereby avoiding the presence of some of the impurities associated with chemical derivatization in yeast-derived products. In particular, these impurities from chemically derived products may be present in API products produced using chemical synthesis processes but may not be present in API products produced using yeast-derived products. Alternatively, when yeast-derived products are mixed with chemically derived products, the resulting impurities may be present, but in amounts less than would be expected in an API that contains only or primarily chemically derived products. In this example, by analyzing the API product for a subset of these impurities, one skilled in the art can determine whether the product originates from the yeast production host or from a traditional chemical derivatization route.

[0187] Non-limiting examples of impurities that may be present in chemically derived tropane alkaloid APIs but not 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.

[0188] However, when both yeast-derived and plant-derived compounds undergo chemical modification via chemical synthesis approaches, the same impurities associated with the chemical synthesis process can be expected in the product. In such situations, the starting material (e.g., CYCM or CNL) can be analyzed as described above.

[0189] Methods for engineering host cells Also included are methods for engineering host cells to produce a desired TA or its precursor. Inserting DNA into a host cell can be accomplished using any convenient method. The method is used to insert a heterologous coding sequence into the host cell such that the host cell functionally expresses an enzyme and converts a desired starting compound into a desired product TA.

[0190] Any convenient promoter can be utilized in the subject host cells and methods. The promoter driving expression of the heterologous coding sequence can be a constitutive promoter or an inducible promoter, provided that the promoter is active in the host cell. The heterologous coding sequences can be expressed from their native promoter, or a non-native promoter can be used. Such promoters can be low to high strength in the host in which they are used. The promoter can be regulated or constitutive. In certain embodiments, a promoter that is not glucose repressed or is only moderately repressed by the presence of glucose in the culture medium is used. Promoters of interest include, for example, B. subtilis These include, but are not limited to, promoters of glycolytic genes, such as the promoter of the tsr gene (encoding the promoter region of the fructose bisphosphate aldolase gene) or promoters derived from the yeast S. cerevisiae gene encoding glyceraldehyde 3-phosphate dehydrogenase (GPD, GAPDH, or TDH3); phosphate starvation-inducible promoters, such as the ADH1 promoter from baker's yeast and the PHO5 promoter from yeast; the alkaline phosphatase promoter from B. licheniformis; yeast-inducible promoters, such as Gal1-10, Gal1, GalL, and GalS; repressible promoters, such as Met25 and tetO; and constitutive promoters, such as the 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), and triosephosphate isomerase (TPI). Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones can also be used, including, but not limited to, glucocorticoid response elements (GREs) and thyroid hormone response elements (TREs). These and other examples are described in U.S. Patent No. 7,045,290, which is incorporated by reference, including the references cited therein.Vectors containing additional constitutive or inducible promoters, such as α-factor, alcohol oxidase, and PGH, can be used. Furthermore, any promoter / enhancer combination (according to the Eukaryotic Promoter Database EPDB) can be used to promote gene expression. Any convenient and suitable promoter can be selected for the host cell, for example, E. coli. Promoter selection can also be used to optimize the transcript and, therefore, the enzyme level, thereby maximizing production while minimizing energy resources.

[0191] Any convenient vector can be utilized in the subject host cells and methods. Vectors of interest include vectors for use in yeast and other cells. Yeast vector types can be divided into four general categories: integrating vectors (YIp), autonomously replicating high-copy-number vectors (YEp or 2μ plasmids), autonomously replicating low-copy-number vectors (YCp or centromeric plasmids), and vectors for large fragment cloning (YAC). Vector DNA is introduced into prokaryotic or eukaryotic cells via convenient transformation or transfection techniques.

[0192] usefulness The host cells and methods of the present invention find use in a variety of applications, for example, as described above. Applications of interest include, but are not limited to, research applications and therapeutic applications. The methods of the present invention find use in a variety of different applications, including any convenient application in which production of a TA is of interest.

[0193] The subject host cells and methods find use in a variety of therapeutic applications. Therapeutic applications of interest include those directed to the preparation of pharmaceuticals containing TAs. The host cells described herein produce tropane alkaloid precursors (TA precursors) and TAs of interest. Tropinone and tropine are important key branching point intermediates in the synthesis of TAs, including engineering efforts to produce end products such as medicinal TA products. The subject host cells can be utilized to produce TA precursors from simple, inexpensive starting materials, which can find use in the production of TAs of interest, including tropinone, tropine, and TA end products. Thus, the subject host cells find use in the delivery of therapeutically active TAs or their precursors.

[0194] In some cases, the host cells and methods find use in producing commercial-scale quantities of TAs or their precursors where chemical synthesis of these compounds is too low-yielding to be a viable means for large-scale production. In certain cases, the host cells and methods are utilized in fermentation facilities containing, for example, bioreactors (fermentors) with capacities of 5,000 to 200,000 liters, enabling rapid production of the TA or its precursor for therapeutic use. Such applications may include industrial-scale production of the TA from fermentable carbon sources such as cellulose, starch, and free sugars.

[0195] The subject host cells and methods find use in a variety of research applications. The subject host cells and methods can be used to analyze the effects of various enzymes on the biosynthetic pathways of various TAs of interest or their precursors. Furthermore, host cells can be engineered to produce TAs or their precursors that find use in testing for biological activities of interest in as yet unproven therapeutic functions. In some cases, engineering host cells to contain various heterologous coding sequences encoding various enzymes elucidates high-yield biosynthetic pathways toward the TAs of interest or their precursors. In some cases, research applications include the production of precursors of therapeutic molecules of interest, which can then be further chemically modified or derivatized to the desired product or screened for increased therapeutic activity of interest. In some cases, host cell strains are used to screen for enzymatic activity of interest in such pathways, potentially leading to the discovery of enzymes mediating the conversion of TA metabolites produced in these strains.

[0196] The subject host cells and methods can be used as a production platform for plant-specific metabolites. The subject host cells and methods can also be used as a platform for drug library development and plant enzyme discovery. For example, the subject host cells and methods can find use in developing natural product-based drug libraries by harvesting yeast strains that produce interesting scaffold molecules, such as hyoscyamine and scopolamine, and further functionalizing the compound structures via combinatorial biosynthesis or by chemical means. By creating drug libraries in this manner, potential drug hits are already associated with production hosts suitable for large-scale cultivation and production. As another example, these subject host cells and methods can 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 and 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.

[0197] Kits and Systems Aspects of the invention further include kits and systems, which may include one or more components used in the methods of the invention, as described herein, e.g., host cells, starting compounds, heterologous coding sequences, vectors, media, etc. In some embodiments, the subject kits include host cells (e.g., as described herein), and one or more components selected from the following: starting compounds, heterologous coding sequences and / or vectors comprising same, vectors, propagation materials, components suitable for use in expression systems (e.g., cells, cloning vectors, multiple cloning sites (MCSs), bidirectional promoters, internal ribosome entry sites (IRESs), etc.), and culture media.

[0198] Any of the components described herein, such as host cells containing one or more modifications, starting compounds, culture media, etc., can be provided in a kit. Various components suitable for use in the creation and use of heterologous coding sequences, cloning vectors, and expression systems can be found for use in the subject kits. Kits may also include tubes, buffers, etc., and instructions for use. The various reagent components of the kit can be present in separate containers, or some or all of them can be pre-combined into a reagent mixture in a single container, as needed.

[0199] Systems for producing a TA of interest are also provided, which may include engineered host cells containing one or more modifications (e.g., as described herein), starting compounds, culture media, fermenters and fermentation equipment, e.g., suitable instrumentation for maintaining growth conditions for the host cells, sampling and monitoring devices and components, etc. A variety of components suitable for use in large-scale fermentation of yeast cells may be found for use in the subject systems.

[0200] In some cases, the system includes components for large-scale fermentation of the engineered host cells and monitoring and purification of the 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 in which the engineered host cells in the fermenter produce one or more desired TA products or precursors thereof. In some cases, the host cells produce a TA of interest (e.g., as described herein). In some cases, the TA product of interest is a medicinal TA product, such as hyoscyamine, N-methylhyoscyamine, anisodamine, scopolamine, N-methylscopolamine, and N-butylscopolamine.

[0201] In some cases, the subject systems include means for monitoring and / or analyzing one or more TA compounds or their precursors produced by the subject host cells. For example, an LC-MS analysis system, a chromatography system, or any convenient system capable of analyzing samples and comparing them to standards, e.g., as described herein. The fermentation medium can be monitored before and at any convenient time during fermentation by sampling and analysis. Upon completion of conversion of the starting compounds to the desired TA product or precursor, fermentation can be stopped and purification of the TA product can occur. Thus, in some cases, the subject systems include purification components suitable for purifying the desired TA product or precursor from the host cell medium in which it is produced. The purification components can include any convenient means that can be used to purify the TA product or precursor 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 systems provide for the production and isolation of the desired TA fermentation product following the input of one or more starting compounds into the system.

[0202] The following examples are offered 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 the examples 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, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. [Example]

[0203] Example Methods The following sections provide examples of methods and procedures that can be used to construct, culture, and test microbial strains, such as yeast strains, for the production of TA precursors and TAs, and to ferment such strains to produce TA precursors and TAs. Also included are examples of methods, procedures, and materials that can be used to generate DNA sequences necessary for the modification of microbial hosts and to introduce the desired DNA sequences into the microbial hosts.

[0204] Compounds and Standards. TA precursors and TA chemical standards for verifying and quantifying the identity of metabolites produced by engineered host cells can be purchased from commercial vendors. For example, putrescine dihydrochloride, N-methylputrescine, hygrine, tropinone, and tropine can be purchased from Santa Cruz Biotechnology (Dallas, TX). 4-(Methylamino)butyric acid hydrochloride can be purchased from Sigma (St. Louis, MO). γ-Methylaminobutyraldehyde (4MAB) diethyl acetal and littorine can be purchased from Toronto Research Chemicals (Toronto, ON). A chemical standard of NMPy can be synthesized by deprotecting 1 volume of diethyl acetal with 5 volumes of 2 M HCl at 60°C for 30 minutes as previously described (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 3 volumes of diethyl ether to remove residual organic impurities.

[0205] Plasmid construction. Oligonucleotides used for generating novel DNA sequences by polymerase chain reaction (PCR) and for DNA sequencing can be obtained from DNA synthesis companies such as IDT DNA, Twist Bioscience, or the Stanford Protein and Nucleic Acid Facility (Stanford, CA). Native yeast genes can be amplified from S. cerevisiae genomic DNA via colony PCR (see Kwiatkowski, TJ, Zoghbi, HY, Ledbetter, SA, Ellison, KA, & Chinault, ACRapid identification of yeast artificial chromosome clones by matrix pooling and crude isate PCR. Nucleic Acids Res. 18, 7191 (1990)). Gene sequences for heterologous enzymes can be codon-optimized to improve expression in S. cerevisiae using suitable codon optimization software, such as GeneArt GeneOptimizer software (Thermo Fisher Scientific). The heterologous gene sequence can then be synthesized as a linear, double-stranded DNA fragment 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) retention plasmids, which provide a template for genomic integration of selected promoter-gene-terminator cassettes.

[0206] DE plasmids contain 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 can be constructed by PCR amplifying the gene of interest, adding 5' and 3' restriction sites using primer overhangs, digesting the PCR product or synthesized gene fragment with the appropriate pair of restriction enzymes (e.g., SpeI, BamHI, EcoRI, PstI, or XhoI), and then ligating the gene fragment using T4 DNA ligase into a similarly digested vector with a suitable yeast promoter, terminator, and replication sequence, such as plasmids pAG414GPD-ccdB, pAG415GPD-ccdB, or pAG416GPD-ccdB (see Alberti, S., Gitler, AD & Lindquist, SA suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast 24, 913-9 (2007)).

[0207] YI plasmids contain a gene of interest flanked by a constitutive promoter and terminator but lack a yeast origin of replication or an auxotrophic selectable marker. YI plasmids can be constructed by linearizing an empty carrier vector with a suitable promoter and terminator using "around-the-horn" PCR with primers designed to bind to the 3' and 5' ends of the promoter and terminator, respectively. The gene of interest can also be PCR-amplified to add 5' and 3' overhangs with 35-40 bp of homology to the ends of the linearized vector backbone. Assembly of the gene into the YI vector can then be performed using Gibson assembly. DE plasmids expressing GFP fusions of biosynthetic enzymes can be prepared by first assembling PCR-amplified DNA fragments encoding GFP, the target enzyme, and the YI vector backbone separately using Gibson assembly, followed by subcloning the fusion construct from the YI plasmid into the DE vector using restriction enzymes and ligation cloning as described.

[0208] PCR amplification can be performed using any high-fidelity recombinant DNA polymerase available from commercial suppliers, and linear DNA can 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 containing carbenicillin (100 μg / mL), kanamycin (50 μg / mL), or on LB agar plates, or using another antibiotic selection. Plasmid DNA can be isolated by alkaline lysis from overnight cultures of E. coli grown in selective LB medium at 37 °C and 250 rpm using a plasmid purification column according to the manufacturer's protocol. Plasmid sequences should be confirmed by Sanger sequencing.

[0209] Construction of yeast strains. Any suitable laboratory strain of yeast can be used as the host organism. The yeast strain described in the examples in the experimental section is derived from the parent strain CEN.PK2-1D, designated CEN.PK2 (see Entian, KD & Kotter, p. 25 Yeast Genetic Strain and Plasmid Collections. Methods Microbiol. 36, 629-666 (2007)). Strains can be grown nonselectively in yeast peptone medium (YPD medium) supplemented with 2% w / v dextrose, yeast nitrogen base basal (YNB) defined medium (YNB-SC) supplemented with synthetic complete amino acid mixture and 2% w / v dextrose, or on agar plates of the aforementioned media. Strains transformed with plasmids carrying auxotrophic selection markers (URA3, TRP1, HIS3, and / or LEU2) can be selectively grown on YNB medium supplemented with 2% w / v dextrose and the appropriate dropout solution (YNB-DO) or on YNB-DO agar plates. Yeast strains deficient in acetate metabolism can be grown in the aforementioned media (i.e., YPAD or YNBA) supplemented with 0.1% w / v potassium acetate.

[0210] Yeast genome modification can be performed using the CRISPRm method (see Ryan, OW 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. They can be constructed by assembly PCR and Gibson assembly of DNA fragments encoding SpCas9, a tRNA promoter and HDV ribozyme, a 20-nt guide RNA sequence, a tracrRNA, and a terminator. For gene insertion, integration fragments containing one or more genes of interest flanked by unique promoters and terminators can be constructed using PCR amplification and cloned into a holding vector by Gibson assembly. Integration fragments are PCR-amplified using a suitable high-fidelity DNA polymerase to contain flanking fragments and / or 40-bp microhomology regions flanking the yeast genome at the integration site. For gene disruptions, the integration fragment contains six to eight stop codons in all three reading frames flanked by 40 bp of microhomology to the disruption site, located within the first half of the open reading frame. For complete gene deletions, the integration fragment contains an auxotrophic marker gene flanked by 40 bp of microhomology to the deletion site. Each integration fragment is co-transformed with a CRISPRm plasmid targeting the desired genomic site. Positive integrants can be identified by yeast colony PCR, Sanger sequencing, and / or functional screening by liquid chromatography and tandem mass spectrometry (LC-MS / MS).

[0211] Yeast transformation. Yeast strains can be transformed using any suitable method, including heat shock, electroporation, and chemical transformation. For example, the yeast strains described in 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 medium and grown overnight at 30°C and 250 rpm. The saturated culture is then inversely diluted 1:10–1:50 in YP(A)D medium and grown for an additional 5–7 h to reach exponential phase. The culture is pelleted by centrifugation at 500 × g for 4 min, and the pellet is then washed twice by resuspending in 50 mM Tris-HCl buffer, pH 8.5. The washed pellet is resuspended in 20 μL of EZ2 solution per transformation and mixed with 100–600 ng of total DNA and 200 μL of EZ3 solution. The yeast suspension is then incubated at 30°C with gentle rotation for 1 h. For plasmid transformation, transformed yeast were plated directly onto YNB(A)-DO agar plates. For Cas9-mediated chromosomal modification, the yeast suspension was mixed with 1 mL of YP(A)D medium, pelleted by centrifugation at 500 x g for 4 min, and resuspended in 250 µL of fresh YP(A)D medium. The suspension was then incubated at 30 °C for an additional 2 h with gentle rotation to allow production of the G418 resistance protein, and spread onto YP(A)D plates containing 400 mg / L G418(geneticin) sulfate. The plates were then incubated at 30 °C for 48–60 h to allow colony formation.

[0212] Spot dilution assay. Strains are inoculated into YNB(A)-DO medium and grown overnight at 30°C and 250 rpm. Saturated overnight cultures are pelleted by centrifugation at 500 x g for 4 min, and the OD 600 Based on 10 7The cells were resuspended in sterile Tris-HCl buffer, pH 8.0, to a concentration of 100 cells / mL. Ten-fold serial dilutions of each strain were then prepared in Tris-HCl buffer, and 10 μL of each dilution was spotted onto prewarmed YNB(A)-DO plates. The plates were incubated at 30°C and imaged after 48 hours.

[0213] Growth conditions for metabolite assays. Small-scale metabolite production studies can be performed in YNB(A)-SC or YNB(A)-DO medium. Yeast colonies can be inoculated into 300–500 μL of medium and grown in 2 mL deep-well 96-well plates covered with gas-permeable film in a shaker at 30°C, 460 rpm, and 80% relative humidity for 48–72 hours.

[0214] Analysis of Metabolite Production. Metabolite profiles and titers can be analyzed using liquid chromatography and tandem mass spectrometry (LC-MS / MS). To separate cells from the medium for analysis, fermentation cultures can be pelleted by centrifugation at 3,500 × g for 5 minutes at 12 °C, after which 100–200 μL aliquots of the supernatant can be removed for direct analysis. Metabolite production can be analyzed by LC-MS / MS using a suitable HPLC device coupled with a triple quadrupole mass spectrometer, such as the Agilent 1260 Infinity Binary HPLC and the Agilent 6420 Triple Quadrupole Mass Spectrometer. Chromatography can 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 mobile phase solvent B. The column is operated at a constant flow rate of 0.4 mL / min at 40 °C with a sample injection volume of 5 µL. Compound separation can 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; and 4.34–5.00 min, equilibrated with 1% B. The LC eluent is delivered to the MS over a 0.01–5 min period, operated under electrospray ionization (ESI) in positive mode, with a source gas temperature of 350 °C, a gas flow rate of 11 L / min, and a nebulizer pressure of 40 psi. Metabolites can be quantified by multiple reaction monitoring (MRM) parameters and integrated peak areas based on standard curves.

[0215] Fluorescence microscopy. Individual colonies of yeast strains transformed with plasmids encoding biosynthetic enzymes fused to fluorescent protein reporters are inoculated into 1 mL of YNB-DO medium and grown overnight at 30 °C and 250 rpm. The overnight cultures are pelleted by centrifugation at 500 x g for 4 min, resuspended in 2 mL of YNB-DO medium containing 2% w / v dextrose, and grown for an additional 4–6 h at 30 °C and 250 rpm to reach exponential phase and allow the expressed fluorescent proteins to fully fold. Approximately 5–10 µL of the culture is then spotted onto a glass microscope slide, covered with a glass coverslip, and imaged using a suitable inverted fluorescence microscope equipped with a 60x oil immersion objective. Fluorescence excitation can 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. The emitted light is captured by a CCD camera and subsequent image analysis can be performed with any suitable scientific image analysis software, such as ImageJ (NIH).

[0216] Identification of novel gene variants from transcriptome databases. Novel genes and their variants can 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 tBLASTn searches of the D. metel and A. belladonna transcriptomes in the 1000 Plants Project database (see Matasci, N. et al., Data access for the 1,000 Plants (1KP) project. Gigascience 3, 17 (2014)). The coding sequences of putative genes identified using these search strategies can then be optimized for yeast expression and cloned into expression vectors as previously described.

[0217] Enzyme structure analysis. Heterologous enzymes can be analyzed for structural features that may be problematic during expression in yeast, such as large unstructured regions, by examining homology models constructed using suitable homology modeling or de novo structure prediction software, such as RaptorX or Rosetta. The resulting protein models can be visualized using three-dimensional molecular display software, such as PyMOL (Schrodinger) or UCSF Chimera. Enzyme affinity for specific substrates can be analyzed using suitable ligand docking simulation software, such as AutoDock, SwissDock, GOLD, or Glide.

[0218] Analysis of yeast protein expression by Western blot. For immunoblot analysis of expressed proteins in yeast, a suitable strain is transformed with an expression vector carrying the epitope-tagged protein of interest. Three days after transformation, a transformed colony is inoculated into 2 mL YNB-DO medium and grown overnight (approximately 16-20 hours) at 30 °C and 460 rpm to stationary phase. Cells are pelleted by centrifugation at 3,000 x g for 5 minutes, resuspended in 200 µL of HO, mixed with 200 µL of 0.2 M NaOH, and incubated at room temperature for 5 minutes to hydrolyze cell wall glycoproteins. Cells are repelleted at 3,000 x g for 5 minutes, resuspended in 75 µL of HO, mixed with 25 µL of 4X NuPAGE LDS sample buffer (Thermo Fisher), and boiled at 95 °C for 3 minutes to lyse the cells. The suspension is centrifuged at 16,000 x g for 5 minutes to pellet the insoluble debris and transfer the supernatant to a pre-chilled tube. For analysis under reducing conditions, the protein lysate is mixed with β-mercaptoethanol (final concentration 10%) and incubated at 70°C for 10 minutes. Approximately 20-40 µg of total protein is loaded onto a NuPAGE Bis-Tris 4-12% acrylamide gel (Thermo Fisher) equipped with Precision Plus Dual Color Protein Molecular Weight Markers (BioRad). Electrophoresis is performed at 150 V for 90 minutes in 1X NuPAGE MOPS SDS running buffer. Protein transfer to a nitrocellulose membrane is performed at 15 V for 15 minutes using a Trans-Blot Semi-Dry apparatus (BioRad) and NuPAGE transfer buffer (Thermo Fisher) according to the manufacturer's instructions. For reducing conditions, NuPAGE antioxidant (Thermo Fisher) is added to both the running buffer and transfer buffer to a final concentration of 1x. Wash the membrane containing the transferred proteins with Tris-buffered saline containing Tween (TBS-T; 137 mM NaCl, 2.7 mM KCl, 19 mM Tris base, 0.1% Tween 20, pH 7.4) for 5 min and block with 5% skim milk in TBS-T for 1 h at room temperature.The membranes were incubated overnight at 4°C with HRP-conjugated antibodies appropriately diluted in TBS-T containing 5% milk, washed three times for 5 min each in TBS-T, and then visualized using Western Pico PLUS HRP substrate (Thermo Fisher) and a suitable imager.

[0219] experiment A series of specific genetic modifications provides a biosynthetic process in Saccharomyces cerevisiae for producing TAs from simple and inexpensive feedstocks or precursor molecules. We describe methods for constructing novel strains capable of producing the initial 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. NMPy is the natural precursor of all known TA molecules. We also describe methods for manipulating the regulation of yeast biosynthetic pathways and optimizing the production of amino acid-derived TA precursors. We also describe methods for constructing novel strains capable of producing non-medicinal TAs, such as pseudotropine alkaloids and calystegine, from simple feedstocks. Furthermore, we describe methods for constructing novel strains capable of producing medicinal TAs, such as hyoscyamine, anisodamine, and scopolamine, from non-TA precursors or simple feedstocks. Furthermore, we describe methods for constructing novel strains capable of producing unnatural TAs, such as cinnamoyltropine, from non-TA precursors or simple feedstocks.

[0220] Example 1. Engineering a platform yeast strain for high-level putrescine production The tropine moiety of TA is derived from the amino acid arginine via the polyamine molecule putrescine. S. cerevisiae strains have been developed with improved flux through the arginine and polyamine biosynthetic pathway to increase intracellular concentrations of TA precursor molecules, including putrescine, NMP, 4MAB, and NMPy. These strains generally combine genetic modifications aimed at increasing carbon and nitrogen flux from central metabolism to arginine and polyamine biosynthesis, and include the introduction of key heterologous enzymes for the additional production of the TA precursor putrescine. Genetic modifications employed include the introduction of feedback inhibition to mitigate mutations in genes encoding native biosynthetic enzymes and regulatory proteins, adjusting the transcriptional regulation of native biosynthetic enzymes, deleting or disrupting genes encoding enzymes that divert precursor molecules from the intended pathway, and introducing heterologous enzymes to convert endogenous molecules into TA precursor molecules.

[0221] 1.1) The biosynthetic pathway of the engineered strain incorporates overexpression of native yeast genes involved in arginine metabolism and polyamine biosynthesis (Figure 4).

[0222] 1.1.1) Examples of native genes that can be overexpressed 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 in the mitochondrial matrix to produce ornithine; mitochondrial membrane transporter (Ort1p), which exports ornithine from the mitochondrial matrix to the cytosol; ornithine decarboxylase (Spe1p), which decarboxylates ornithine to putrescine in the cytosol; and polyamine oxidase (Fms1p), which dealkylates spermine and spermidine to putrescine.

[0223] 1.1.2) The effect of overexpression of these native enzymes on putrescine production was investigated by cotransforming yeast strains 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. After 48 h of growth in selective medium, the titer of putrescine accumulated in the extracellular medium of cotransformed cells was quantified by LC-MS / MS (Figure 5). Overexpression of SPE1 alone increased putrescine titer by 13.4-fold to 23 mg / L. Co-overexpression of CAR1 or ARG2 with SPE1 resulted in a 27% and 12% increase in putrescine production compared to SPE1 alone, whereas overexpression of ORT1 and SPE1 caused a 35% decrease in putrescine titer compared to SPE1 alone. Overexpression of any three of SPE1, CAR1, ARG2, and FMS1 resulted in an overall increase in extracellular putrescine titer to 34–35 mg / L.

[0224] 1.2) The biosynthetic pathway of the engineered strain incorporates the expression of heterologous enzymes from polyamine production pathways found in organisms other than yeast to further increase putrescine production (Figure 4).

[0225] 1.2.1) In addition to the ornithine-dependent pathway found in most plants, animals, and fungi, where putrescine is synthesized by deguanidination of arginine followed by ornithine decarboxylation, many bacteria and plants also express an alternative pathway where arginine is first decarboxylated by arginine decarboxylase (ADC) to produce agmatine. In plants, the guanidine group of agmatine is converted to urea by iminohydrolase (AIH) to produce N-carbamoylputrescine (NCP), from which the amide group is then removed by amidase (CPA) to produce 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 agmatine ureohydrolase (AUH) enzymes that allow the direct removal of the guanidine group from agmatine to produce putrescine without an N-carbamoylation intermediate (see Klein, RD et al. Reconstitution of a bacterial / plant polyamine biosynthesis pathway in Saccharomyces cerevisiae. Microbiology 145(Pt 2, 301-7 (1999)).

[0226] 1.2.2) The following enzymes can be used to reconstruct a heterologous putrescine biosynthetic pathway in yeast: ADC, AIH, CPA, and AUH. As examples of engineered strains with these enzyme activities, ADC from oat (Avena sativa; AsADC), previously shown to be active in S. cerevisiae (see Klein, RD et al. Reconstitution of a bacterial / plant polyamine biosynthesis pathway in Saccharomyces cerevisiae. Microbiology 145 (Pt 2, 301-7 (1999))), AIH from Arabidopsis thaliana (AtAIH), two CPA orthologs from tomato (Solanum lycopersicum; SlCPA) and A. thaliana (AtCPA), and two AUHs from E. coli (speB) and A. thaliana (AtARGAH2) were selected for expression in yeast.

[0227] 1.2.3) To establish the function of each heterologous enzyme in yeast, the three-step (arginine → agmatine → NCP → putrescine) or two-step (arginine → agmatine → putrescine) putrescine pathway was reconstituted in a stepwise manner by co-transforming a wild-type yeast strain with low-copy plasmids expressing AsADC, AtAIH, and either SlCPA or AtCPA; or AsADC and either speB or AtARGAH2. To eliminate effects on cell growth and metabolite production resulting from different levels of auxotrophy, all transformations were performed with three low-copy plasmids carrying different auxotrophic markers, using BFP as a negative control instead of a blank or no plasmid. The relative accumulation of agmatine, NCP, and putrescine in the extracellular medium of transformed cells after 48 h of growth in selective medium was analyzed by LC-MS / MS, showing that all enzymes, except SlCPA and AtARGAH2, retained activity in yeast (Figures 6 and 7). Reconstitution of the plant-specific pathway containing AsADC, AtAIH, and AtCPA enabled putrescine production at a titer of 23 mg / L, a 22-fold improvement over the wild-type titer. The orthologous CPA from tomato (SlCPA) enabled putrescine production at a titer of 4.5 mg / L when combined with AsADC and AtAIH, similar to the putrescine levels in cells expressing AsADC and AtAIH. Reconstitution of the bacterial shortcut pathway via AsADC and E. coli ureohydrolase (speB) enabled putrescine production at a titer of 34 mg / L, 32-fold higher than the wild-type.

[0228] 1.3) The biosynthetic pathway of the engineered strain incorporates overexpression of native yeast genes involved in arginine and polyamine biosynthesis, as well as expression of heterologous biosynthetic enzymes from polyamine production pathways found in organisms other than yeast, further increasing putrescine production.

[0229] 1.3.1) The best-performing triad of overexpressed native genes for putrescine biosynthesis (SPE1, ARG2, CAR1; 1.1.2) was combined with the best-performing heterologous putrescine pathway (AsADC, speB; 1.2.3) by co-transforming a wild-type yeast strain with a low-copy plasmid encoding SPE1, AsADC, and speB and a low-copy plasmid encoding ARG2 and CAR1. The putrescine titer in the culture medium of transformed cells was measured by LC-MS / MS analysis after 48 h. The resulting strain produced putrescine at a titer of 47 mg / L (Figure 10).

[0230] 1.4) Polyamine biosynthesis in yeast is regulated by several mechanisms (Figure 8). The biosynthetic pathway in the engineered strain incorporates disruption of one or more of these regulatory mechanisms to reduce feedback inhibition of putrescine production.

[0231] 1.4.1) Native yeast genes involved in regulating polyamine biosynthesis and therefore potentially disrupted to improve intracellular putrescine accumulation include, but are not limited to, the following examples (Figure 8): Methylthioadenosine phosphorylase (Meu1p) catalyzes the driving step in the recycling pathway of decarboxylated S-adenosylmethionine (dcSAM), which constitutes the alkyl group donor for the conversion of putrescine to spermidine and spermine, catalyzed by spermidine synthase (Spe3p) and spermine synthase (Spe4p) (see Chattopadhyay, M.K., Tabor, C.W. & Tabor, H. Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1Δ mutant. Biochem. Biophys. Res. Commun. 343, 203-207 (2006)). Methylthioadenosine is known to inhibit the activity of spermidine synthase (see Chattopadhyay, M.K., Tabor, C.W., & Tabor, H. Studies on the regulation of ornithine decarboxylase in yeast: Effect of deletion in the MEU1 gene. Proc. Natl. Acad. Sci. 102, 16158-16163 (2005)). Polyamine biosynthesis is regulated by an antizyme-mediated negative feedback loop that is conserved between fungi and metazoans (see Pegg, A.E. Regulation of ornithine decarboxylase. Journal of Biological Chemistry 281, 14529-14532 (2006)). In yeast, the OAZ1 gene contains two exons separated by a single nucleotide that collectively encode antizyme-1, a competitive inhibitor of ornithine decarboxylase (Spe1p). The polyamine-induced ribosomal frameshifting mechanism allows translation of full-length antizymes only at high polyamine levels, thereby imposing feedback inhibition of their biosynthesis.Finally, the uptake of polyamines from the extracellular environment is mediated by a signaling pathway involving Agp2p, a plasma membrane permease with affinity for carnitine, spermidine, and spermine, and Sky1p, a protein kinase that is thought to interact with Agp2p.

[0232] 1.4.2) Yeast single-gene disruption strains for MEU1, OAZ1, SPE4, SKY1, and AGP2 were constructed by inserting a series of tandem nonsense mutations within the first one-third of each open reading frame in wild-type yeast. To characterize the impact of each regulatory disruption relative to the native and heterologous putrescine production pathways, yeast ODC (SPE1) was overexpressed or AsADC and speB were coexpressed 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 (Figure 9). Disruption of MEU1 improved putrescine titers by 68% when the native putrescine production pathway via SPE1 was overexpressed. Similarly, disruption of OAZ1, when combined with overexpression of SPE1, significantly improved putrescine production by 174%. Disruption of OAZ1 increased putrescine titers 21-fold in untransformed cells, with neither native nor heterologous overexpression of the putrescine pathway. Disruption of SKY1 and AGP2 increased putrescine titers by 29% and 14%, respectively, when overexpressed in SPE1. Disruption of SKY1 reduced putrescine titers by 41% when combined with heterologous expression of AsADC and speB.

[0233] 1.5) The biosynthetic pathway of the engineered strain was further enhanced by combining MEU1 and OAZ1 regulatory gene knockout with overexpression of native and heterologous putrescine biosynthetic genes 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 the 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 showed that the putrescine titer reached 86 mg / L after 48 hours of growth in selective medium (Figure 10).

[0234] Example 2. Engineering of yeast strains for the production of NMPy S. cerevisiae strains are developed by modifying the putrescine-overproducing strain developed in Example 1 for the production of the TA precursor NMPy. These strains incorporate a combination of genetic modifications aimed at increasing carbon and nitrogen flux from putrescine toward NMPy biosynthesis, including the introduction of heterologous enzymes key to the production of the TA precursors NMP, 4MAB, and NMPy. Genetic modifications are employed, including modification of the N- and / or C-terminal domains of the enzymes of interest to improve activity in heterologous hosts, and deletion or disruption of genes encoding enzymes that divert precursor molecules from their intended pathway.

[0235] 2.1) The biosynthetic pathway of the engineered strain enables the production of NMPy from endogenous putrescine. Putrescine is first converted to N-methylputrescine (NMP) by a SAM-dependent N-methyltransferase (PMT), which is then 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 by base-catalyzed nucleophilic attack to form NMPy (Figure 11).

[0236] 2.1.1) The putrescine-overproducing strain from Example 1.5, harboring a low-copy plasmid expressing SPE1, AsADC, and speB for putrescine overproduction, was cotransformed with an additional low-copy plasmid expressing a PMT from A. belladonna (AbPMT1) followed by an additional low-copy plasmid expressing the MPO enzyme from Nicotiana tabacum (NtMPO1). The accumulation of intermediates in the extracellular medium of transformants expressing each enzyme in succession between putrescine and NMPy was compared via LC-MS / MS analysis after 48 hours of growth. The direct product of NtMPO1 (4MAB) and its spontaneous cyclization product (NMPy) were produced by expression of AbPMT1 and NtMPO1 (Figure 11), as well as their precursors, NMP and putrescine (Figure 12).

[0237] 2.1.2) NMP accumulation 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 showed that the previous disruption of MEU1 in the putrescine-overproducing strain and the resulting effect on SAM recycling did not inhibit putrescine N-methylation by AbPMT1 (Figure 13).

[0238] 2.2) When expressed heterologously from the original host organism, enzymes may localize to different subcellular compartments, resulting in reduced function. The biosynthetic pathways of engineered strains may incorporate modifications to the polypeptide sequences of native and heterologous enzymes to induce the localization of these engineered enzymes to subcellular compartments other than their natural localization. For example, previous studies have shown that NtPMT is expressed in the cytosol of tobacco cells, while NtMPO1 is localized to the peroxisomal 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)).

[0239] 2.2.1) The subcellular localization of NtMPO1 was investigated 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 showed that NtMPO1 possesses a strong yeast consensus peroxisome-targeting sequence (PTS) at its C-terminus (Ala-Lys-Leu, denoted PTS1), suggesting that NtMPO1 may localize to peroxisomes when heterologously expressed in yeast (Figure 14).

[0240] 2.2.2) Fluorescence microscopy of wild-type yeast cells expressing N- or C-terminally GFP-tagged AbPMT1 and NtMPO1 from low-copy plasmids reveals that AbPMT1 is found primarily in the cytosol, whereas NtMPO1 localization to peroxisomes depends on the exposed C-terminal PTS (Figures 15a, 16).

[0241] 2.2.3) Cytosolic expression of NtMPO1, achieved by masking the C-terminal PTS with a GFP fusion, did not significantly affect extracellular 4MAB or NMPy levels (Figure 15b).

[0242] 2.3) The biosynthetic pathways of engineered strains may incorporate orthologs of biosynthetic enzymes other than those listed in Table 1. Different orthologs of enzymes may exhibit significant differences in activity when expressed in heterologous hosts. Thus, 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 transformations.

[0243] 2.3.1) tBLASTn searches 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)) were performed using the amino acid sequence of NtMPO1 as a query and an E-value threshold of 10 -150 Two full-length orthologous sequences, designated AbMPO1 and DmMPO1, were identified, each sharing 91% sequence identity with NtMPO1 (Figure 17a).

[0244] 2.3.2) The yeast codon-optimized sequences of AbMPO1 and DmMPO1 were obtained and cloned into low-copy expression plasmids. To assess their activity, each of the three MPO variants was coexpressed with AbPMT1 from a low-copy plasmid in the putrescine-overproducing strain of Example 1.5, and 4MAB and NMPy accumulation was measured in the extracellular medium by LC-MS / MS after 48 hours of growth in selective medium. DmMPO1 showed comparable levels of 4MAB and NMPy production to the original NtMPO1 variant (Figure 17b).

[0245] 2.3.3) Differences in activity between orthologous enzymes can often be attributed, at least in part, to differences in their active site structures. Template-based homology models of NtMPO1, AbMPO1, and DmMPO1 were constructed based on the crystal structure of Pisum sativum copper-containing aminooxidase (PDB:1KSI) using the RaptorX web server (see Kallberg, M. et al. Template-based protein structure modeling using the RaptorX web server. Nat. Protoc. 7, 1511-22 (2012)). The homology models showed that the orthologs possess long, unstructured N- and C-terminal tail regions (Figure 17c).

[0246] 2.3.4) Truncated forms of 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 in which the unstructured tail was removed but the PTS was retained ( ΔC-PTS1 (Indicated as ). Each MPO truncation was coexpressed with AbPMT1 from a low-copy plasmid in the putrescine-overproducing strain of Example 1.5, and accumulation of 4MAB and NMPy in the medium after 48 hours of growth was quantified by LC-MS / MS. No significant differences in activity were observed between the NtMPO1 truncations (Figure 18). Removal of the C-terminal unstructured region from DmMPO1 while retaining the C-terminal PTS tripeptide increased extracellular 4MAB levels by 31% compared to the wild-type DmMPO1 enzyme.

[0247] 2.4) The biosynthetic pathway of the engineered strain incorporates one or more genetic modifications to reduce or eliminate metabolic flux of undesired side reactions. Biosynthetic enzymes expressed in heterologous hosts may be involved in undesired side reactions that divert metabolic flux away from the biosynthesis of the desired compound. For example, yeast aldehyde dehydrogenase may oxidize heterologous aldehyde molecules, such as 4MAB, to their cognate carboxylic acids. Based on LC-MS / MS analysis, AbPMT1 and DmMPO1 ΔC-PTS1 Accumulation of 4MAB acid was observed in the growth medium of the putrescine overproducing strain of Example 1.5 when co-expressed from a low copy plasmid, but not in the absence of the MPO enzyme (Figure 11).

[0248] 2.4.1) Six yeast genes (ALD2-ALD6 and HFD1) have been documented to encode enzymes with aldehyde dehydrogenase activity (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 see 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 that 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 encode two mitochondrial and one cytosolic acetaldehyde dehydrogenase, respectively, which, in addition to oxidizing acetaldehyde to acetate during fermentative growth on glucose and ethanol (Saint-Prix, F., Bonquist, 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 a diverse set of aliphatic and aromatic aldehydes to carboxylic acids (Datta, S., Annapure, U.S. & Timson, D.J. Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae). var. boulardii. Biosci. Rep. 37, BSR20160529 (2017)). Knockout strains of each of these four target genes were constructed by inserting a series of tandem nonsense mutations within the first third of the open reading frame of the putrescine-overproducing strain described in Example 1.5. The contribution of each of the four dehydrogenases to 4MAB oxidation was determined by inserting AbPMT1 and DmMPO1 from low-copy plasmids in each single disruption strain. ΔC-PTS1 The effects of HFD1 and ALD4-6 on 4MAB acid accumulation in the culture medium were assessed by co-expression and LC-MS / MS after 48 h of growth. A slight decrease in 4MAB acid levels was observed upon individual disruption of HFD1 and ALD4-6 (Figure 19).

[0249] 2.4.2) Although ALD4-6 are considered essential genes due to their role in acetate and acetyl-CoA production, previous studies have shown that the three genes are at least partially redundant and that the lethal phenotypes of double and triple knockouts can be rescued by supplementing the medium with acetate (Saint-Prix, F., Bonquist, 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. 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 by disrupting the open reading frames of HFD1 and ALD4-6, and expressing AbPMT1 and DmMPO1 from a low-copy plasmid. ΔC-PTS1 This strain showed a 45% reduction in 4MAB acid levels (Fig. 20a) and a concomitant 46% increase in NMPy production (Fig. 20b) compared to the undisrupted strain.

[0250] 2.4.3) The tandem ALD2-ALD3 genes were deleted from the genome of the quadruple knockout strain of Example 2.4.2, and AbPMT1 and DmMPO1 were introduced from a low copy plasmid. ΔC-PTS1An ALD-null strain was constructed by co-expressing ALD2 and ALD3. After 48 h of growth, LC-MS / MS analysis showed that deletion of ALD2 and ALD3 completely eliminated the 4MAB acid byproduct and increased the production of 4MAB and NMPy by 83% and 75%, respectively, compared to the intact strain containing all six ALD genes (Figure 20a, b).

[0251] 2.4.4) The NMPy-producing yeast strain was constructed by integrating the putrescine overproduction gene cassette (SPE1, AsADC, speB) from the previous plasmid into the genome of the ALD null strain from Example 2.4.3, and further integrating AbPMT1 and DmMPO1 ΔC-PTS1 LC-MS / MS analysis showed that NMPy production in this strain after 48 h of growth in non-selective medium was mediated by the incorporation of the necessary putrescine production genes, AbPMT1 and DmMPO1, from a low-copy plasmid. ΔC-PTS1 It was confirmed that the expression of the ALD gene was equivalent to that of the ALD null strain of Example 2.4.3, which was cultured in a selective medium (Figure 21).

[0252] Example 3. Yeast strains engineered to produce tropine from simple sugars and nutrients Type III polyketide synthases (PKSs) and cytochrome P450s enable the conversion of NMPy to tropinone via the TA precursor MPOB. Tropinone is reduced by a stereospecific reductase called 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)) (Figure 22).

[0253] 3.1) The biosynthetic pathway of the engineered strain incorporates a pyrrolidine ketide synthase, tropinone synthase CYP82M3, one or more cytochrome P450 reductases, and tropinone reductase 1, which converts NMPy to tropine.

[0254] 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. P450 enzymes require NADP for continuous electron exchange. + Because they require a cytochrome P450 reductase (CPR) partner, 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 (poppy), and a native yeast CPR (NCP1), were also obtained for expression in yeast. The yeast strain was constructed by integrating DsTR1 into the genome of the NMPy-producing strain from Example 2.4.4 and expressing AbPYKS, AbCYP82M3, and each of the four CPRs from low-copy plasmids. To verify enzyme activity and identify potential bottlenecks, the accumulation of NMPy, MPOB, tropinone, and tropine was monitored by LC-MS / MS in the medium of the transformed strains after 48 hours of growth (Figure 23). Under the assay conditions, comparable levels of de novo tropin production (175–210 μg / L) were observed with all four CPR partners.

[0255] 3.2) The presence of metabolic bottlenecks, defined as spontaneous steps where a biosynthetic enzyme or its low activity limits flux through part of a biosynthetic pathway, can result in suboptimal production of desired TAs and precursors.

[0256] 3.2.1) For example, analysis of the accumulation of TA intermediates in the culture medium of the engineered strains of Example 3.1.1 showed that while accumulation of the AbCYP82M3 product, tropinone, was minimal, a significant portion of the MPOB produced by AbPYKS remained unconsumed by AbCYP82M3 (Figure 24).

[0257] 3.2.2) Integration of tropine biosynthesis genes into the yeast genome can improve tropine production by enabling more stable AbCYP82M3 expression. A tropine production platform strain was constructed by integrating AtATR1, along with AbPYKS and AbCYP82M3, into the genome of the NMPy-producing strain from Example 3.1.1. Tropine and hyglin accumulation in the integrated strain was compared to plasmid-based expression of the same genes via LC-MS / MS analysis after 48 hours (Figure 28). Genomic expression of AbPYKS, AbCYP82M3, and AtATR1 increased tropine titer by nearly three-fold (565 μg / L) compared to plasmid-based expression (189 μg / L). The engineered strain also showed a 2.6-fold increase in hyglin accumulation.

[0258] 3.3) Accumulation of by-products in the biosynthetic pathway of engineered strains may result in suboptimal production of desired TAs and precursors.

[0259] 3.3.1) For example, analysis of TA intermediate accumulation in the medium of the engineered strains of Example 3.1.1 showed substantial accumulation of hyglin, a derivative of NMPy, at titers nearly four-fold greater than tropine (775-900 μg / L). In related literature, hyglin has been observed to accumulate via spontaneous decarboxylation of MPOB (see Bedewitz, MA, Jones, AD, D'Auria, C. & Barry, C.S. Tropinone synthesis via an atypical polyketide synthase and P450-mediated cyclization. Nat. Commun. 9, 5281 (2018)) (Figure 22). As another example, LC-MS / MS analysis of the growth medium of the engineered strains of Example 3.1.1 showed that hyglin also accumulated in a negative control strain lacking AbPYKS and AbCYP82M3 due to decarboxylation condensation with NMPy (Figure 22).

[0260] 3.3.2) Adjustment of growth temperature can be used to reduce by-product accumulation in the biosynthetic pathway of engineered strains and increase flux to the desired TA and precursor. In one example, the effect of temperature on spontaneous hyglin production was evaluated by utilizing the kinetic principle that the rates of enzymatic and spontaneous reactions decrease at lower temperatures. Because A. belladonna and other TA-producing Solanaceae species are adapted for optimal growth in cooler climates, growth of yeast strains expressing Solanaceae genes at 25°C improves enzyme folding and / or activity, allowing for production of enzymatically produced tropine equivalent to growth at 30°C, while simultaneously reducing the rate of spontaneous hyglin production. Cultures of the tropine-producing strain from Example 3.2.2 were grown in non-selective defined medium at 30°C and 25°C, and the accumulation of tropine and hyglin was compared by LC-MS / MS analysis of the growth medium after 48 hours. Tropine titer was minimally affected by the decrease in temperature. Hyglin accumulation was reduced by 42% at 25°C compared to 30°C, with a 60% increase in the ratio of tropin to hyglin produced (Figure 25).

[0261] 3.3.3) Reduction or elimination of undesired side reactions can be used to improve metabolite flux to desirable TAs and TA precursors in the biosynthetic pathway of engineered strains. In one example, flux to the TA precursor tropine can be improved by reducing hygrine production, which results from spontaneous decarboxylative condensation with acetate. The effect of removing supplied acetate from the medium of the NMPy-producing strain of Example 2.4.4 on hygrine and tropine production was evaluated. The effect of eliminating acetate requirement in the engineered strain of Example 2.4.4 was assessed by expressing functional copies of ALD4 and ALD6 carried on low-copy plasmids and monitoring the accumulation of hygrine and 4MAB acids via LC-MS / MS analysis after 48 hours of growth. Reconstitution of ALD4 or ALD6 enabled growth on selective medium without acetate supply (Figure 26a), while addition of ALD4 increased 4MAB acid accumulation five-fold, while no significant increase was observed with ALD6 (Figure 26b). Furthermore, elimination of acetate supply in either ALD4 or ALD6 reduced hygrin accumulation by 38% and 59%, respectively (Fig. 26b).

[0262] 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 effect of this integration on the accumulation of all metabolites, including NMPy and tropine, was measured by LC-MS / MS analysis after 48 hours of growth in non-selective medium. Restoration of Ald6p-mediated acetate metabolism resulted in a 2.7-fold increase in tropine titer and a 1.6-fold increase in hygrine accumulation (Figure 28). Furthermore, integration of ALD6 significantly increased the production of NMPy and tropinone, as well as MPOB consumption (Figure 27).

[0263] 3.3.5) Additional copies of the putrescine and tropine biosynthetic enzyme genes (i.e., AbPMT1, DmMPO1) ΔC-PTS1, AbPYKS, and AbCYP82M3) were expressed from low-copy plasmids 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 medium. 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 (Figure 29). Expression of an additional copy of AbPMT1 significantly improved production of all TA precursors between NMP and tropinone, increasing tropine production by 2.4-fold (Figure 29). Therefore, additional copies of PMTs (AbPMT1 and DsPMT1) and PYKS (AbPYKS) were integrated into the genome of the tropine-producing strain of Example 3.3.4 (CSY1249) at the PAD1 locus. The resulting engineered strain (CSY1251) was grown in non-selective medium at 25°C for 48 hours, resulting in tropine production at a titer of 3.4 mg / L, 2.2-fold higher than the tropine-producing strain (CSY1249) from Example 3.3.4 (Figure 30).

[0264] Example 4: Yeast engineered to produce pseudotropine alkaloids from L-arginine Yeast strains can be engineered to produce non-medicinal TAs from initial amino acid precursors such as L-arginine. As an example, the platform yeast strain described in Example 3 can be further engineered to produce pseudotropine alkaloids from L-arginine (Figure 1).

[0265] Platform yeast strains that produce tropinone from L-arginine (see the description in Example 3) can be further engineered to incorporate a stereospecific reductase, such as tropinone reductase 2 (TR2; EC 1.1.1.236), to convert the biosynthesized tropinone to pseudotropine. An expression cassette carrying a strong constitutive promoter, such as TDH3, and the coding sequence of a TR2 variant, such as 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, such as calystegine, by incorporating one or more expression cassettes carrying 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 at a different position on the pseudotropine backbone, various calystegine and its derivatives can be biosynthesized. The engineered strains are then cultured in non-selective synthetic complete medium at 30 °C or 25 °C for 48–96 h, after which the accumulation of pseudotropine alkaloids in the culture medium can be analyzed by LC-MS / MS.

[0266] Example 5: Yeast engineered for overproduction of phenylpyruvate and related TA precursors Yeast strains can be engineered for the overproduction of phenylpyruvate, the precursor of the acyl donor molecule required for the production of medicinal TAs, with the goal of increasing carbon and nitrogen flux from central metabolism to the desired TA and TA precursor (Figure 2). Yeast strains can be engineered for the overproduction of phenylpyruvate by incorporating genetic modifications, including, but not limited to, adjusting the transcriptional regulation of native biosynthetic enzymes, deleting or disrupting genes encoding enzymes that divert precursor molecules from the intended pathway, and introducing heterologous enzymes to convert endogenous molecules into TA precursor molecules.

[0267] In one example, yeast strains can be engineered to increase phenylpyruvate production by incorporating additional copies of native genes encoding 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, and 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 example, one or more additional copies of ARO8 can be incorporated into the engineered strain under the control of a strong constitutive promoter. In one example, one or more additional copies of ARO9 can be incorporated into the engineered strain under the control of a strong constitutive promoter. In another example, 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 present 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 a unique, strong constitutive promoter.

[0268] Example 6: Yeast engineered for the production of acyl donors from L-phenylalanine or L-tyrosine for the biosynthesis of TA scaffolds Yeast strains can be engineered for the production of a variety of phenylpropanoid acyl donor compounds from L-phenylalanine and L-tyrosine, including PLA, cinnamic acid, coumaric acid, ferulic acid, benzoic acid, and the coenzyme A thioester and glycoside derivatives of these compounds, which can be esterified with tropine, pseudotropine, or their derivatives to biosynthesize medicinal, non-medicinal, and unnatural TAs (Figures 1-3).

[0269] 6.1) Because wild-type yeast only produces trace amounts of PLA, increased production of this TA precursor is necessary to fully accumulate downstream TAs. To improve PLA production, heterologous phenylpyruvate reductases (PPRs) can be expressed in engineered host cells. PPR orthologs from E. coli, Lactobacillus, A. belladonna, and Wickerhamia fluorescens, as well as lactate dehydrogenases (LDHs) from Bacillus and Lactobacillus, which have reported activity toward 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 hours of growth in selective medium. All LDH candidates and PPRs from L. plantarum, E. coli, and A. belladonna resulted in a moderate (1.3- to 3.5-fold) improvement in PLA production compared to the control, whereas expression of PPR from W. fluorescens resulted in an approximately 80-fold increase in PLA production to approximately 250 mg / L (Figure 31). Therefore, WfPPR was selected for integration into CSY1251 to create strain CSY1287.

[0270] 6.2) As another example, yeast strains can be engineered for the production of cinnamic acid and coumaric acid, phenylpropanoids that can be used as acyl donor compounds for esterification with tropine or pseudotropine to form unnatural TAs, from L-phenylalanine and L-tyrosine, respectively. Yeast can be engineered to produce cinnamic acid from L-phenylalanine by incorporating an ammonia lyase such as phenylalanine ammonia lyase (PAL; EC 4.3.1.24). Similarly, yeast can be engineered to produce coumaric acid from L-tyrosine by incorporating an ammonia lyase such as tyrosine ammonia lyase (TAL; EC 4.3.1.23). Yeast strains were engineered to produce cinnamic acid from L-phenylalanine by transformation with a low-copy CEN / ARS plasmid containing a TRP1 selectable marker, a TEF1 promoter, and the coding sequence for a PAL variant from Arabidopsis thaliana (AtPAL1). The resulting strains carrying the low-copy plasmid were grown in synthetic complete medium containing the appropriate amino acid dropout solution (-Ura) at 30° C. After 48 h of growth, the medium was analyzed for cinnamic acid content by LC-MS / MS analysis (FIG. 32).

[0271] 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)). Because plant UGTs are involved in the biosynthesis of diverse phenylpropanoids and often exhibit a broad substrate range (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 UGTs with sufficiently high activity toward the desired acyl donor.

[0272] 6.3.1) As an example, the activity of AbUGT toward various phenylpropanoid acyl donors, including the standard substrate PLA, was evaluated by expressing AbUGT from a low-copy plasmid in CSY1251 and measuring the conversion of each of the three phenylpropanoid acyl donors (PLA, cinnamic acid, and ferulic acid) to their respective glucosides. AbUGT glucosylated approximately 60% and 90% of cinnamic acid and ferulic acid, respectively, while PLA glucosylation was the lowest of the substrates tested, with <3% conversion (Figure 33).

[0273] 6.3.2) Orthologs of AbUGTs from other TA-producing Solanaceae species can be evaluated for activity against PLA and other phenylpropanoids. In this example, a tBLASTn search was used to identify transcripts encoding UGT84A27 from the transcriptomes of Brugmansia sanguinea (BsUGT) and D. metel (DmUGT) in the 1000Plants database. Yeast codon-optimized sequences encoding these orthologous UGTs were screened for activity by expressing AbUGT, BsUGT, DmUGT, or a BFP negative control from a low-copy plasmid in CSY1251. After 72 hours of growth in selective medium supplemented with 500 μM PLA, cinnamic acid (CA), or ferulic acid (FA) as glucose acceptors, glucoside production in cultures of transformed strains was measured via LC-MS / MS. All three UGT orthologs showed substantial glycosylation of CA (34–65% conversion) and FA (85–90% conversion) and only minor activity toward PLA (<3% conversion), with AbUGT showing the greatest conversion of PLA (2.7%) (Figures 33, 34).

[0274] 6.3.3) Given the disproportionate variation in activity of AbUGT toward the structurally similar substrates cinnamic acid, ferulic acid, and PLA, a structure-based rational mutagenesis approach can be performed to engineer the active site of AbUGT to improve activity toward PLA. In this example, a homology model of AbUGT bound to UDP-glucose was first constructed based on the crystal structure of Arabidopsis thaliana salicylic acid UDP-glucosyltransferase UGT74F2 (PDB:5V2K) using the RaptorX web server (Figure 35). Then, the docking of D-PLA in the active site was simulated using the Maestro / GlideXP software suite. Based on the energy-minimized binding mode, the aryl ring of D-PLA is likely stabilized by π-stacking interactions with F130, and its α-hydroxyl and carboxylate groups are stabilized by hydrogen bonds with Q151 and H24, respectively, so that the nucleophilic carboxylate oxygen is within 4 Å of the electrophilic C1 carbon of UDP-glucose (Figure 35). D-PLA is further adjacent to residues L205 and I292, neither of which appear to interact with either substrate. This suggests that (i) mutation of F130 to tyrosine may provide an additional hydrogen bond that stabilizes the α-hydroxyl oxygen of D-PLA, which is not present in cinnamic and ferulic acids, while maintaining π-stacking with the aryl ring of D-PLA; (ii) mutation of L205 to phenylalanine may increase the π-stacking stabilization of D-PLA by F130Y; and (iii) mutation of I292 to glutamine may generate two additional stabilizing hydrogen bonds with D-PLA and UDP-glucose (Figure 35). AbUGT F130Y, L205F, and I292Q point mutants were screened for activity by expressing each mutant, wild-type AbUGT, or a BFP control from a low-copy plasmid in CSY1251, and measuring glucoside production by LC-MS / MS after 72 h of growth in selective medium supplemented with 500 μM PLA, CA, or FA.Although the F130Y and I292Q mutations significantly reduced UGT activity in CA, all three mutants exhibited relatively low (and statistically indistinguishable) activity in PLA compared to wild-type AbUGT (<3% conversion) (Figure 36).

[0275] 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 and verified by confirmation of PLA production (66 mg / L) and minimal PLA glucoside accumulation (Figure 37).

[0276] 6.4) Because low AbUGT activity toward PLA may limit the flux of TA precursors to downstream TAs, incorporating genetic modifications that promote UDP-glucose accumulation and reduce glycoside degradation could increase the flux of phenylalanine to PLA glucosides.

[0277] 6.4.1) UDP-glucose is important for the formation of storage polysaccharides, cell wall glucans, and glycoproteins, and therefore its biosynthesis is tightly regulated (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 derives glucose-6-phosphate along two major metabolic pathways: glycolysis and starch biosynthesis. Because citrate is an allosteric inhibitor of phosphofructokinase, the rate-limiting enzyme in glycolysis (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 by citrate supplementation may increase UDP-glucose availability and glucoside production (Figure 38). Strain CSY1288, encoding genomic WfPPR and AbUGT for endogenous PLA glucoside production, was cultured in medium supplemented with 2% citrate and 500 μM CA or FA, and glucoside production was compared by LC-MS / MS after 72 h of growth. Citric acid supplementation reduced the glucosylation of PLA, CA, and FA by 83%, 56%, and 78%, respectively (Figure 39).

[0278] 6.4.2) Overexpression of PGM2 and UGP1, whose gene products catalyze the isomerization of glucose-6-phosphate to glucose-1-phosphate and the conversion of glucose-1-phosphate to UDP-glucose, respectively, can be used to increase UDP-glucose supply.

[0279] 6.4.2.1) Extra copies of PGM2 and UGP1 were expressed from low-copy plasmids in CSY1288, and PLA glucoside production was measured after 72 hours of growth in selective medium. While overexpression of PGM2 did not result in improvement compared to the control, overexpression of UGP1 resulted in an approximately 1.8-fold increase in PLA glucoside production (Figure 40), supporting that increasing the UDP-glucose pool improves PLA utilization by AbUGT.

[0280] 6.4.2.2) It is possible that natural glucosides may act on PLA and other TA precursor glucosides to reduce their accumulation, as other heterologous glucosides have been shown to be hydrolyzed in this manner in yeast (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 after 72 hours of growth of the disruption mutants on nonselective medium. Disruption of EGH1 more than doubled PLA-glucoside production (Figure 41), indicating that hydrolysis by Egh1p constitutes a substantial loss of TA precursor flux.

[0281] Example 7: Yeast engineered to convert littorine to hyoscyamine aldehyde Yeast strains can be engineered to convert littorine to hyoscyamine aldehyde (Figure 2). For example, the tropine and PLA glucoside-producing yeast strain described in Example 6 can be further engineered to express cytochrome P450 CYP80F1 (EC 1.14.19.-), which catalyzes the rearrangement of littorine to hyoscyamine aldehyde, and cytochrome P450 reductase (CPR, EC 1.6.2.4), which supports the activity of the P450 enzyme. Yeast strains were engineered to convert supplied littorine to hyoscyamine aldehyde by transforming them with a low-copy CEN / ARS plasmid containing a LEU2 selectable marker, a TDH3 promoter, and the coding sequence of a CYP80F1 variant from A. belladonna (AbCYP80F1), as well as with a low-copy CEN / ARS plasmid containing a TRP1 selectable marker, a TEF1 promoter, and the coding sequence of a cytochrome P450 reductase (CPR) from S. cerevisiae (NCP1) or A. thaliana (AtATR1). The resulting strains carrying the low-copy plasmids were grown at 30°C in synthetic complete medium supplemented with the appropriate amino acid dropout solution (-Leu-Trp) and 1 mM littorine. After 48 h of growth, the medium was analyzed for hyoscyamine aldehyde content by LC-MS / MS analysis (Figure 42).

[0282] Example 8: Yeast engineered to convert hyoscyamine to scopolamine Yeast strains can be engineered to convert hyoscyamine to scopolamine (Figure 2). For example, the yeast strain described in Example 7 can be further engineered to incorporate an enzyme with hydroxylase activity at the 6β-position of hyoscyamine to form anisodamine, an enzyme with dioxygenase activity at the 6β-hydroxyl position of anisodamine to form scopolamine, or an enzyme with both of these activities (EC 1.14.11.11). Yeast strains were engineered to convert supplied hyoscyamine to scopolamine by transformation with a low-copy CEN / ARS plasmid containing a LEU2 selectable marker, a TDH3 promoter, and the coding sequence for hyoscyamine 6β-hydroxylase / dioxygenase (H6H) from D. stramonium (DsH6H), Anisodus acutangulus (AaH6H), Brugmansia arborea (BaH6H), or Datura metel (DmH6H). The resulting strain carrying the low-copy plasmid was grown at 30°C in synthetic complete medium supplemented with the appropriate amino acid dropout solution (-Leu) and 1 mM hyoscyamine. After 72 h of growth, the medium was analyzed for scopolamine content by LC-MS / MS analysis (Figure 43). While all variants tested exhibited H6H activity in vivo, the strain expressing the H6H variant from D. stramonium exhibited the greatest conversion of supplied hyoscyamine to scopolamine. Further optimization of cofactor requirements was performed by supplementing the culture medium of this engineered yeast strain with different cofactors and analyzing the medium by LC-MS / MS after 72 h of growth. This analysis confirmed that ferrous iron supplementation increased the conversion of hyoscyamine to scopolamine (Figure 44).

[0283] Example 9. Identification of candidate hyoscyamine dehydrogenase enzymes and reduction of hyoscyamine aldehyde to hyoscyamine in engineered non-plant cells To identify suitable dehydrogenase enzymes for carrying out the TA alcohol-aldehyde interconversion of the methods disclosed herein, and in particular for reducing hyoscyamine aldehyde to hyoscyamine, hyoscyamine dehydrogenase (HDH) open reading frames were identified from publicly available plant RNA sequencing data.

[0284] 9.1) For each of the 43,861 unique transcripts identified in the A. belladonna transcriptome, tissue-specific abundance (fragments per kilobase of contig per million mapped reads, FPKM) and putative protein structural and functional annotations were obtained from the Michigan State University Medicinal Plant Genomics Resource. Transcripts encoding candidate hyoscyamine dehydrogenases were identified based on clustering of tissue-specific expression profiles of the bait genes CYP80F1 (littorine mutase) and H6H (hyoscyamine 6β-hydroxylase / dioxygenase), which are located before and after the dehydrogenase step of the TA biosynthesis pathway, respectively, using the following computational filtering algorithm:

[0285] 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 the bait genes. Next, average tissue-specific expression profiles of the CYP80F1 and H6H bait genes were generated. For each of the two bait genes, a linear regression model was constructed to express the bait gene expression profile (FPKM) as a linear function of each candidate gene profile, and a correlation p-value was calculated for each candidate. Candidates identified using each of the two bait genes were pooled, and duplicates were removed. The combined p-value for each candidate was calculated as the sum of the log10 p-values ​​of the correlation with each of the two bait genes. Transcripts matching known dehydrogenases in the TA biosynthesis pathway (i.e., tropinone reductase I and II) were removed, and the remaining candidates were ranked by their combined p-value and distance from the bait gene by hierarchical clustering of tissue-specific expression profiles (Figure 45).

[0286] 9.2) Nearly all of the 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 approximately 30 candidates against the UniPROT / SwissPROT database revealed that many transcripts were missing terminal or internal sequence regions. To address this, we repeated de novo transcriptome assembly from deposited raw RNA-seq reads using the Trinity software package (see Haas, BJ 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)). We then reconstructed all missing sequence fragments for the 12 HDH candidat...

Claims

1. 1. An engineered non-plant cell that produces a tropane alkaloid product, comprising: the engineered non-plant cell comprises a heterologous coding sequence encoding a hyoscyamine dehydrogenase (HDH), the hyoscyamine dehydrogenase comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 13, 14 or 15; the tropane alkaloid product is hyoscyamine; The engineered non-plant cell.

2. The cell of claim 1, wherein the cell comprises one or more alterations to one or more endogenous metabolic pathways or regulatory mechanisms selected from the group consisting of endogenous arginine metabolism, endogenous phenylalanine and phenylpropanoid metabolism, endogenous polyamine regulatory mechanisms and metabolism, endogenous glycoside metabolism, and endogenous acetate metabolism.

3. The cell of claim 1 , wherein the cell comprises one or more alterations to endogenous glycoside metabolism.

4. The cell according to any one of claims 1 to 3, wherein the cell is a microbial cell.

5. The cell of claim 4 , wherein the cell is a fungal cell.

6. the engineered cell further comprises one or more heterologous coding sequences for one or more enzymes; 6. The cell of any one of claims 1 to 5, 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, phenylalanine ammonia-lyase, tyrosine ammonia-lyase, phenylpyruvate reductase, 4-coumarate-CoA ligase, 3-phenyllactate UDP-glucosyltransferase 84A27, littorine synthase, littorine mutase, hyoscyamine 6β-hydroxylase / dioxygenase, and cocaine synthase.

7. endogenous arginine metabolism is altered in the cell by modifications to one or more coding sequences for one or more endogenous enzymes; 7. The cell of any one of claims 1 to 6, wherein at least one of the enzymes is selected from the group consisting of glutamate N-acetyltransferase, acetylglutamate kinase, N-acetyl-γ-glutamylphosphate reductase, acetylornithine aminotransferase, ornithine acetyltransferase, ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lyase, and arginase.

8. endogenous phenylalanine and phenylpropanoid metabolism is altered in said cell by modifications to one or more coding sequences for one or more endogenous enzymes; 8. The cell of any one of claims 1 to 7, wherein at least one of the enzymes is selected from the group consisting of a pentafunctional AROM polypeptide, chorismate synthase, chorismate mutase, prephenate dehydratase, aromatic aminotransferase, and phenylacrylic acid decarboxylase.

9. endogenous polyamine regulatory mechanisms are altered in said cell by modifications to one or more coding sequences of one or more endogenous proteins; 9. The cell of claim 1, wherein at least one of the proteins is selected from the group consisting of methylthioadenosine phosphorylase, ornithine decarboxylase, ornithine decarboxylase antizyme, polyamine oxidase, spermidine synthase, spermine synthase, polyamine transporter, and polyamine permease.

10. endogenous acetate metabolism is altered in the cell by modifications to one or more coding sequences for one or more endogenous enzymes; at least one of the enzymes is selected from the group consisting of alcohol dehydrogenase and aldehyde dehydrogenase; A cell according to any one of claims 1 to 9.

11. endogenous glycoside metabolism is altered in said cell by modifications to one or more coding sequences for one or more endogenous enzymes; At least one of the enzymes is selected from the group consisting of glucan 1,3-β-glucosidase and steryl-β-glucosidase; A cell according to any one of claims 1 to 10.

12. 12. The cell of any one of claims 7 to 11, wherein the modification to one or more coding sequences is selected from the group consisting of feedback inhibition that alleviates a mutation in a biosynthetic enzyme or regulatory protein gene native to the cell, and an inactivating mutation in the native enzyme or protein.

13. the engineered cells are one or more heterologous coding sequences encoding one or more enzymes, including one or more soluble protein domains fused to the N-terminus of a serine carboxypeptidase-like acyltransferase domain to enable functional expression of the acyltransferase domain in an intracellular compartment of the engineered cell; 13. The cell of claim 1, wherein the serine carboxypeptidase-like acyltransferase domain is littorine synthase.

14. 14. The cell of any one of claims 1 to 13, wherein the transport of one or more tropane alkaloids (TAs) across intracellular membranes or across the plasma membrane is modified in the cell.

15. the altered transport is enabled by one or more heterologous coding sequences encoding one or more transporters; At least one of the transporters is selected from the group consisting of multidrug and toxin efflux transporters, nitrate / peptide family transporters, ATP-binding cassette transporters, and pleiotropic drug resistance transporters. The cell of claim 14.

16. 1. A method for producing a tropane alkaloid product, comprising: (a) culturing the cell of any one of claims 1 to 15 under conditions suitable for protein production; (b) adding a starting compound to the cell culture; (c) recovering the tropane alkaloid product from the culture; and The method comprising:

17. 17. The method of claim 16, wherein the cells are cultured in fed-batch or batch fermentation.

18. 18. The method of claim 16 or 17, wherein the starting compound added to the cell culture is a sugar or a substrate that contains one or more sugars or is converted to one or more sugars during microbial fermentation.

19. 18. The method of claim 16 or 17, wherein the starting compound added to the cell culture is an amino acid or a mixture comprising one or more amino acids, or a substrate that is converted into one or more amino acids during microbial fermentation.

20. The starting compound added to the cell culture is A precursor of a tropane alkaloid product selected from the group consisting of tropine, phenyllactic acid (PLA), arginine, ornithine, agmatine, N-carbamoylputrescine (NCP), putrescine, N-methylputrescine (NMP), 4-methylaminobutanal, N-methylpyrrolinium (NMPy), 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid (MPOB), tropinone, phenylalanine, prephenic acid, and phenylpyruvic acid (PPA).

18. The method of claim 16 or 17, wherein

21. 21. The method of any one of claims 16 to 20, wherein the tropane alkaloid product is recovered via a process comprising liquid-liquid extraction, chromatographic separation, distillation, or recrystallization.

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