NON-PLANT HOST CELLS PRODUCING TROPANE ALKALOIDS (TA), AND METHODS OF PREPARATION AND USE THEREOF

Abstract

This paper provides, among other things, an engineered non-plant cell that produces a tropane alkaloid product, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product. It also describes a method for producing a tropane alkaloid, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product using this cell.

Description

NON-PLANT HOST CELLS PRODUCING TROPANE ALKALOIDS (TA), AND METHODS OF PREPARATION AND USE THEREOF Cross-references This application claims the benefit of U.S. provisional patent applications serial numbers 62 / 815,709, filed March 8, 2019, 62 / 848,419, filed May 15, 2019, and 62 / 891,771, filed August 26, 2019. 2019, the applications for which are incorporated herein by reference. Government rights The present invention was carried out with government support under contract numbers GM110699 and AT007886 awarded by the National Institutes of Health (National Institutes of Health, United States). The government has certain rights over the invention. Introduction Tropane alkaloids (TAs) are a class of anticholinergic secondary metabolites produced by plants of the nightshade family (Solanaceae). Various TAs, including atropine, hyoscyamine and scopolamine, are classified as essential medicines by the World Health Organization for the treatment of various neurological disorders such as organophosphate and nerve agent poisoning, gastrointestinal spasms and cardiac arrhythmias, as well as to control symptoms of Parkinson's disease. As such, an adequate and constant supply of these TA molecules to make them available to researchers and clinicians is of interest. Current supply chains for medicinal TAs depend on the extraction of unsustainable and geographically restricted plant monocultures, in which TAs accumulate to only 0.2-4% of dry weight, and which are susceptible to pests, changes in the land use and climate. No total chemical synthesis for TAs from simple raw materials has proven to be sufficiently economical for industrial use due to difficulties arising from the stereochemistry of TAs. Furthermore, poor economies of scale and long generation times have made engineering transgenic plants or plant crops with improved TA production an unviable strategy to obtain these compounds. As such, methods for preparing TAs are of interest. Brief description of the invention This invention includes non-plant organisms designed for the production of various tropane alkaloids (TA) from precursors and sugar. For example, included in this invention are microbial strains designed for the production of medicinal TAs, defined here as naturally occurring TAs with established uses in current medical practice, including hyoscyamine, atropine, anisodamine and scopolamine, and precursors and derived from them. Also included in this invention are microbial strains designed for the production of non-medicinal TA, which are defined here as ΤΑ of natural origin without established uses in current medical practice, but that may possess bioactivities of medicinal interest, including calistegins, cocaine and precursors. and derivatives thereof. This invention further includes microbial strains engineered for the production of unnatural TAs, defined herein as TAs not produced by unmodified organisms, such as TAs produced by esterification of acyl donor compounds and acyl acceptors that are not esterified in organisms. natural origin, including those derived from medicinal TAs and those derived from non-medicinal TAs. An example of the schemes included in this invention is detailed in Figures 1 to 3. The invention encompasses methods for producing pseudotropin and pseudotropine-derived alkaloids, for example, calistegins, using microorganisms designed to express at least one heterologous enzyme as microbial catalysts. This invention further includes methods for producing various 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 microbial catalysts. This invention also includes methods for esterifying acyl donors and acceptors for the production of TA scaffolds using microorganisms designed to express at least one heterologous enzyme as microbial catalysts. The invention further includes methods for modifying and cultivating microbial strains designed for the production of medicinal TAs such as hyoscyamine and scopolamine, non-medicinal TAs such as calistegins, and non-natural TAs such as those derived from the esterification of tropine with acyl donor compounds, other than 3-phenylactic acid (PLA). Host cells are provided that are designed to produce tropane alkaloids (TA) that are of interest, such as hyoscyamine and scopolamine. TAs of interest may include TA precursors, TAs, and modifications of TAs, including TA derivatives. Host cells may have one or more modifications selected from: a mutation that mitigates feedback inhibition in an enzyme gene; a transcriptional modulation modification of a biosynthetic enzyme gene; an inactivating mutation in an enzyme; and a heterologous coding sequence. Also provided are methods 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. One aspect of the invention provides a method of forming a product stream having a tropane alkaloid (TA) product. The method comprises providing engineered non-plant cells and a feedstock including nutrients and water to a batch reactor, which engineered non-plant cells have at least one modification selected from the group consisting of: a mutation that mitigates feedback inhibition in a cell-native biosynthetic enzyme gene; a transcriptional modulation modification of a cell-native biosynthetic enzyme gene; and an inactivating mutation in an enzyme native to the cell. Furthermore, the method comprises, in the batch reactor, subjecting the engineered non-plant cells to fermentation by incubating the engineered non-plant cells for a period of time of at least about 5 minutes to produce a solution comprising the TA product and the material. cell phone. The method also comprises using at least one separation unit to separate the TA product from the cellular material to provide said product stream comprising the TA product. In another aspect, the invention provides a method of forming a product stream having a TA product. The method comprises providing engineered non-plant cells and a feedstock including nutrients and water to a reactor. The method also comprises, in the reactor, subjecting the engineered non-plant cells to fermentation by incubating the engineered yeast cells for a period of time of at least about 5 minutes (e.g., 5 minutes or more) to produce a solution comprising material cell and TA product. Furthermore, the method comprises using at least one separation unit to separate the TA product from the cellular material to provide the product stream comprising the TA product. Another aspect of the invention provides an engineered non-plant cell that produces a tropane alkaloid (TA) product, the engineered non-plant cell having at least one modification selected from the group consisting of: a mutation that mitigates feedback inhibition in a gene biosynthetic enzyme native to the cell; a transcriptional modulation modification of a cell native biosynthetic enzyme gene; and an inactivating mutation in an enzyme native to the cell. The designed non-plant cell comprises at least one heterologous coding sequence that encodes at least one enzyme that is selected from the group of arginine decarboxylase, agmatine ureohydrolase, agmatinase, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidine ketide synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylpyruvate reductase, 3-phenyl lactic acid UDPglucosyltransferase 84A27, littorin synthase, littorin mutase, hyoscyamine dehydrogenase, hyoscyamine 6βhydroxylase / dioxygenase and cocaine synthase. In some examples, the engineered non-plant cell comprises a plurality of heterologous coding sequences encoding an enzyme selected from the group of arginine decarboxylase, agmatine ureohydrolase, agmatinase, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidine ketide synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylpyruvate reductase, 3-phenyl lactic acid UDP-glucosyltransferase 84A27, littorin synthase, littorin mutase, hyoscyamine dehydrogenase, hyoscyamine 6β - hydroxylase / dioxygenase and cocaine synthase. In some examples, the heterologous coding sequences may be operably linked. Heterologous coding sequences that are operably connected may be within the same pathway of production of a particular tropane alkaloid product. In some examples, the engineered non-plant cell comprises one or more intracellular compartmentalization modifications that are selected from the group including, but not limited to, modified intracellular trafficking of enzymes, modified intracellular localization of enzymes, and modified intracellular transport of metabolites. In another aspect of the invention, a therapeutic agent is provided. The therapeutic agent comprises a tropane alkaloid product. Brief description of the figures The invention is better understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, in accordance with common practice, the various features in the drawings are not to scale. Rather, the dimensions of the various features are arbitrarily enlarged or reduced for clarity. The following figures are included in the drawings. Figure 1 illustrates an exemplary biosynthetic scheme for converting L-arginine into non-medicinal TAs. ADC, arginine decarboxylase; ARG, arginase; AUH, agmatine ureohydrolase; ODC, ornithine decarboxylase; PAO, polyamine oxidase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; spont., spontaneous (non-enzymatic) step; PYKS, pyrrolidine ketode 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 do not occur naturally in yeast. The final products, indicated within the table, are examples of the non-medicinal TAs. Figure 2 illustrates an exemplary biosynthetic pathway by which amino acids can be converted into medicinal TA molecules of interest and precursor molecules thereof. 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; spont., spontaneous (non-enzymatic) step; PYKS, pyrrolidine ketode synthase; CYP82M3, tropinone synthase; CPR, cytochrome P450-NADP+ reductase; TR1, tropinone reductase 1; ArAT, aromatic aminotransferase; PPR, phenylpyruvate reductase; UGT84A27, 3-phenyl lactate UDP-glucosyltransferase; LS, littorin synthase; CYP80F1, littorin mutase; HDH, (S)-hyoscyamine dehydrogenase; H6H, (S)-hyoscyamine 63-hydroxylase / dioxygenase. Arginine, ornithine, spermine, spermidine, putrescine, phenylalanine, 3-phenylpyruvic acid and traces of 3-phenyl lactic acid are synthesized naturally in yeast. All other metabolites shown do not occur naturally in yeast. The final products, indicated within the table, are examples of medicinal TAs. Figure 3 illustrates an exemplary biosynthetic pathway by which amino acids can be converted into unnatural TA and precursor molecules thereof. In this example, L-arginine and Lphenylalanine become the unnatural TAs. ADC, arginine decarboxylase; ARG, arginase; AUH, agmatine ureohydrolase; ODC, ornithine decarboxylase; PAO, polyamine oxidase; PMT, putrescine Nmethyltransferase; MPO, N-methylputrescine oxidase; spont., spontaneous (non-enzymatic) step; PYKS, pyrrolidine ketode synthase; CYP82M3, tropinone synthase; CPR, cytochrome P450-NADP+reductase; TR1, tropinone reductase 1; PAL, phenylalanine ammonium lyase; 4CL, 4-coumarate-CoA ligase; CS, cocaine synthase. Arginine, ornithine, spermine, spermidine, putrescine and phenylalanine are synthesized naturally in yeast. All other metabolites shown do not occur naturally in yeast. The final product, indicated inside the box, is an example of an unnatural TA. Figure 4 illustrates exemplary biosynthetic pathways for the production of putrescine from amino acids and other polyamine molecules. This figure shows how endogenous yeast and heterologous biosynthetic pathways can be used to produce putrescine from core metabolites. Figure 5 shows 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 native genes were expressed from low-copy plasmids in wild-type yeast (CEN.PK2). The transformed strains were grown in selective medium with 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 relative to the corresponding control (i.e., CEN.PK2). Figure 6 shows that yeast strains engineered for heterologous expression of biosynthetic enzymes from organisms other than yeast that are involved in arginine and polyamine metabolism can produce putrescine production in liquid culture. In this example, the yeast strains are engineered to express a heterologous plant and bacterial biosynthetic pathway. Heterologous enzymes were expressed from low-copy plasmids in wild-type yeast. The transformed strains were grown in selective medium with 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 relative to the corresponding control (i.e., CEN.PK2). Figure 7 shows that yeast strains engineered for heterologous expression of biosynthetic enzymes involved in arginine and polyamine metabolism from organisms other than yeast can produce TA precursors and agmatine, N-carbamoylputrescine, and putrescine intermediates in liquid culture. This figure shows the functional validation of the genes of the agmatine / putrescine biosynthetic pathway in yeast. The wild-type yeast strain CEN.PK2 was transformed with three low-copy plasmids to co-express between zero (negative control) and 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 grown in selective medium with 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 relative to the negative control (CEN.PK2). Data represent the mean of three biological replicates and error bars show standard deviation. Figure 8 illustrates the endogenous regulatory pathways that tightly control intracellular putrescine levels during normal yeast growth. Figure 9 shows a heat map of putrescine production in yeast strains with alterations in the regulatory mechanisms of endogenous polyamine biosynthesis. For overexpression of the native or heterologous putrescine pathways, the indicated genes were expressed from low-copy plasmids in wild-type (WT) yeast or in each individual disruption strain. Strains were grown in selective medium (YNB-DO) with 2% dextrose at 30 °C for 72 h before LC-MS / MS analysis. All data represent the average of at least three biological replicates. This figure shows that yeast strains that have unique alterations of polyamine metabolism genes and overexpressed endogenous or heterologous putrescine biosynthetic pathways can produce putrescine in liquid culture. Figure 10 provides a summary of the design efforts to increase putrescine production in yeast. The '+' symbol indicates the expression of at least one pathway gene, while indicating no expression of any pathway gene. Strains were grown in selective medium with 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 relative to the corresponding control (i.e., CEN.PK2). Figure 11 shows LC-MS / MS chromatograms illustrating the step-by-step conversion of putrescine to the TA intermediate NMPy and the acidic side product 4MAB through the intermediates NMP and 4MAB in the engineered yeast, according to embodiments of the invention. The proposed mechanism for the formation of the acidic side product 4MAB through the activity of an endogenous yeast enzyme (ALD) is shown. MRM traces of the extracted ion chromatogram are shown for each metabolite along the pathway and for authentic standards using the highest precursor ion / product ion transition for each metabolite. The control represents strain CSY1235 (see Example 1.5) that expresses SPE1, AsADC, and speB on a low-copy plasmid. The chromatograms are representative of three biological replicates. Enzyme symbols: PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; ALD, aldehyde dehydrogenase. Figure 12 shows LC-MS / MS chromatograms illustrating the relative production of TA precursors (A) putrescine, (B) NMP, (C, E) 4MAB, and (D, F) NMPy in engineered yeast liquid cultures. that express AbPMTI and an MPO enzyme, according to embodiments of the 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 AbPMTI 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 AbPMTI and NtMPOI 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 AbPMTI and DmMPO1AC-PTS1 from low copy plasmids. The y-axes of the plots are raw MRM ion counts. All chromatograms were generated by LC-MS / MS analysis of the extracellular medium after 48 hours of growth at 30 °C in selective medium with 2% dextrose. Traces are representative of at least three biological replicates. Figure 13 shows the effect of MEU1 disruption on SAM-dependent N-methylation of putrescine by AbPMTI. Wild type strain CEN.PK2 or meu1 disruption strain CSY1229 were co-transformed with low copy plasmids expressing SPE1, AsADC, and speB and AbPMTI. Data indicates mean MPN titer relative to control CEN.PK2 quantified by LC-MS / MS peak area for three biological replicates after 48 hours of growth at 30°C in 2% dextrose selective medium. Error bars indicate standard deviation. Two-tailed Student's t test: * p < 0.05, ** p < 0.01, *** p < 0.001. Figure 14 shows an in silico prediction of the subcellular localization of NMPy biosynthetic genes in plant and yeast / fungal cells using the SherLoc2 web server. Values ​​and colors indicate probability scores (0 to 1) for localization in each compartment: CYT, cytosol; nuc, nucleus; VAC, vacuole; CHL, chloroplast; MIT, mitochondria; POX, peroxisome. Figure 15 illustrates (A) the colocalization of N- and C-terminal GFP-tagged NtMPOI with a peroxisomal marker PEX3 and (B) the effect of N- and C-terminal GFP-tagged NtMPOI on the production of precursors. of ΤΑ 4MAB and NMPy in liquid cultures of engineered yeast, according to embodiments of the invention. This figure shows an experimental validation of the subcellular localization of NtMPOI. (A) Fluorescence microscopy of the N- and C-terminal GFP fusions of NtMPOI co-expressed with the peroxisome marker mCherry-PEX3 in wild-type yeast (CEN.PK2). White arrows indicate colocalization of GFP-tagged NtMPOI with peroxisomes. Scale bar, 10 pm. (B) Effect of forcing the cytosolic localization of NtMPOI on the production of 4MAB or NMPy. Wild-type yeast (CEN.PK2) was co-transformed with low-copy plasmids expressing wild-type NtMPOI or N- or C-terminal GFP fusions together with low-copy plasmids expressing SPE1, AsADC, and speB and AbPMTI. . LC-MS / MS analysis was performed after 48 h of growth at 30 °C in selective medium with 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 provides fluorescence microscopy data depicting the subcellular localization of AbPMTI and NtMPOI when heterologously expressed in yeast. Microscopy was performed on wild-type yeast expressing AbPMT 1 or NtMPOI tagged with N- or C-terminal GFP from low copy plasmids. Scale bar, 10 pm. Figure 17 illustrates (A) a sequence alignment of NtMPOI and the putative MPO enzymes AbMPOI and DmMPOI identified from the plant transcriptome data (top to bottom: SEQ ID NO: 27-29), (B ) a comparison of the production of the ΤΑ precursors 4MAB and NMPy in liquid cultures of engineered yeast strains expressing NtMPOI, AbMPOI or DmMPOI, and (C) a comparison of the predicted three-dimensional structures of NtMPOI, AbMPOI and DmMPOI determined from of homology models, according to embodiments of the invention. (A) Alignment of the query NtMPOI sequence against the AbMPOI and DmMPOI candidates from the 1000 Plants Project database. Blue indicates conservation of amino acid structure; red indicates discordances. (B) Comparison of relative activities of MPO orthologs. The putrescine-overproducing strain CSY1235 (see Example 1.5) was co-transformed with low-copy plasmids expressing SPE1, AsADC, and speB, AbPMTI, and one of three MPO variants. LCMS / MS analysis was performed after 48 hours of growth in selective medium at 30 °C. Data represent the mean of three biological replicates; error bars indicate standard deviation. (C) MPO enzyme homology models (pink) constructed from the crystal structure of copper-containing amino oxidase from Pisum sativum (PDB: 1KSI, blue) using the RaptorX web server. Top: NtMPOI; center: AbMPOI; bottom: DmMPOI. Figure 18 illustrates the production of 4MAB in liquid culture of engineered yeast strains that overproduce putrescine and express AbPMTI and N- and C-terminal truncations of NtMPOI and DmMPOI. This figure shows the effect of N- and C-terminal truncations of methylputrescine oxidase on 4MAB production in engineered yeast. The wild-type (WT) enzymes and indicated truncations were expressed from low-copy plasmids in the putrescine-overproducing strain CSY1235 (see Example 1.5). Strains were grown in selective medium with 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. Figure 19 illustrates the production of the ΤΑ precursors 4MAB and NMPy and the acidic secondary product 4MAB in liquid cultures of engineered yeast strains harboring unique alterations of one of the four native aldehyde dehydrogenase genes. This figure shows the effect of altering individual aldehyde dehydrogenases on 4MAB acid accumulation. The putrescine-overproducing strain CSY1235 (control) or the daughter strains impaired by nonsense mutations of hfd 1, ald4, ald5 or ald6 were transformed with low-copy plasmids expressing SPE1, AsADC and speB, AbPMTI and DmMP01ACPTS1. Bars indicate the relative titer of 4MAB acid measured by LC-MS / MS peak area normalized to CSY1235 (without ALD alterations) after 48 hours of growth in selective medium at 30 °C. 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 20 illustrates the production of (A) the acid side product 4MAB as well as (B) the precursors of TA4MAB and NMPy in liquid cultures of engineered yeast strains harboring one or more alterations of native aldehyde dehydrogenases. This figure shows the effect of aldehyde dehydrogenase gene alterations on the production of (A) the acidic side product 4MAB and (B) 4MAB and NMPy in engineered yeast. The symbols + and - indicate the presence or absence of functional enzyme, respectively. Strains were grown in selective medium (YNB-DO) with 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 the error bars indicate the standard deviation. Two-tailed Student's t-test: * p < 0.05, “ p < 0.01, p < 0.001. Unless otherwise stated, statistical significance is shown relative to the corresponding control (CSY1235). Figure 21 illustrates a comparison of the production of the TA precursor NMPy in liquid cultures of engineered yeast strains with genomic or low-copy plasmid-based expression of putrescine overproduction genes, AbPMTI and a truncation of DmMPOI, according to the modalities of the invention. This figure provides a comparison of 4MAB and NMPy production with plasmid-based (CSY1241) and genomic-based (CSY1243) expression of NMPy biosynthetic genes. Strain CSY1241 was transformed with low-copy plasmids expressing putrescine overproduction genes (SPE1, AsADC, speB), AbPMTI and DmMP01ÜC~PTS1. Strain CSY1243 expressed all of the aforementioned genes from integrated genomic copies. NMPy levels were quantified by LC-MS / MS after growth on selective (CSY1241) or non-selective (CSY1243) media. MA / 1 1 at 30 °C for 48 h. Data represent the mean of at least two biological replicates and error bars indicate standard deviation. Figure 22 illustrates the biosynthetic pathways for the production of the secondary product hygrin from NMPy and MPOB, according to embodiments of the invention. Putative major and minor side reactions in yeast are indicated by bold and dotted arrows, respectively. Figure 23 illustrates a comparison of the production of the TA precursors tropinone and tropin and the secondary product hygrin in liquid cultures of engineered yeast strains expressing AbPYKS, AbCYP82M3, DsTR1 and one of four different CPRs based on low-density plasmids. copy. This figure shows the production of tropin and intermediates related to the expression of AbPYKS, AbCYP82M3 and DsTR1 in engineered yeast. The indicated genes were expressed from low-copy plasmids in CSY1246; The + and symbols indicate the presence or absence of enzyme. Strains were grown in selective medium with 2% dextrose 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 24 illustrates (A) an LC-MS / MS chromatogram illustrating the characteristic triple peak of the TA precursor MPOB produced in liquid cultures of engineered yeast strains, and (B) the production of the TA precursors NMPy and MPOB in liquid cultures of yeast strains engineered to express AbPYKS, AbCYP82M3, and one of the four CPR plasmids. This figure shows the accumulation of NMPy and MPOB in the media of engineered strains expressing AbPYKS. (A) Representative LC-MS / MS multiple reaction monitoring (MRM) chromatogram for the detection of MPOB in the extracellular medium of CSY1246 expressing AbPYKS only from a low-copy plasmid. The three characteristic peaks of the MPOB isoform are marked (I), (II), and (III). LC-MS / MS analysis was performed after growth in selective medium at 30 °C for 48 h. (B) Relative abundance of NMPy and MPOB (all 3 peaks) in the extracellular medium of CSY1246 expressing AbPYKS, AbCYP82M3, and one of the four CPR low-copy plasmids after 48 h of growth at 30°C on selective media. The + and symbols indicate the presence or absence of a gene. Data represent the mean of three biological replicates; error bars indicate standard deviation. Figure 25 illustrates the effect of growth temperature on the production of the TA precursor, tropin, and the secondary product, hygrin, in liquid cultures of engineered yeasts. This figure shows the effect of growth temperature on spontaneous hygrin production in the tropin-producing yeast strain (CSY1248). Relative selectivity represents the ratio between the relative tropine titer and the relative hygrin titer. Strains were grown in nonselective media with 2% dextrose at 30°C or 25°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 26 illustrates (A) the effect of reconstitution of ALD4 and ALD6 on the growth of engineered tropine-producing yeast strains in media with or without acetate supplement, and (B) the effect of eliminating acetate auxotrophy on the production of the secondary products 4MAB acid and hygrin ΜΛ / t / zuz i / υοό^υ i in liquid cultures of engineered tropine-producing yeast strains, according to embodiments of the invention. This figure shows the effect of eliminating acetate auxotrophy in a tropine-producing engineered yeast strain. (A) Effect of reconstitution of functional ALD4 or ALD6 genes on the growth of the NMPy-producing yeast strain (CSY1246) with and without acetate supplement. ALD4 and ALD6 were expressed from low-copy plasmids. WT indicates CSY1246 with control plasmid (BFP). Adjacent columns show ten-fold dilutions. (B) Production of 4MAB acid and hygrin secondary products with reconstituted acetate metabolism in the engineered yeast. + and symbols indicate the presence or absence of fed metabolite (acetate) or ALD4 and ALD6 genes expressed from low-copy plasmids. Strains were grown in selective medium (YNB-DO) with 2% dextrose 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 illustrates (A) the effect of acetate auxotrophy on the accumulation of TA precursors between NMPy and tropinone in liquid cultures of yeast strains engineered to produce tropin, and (B) LC-MS / MS chromatograms representative of the ΤΑ MPOB precursor produced in liquid cultures of yeast strains designed to produce tropine with and without acetate auxotrophy, according to embodiments of the invention. This figure shows the effect of reconstituting ALD6 activity on metabolite flux through NMPy toward tropin in engineered yeast. (A) Production of intermediates between NMPy and tropinone in engineered strains with and without functional Ald6p. The abundances of intermediates were measured by LC-MS / MS MRM in the extracellular medium of the integrated tropin-producing strain (CSY1248) grown in non-selective medium supplemented with 0.1% w / v potassium acetate (gray) or the tropin with reconstituted ALD6 (CSY1249) cultured in non-selective medium without acetate supplement (pink) at 25 °C for 48 h. Data represent the mean of three biological replicates; error bars indicate standard deviation. Two-tailed Student's t-test: * p < 0.05, “ p < 0.01, p < 0.001. (B) Representative MRM chromatograms for MPOB production from CSY1248 (gray) and CSY1249 (red) grown as described in (A). Figure 28 illustrates the progression of improvements in the production of the TA precursor, tropin, and the secondary product, hygrin, in liquid cultures of engineered yeast strains. This figure provides a summary of strains designed to increase tropin production in yeast. The symbol indicates absence of gene; p and i indicate low-copy plasmid gene expression or genomic integration, respectively. Strains were grown in selective or nonselective media with 2% dextrose at 30°C or 25°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 29 illustrates the effect of expressing additional copies of the heterologous biosynthetic enzymes PMT, MPO, PYKS and CYP82M3 on the production of each TA precursor between putrescine and tropine in liquid cultures of engineered yeast, according to embodiments of the invention. This figure identifies metabolic bottlenecks in the optimized tropin-producing strain (CSY1249). Strain CSY1249 was transformed with a control plasmid expressing BFP (no overexpression) or a low-copy plasmid expressing an additional copy of AbPMTI, DmMP01ÚCPTS1, AbPYKS, or AbCYP82M3. The levels of intermediates in the extracellular medium were quantified by LC-MS / MS after growth at 25 °C in selective medium for 48 h. Data indicate the mean of three biological replicates and error bars indicate standard deviation. Figure 30 illustrates the impact of additional copies of the bottleneck enzymes PMT and PYKS on tropine production in engineered yeast. This figure shows the alleviation of metabolic bottlenecks by genomic integration of additional copies of the PMT and PYKS enzymes. Tropin-producing strains CSY1249 and CSY1251 were grown in non-selective media at 25 °C for 48 h 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 illustrates the production of the TA precursor acyl donor compound, PLA, in liquid cultures of engineered yeast strains expressing the heterologous lactate dehydrogenase and phenylpyruvate reductase enzymes. This figure shows LC-MS / MS analysis of yeast strains engineered to convert L-phenylalanine to 3-phenyl lactic acid. The yeast strains are engineered to have a low-copy CEN / ARS plasmid harboring a LEU2 selection marker, a TDH3 promoter, and a BFP coding sequence as a negative control; an LDH variant from B. coagulans (BcLLDH), L. casei (LcLLDH), L. plantarum (LpLLDH); or a PPR variant of A. belladonna (AbPPR), L. plantarum (LpPPR), Escherichia coü (hcxB) or W. fluorescens (WfPPR). Yeast were grown from freshly transformed colonies in 300 pL of selective medium (-Leu) in 96-deep-well microtiter plates. After 72 h of growth in a shaking incubator at 25 °C and 460 rpm, the yeast was pelleted and the medium supernatant was analyzed by LC-MS / MS. Data show relative titers of 3-phenyl lactic acid normalized to trace levels present in the negative control based on extracted ion chromatograms (ammonium adduct, EIC m / z+= 184). 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 shows LC-MS / MS chromatograms illustrating the production of the TA precursor acyl donor compound, cinnamic acid, in liquid cultures of engineered yeast strains expressing phenylalanine ammonium lyase. This figure shows LC-MS / MS analysis of yeast strains engineered to convert L-phenylalanine to cinnamic acid. The yeast strains are engineered to have a low-copy CEN / ARS plasmid harboring a TRP1 selective marker, a TEF1 promoter, and a coding sequence for (i) BFP or (ii) A. thaliana phenylalanine ammonium lyase (AtPALI). Yeast were grown from freshly transformed colonies in 300 pL of selective medium (-Trp) in 96-deep-well microtiter plates. After 48 h of growth in a shaking incubator at 30 °C and 460 rpm, the yeast was pelleted and the medium supernatant was analyzed by LC-MS / MS. The chromatogram traces show the cinnamic acid produced by these strains based on the most abundant multiple reaction monitoring (MRM) transition for cinnamic acid (m / z+ 149 ^131). Each trace is representative of three samples. Figure 33 illustrates the substrate specificity of orthologs of UDP-glucosyltransferase 84A27 (UGT84A27) from TA-producing Solanaceae expressed in engineered yeast. This figure shows a comparison of the activity of UGT84A27 orthologs on three different phenylpropanoid compounds expressed in engineered yeast. (A) Phenylpropanoids tested as glucose (Glu) acceptors for UGT84A27 in engineered yeast. Top, (D)-3-phenyl lactic acid (PLA); medium, transcinnamic acid (CA); lower, frans-ferulic acid (FA). (B) Heat map of the percentage conversion of fed phenylpropanoids to glycosides by yeast engineered for UGT84A27 expression. UGT84A27 orthologs or a BFP negative control were expressed from low-copy plasmids in CSY1251. Transformed cells were cultured in selective medium supplemented with PLA, CA, or 500 μΜ FA for 72 h before LC-MS / MS analysis. Data represent the mean of n = 3 biologically independent samples ± standard deviation. Figure 34 illustrates an example of chromatographic and mass spectrometric analysis of UGT84A27 activity. This figure shows representative LC-MS / MS traces showing the conversion of PLA, CA, and FA into cognate glycosides by AbUGT in CSY1251 grown as in Figure 33B for 120 h to allow for more complete glycosylation. For PLA, the acid (upper trace in each panel) and the glycoside (lower trace in each panel) were distinguished by different parental masses of ΝΗΛ adducts as well as different retention times. For CA and FA, rapid fragmentation required detection of the glycosides based on the lower retention peaks produced by their phenylpropanoid fragments. Figure 35 illustrates the structure-guided active site design of AbUGT to alter substrate specificity. This figure shows structural analysis of the 3D structure of AbUGT to identify potential mutations that increase activity in PLA. (A) AbUGT84A27 homology model constructed from the crystal structure of Arabidopsis thaliana salicylate UDP-glucosyltransferase UGT74F2 with bound UDP (PDB: 5V2K). PLA (orange) is shown in the preferred binding pose with UDP-glucose (pink) based on docking simulations. (B) Magnified view of the active site of AbUGT with coupled D-PLA and UDP-glucose. Potential mutations identified to improve PLA selectivity are shown (F130Y, L205F, I292Q); dashed lines indicate putative polar / hydrogen bonding interactions. Figure 36 illustrates the substrate specificity of the AbUGT84A27 active site mutants. This figure shows a heat map of the percentage conversion of fed phenylpropanoids to glycosides by yeast engineered for the expression of AbUGT mutants. Wild-type AbUGT, active site mutants, or a BFP negative control were expressed from low-copy plasmids in CSY1251. Transformed cells were cultured in selective medium supplemented with PLA, CA, or 500 μΜ FA for 72 h before LC-MS / MS analysis. Data represent the mean of n = 3 biologically independent samples ± standard deviation. Figure 37 shows LC-MS / MS chromatograms validating the stepwise biosynthesis of PLA glycoside in yeast engineered for tropin production. This figure shows the multiple reaction monitoring (MRM) and extracted ion chromatogram (EIC) traces of the culture media of the yeast strains designed for stepwise reconstitution of PLA glycoside. The strains were grown in non-selective media for 72 h before LC-MS / MS analysis of the culture supernatant. The chromatograms are representative of three biological replicates. Figure 38 shows a schematic of the glucose metabolic dual fate biosynthetic pathway in yeast. This figure illustrates the effect of citrate on glycoside production by inhibiting glycolysis. Abbreviations: HXK, hexokinase; GPI, glucose-6-phosphate isomerase; PFK, phosphofructokinase; PGM, phosphoglucomutase; UGP, UDP-glucose pyrophosphorylase. Figure 39 illustrates the effect of citrate supplementation on heterologous glycoside production in engineered yeast. This figure shows the effect of 2% citrate supplementation on the conversion of phenylpropanoid acids to glycosides by yeast engineered for AbUGT expression. Strain CSY1288 was grown in non-selective medium with or without 2% citrate and without additional supplementation to evaluate glycosylation of endogenously produced PLA, or with supplementation of 500 μΜ of frans-cinnamic acid (CA) or trans-ferulic acid (FA ). 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 shows the relative production of PLA glycosides in yeast strains engineered for overexpression of UDP-glucose biosynthetic enzymes. This figure illustrates the effect of overexpressing native enzymes involved in the biosynthesis of the UDP-glucose glycoside precursor on the production of PLA glycoside in engineered yeast. The enzymes or negative control (BFP) were expressed from low-copy plasmids in strain CSY1288. Strains were grown for 72 h in selective medium before LC-MS / MS analysis of metabolites in the culture supernatant. 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. Statistical significance is shown relative to the corresponding control. Figure 41 shows the relative production of PLA glycosides in CSY1288 with alterations of endogenous glycosidases. This figure illustrates the effect of altering each of the three native glucosidase genes on PLA glycoside accumulation in engineered yeast. The strains were grown in non-selective media for 72 h before LC-MS / MS analysis of the culture supernatant. 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. Statistical significance is shown relative to the corresponding control. Figure 42 shows LC-MS / MS chromatograms illustrating the production of the medicinal TA precursor hyoscyamine aldehyde from littorin in liquid culture for engineered yeast cells expressing AbCYP80F1. This figure shows LC-MS / MS analysis of yeast strains engineered to convert (R)-littorin to hyoscyamine aldehyde. The yeast strains are engineered to have a low-copy CEN / ARS plasmid that harbors a LEU2 selection marker, a promoter TDH3 and a coding sequence for littorin mutase CYP80F1 from A. belladonna (AbCYP80F1). Additionally, the strains have a second low-copy plasmid harboring a TRP1 selection marker, a TDH3 promoter, and a coding sequence for (i) BFP as a negative control, (i) S. cerevisiae CPR (NCP1), or ( iii) CPR of A. thaliana (AtATRI). Yeast were grown from freshly transformed colonies in 300 μL of selective medium (-Leu—Trp) supplemented with 1 mM littorin in 96-deep-well microtiter plates. After 48 h of growth in a shaking incubator at 30 °C and 460 rpm, the yeast was pelleted and the medium supernatant was analyzed by LC-MS / MS. Chromatogram traces show hyoscyamine aldehyde produced by these strains based on the most abundant MRM transition (m / z+ 288 —> 124). Arrowheads indicate the putative hyoscyamine aldehyde peak. Each trace is representative of three samples. Figure 43 illustrates the production of medicinal scopolamine TA from medicinal hyoscyamine TA in liquid culture for genetically modified yeast cells expressing hyoscyamine δβ-hydroxylase / dioxygenase (H6H) orthologs. This figure shows the conversion of (S)-hyoscyamine to (S)-scopolamine by engineered yeast strains expressing H6H orthologs. Yeast strains are engineered to have a low copy CEN / ARS plasmid harboring a LEU2 selection marker, a TDH3 promoter, and a BFP coding sequence as a negative control or a D. stramonium H6H (DsH6H) variant, A. acutangulus (AaH6H), B. arborea (BaH6H) or D. metel (DmH6H). Yeast from newly transformed colonies were grown in 300 µL of selective medium (-Leu) supplemented with 1 mM hyoscyamine in 96-well deep microtiter plates. After 48 hours of growth in a shaking incubator at 30 °C and 460 rpm, the yeast was pelleted and the medium supernatant was analyzed by LC-MS / MS. Data represent the mean of three biological replicates and are normalized to the amount of scopolamine contaminant in the hyoscyamine feed. Error bars represent the standard deviation. The relative titer of scopolamine was quantified based on the peak area of ​​the MRM transition m / z+ 304 —► 138. Figure 44 illustrates the effect of cofactor availability and medium 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 are engineered to have a low copy CEN / ARS plasmid harboring a LEU2 selection marker, a TDH3 promoter, and a coding sequence for (i) BFP as a negative control or (iii) hyoscyamine 6βhydroxylase / D dioxygenase. stramonium (DsH6H). Yeast was grown from newly transformed colonies in 300 pL of selective medium (-Leu) supplemented with the indicated substrates and / or cofactors in 96-well deep microtiter plates. After 48 hours of growth in a shaking incubator at 30 °C and 460 rpm, the yeast was pelleted and the medium supernatant was analyzed by LC-MS / MS. Relative (S)-scopolamine titers were quantified based on the integrated peak area of ​​the MRM transition m / z+ 304 —> 138 and normalized to the strain expressing DsH6H and all supplemented cofactors and substrates. Data represent the mean of three biological replicates and error bars indicate standard deviation. Hyo, (S)-hyoscyamine; 2-OG, 2-oxoglutaramate; MA / t / ZUZ 1 1 L-AA, L-ascorbic acid. Figure 45 shows a hierarchical clustering heat map of hyoscyamine dehydrogenase gene candidates identified from the A. belladonna transcriptome by analysis of tissue coexpression data. This figure shows the clustering of tissue-specific expression profiles of transcripts in the A. belladonna transcriptome that potentially encode enzymes with hyoscyamine dehydrogenase activity. Transcript expression for each candidate is scaled by row using a normal distribution. The dendrogram indicates the hierarchical grouping of candidates by tissue-specific expression profile. Known TA pathway genes are identified by name; putative HDH candidates are indicated by the ID locus. Black triangles indicate selected candidates for activity; the double black triangle indicates a candidate with experimentally verified HDH activity. Figure 46 illustrates the production of the medicinal scopolamine TA from littorin in liquid cultures of engineered yeast cells expressing hyoscyamine dehydrogenase (HDH) candidates. This figure illustrates the experimental selection of the activity of HDH candidates identified from the A. belladonna transcriptome in engineered yeast. The yeast strains are engineered to express A. belladonna littorin mutase (AbCYP80F1) and D. stramonium hyoscyamine 63-hydroxylase / dioxygenase (DsH6H) from constitutive promoters within expression cassettes integrated into the genome, as well as one of each of the 13 HDH candidates from a low-copy CEN / ARS plasmid harboring a TRP1 selection marker and a TDH3 promoter. Yeast were grown from freshly transformed colonies in 300 pL of selective medium (-Trp) supplemented with 1 mM littorin in 96-deep-well microtiter plates. After 72 h of growth in a shaking incubator at 30 °C and 460 rpm, the yeasts were pelleted and the medium supernatant was analyzed by LC-MS / MS. Relative hyoscyamine aldehyde titers were quantified based on the integrated peak area of ​​the MRM transition m / z+288 —> 124 and normalized to that of the engineered strain expressing BFP instead of a HDH candidate. (S)-scopolamine titers were quantified based on the integrated peak area of ​​the MRM transition m / z+304 —> 138 and a standard curve of a genuine scopolamine standard. Data represent the mean of three biological replicates and error bars indicate standard deviation. Figure 47 illustrates the three-dimensional structure of hyoscyamine dehydrogenase from A. belladonna. This figure shows an animated representation of the structure of AbHDH as a homology model constructed from the crystal structure of Populas tremuloides sinapil alcohol dehydrogenase (PtSAD; PDB: 1YQD) as a template. NADPH and Zn2+are shown in the active site. The inset shows an enlarged view of the active site of AbHDH with NADPH and the coupled hyoscyamine aldehyde. Dashed lines indicate interactions important for catalysis. Figure 48 shows a phylogenetic tree of the three identified HDH orthologs (AbHDH, DiHDH, DsHDH) along with the closest protein matches in the UniProt / SwissProt database. This figure shows the clustering of the three HDH enzyme orthologs identified 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 value as well as 10 additional matches selected from the next 100 ranks. Phylogenetic relationships were obtained through bootstrap neighbor joining with n = 1000 trials 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 illustrates the production of medicinal scopolamine TA from littorin in liquid cultures of engineered yeast cells expressing hyoscyamine dehydrogenase orthologs. This figure illustrates a comparison of activities between identified HDH enzyme orthologs expressed in engineered yeast. The yeast strains are engineered to express A. belladonna littorin mutase (AbCYP80F1) and D. stramonium hyoscyamine 6P-hydroxylase / dioxygenase (DsH6H) from constitutive promoters within expression cassettes integrated into the genome, one of each of three HDH orthologs (AbHDH, DiHDH, DsHDH) from a low-copy CEN / ARS plasmid harboring a TRP1 selection marker and a TDH3 promoter, and an additional copy of DsH6H from a low-copy CEN / ARS plasmid. copy harboring a LEU2 selection marker and a TDH3 promoter. Yeast were grown from freshly transformed colonies in 300 pL of selective medium (-Leu -Trp) supplemented with 1 mM littorin in 96-deep-well microtiter plates. After 72 hours of growth in a shaking incubator at 30”C and 460 rpm, the yeasts were pelleted and the medium supernatant was analyzed by LC-MS / MS. Relative hyoscyamine aldehyde titers were quantified based on the integrated peak area of ​​the MRM transition m / z+288 —> 124 and normalized to that of the engineered strain expressing AbHDH and BFP instead of DsH6H. (S)scopolamine titers were quantified based on the integrated peak area of ​​the MRM transition m / z+304 —>138 and a standard curve of a genuine scopolamine standard. Data represent the mean of three biological replicates and error bars indicate standard deviation. Figure 50 illustrates experimental validation of the conversion of fed littorin to scopolamine by engineered yeast for expression of CYP80F1, HDH and H6H. This figure shows multiple reaction monitoring (MRM) LC-MS / MS traces of culture media of engineered yeast strains for the conversion of littorin to scopolamine. Strains were grown for 72 h in nonselective medium supplemented with 1 mM littorin before LC-MS / MS analysis of metabolites in the culture supernatant. The dark trace in the lower right panel (CSY1294, scopolamine) represents the 125 nM scopolamine standard (38 pg / L). The chromatograms are representative of three biological replicates Figure 51 illustrates the canonical ER-to-vacuole trafficking and maturation pathway for plant SCPL acyltransferases (SCPL-AT). This figure shows as an example, a schematic representation of a canonical ER-to-vacuole trafficking and maturation pathway followed by SCPL-AT in plants, with A. belladonna littorin synthase (AbLS). Circled numbers indicate major steps in SCPL-AT expression and activity, including maturation in the (1) ER lumen and (2) Golgi, (3) trafficking to the vacuole and (4) vacuolar import. substrate and (5) export of product. Figure 52 shows the co-localization of wild-type A. belladonna llttorin synthase expressed in engineered yeast. This figure shows epifluorescence microscopy of yeast engineered for expression of N-terminal GFP-tagged AbLS (GFP-AbLS) and stained with the vacuolar membrane stain FM4-64. Microscopy was performed on CSY1294 expressing GFP-AbLS from a low-copy plasmid. Scale bar, 5 pm. Figure 53 illustrates a strategy for forced localization of littorin synthase in different subcellular compartments of yeast by signal sequence replacement. This figure illustrates a protein design approach to modify the subcellular localization of AbLS to address potential constraints on substrate availability in different compartments. (A) Schematic of the yeast subcellular compartments targeted by AbLS localization through signal sequence exchange. The source proteins of the signal sequence are indicated for each compartment. (B) Terminals and residues selected for the replacement of the AbLS signal sequence. Residues comprising each signal sequence domain were selected based on structural annotations in the UniProt / SwissProt database. Figure 54 shows a Western blot of wild-type AbLS expressed in tobacco and treated with deglycosylases. This figure illustrates the identification of the types of glycosylation modification for AbLS expressed in plants. HA C-terminal tagged AbLS was transiently expressed in N. benthamiana leaves by agroinfiltration. Extracts from raw leaves 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 an HRP-conjugated chimeric rabbit IgGK anti-HA antibody. All electrophoresis and transfer steps were carried out under reduced disulfide conditions (see Methods online). Lane L, Bio-Rad Precision Plus Dual Color protein marker. Figure 55 shows Western blots of AbLS glycosylation site mutants expressed in yeast and tobacco. This figure shows a comparison of the N-glycosylation patterns present for AbLS expressed in yeast and in tobacco. They were transiently expressed by agroinfiltration in N. benthamiana (Nb) (A) or from low-copy plasmids in CSY1294 (Yeast) (B), single glycosylation site point mutants (N —► Q) or a quadruple mutant of wild-type AbLS tagged with C-terminal HA. Preparation of crude tobacco and yeast extracts was performed under denaturing and disulfide-reducing conditions (see Online Methods). Crude extracts were separated by electrophoresis on a NuPAGE 4-12% BisTris gel and then transferred to a nitrocellulose membrane for immunodetection using an HRP-conjugated chimeric rabbit IgGK anti-HA antibody. All electrophoresis and transfer steps were carried out under reduced disulfide conditions (see Methods online). For (A) and (B), the corresponding controls expressed in yeast and tobacco are included for comparison. ivia / t / zuz ι / υοο^υ i Lane L, Bio-Rad Precision Plus Dual Color protein marker. Figure 56 shows the phylogenetic identification of the putative deletion of the endoproteolytic propeptide in littorin synthase. This figure shows an AbLS sequence alignment with characterized serine carboxypeptidases and SCPL acyltransferases that are known to possess (AtSCT, AsSCPLI, TaCBP2) or lack (AtSMT, yPRC1) proteolytically removed internal propeptide linkers (bold, gray). Putative N-terminal signal peptides are indicated in bold (black); disulfide bonds are indicated as connecting lines. AtSCT, sinapoylglucose:choline sinapoyltransferase from Arabidopsis thaliana, AtSMT, sinapoylglucose:malate sinapoyltransferase from A. thaliana, AbLS, littorin synthase from Atropa belladonna; AsSCPLI, avenacin synthase from Avena strigosa-, TaCBP2, carboxypeptidase 2 from Triticum aestivum; yPRC1, yeast carboxypeptidase Y. From top to bottom: SEQ ID NO: 30-35. Figure 57 shows the structural identification of the putative deletion of the endoproteolytic propeptide in littorin synthase. This figure shows a comparison of the three-dimensional structures of two SCPL-ATs, one of which is known to contain a proteolytically removed internal propeptide sequence. Left: Crystal structure of TaCBP2 (PDB: 1WHT) in mapped (top) and surface (bottom) representation showing disulfide bonds and internal propeptide removal sites. Right: AbLS homology model based on the crystal structure of TaCBP2 in (top) drawing and (bottom) surface representation showing the Nterminal signal peptide, disulfide bonds, and the putative internal propeptide that appears to block access to the active site. . Figure 58 shows analysis of proteolytic cleavage patterns for AbLS cleavage controls and putative propeptide interchange variants in yeast. This figure shows Western blot analysis of the sizes of the protein fragments produced by the AbLS cleavage controls and the propeptide variants expressed in engineered yeast. HA C-terminal tagged AbLS variants were expressed from low copy plasmids in CSY1294 (lanes 1-6); 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 reduced disulfide conditions and detection was carried out using an anti-HA antibody (see Methods online). Lanes symbols: L, protein molecular weight marker; WT, wild-type AbLS; SPL, AbLS cleaved on the putative propeptide with signal peptides on both fragments; SPLT, AbLS cleaved on the putative propeptide with no signal peptides on any of the fragments; GS, variant of AbLS with the wild-type propeptide exchanged for the flexible linker Gly-Ser; SCT, AbLS variant with the wild-type propeptide swapped for the AtSCT propeptide sequence; CUT, AbLS variant with the wild-type propeptide exchanged for the synthetic poly-arginine site recognized and cleaved by the Kex2p protease. Figure 59 illustrates the de novo production of hyoscyamine and scopolamine in yeast strains engineered for expression of N-terminal AbLS fusions. 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. Transformed strains were grown for 96 h in selective media before LC-MS / MS analysis of metabolites in the culture supernatant. 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 60 shows fluorescence microscopy of tobacco alkaloid transporters expressed in CSY1296 to mitigate the limitations of TA vacuolar transport. This figure shows fluorescence microscopy images of engineered yeasts expressing tobacco alkaloid transporters fused at their C-terminus to GFP, to allow identification of their subcellular localization. C-terminal GFP fusions of (A) NtJATI and (B) NtMATE2 were expressed from low copy plasmids in CSY1296. Scale bar, 5 pm. Figure 61 shows the production of tropine, hyoscyamine and scopolamine in CSY1296 designed for the expression of heterologous alkaloid transporters. This figure illustrates the utility of different plant alkaloid transporters to mitigate intracellular substrate transport limitations in yeast engineered for TA production. Nicotiana tabacum jasmonate-inducible alkaloid transporter 1 (NtJATI), multidrug and toxin transporters 1 or 2 (MATE), or a negative control (BFP) were expressed from low-copy plasmids in CSY1296. Transformed strains were grown for 96 h in selective media before LC-MS / MS analysis of metabolites in the culture supernatant. 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 62 shows LC-MS / MS chromatograms in (A) product ion mode and (B) multiple reaction monitoring mode illustrating de novo production of the unnatural TA cinnamoyltropin in engineered yeast. This figure shows LC-MS / MS analysis of engineered yeast strains that produce the unnatural TA cinnamoyltropin. (A) Tandem MS / MS spectra of the extracellular medium of (i) the tropin-producing strain CSY1251; (i) CSY1251 expressing phenylalanine ammonium lyase (AtPALI), 4coumarate-CoA ligase 5 (At4CL5) and cocaine synthase (EcCS), denoted CSY1282; or (iii) a genuine cinnamoyltropin standard for a parent mass of m / z+ = 272. The blue diamond indicates the peak of the parent compound. (B) Validation of EcCS acyltransferase activity on cinnamic acid and α-tropine by substrate feeding. Strains were transformed with combinations of plasmids expressing AtPALI (low-copy plasmid pCS4252) and / or At4CL5 and EcCS (high-copy plasmid pCS4207), and then grown in media with different supplemented substrates, as follows: (i) CEN .PK2 + At4CL5 + EcCS + 0.1 mM trans-cinnamic acid; (ii) CEN.PK2 + At4CL5 + EcCS + 0.5 mM α-tropine; (iii) CEN.PK2 + AtPALI + At4CL5 + EcCS; (v) CEN.PK2 + AtPALI + At4CL5 + EcCS + 0.5 mM atropine; (v) CSY1251 + At4CL5 + EcCS; (vi) CSY1251 + At4CL5 + EcCS + 0.2 mM trans-cinnamic acid; (vii) CSY1251 + AtPALI + At4CL5 + EcCS; (viii) 25 nM cinnamoyltropin standard. For (A) and (B), yeast strains were grown in selective media (YNB-DO + 2% dextrose + 5% glycerol) at 25 °C for 72 h before LC-MS / MS analysis. Figure 63 illustrates the impact of various carbon sources fed (A) alone or (B) together with dextrose on the production of tropin and related TA precursors in engineered yeast liquid cultures. This figure shows the optimization of the carbon source to improve tropine production in the engineered yeast. Overnight cultures of the tropin-producing strain CSY1249 (see Example 3.3.4) were performed in rich non-selective (YPD) medium. Overnight cultures were pelleted and resuspended in defined non-selective medium (YNB-SC) with all amino acids and (A) 2% of each carbon source or (B) 2% dextrose and 2% of each additional carbon source. including dextrose. Cultures were grown at 25 °C for 48 h before the growth medium was analyzed 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 mean of three biological replicates and error bars indicate standard deviation. Figure 64 illustrates the metabolic bottleneck analysis of the scopolamine-producing strain CSY1296. This figure shows the effect of expressing additional copies of flux-limiting enzymes on the production of TAs and TA precursors in engineered yeast. An additional copy of each biosynthetic enzyme between tropine and scopolamine was expressed from the following low-copy plasmids in strain CSY1296: (A) WfPPR, pCS4436; (B) AbUGT, pCS4440; (C) DsRedAbLS, pCS4526; (D) AbCYP80F1, pCS4438; (E) DsHDH, pCS4478; (F) DsH6H, pCS4439; or a BFP control (pCS4208, pCS4212 or pCS4213) corresponding to the same auxotrophic marker as each biosynthetic gene plasmid. The transformed strains were grown in appropriate selective media at 25 °C for 96 h before quantification of metabolites in the growth medium by LC-MS / MS. Data indicate 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 65 shows the effect of mitigating flow and transport limitations on hyoscyamine and scopolamine production in engineered yeast. This figure shows a comparison of de novo production of hyoscyamine and scopolamine in yeast strain CSY1296 and CSY1297, where the latter possesses additional genomic copies of flux-limiting enzymes (WfPPR and DsH6H) as well as an importer of tobacco vacuolar alkaloids. (NtJATI). Strains were grown in nonselective media for 96 h before LC-MS / MS analysis of metabolites in the culture supernatant. 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. Definitions Before describing the exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the description. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as would normally be understood by one skilled in the art to which the present invention pertains. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2ND ED„ John Wiley and Sons, New York (1994), and Hale and Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide a skilled person with the general meaning of many of the terms used herein. However, some terms are defined below for the sake of clarity and ease of reference. It should be noted that, as used herein and in the accompanying claims, the singular forms an, an, and the / the include plural referents, unless the context clearly dictates otherwise. For example, the term a primer refers to one or more primers, that is, a single primer and multiple primers. Furthermore, it is noted that the claims are drafted to exclude any optional elements. As such, this statement is intended to serve as a basis for the use of proprietary terminology such as solely, only, and the like in connection with the reading of the elements of the claim, or the use of a negative limitation. The terms determine, measure, evaluate, and test are used interchangeably herein and include quantitative and qualitative determinations. 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 polymers of encoded and non-encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having modified peptide backbones in which the conventional backbone has been replaced by synthetic or unnatural backbones. A polypeptide may have any convenient length, for example, 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, such as up to 500 or 1000 or more amino acids. The peptides may be 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, such as up to 50 amino acids. In some embodiments, the peptides are between 5 and 30 amino acids in length. As used herein, the term isolated refers to a portion of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free and even at least 99% free of other components with which the portion is associated before purification. As used herein, the term encoded by refers to a nucleic acid sequence that encodes a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of 3 or more amino acids, such as such as 5 or more, 8 or more, 10 or more, 15 or more, or 20 or more amino acids of a polypeptide encoded by the nucleic acid sequence. The term also encompasses polypeptide sequences that are immunologically identifiable with a polypeptide encoded by the sequence. A “vector” is capable of transferring gene sequences to target cells. As used herein, the terms vector construct, expression vector, and gene transfer vector are used interchangeably to mean any nucleic acid construct capable of directing the expression of a gene of interest and that can transfer gene sequences to target cells, which is achieved through genomic integration of all or part of the vector, or the transient or heritable maintenance of the vector as an extrachromosomal element. Therefore, the term includes cloning and expression vehicles, as well as integration vectors. An expression cassette includes any nucleic acid construct capable of directing the expression of a gene / coding sequence of interest, which is operably linked to a promoter of the expression cassette. Said cassette is constructed into a vector, vector construct, expression vector or gene transfer vector, in order to transfer the expression cassette to target cells. Therefore, the term includes cloning and expression vehicles as well as viral vectors. A plurality contains at least 2 members. In certain cases, a plurality may have 10 or more, such as 100 or more, 1000 or more, 10,000 or more, 100,000 or more, 106 or more, 107 or more, 108 or more, or 109 or more members. In any modality, a plurality can have 2-20 members. The term tropane alkaloid product refers to any molecule whose skeleton contains an 8-azabicyclo[3.2.1]octane core group comprising a cycloheptane ring and a nitrogen bridge connecting carbon atoms 1 and 5, where the 8-azabicyclo[3.2.1]octanyl group is covalently linked to an acyl group via an ester bond at the 3-position, and / or wherein the 8-azabicyclo[3.2.1]octanyl group is functionalized with a group hydroxyl at position 3 and one or more hydroxyl groups at positions 2, 4, 5, 6 and / or 7. Alkaloid products of tropane include, but are not limited to, littorine, hyoscyamine, atropine, anisodamine, scopolamine, cocaine and any other similar tropine / pseudotropin + natural or unnatural acyl group of tropane alkaloids (e.g. calistegins). 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 that can be converted to a tropane alkaloid product in one or more (e.g. example, one or two) biosynthetic steps; wherein the carbon source is a carbohydrate, a non-carbohydrate sugar, a sugar alcohol, a lipid, a fatty acid or a substrate that is converted to one or more of the above carbon sources through a metabolic pathway; and wherein the nitrogen source is ammonia, urea, nitrate, nitrite, any amino acid excluding glutamic acid, arginine, ornithine and citrulline, a peptide, a protein or any substrate that is converted to one or more of the above nitrogen sources through a metabolic pathway. The term derived from a tropane alkaloid product is intended to refer to any molecule not naturally produced by an unmodified organism, wherein the backbone of the molecule comprises a tropane alkaloid product and which is differentiated from said tropane alkaloid product. by the union of functional groups without modification of the skeleton itself. As used herein, attachment of functional groups includes, but is not limited to, hydroxylation, alkylation and N-alkylation, acetylation and N-acetylation, acylation and N-acylation and halogenation. Numeric intervals include the numbers that define the interval. The methods described herein include multiple steps. Each step can be performed after a predetermined amount of time has passed between steps, as desired. As such, the time between completing 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 even 5 hours or more. In certain embodiments, each subsequent step is performed immediately after completing the previous step. In other embodiments, a step may be performed after an incubation or waiting time after completing the previous step, for example, from a few minutes to a waiting time of overnight. Other definitions of terms may appear throughout the specification. Detailed description of the invention Host cells are provided that are designed to produce tropane alkaloids (TA) that are of interest, such as hyoscyamine and scopolamine. Host cells may have one or more design modifications selected from: a mutation that mitigates feedback inhibition in an enzyme gene; a transcriptional modulation modification of a biosynthetic enzyme gene; an inactivating mutation in an enzyme; and a heterologous coding sequence. Also provided are methods 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. Before the present invention is described in greater detail, it should be understood that this invention is not limited to the particular embodiments described and, as such, may vary. It should also be understood that the terminology used herein is solely intended to describe particular embodiments and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims. Where a range of values ​​is given, it is understood that each intermediate value, up to one-tenth of a unit of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limit of that range and any other stated value or intermediate in that indicated range, is included within the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included within the invention, subject to any limits specifically excluded in the indicated range. Where the indicated range includes one or both of the limits, ranges that exclude one or both of the included limits are also included in the invention. Certain intervals with numerical values ​​preceded by the term approximately are presented in this document. The term approximately is used herein to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number that the term precedes. To determine whether a number is close to or approximately a specifically referred number, the near or approximate non-referenced number may be a number that, in the context in which it is presented, provides the substantial equivalent of the specifically referred number. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as would normally be understood by one skilled in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described. All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in relation to which publications are cited. Citation of any publication is for disclosure prior to the filing date and should not be construed as an admission that the present invention has no right to predate such publication by virtue of the prior invention. Additionally, the release dates provided may be different from the actual release dates, which may need to be confirmed independently. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and characteristics that can be easily separated or combined with the characteristics of any of the other embodiments without departing from the scope or spirit of the present invention. Any referred method is carried out in the order of the referred events or in any other order that is logically possible. In the further description of the present invention, the TA precursors of interest, TAs, and modifications of TAs, including derivatives of TAs, are first described in greater detail, followed by the host cells for producing the same. Methods of interest in which host cells find use are reviewed below. Also described are kits that can be used in practicing methods of the invention. Tropane alkaloid (TA) precursors As summarized above, host cells that produce tropane alkaloid precursors (TA precursors) are provided. The TA precursor may be any intermediate compound or precursor in a synthesis 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 certain cases, the TA precursor has a structure that can be characterized as a fragment of a TA. In some cases, the precursor to TA is an early TA. As used herein, early TA means an early intermediate in the synthesis of a TA of interest in a cell, where the early TA is produced by a host cell from a host cell raw material or a compound. simple start. In some cases, early TA is a TA intermediate that is produced by the host cell in question solely from a host cell raw material (e.g., a carbon and nutrient source) without the need to add a compound. starting to the cells. The term early TA may refer to a precursor of a final TA product of interest, whether the early TA can be characterized as a tropane alkaloid or not. In some cases, the TA precursor is an early TA, such as a pre-tropine tropane alkaloid. ΜΛ / t / ZUZ 1 1 or a pre-littorin trephine alkaloid. As such, host cells are provided that produce pre-tropine tropane alkaloids (pre-tropine TA) and pre-littorine tropane alkaloids (pre-littorine TA). Tropine is an important intermediate branch point of interest in the synthesis of downstream TAs through design efforts to produce end products such as littorin-derived medicinal TA products (Figure 2). The host cells in question can produce TA precursors from simple and inexpensive starting materials that can find use in the production of tropin, littorin, and downstream TA end products. As used herein, the terms preesterification tropane alkaloid, preesterification TA and preesterification TA precursor are used interchangeably and refer to a biosynthetic precursor of littorin, cinnamoyltropine or other acyl donor and esterification product of the acceptor. acyl, whether the structure of the esterification precursor itself is characterized as a tropane alkaloid or not. The term pre-esterification TA is intended to include biosynthetic precursors, intermediates and metabolites thereof, from any suitable member of a host cell biosynthetic pathway that can lead to esterification products such as littorin. In some cases, the pre-esterification TA includes a tropane alkaloid fragment, such as a tropine fragment, a phenylpropanoid fragment, or a precursor or derivative thereof. In certain cases, the pre-esterification TA has a structure that can be characterized as a tropane alkaloid or a derivative thereof. TA precursors of interest include, but are not limited to, tropine and phenyl lactic 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-2pyrrodinyl)-3-oxobutanoic acid (MPOB), tropinone, phenylalanine, prephenic acid and phenylpyruvic acid (PPA). In some embodiments, the one or more TA precursors are tropine and PLA. In certain cases, one or more TA precursors are tropine and a phenylpropanoid carboxylic acid other than PLA, such as cinnamic acid. Figures 1, 2 and 3 illustrate the biosynthesis of non-medicinal, medicinal and non-natural TAs, respectively, from various TA and non-TA precursor molecules. Synthetic pathways toward a TA precursor can be generated in host cells and can begin with any convenient compound(s) or starting materials. Figures 1 to 4 illustrate a synthesis pathway of interest for TA precursors from amino acids. The starting material may be of non-natural origin or the starting material may be of natural origin in the host cell. Any suitable compound and material can be used as starting material, based on the synthesis pathway present in the host cell. The source of the starting material may be from the host cell itself, for example, arginine or phenylalanine, or the starting material may be added or supplemented to the host cell from an external source. As such, in some cases, the starting compound refers to a compound in a cell synthesis pathway that is added to the host cell from an external source that is not part of a growth starting material or cell growth medium. . Starting compounds of interest include, but are not limited to, N-methylputrescine, 4-methylaminobutanal, tropinone, tropin, PLA, cinnamic acid, as well as any of the compounds shown in Figures 1 to 4. For example, if the host cells are growing in liquid culture, the cell medium can be supplemented with the starting material, which is transported into the cells and converted by the cell into the desired products. Starting materials of interest include, but are not limited to, inexpensive raw materials and simple precursor molecules. In some cases, the host cell uses a feedstock that includes a simple carbon source as a starting material, which the host cell uses to produce compounds from the cell's synthesis pathway. The feedstock for host cell growth may include one or more components, such as a carbon source such as cellulose, starch, free sugars, and a nitrogen source, such as ammonium salts or inexpensive amino acids. In some cases, a growing feedstock used as a starting material may be derived from a sustainable source, such as biomass grown on marginal lands, including switchgrass and algae, or biomass waste products from other industrial or agricultural activities. . Tropane alkaloids (TA) As summarized above, host cells that produce tropane alkaloids (TA) of interest are provided. In some embodiments, the engineered strains of the invention will provide a platform for producing tropane alkaloids of interest and modifications thereof in various classes including, but not limited to, medicinal TAs such as tropine derivatives and PLA; non-medicinal TAs such as those derived from tropinone, pseudotropin or norpseudotropin; and non-natural TAs such as those derived from the esterification of TA precursors (e.g., acyl donor and acyl acceptor compounds) other than tropine and PLA. Each of these classes is intended to include biosynthetic precursors, intermediates and metabolites thereof, of any suitable 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 given below. In some embodiments, the structure of a given example may or may not be characterized as a tropane alkaloid. The present chemical entities are intended to include all possible isomers, including individual enantiomers, racemic mixtures, optically pure forms, mixtures of diastereomers and intermediate mixtures. Medicinal TAs may include, but are not limited to, littorin, hyoscyamine, atropine, anisodamine, scopolamine and derivatives thereof that are naturally produced by plants. Non-medicinal TAs may include, but are not limited to, calistegins, cocaine and their derivatives that are produced naturally by plants. Unnatural TAs may include, but are not limited to, cinnamoyltropin, cinnamoyl-3p-tropine, coumaroyltropin, coumaroyl-3p-tropine, benzoyltropine, benzoyl-33-tropine, caffeolItropin, caffeoyl-33-tropine, feruloyltropine, and feruloyl. -3P-tropine. Modifications to TAs, including derivatives As summarized above, host cells that produce modified derivatives of tropane alkaloids (TA) of interest are provided. In some embodiments, the engineered strains of the invention will provide a platform for derivatizing TAs of interest, including the derivatization of ΤΑ precursors, medicinal TAs, non-medicinal TAs, and non-natural TAs that are produced by engineered host cells or that are fed to cells. designed hosts in the growth medium. As used herein, the terms derivatization, functionalization, derivatization modification, and functionalization modification refer to the modification of TAs or TA precursors by attaching functional groups without modification of the TA backbone itself. As used herein, attachment of functional groups includes, but is not limited to, hydroxylation, alkylation and nalkylation, acetylation and N-acetylation, acylation and N-acylation and halogenation. In some embodiments of the invention, derivatization of the TAs of interest can be achieved enzymatically by feeding pre-functionalized TA precursors, for example, halogenated or alkylated amino acids, to host cells designed to uptake and then convert fed TA precursors into the TAs of interest. interest. In other embodiments of the invention, derivatization of the TAs of interest can be achieved enzymatically by engineering the host cells to express enzymes that possess the desired activity to attach a functional group to a target TA, in addition to the enzymes and cellular modifications necessary to produce the TA. TA not modified. In other embodiments of the invention, derivatization of the TAs of interest can be achieved enzymatically by treating unmodified TAs produced by engineered host cells with purified enzymes capable of binding the desired functional groups, or with crude lysate from host cells engineered to express enzymes that have the desired derivatization activity. In other embodiments of the invention, derivatization of the TAs of interest can be achieved non-enzymatically by treating unmodified TAs produced by engineered host cells with chemical agents with the desired functional groups. Modified derivatives of TAs include, but are not limited to, p-hydroxyatropine, phydroxyhyoscyamine, p-fluorohioscyamine, p-chlorohioscyamine, p-bromohyoscyamine, p-fluoroscopolamine, p-chloroscopolamine, p-bromoscopolamine, N-methylhyoscyamine butylhyoscyamine, N -methylscopolamine, N-butylscopolamine, N-acetylhyoscyamine and N-acetylscopolamine. Host cells As summarized above, one aspect of the invention is a host cell that produces one or more TAs of interest. Any suitable cell can be used in the target host cells and methods. In some cases, the host cells are non-plant cells. In some cases, the host cells can be characterized as microbial cells. In certain cases, the host cells are insect cells, mammalian cells, bacterial cells or fungal cells. Any convenient type of host cell can be used to produce the target TA-producing cells, see, for example, US2008 / 0176754 now published as US Patent No. 8,975,063, US2014 / 0273109 and WO2014 / 143744); whose disclosures are incorporated by reference in their entirety. Host cells of interest include, but are not limited to, bacterial cells, such as Bacillus subtilis, Escherichia coli, Streptomyces, Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria, Lactobacillus, Leucononstoc, Macrococcus, Methylomonas, Methylobacter, Methylocella, Methylococcus, Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus, Propionibacterium, Proteus, Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystís, Tetragenococcus, Weissella, Zymomonas and Salmonella typhimuium, insect cells such as Drosophila melanogaster S2 and Spodoptera frugiperda Sf9, and yeast cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Yarrowia lipolytica, Candida albicans, Aspergillus spp., Rhizopus spp. , Penicillium spp. and Trichoderma reesei. In some embodiments, the host cells are yeast cells or E. coli cells. In some cases, the host cell is a yeast cell. In some cases, the host cell is a yeast strain engineered to produce a TA of interest. Any of the host cells described in US2008 / 0176754, now published as US Patent No. 8,975,063, US2014 / 0273109 and WO2014 / 143744, can be adapted for use in the target cells and methods. In certain embodiments, the yeast cells may be of the species Saccharomyces cerevisiae (S. cerevisiae). In certain embodiments, the yeast cells may be of the species Schizosaccharomyces pombe. In certain embodiments, the yeast cells may be of the species Pichia pastoris. Yeast is of interest as a host cell because the cytochrome P450 proteins, which are involved in some biosynthetic pathways of interest, are able to fold properly in the endoplasmic reticulum membrane to maintain their activity. Yeast strains of interest that find use in the invention include, but are not limited to, CEN.PK (Genotype: MATa / α ura3-52 / ura3-52 trp1-289 / trp1-289 Ieu2-3_112 / leu2-3_112 his3 A1 / his3 Δ1 MAL2-8C / MAL2-8C SUC2 / SUC2), S288C, W303, D273-10B, X2180, A364A, Σ1278Β, AB972, SK1 and FL100. In certain cases, the yeast strain is any of S288C (MATa; SUC2 mal mel gal2 CUP1 flo1 flo8-1 hap1), BY4741 (MATa; his3A1; leu2A0; met15A0; ura3A0), BY4742 (MATa; his3A1; leu2A0; lys2A0; ura3A0), BY4743 (MATa / MATa; his3A1 / his3A1; leu2A0 / leu2A0; met15A0 / MET15; LYS2 / lys2A0; ura3A0 / ura3A0), and WAT11 or W(R), derived from strain W303-B (MATa; ade2- 1; his3-11,-15; leu2-3,-112; ura3-1; canR; cyr+) that express the Arabidopsis thaliana NADPH-P450 reductase ATR1 and the yeast NADPHP450 reductase CPR1, respectively. In another embodiment, the yeast cell is W303alpha (MATa; his3-11,15 trp1-1 leu2-3 ura3-1 ade2-1). The identity and genotype of additional yeast strains of interest can be found at EUROSCARF (web.unifrankfurt.de / fb15 / mikro / euroscarf / col_index.html). In some cases, the host cell is a fungal cell. In certain embodiments, the fungal cells may be of the Aspergillus species and the strains include Aspergillus niger (ATCC 1015, ATCC 9029, CBS 513.88), Aspergillus oryzae (ATCC 56747, RIB40), Aspergillus terreus (NIH 2624, ATCC 20542) and Aspergillus nidulans (FGSC A4). In certain embodiments, the heterologous coding sequences may be codon optimized for expression in Aspergillus sp. and expressed from an appropriate promoter. In certain embodiments, the promoter can be selected from phosphoglycerate kinase (PGK) promoter, MbfA promoter, cytochrome c oxidase subunit (CoxA) promoter, SrpB promoter, TvdA promoter, malate dehydrogenase (MdhA) promoter, beta-mannosidase (ManB) promoter. In certain embodiments, a terminator may be selected from the glucoamylase (GlaA) terminator or the TrpC terminator. In certain embodiments, the expression cassette consisting of a promoter, a heterologous coding sequence and a terminator can be expressed from a plasmid or integrated into the host genome. In certain embodiments, selection of cells that maintain the plasmid or integration cassette can be performed with the selection of antibiotics, such as hygromycin, or the use of nitrogen sources, such as the use of 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. In some cases, the host cell is a bacterial cell. The bacterial cell can be selected from any bacterial genus. Some examples of genera from which the bacterial cell can come are Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria , Lactobacillus, Leucononstoc, Macrococcus, Methylomonas, Methylobacter, Methylocella, Methylococcus, Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus, Propionibacterium, Proteus, Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopse udomonas, Serratia, Staphylococcus, Streptococcus , Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella and Zymomonas. Examples of bacterial species that can be used with the methods of this disclosure include Arthrobacter nicotianae, Acetobacter aceti, Arthrobacter arilaitensis, Bacillus cereus, Bacillus coagulaos, Bacillus licheniformis, Bacillus pumilus, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium adolescentis, Brachybacterium tyrofermentans , Brevibacterium linens, Carnobacterium divergens, Corynebacterium flavescens, Enterococcus faecium, Gluconacetobacter europaeus, Gluconacetobacter johannae, Gluconobacter oxydans, Hafnia alvei, Halomonas elongata, Kocuria rhizophila, Lactobacillus acidifarinae, Lactobacillus jensenii, Lactococcus lactis, Lacto bacillus yamanashiensis, Leuconostoc citreum, Macrococcus caseolyticus, Microbacterium foliorum, Micrococcus lylae, Oenococcus oeni, Pediococcus acídilactici, Propionibacterium acidipropionici, Proteus vulgaris, Pseudomonas fluorescens, Psychrobacter celer, Staphylococcus condimenti, Streptococcus thermophilus, Streptomyces griseus, Tetragenococcus halophilus, Weissella cibaria, Weissella koreensis, Zymomona s mobilis, Corynebacterium glutamicum, Bifidobacterium bifidum / Breve / longum, Streptomyces lividans, Streptomyces coelicolor, Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus case!, Pseudoalteromonas citrea, Pseudomonas putida, Clostridium Ijungdahlii / aceticum / acetobutylicum / beijerinckii / butyricum and Moorella themocellum / thermoacetica. In certain embodiments, the bacterial cells may be from a strain of Escherichia coli. In certain embodiments, the E. coli strain can be selected from BL21, DH5a, XL1-Blue, HB101, BL21 and K12. In certain embodiments, heterologous coding sequences can be codon optimized for expression in E. coli and expressed from an appropriate promoter. In certain embodiments, the promoter can be selected from T7 promoter, tac promoter, trc promoter, tetracycline inducible promoter (tet), lac operon promoter (lac), lacO1 promoter. In certain embodiments, the expression cassette consisting of a promoter, heterologous coding sequence and a terminator can 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 integration cassette can be performed with selection of antibiotics such as kanamycin, chloramphenicol, streptomycin, spectinomycin, gentamicin, erythromycin or ampicillin. In certain embodiments, DNA constructs can be introduced into host cells using established transformation methods such as conjugation, heat shock chemical transformation, or electroporation. In certain embodiments, cells can be cultured in liquid Luria-Bertani (LB) medium at about 37°C with or without antibiotics. In certain embodiments, the bacterial cells may be a strain of Bacillus subtilis. In certain embodiments, the B. subtilis strain can be selected from 1779, GP25, RO-NN-1, 168, BSn5, BEST195, 1A382 and 62178. In certain embodiments, heterologous coding sequences can be codon optimized for expression. in Bacillus sp. and expressed from an appropriate promoter. In certain embodiments, the promoter can be selected from the grac promoter, the p43 promoter or the trnQ promoter. In certain embodiments, the expression cassette consisting of the promoter, the heterologous coding sequence and the terminator can 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, integration vectors such as pDG364 or pDG1730 can be used to integrate the expression cassette into the genome. In certain embodiments, selection of cells that maintain the plasmid or integration cassette can be performed with selection of antibiotics such as erythromycin, kanamycin, tetracycline and spectinomycin. In certain embodiments, DNA constructs can be introduced into host cells using established transformation methods such as natural competition, heat shock, or chemical transformation. In certain embodiments, cells can be cultured in liquid Luria-Bertani (LB) medium at 37°C or M9 medium plus glucose and tryptophan. Genetic modifications of host cells The host cells can be designed to include one or more modifications (such as two or more, three or more, four or more, five or more, or even more modifications) that provide for the production of the TAs of interest. In some cases, modification means a genetic modification, such as a mutation, addition or deletion of a gene or fragment thereof, or regulation of transcription of a gene or fragment thereof. In some cases, the one or more (such as two or more, three or more, or four or more) modifications are selected from: a mutation that mitigates feedback inhibition in a native biosynthetic enzyme gene of the cell; a transcriptional modulation modification of a cell-native biosynthetic enzyme gene; an inactivating mutation in an enzyme native to the cell; a heterologous coding sequence that encodes an enzyme; and a heterologous coding sequence that encodes a protein that modifies the trafficking and / or subcellular localization of an enzyme or metabolite. A cell that includes one or more modifications may be referred to as a modified cell. A modified cell can overproduce one or more modified ΤΑ, ΤΑ, or TA precursor molecules. By overproduction it is meant that the cell has an improved or increased production of a TA molecule of interest relative to a control cell (e.g., an unmodified cell). By improved or increased production is meant both the production of some amount of the TA of interest when the control has no production of TA precursors, as well as an increase of about 10% or more, such as about 20% or more, about 30%. or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, such as 2 times or more, such as 5 times or more, including 10 times or more in situations where the control has some TA production of interest. In some cases, the host cell is capable of producing a greater amount of putrescine relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain cases, the increased amount of putrescine is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell. In some cases, the host cell is capable of producing a greater amount of N-methylpyrrolinium relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain cases, the increased amount of N-methylpyrrolinium is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more , about 60% or more, about 80% or more, about 100% or more, 2 times or more, 5 times or more, or even 10 times or more relative to the control host cell. In some cases, the host cell is capable of producing a greater amount of tropin relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain cases, the increased amount of tropin is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, approximately 80% or more, approximately 100% or more, 2 times or more, 5 times or more, or MA / t / ZUZ1 1 even 10 times or more relative to the control host cell. In some cases, the host cell is capable of producing a greater amount of phenylpyruvic acid relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain cases, the increased amount of phenylpyruvic acid is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell. In some cases, the host cell is capable of producing a greater amount of phenyl lactic acid relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain cases, the increased amount of phenyl lactic acid is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more , about 60% or more, about 80% or more, about 100% or more, 2 times or more, 5 times or more, or even 10 times or more relative to the control host cell. In some cases, the host cell is capable of producing a greater amount of littorin relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain cases, the increased amount of littorin is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell. In some cases, the host cell is capable of producing a greater amount of hyoscyamine relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain cases, the increased amount of hyoscyamine is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell. In some cases, the host cell is capable of producing a greater amount of scopolamine relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain cases, the increased amount of scopolamine is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell. In some embodiments, the host cell is capable of producing a yield of 10% or more of tropin from a starting compound such as arginine, such as 20% or more, 30% or more, 40%. MA / 1 1 or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more tropine yield from a starting compound. In some embodiments, the host cell is capable of producing a yield of 10% or more of phenyl lactic acid from a starting compound such as phenylalanine, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more yield of phenyl lactic acid from a starting compound. In some embodiments, the host cell is capable of producing a yield of 10% or more of hyoscyamine from a starting compound such as arginine or phenylalanine, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more yield of hyoscyamine from a starting compound. In some embodiments, the host cell is capable of producing a yield of 10% or more of scopolamine from a starting compound such as arginine or phenylalanine, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more yield of scopolamine from a starting compound. In some embodiments, the host cell overproduces one or more TA molecules of interest selected from the group consisting of arginine, ornithine, agmatine, putrescine, N-methylputrescine, 4-methylaminobutanal, N-methylpyrrolinium, 4-(1-methyl-2- pyrrodinyl)- 3-oxobutanoic acid, tropinone, tropine, phenylalanine, prephenic acid, phenylpyruvic acid, phenyl lactic acid, glucose-1-O-phenyl lactate, littorin, hyoscyamine aldehyde, hyoscyamine, anisodamine and scopolamine. Any suitable combination of one or more modifications can be included in the target host cells. In some cases, two or more (such as two or more, three or more, or four or more) different types of modifications are included. In certain cases, two or more (such as three or more, four or more, five or more, or even more) different modifications of the same type of modification are included in the target cells. In some embodiments of the host cell, when the cell includes one or more heterologous coding sequences that encode one or more enzymes, it includes at least one additional modification selected from the group consisting of: a feedback inhibition mitigating mutation in a biosynthetic enzyme native to the cell; a transcriptional modulation modification of a biosynthetic enzyme gene native to the cell; and an inactivating mutation in an enzyme native to the cell. In certain host cell embodiments, when the cell includes one or more mutations that mitigate feedback inhibition in one or more of the cell's native biosynthetic enzyme genes, it includes at least one additional modification selected from the group consisting of: a modification transcriptional modulation of a cell-native biosynthetic enzyme gene; an inactivating mutation in an enzyme native to the cell; and a heterologous coding sequence that encodes an enzyme. In some embodiments of the host cell, when the cell includes one or more transcriptional modulation modifications of one or more of the cell's native biosynthetic enzyme genes, it includes at least one additional modification selected from the group consisting of: a mutation that mitigates the feedback inhibition in a cell's native biosynthetic enzyme gene; an inactivating mutation in a native enzyme of the cell; a heterologous coding sequence that encodes an enzyme; and a heterologous coding sequence that encodes a protein that modifies the subcellular trafficking and / or localization of an enzyme or metabolite. In certain host cell instances, when the cell includes one or more inactivating mutations in one or more enzymes native to the cell, it includes at least one additional modification selected from the group consisting of: a mutation that mitigates feedback inhibition in a biosynthetic enzyme gene native to the cell; a transcriptional modulation modification of a biosynthetic enzyme gene native to the cell; a heterologous coding sequence that encodes an enzyme; and a heterologous coding sequence that encodes a protein that modifies the trafficking and / or subcellular localization of an enzyme or metabolite. In certain embodiments of the host cell, the cell includes one or more mutations that mitigate feedback inhibition in one or more biosynthetic enzyme genes native to the cell; and one or more transcriptional modulation modifications of one or more native biosynthetic enzyme genes of the cell. In certain embodiments of the host cell, the cell includes one or more mutations that mitigate feedback inhibition in one or more biosynthetic enzyme genes native to the cell; and one or more inactivating mutations in a native enzyme of the cell. In certain embodiments of the host cell, the cell includes one or more mutations that mitigate feedback inhibition in one or more biosynthetic enzyme genes native to the cell; and one or more heterologous coding sequences. In some embodiments, the host cell includes one or more modifications (e.g., as described herein) that include one or more of the genes of interest described in Table 1. Mutations that mitigate feedback inhibition In some cases, host cells are cells that include one or more mutations that mitigate feedback inhibition (such as two or more, three or more, four or more, five or more, or even more) in one or more genes of biosynthetic enzymes of the cell. In some cases, 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 mitigating mutation" refers to a mutation that mitigates a feedback inhibition control mechanism of a host cell. Feedback inhibition is a cell control mechanism in which an enzyme in the synthesis pathway of a regulated compound is inhibited when that compound has accumulated to a certain level, thus balancing the amount of the compound in the cell. In some cases, the one or more mutations that mitigate feedback inhibition are found in an enzyme described in a biosynthetic pathway of Figures 1 to 4 or in the scheme of Figure 8. A mutation that mitigates feedback inhibition reduces the inhibition of a regulated enzyme in the cell of interest relative to a control cell and provides an increased level of the regulated compound or a biosynthetic product downstream thereof. In some cases, mitigating the inhibition of the regulated enzyme means that the inhibition ICso increases by 2 times or more, such as, 3 times or more, 5 times or more, 10 times or more, 30 times or more, 100 times or more, 300 times or more, 1000 times or more, or even more. By increased level is meant a level that is 110% or more of the regulated compound in a control cell or a product downstream thereof, such as 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, such as at least 3 times or more, at least 5 times or more, at least 10 times or more or even more of the compound regulated in the host cell or a downstream product thereof. A variety of feedback inhibition control mechanisms and host cell native biosynthetic enzymes that are directed at regulating TA precursor levels can be targeted for mitigation in the host cell. The host cell may include one or more mutations that mitigate feedback inhibition in one or more biosynthetic enzyme genes native to the cell. The mutation may 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 an ornithine decarboxylase (ODC), an ornithine decarboxylase antizyme, and a putrescine Nmethyltransferase. In some embodiments, the one or more biosynthetic enzyme genes encode an ornithine decarboxylase. In some cases, one or more biosynthetic enzyme genes encode an ornithine decarboxylase antizyme. In some embodiments, one or more biosynthetic enzyme genes encode a putrescine N-methyltransferase. In certain cases, one or more mutations that mitigate feedback inhibition are present in a biosynthetic enzyme gene selected from SPE1, OAZ1 and PMT. In certain cases, the one or more mutations that mitigate feedback inhibition are present in a biosynthetic enzyme gene which is SPE1. In certain cases, the one or more mutations that mitigate feedback inhibition are present in a biosynthetic enzyme gene which is OAZ1. In certain cases, the one or more mutations that mitigate feedback inhibition are present in a biosynthetic enzyme gene that is PMT. In some embodiments, the host cell includes one or more mutations that mitigate feedback inhibition in one or more biosynthetic enzyme genes, such as one of the genes described in Table 1. Any convenient number and type of mutations can be used to mitigate a feedback inhibition control mechanism. As used herein, the term mutation refers to a deletion, insertion or substitution of an amino acid residue(s) or nucleotide residue(s) with respect to a reference sequence or motif. The mutation can be incorporated as a mutation targeting the native gene at the original locus. In some cases, the mutation can be incorporated as an additional copy of the gene introduced as a genetic integration at a separate locus, or as an additional copy in an episomal vector such as a 2μ or centromeric plasmid. In certain cases, feedback-inhibited copying of the enzyme is under the transcriptional regulation of the native cell. In some cases, feedback-inhibited copying of the enzyme is introduced with constitutive or dynamic designer regulation of protein expression by placing it under the control of a synthetic promoter. In certain embodiments, the host cells of the present invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more mutations that mitigate feedback inhibition, such as 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mutations that mitigate feedback inhibition in one or more biosynthetic enzyme genes native to the host cell. Transcriptional modulation modifications The host cells may include one or more transcriptional modulation modifications (such as two or more, three or more, four or more, five or more, or even more modifications) of one or more biosynthetic enzyme genes of the cell. In some cases, the one or more biosynthetic enzyme genes are native to the cell. Any suitable biosynthetic enzyme gene in the cell can be targeted for modulation of transcription. Transcription modulation means that the expression of a gene of interest in a modified cell is modulated, for example, increased or decreased, enhanced or repressed, relative to a control cell (for example, an unmodified cell). . In some cases, transcriptional modulation of the gene of interest includes increasing or enhancing expression. By increasing or enhancing expression is meant that the expression level of the gene of interest increases 2-fold or more, such as 5-fold or more, and sometimes 25, 50, or 100-fold or more, and in certain embodiments 300-fold or more. more or more, compared to a control, that is, expression in the same unmodified cell (for example, using any convenient gene expression assay). Alternatively, in cases where the expression of the gene of interest in a cell is so low as to be undetectable, the expression level of the gene of interest is considered to increase if the expression increases to a level that is easily detectable. In certain cases, transcriptional modulation of the gene of interest includes decreasing or repressing expression. Decreasing or repressing expression means that the expression level of the gene of interest decreases 2-fold or more, for example, 5-fold or more and sometimes 25, 50 or 100-fold or more and in certain embodiments 300-fold or more, compared to a control. In some cases, expression is reduced to a level that is undetectable. Modifications of host cell processes of interest that can be adapted for use in target host cells are described in US Publication No. 20140273109 (14 / 211,611) by Smolke et al., the disclosure of which is incorporated herein. for reference in its entirety. Any suitable biosynthetic enzyme gene can be transcriptionally modulated and includes, but is not limited to, those biosynthetic enzymes described in Figures 1 to 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, one or more biosynthetic enzyme genes is ARG2. In certain cases, the one or more biosynthetic enzyme genes is CAR1. In some embodiments, the one or more biosynthetic enzyme genes is SPE1. In some embodiments, the one or more biosynthetic enzyme genes is FMS1. In some embodiments, the host cell includes one or more transcriptional modulation modifications to one or more genes, such as one of the genes described in Table 1. In some embodiments, the host cell includes one or more transcriptional modulation modifications to one or more genes such as one of the genes described in a biosynthetic pathway of one of Figures 1 to 4 or in the scheme of Figure 8. In some embodiments, modification of transcriptional modulation includes the replacement of a strong promoter with a native promoter of one or more biosynthetic enzyme genes or the expression of an additional copy or copies of the gene or genes under the control of a strong promoter. The promoters that drive the expression of the genes of interest can be constitutive or inducible promoters, as long as the promoters can be active in the host cells. The genes of interest can be expressed from their native promoters or non-native promoters can be used. Although not required, such promoters must have medium to high strength in the host on which they are used. Promoters can be regulated or constitutive. In some embodiments, promoters are used that are not repressed by glucose, or only slightly repressed by the presence of glucose in the culture medium. There are numerous suitable promoters, examples of which include promoters of glycolytic genes such as the promoter of the tsr gene of B. subtilis (encoding fructose bisphosphate aldolase) or the GAPDH promoter of the yeast S. cerevisiae (encoding glyceraldehyde-phosphate dehydrogenase). ) (Bitter G.A., Meth, Enzymol. 152:673-684 (1987)). Other strong promoters of interest include, but are not limited to, the baker's yeast ADHI promoter (Ruohonen L., et al, J. Biotechnol. 39:193-203 (1995)), promoters induced by phosphate starvation such such as the yeast PHO5 promoter (Hinnen, A., et al, in Yeast Genetic Engineering, Barr, P. J., et al. eds, Butterworths (1989)), the B. licheniformis alkaline phosphatase promoter (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, GalS, Met25 repressible promoter, teto and constitutive promoters such as the glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter, alcohol dehydrogenase (ADH) promoter, translation elongation factor-1-alpha (TEF) promoter, cytochrome c-oxidase (CYC1) promoter, MRP7 promoter, phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI) ), etc. In some cases, the strong promoter is GPD1. In certain cases, the strong promoter is TEF1. Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, spheroids, and thyroid hormones are also known and include, but are not limited to, the glucorticoid-responsive element (GRE) and the thyroid hormone responsive (TRE), see, for example, the promoters described in US Pat. 7,045,290. Vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used. Furthermore, any promoter / enhancer combination (based on the EPDB eukaryotic promoter database) could also be used to drive expression of genes of interest. It is understood that any convenient promoter specific to the host cell, eg, E. coli, may be selected. In some cases, promoter selection can be used to optimize transcription and therefore enzyme levels to maximize production while minimizing energy resources. Inactivating mutations Host cells may include one or more inactivating mutations of an enzyme in the cell (such as two or more, three or more, four or more, five or more, or even more). The inclusion of one or more inactivating mutations can modify the flow of a host cell synthesis pathway to increase levels of a TA of interest or a desirable enzyme or precursor leading thereto. In some cases, the one or more inactivating mutations are from an enzyme native to the cell. Figure 8 illustrates native regulatory mechanisms in yeast that act on polyamine production pathways and Figure 9 shows the effects of disruptions of these native regulatory systems on putrescine production. As used herein, inactivating mutation means one or more mutations to a regulatory gene or DNA sequence of the cell, where the mutation or mutations inactivate a biological activity of the protein expressed by that gene of interest. In some cases, the gene is native to the cell. In some cases, the gene encodes an inactivated enzyme that is part of or connected to the synthesis pathway of a TA of interest produced by the host cell. In some cases, an inactivating mutation is located in a regulatory DNA sequence that controls a gene of interest. In certain cases, the inactivating mutation is from a promoter of a gene. Any convenient mutation (e.g., as described herein) can be used to inactivate a gene or regulatory DNA sequence of interest. By inactivated or inactive it is meant that the biological activity of the protein expressed by the mutated gene is reduced by 10% or more, such as 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, relative to a 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. In some embodiments, the cell includes an inactivating mutation in an enzyme native to the cell. Any convenient enzyme can be targeted for inactivation. Enzymes of interest include, but are not limited to, those enzymes described in Figures 1 to 4, 8, 11, 22 and 41 whose action on host cell biosynthetic pathways tends to reduce levels of a TA of interest. In some cases, the enzyme has methylthioadenosine phosphorylase activity. In certain embodiments, the enzyme that includes an inactivating mutation is MEU1 (see, for example, Figures 8, 9 and 13). In some cases, the enzyme has antizyme ornithine decarboxylase activity. In certain embodiments, the enzyme that includes an inactivating mutation is OAZ1. In some cases, the enzyme has spermidine synthase activity. In certain embodiments, the enzyme that includes an inactivating mutation is SPE3. In some cases, the enzyme has spermine synthase activity. In some embodiments, the enzyme that includes an inactivating mutation is SPE4. In some cases, the enzyme is a membrane transporter with polyamine export activity. In certain embodiments, the enzyme or protein that includes an inactivating mutation is TPO5. In some cases, the enzyme has phenylacrylic acid decarboxylase activity. In certain embodiments, the enzyme that includes an inactivating mutation is PAD1. In some cases, the enzyme has alcohol dehydrogenase activity. In some embodiments, the enzyme that includes an inactivating mutation is selected from ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and SFA1. In certain embodiments, the enzyme that includes an inactivating mutation or mutations is ADH2. In certain embodiments, the enzyme that includes an inactivating mutation or mutations is ADH3. In certain embodiments, the enzyme that includes an inactivating mutation or mutations is ADH4. that they inactivate is ADH5. that they inactivate is ADH6. In certain modalities, In certain modalities, In certain embodiments, the enzyme that includes an inactivating mutation or mutations is ADH7. In some cases, the enzyme has aldehyde oxidoreductase activity. In certain embodiments, the enzyme that includes an inactivating mutation is selected from HFD1, ALD2, ALD3, ALD4, ALD5 and ALD6. In certain embodiments, the enzyme that includes an inactivating mutation or mutations is HFD1. In certain embodiments, the enzyme that includes an inactivating mutation or mutations is ALD2. mutations that inactivate esALD3. mutations that inactivate esALD4. mutations that inactivate esALD5. In certain MA / 1 1 modalities, the enzyme that includes a mutation or In certain embodiments, the enzyme that includes a mutation In certain embodiments, the enzyme that includes a mutation In certain embodiments, the enzyme that includes an inactivating mutation(s) is ALD6. In some cases, the enzyme has glucosidase activity. In certain embodiments, the enzyme that includes an inactivating mutation is selected from EXG1, SPR1 and EGH1. In certain embodiments, the enzyme that includes an inactivating mutation or mutations is EXG1. In certain embodiments, the enzyme that includes an inactivating mutation or mutations is SPR1. In certain embodiments, the enzyme that includes an inactivating mutation or mutations is EGH1. In some embodiments, the host cell includes one or more inactivating mutations in one or more genes described in Table 1. Methods for performing TA acyl transfer reactions using functional expression of acyltransferases in non-plant hosts Some methods, processes and systems provided herein describe the concerted reaction of one or more TA precursors comprising an acyl donor group with one or more TA precursors comprising an acyl acceptor group to produce one or more TA within a non-plant cell (hereinafter TA acyl transfer reactions). Some of these methods, processes and systems may comprise an engineered host cell. In some examples, the TA acyl transfer reaction is a key step in the conversion of a substrate to a wide range of alkaloids. In some examples, the TA acyl transfer reaction comprises a condensation reaction. In some examples, the acyl transfer from TA may involve at least one condensation reaction. In some cases, at least one of the condensation reactions is carried out in the presence of an enzyme. In some cases, at least one of the condensation reactions is catalyzed by an enzyme. In some cases, at least one enzyme is useful to catalyze the condensation reaction. In some methods, processes and systems described herein, a condensation reaction can be performed in the presence of an enzyme. In some examples, the enzyme may be an acyltransferase. The acyltransferase can use a TA with an alcohol or carboxylate functional group as a substrate. The acyltransferase can use a TA containing a carboxylate group activated by a 1-Ο-β glycosidic linkage to a sugar (hereinafter referred to as glycoside) as a substrate. Acyltransferase can convert the alcohol and carboxylate / glucoside functional groups of TA to a corresponding ester derivative. Non-limiting examples of enzymes suitable for condensation of TA precursors in this disclosure include serine carboxypeptidase-type acyltransferases (SCPL AT). For example, littorin synthase (EC 2.3.1.-) can condense tropine and other TA precursors containing alcohol functional groups with 1-Ο-β-phenyl lactoll-glucose and other TA glycoside precursors to littorin and other products. corresponding ester. In some examples, a protein comprising a SCPL-AT domain from any of the above examples can perform condensation. In some examples, SCPL-AT can catalyze the condensation reaction within a host cell, such as an engineered host cell, as described herein. In still other examples, the SCPLAT can catalyze the condensation reaction within a sub-cellular compartment within a host cell, such as an engineered host cell, as described herein. In some embodiments of the invention, the amino acid sequence of an acyltransferase enzyme that is used to perform a TA acyl transfer reaction, such as a SCPL-AT enzyme, is subject to one or more modifications that alter post-translational processing, trafficking, folding, oligomerization and / or sub-cellular localization of the enzyme. Since some acyltransferase enzymes, including SCPL-AT enzymes, have never been shown to exhibit catalytic activity in living non-plant cells, such modifications may be useful, or may be necessary, for activity in non-plant host cells. Examples of such modifications include, but are not limited to: addition, deletion or replacement of N-terminal signal peptide sequences; addition, deletion or replacement of internal propeptide sequences; addition or removal of asparagine-linked Nglycosylation sites; addition or removal of serine-linked O-glycosylation sites; and fusion of protein domains to the N- and / or C-terminus of the acyltransferase domain. In one embodiment of the invention, a SCPL-AT enzyme domain is modified at its N-terminus by fusion to a soluble protein domain. This soluble domain masks any internal signal sequences in the acyltransferase domain, thus modifying the trafficking and / or subcellular localization of the fused SCPL-AT domain. In some examples, the N-terminal fused domain induces trafficking of the SCPL-AT domain to subcellular compartments including, but not limited to, the ER membrane, ER lumen, cis-Golgi, trans-Golgi, lysosome, membrane of the vacuole and lumen of the vacuole. The N-terminally fused soluble domain can also modify the oligomerization state of the SCPL-AT domain from its native state (monomer) to any state including, but not limited to, homodimer, heterodimer, homotrimer, heterotrimer, homotetramer, heterotetramer, homohexamer, heterohexamer, homooctamer, heterooctamer or higher degrees of oligomerization. In one example, the N-terminally fused soluble protein domain is a fluorescent protein selected from the group including, but not limited to, fluorescent proteins derived from Aequoria sp. and fluorescent proteins derived from Discosoma sp. In one example, the N-terminally fused soluble protein domain is the red fluorescent protein of Discosoma sp. (DsRed). In other examples, the N-terminally fused soluble protein domain is another enzyme in the TA biosynthetic pathway, including, but not limited to, ornithine decarboxylase, putrescine N-methyltransferase, pyrrolidine ketide synthase, tropinone reductase, phenylpyruvate reductase, phenyl lactate UDP-glucosyltransferase 84A27 and hyoscyamine dehydrogenase. Examples of amino acid sequences of soluble protein domains that can be fused to the N-terminus of a SCPL-AT domain that can then be used to perform a TA acyl transfer reaction within a non-plant cell are provided in Table 3. . An amino acid sequence for a SCPL-AT enzyme comprising a fused N-terminal domain and used in TA acyl transfer reactions in non-plant cells may be 50% or more identical to a given amino acid sequence, as follows: listed in Table 3. For example, an amino acid sequence for such an acyltransferase may comprise an amino acid sequence that is at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more , 75% or more, 80% or more, 81% or more, % or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, % or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, % or more, or 99 % or more identical to an amino acid sequence as provided herein. Furthermore, in certain embodiments, an identical amino acid sequence contains at least 80%-99% identity at the amino acid level with the specific amino acid sequence. In some cases, an identical amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more in certain cases, at least 95%, 96%, 97%, 98% and 99% identity, at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence is altered to optimize codon usage for the host organism, for example. An engineered non-plant host cell may be provided that produces an acyltransferase that catalyzes a TA acyl transfer reaction, wherein the acyltransferase comprises an amino acid sequence whose N-terminal end is fused to the amino acid sequence of a soluble protein domain. selected from the group consisting of the sequences in Table 3. The acyltransferase that is produced within the engineered host cell can be recovered and purified to form a biocatalyst. The one or more enzymes that are recovered from the engineered host cell that produce the acyltransferase can be used in a process to carry out a TA acyl transfer reaction. The process may include contacting TA precursors possessing an alcohol and / or carboxylate / glucoside functional group with an acyltransferase in an amount sufficient to convert the alcohol and / or carboxylate / glucoside group to a corresponding ester group. In examples, TA precursors possessing an alcohol and / or carboxylate / glucoside functional group may be contacted with a sufficient amount of one or more enzymes such that at least 5% of said TA precursors are converted to the ester. correspondent. In other examples, the TA possessing an alcohol and / or a carboxylate / glucoside functional group may 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 said TA precursors are converted into the corresponding ester. The one or more enzymes that can be used to carry out a TA acyl transfer reaction can be contacted with the TA precursors 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 the TA precursors in vivo. Furthermore, the one or more enzymes that can be used to carry out a TA acyl transfer reaction can be provided to a cell that has the TA precursors within, or can be produced within an engineered non-plant host cell. In some examples, the methods provide engineered non-plant host cells that produce an alkaloid product, wherein the TA acyl transfer reaction may comprise a key step in the production of an alkaloid product. In some examples, the alkaloid produced is a medicinal TA. In still other embodiments, the alkaloid produced is derived from a medicinal TA, including, for example, non-natural TA. In still other embodiments, the alkaloid product is selected from the group consisting of medicinal TA, non-medicinal TA and non-natural TA. In some examples, the substrates are TA precursors selected from the group consisting of tropine, pseudotropin, ecgonine, methylecgonine, phenyl lactic acid, cinnamic acid, ferulic acid, coumanic acid and glycosides of the listed compounds. In some examples, the methods provide engineered non-plant host cells that produce alkaloid products from tropine and 1-Ο-β-phenyl lactoylglucose. The condensation of tropine and Ι-Ο-β-phenyl lactoylglucose to littorin may comprise a key step in the production of various alkaloid products from a precursor. In some examples, the precursor is an L-amino acid or a sugar (e.g., glucose). The various alkaloid products may include, without limitation, medicinal TAs, non-medicinal TAs, and non-natural TAs. Any suitable carbon source can be used as a precursor for a TA acyl transfer reaction. Suitable precursors may include, without limitation, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or a combination thereof. . In some examples, unpurified mixtures of renewable feedstocks (e.g., corn liquor, sugar beet molasses, barley malt, biomass hydrolyzate) may be used. In still other embodiments, the carbon precursor may 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 can be used, for example, methylamine, glucosamine and amino acids (for example, L-arginine and L-phenylalanine). In some examples, a TA or a precursor of a TA possessing an alcohol and / or carboxylate / glucoside functional group can be added directly to a designed host cell of the invention, including, for example, tropine, pseudotropin, ecgonine, methylecgonine, phenyl lactic acid, cinnamic acid, ferulic acid, coumaric acid and glycosides of the listed compounds. In some embodiments, the substrate used to carry out the vacuolar TA acyl transfer reaction may comprise one or more alcohol and / or carboxylate / glucoside functional groups, wherein only one of said functional groups is condensed into the corresponding ester. TA alcohol-aldehyde interconversions Some methods, processes and systems provided herein describe the conversion of TAs with aldehyde functional groups to TAs with alcohol (hydroxyl) functional groups, and the conversion of TAs with alcohol functional groups to TAs with aldehyde functional groups (in (hereinafter referred to as TA alcohol-aldehyde interconversions). Some of these methods, processes and systems may comprise an engineered host cell. In some examples, the alcoholaldehyde interconversion of TA is a key step in the conversion of a substrate to a diverse range of alkaloids. In some examples, the conversion of an aldehyde group of TA to an alcohol group TA comprises a reduction reaction. In some cases, the reduction of an aldehyde substrate from TA to an alcohol can be accomplished by reducing an aldehyde substrate to the corresponding tetrahedral oxyanion intermediate, then protonating this intermediate to a hydroxyl as provided in Figure 2 and as generally depicted in Fig. Scheme 1. As given in Scheme 1, R1 may be H, CH3 or a higher order alkyl group; R2 and R3 can be H, OH or OCH3; R4can be H; and R5 can be H, OH, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 acyl, F, Cl or Br. MA / 1 1 TA with aldehyde functional group TA with tetrahedral intermediate TA with alcohol functional group (e.g. hyoscyamine aldehyde) (e.g. hyoscyamine) Scheme 1 In some examples, the alcohol-aldehyde interconversion of TA may involve at least one oxidation reaction or at least one reduction reaction. In some cases, at least one of the oxidation or reduction reactions is carried out in the presence of an enzyme. In some cases, at least one of the oxidation or reduction reactions is catalyzed by an enzyme. In some cases, oxidation and reduction reactions are carried out in the presence of at least one enzyme. In some cases, at least one enzyme is useful to catalyze oxidation and reduction reactions. Oxidation and reduction reactions can be catalyzed by the same enzyme. In some methods, processes and systems described herein, an oxidation or reduction reaction can be performed in the presence of an enzyme. In some examples, the enzyme may be a dehydrogenase. The dehydrogenase can use a TA with an alcohol or aldehyde functional group as a substrate. Dehydrogenase can convert the alcohol or aldehyde functional group of TA to a corresponding aldehyde or alcohol derivative. The dehydrogenase may be called hyoscyamine dehydrogenase (HDH). Non-limiting examples of enzymes suitable for the oxidation and / or reduction of TAs in this disclosure include a cytochrome P450 oxidase, a 2oxoglutarate-dependent oxidase, a flavoprotein oxidase, a short chain dehydrogenase-reductase (SDR), a dehydrogenase -medium chain reductase (MDR), a cinnamyl alcohol dehydrogenase (CAD) and an aldocete reductase (AKR). For example, tropinone reductase 1 (EC 1.1.1.206) can oxidize tropinone and other TA precursors with ketone functional groups to tropine (3a-tropanol) and other corresponding alcohol products. In some examples, a protein comprising a dehydrogenase domain of any of the above examples may perform oxidation or reduction. In some examples, the dehydrogenase can catalyze oxidation and / or reduction reactions within a host cell, such as an engineered host cell, as described herein. Examples of amino acid sequences of a dehydrogenase enzyme that can be used to perform an alcohol-aldehyde interconversion of TA are provided in Table 2. An amino acid sequence for a dehydrogenase that is used in TA alcoholaldehyde interconversions may be 50% or more identical to a given amino acid sequence as listed in Table 2. For example, an amino acid sequence for such a dehydrogenase may comprise a amino acid sequence that is at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to an amino acid sequence as provided herein. Furthermore, in certain embodiments, an identical amino acid sequence contains at least 80%-99% identity at the amino acid level with the specific amino acid sequence. In some cases, an identical amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more in certain cases, at least 95%, 96%, 97%, 98% and 99% identity, at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence is altered to optimize codon usage for the host organism, for example. An engineered host cell can be provided to produce a dehydrogenase that catalyzes an alcohol-aldehyde interconversion of TA, wherein the dehydrogenase comprises an amino acid sequence selected from the group consisting of the sequences in Table 2. The dehydrogenase that is produced within the Engineered host cell can be recovered and purified to form a biocatalyst. The one or more enzymes that are recovered from the engineered host cell that produces the dehydrogenase can be used in a process for carrying out an alcohol-aldehyde interconversion of TA. The process may include contacting the TA possessing an alcohol and / or aldehyde functional group with a dehydrogenase in an amount sufficient to convert the alcohol and / or aldehyde group of the TA to a corresponding aldehyde and / or alcohol group. In examples, TA possessing an alcohol and / or aldehyde functional group may be contacted with a sufficient amount of one or more enzymes such that at least 5% of said TA is converted to its corresponding aldehyde and / or alcohol group. In other examples, the TA possessing an alcohol and / or aldehyde functional group may 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 said TA is converted into its corresponding aldehyde group and / or alcohol. The one or more enzymes that can be used to carry out an alcohol-aldehyde interconversion of the TA can come into contact with the TA in vitro. Additionally, or alternatively, one or more enzymes that can be used to carry out an alcohol-aldehyde interconversion can come into contact with the TA in vivo. Furthermore, the one or more enzymes that can be used to carry out an alcohol-aldehyde interconversion of the TA can be provided to a cell that has the TA within it, or can be produced within an engineered host cell. In some examples, the methods provide engineered host cells that produce an alkaloid product, wherein alcohol-aldehyde interconversion may comprise a key step in the production of an alkaloid product. In some examples, the alkaloid produced is a medicinal TA. In still other embodiments, the alkaloid produced is derived from a medicinal TA, including, for example, non-natural TA. In another embodiment, a TA possessing an alcohol and / or aldehyde functional group is an intermediate of the engineered host cell product. In still other embodiments, the alkaloid product is selected from the group consisting of medicinal TA, non-medicinal TA and non-natural TA. In some examples, the substrate is a TA or a precursor of a TA selected from the group consisting of littorin, hyoscyamine aldehyde, hyoscyamine, anisodamine and scopolamine. In some examples, the methods provide engineered host cells that produce alkaloid products from hyoscyamine aldehyde. The reduction of the hyoscyamine aldehyde to hyoscyamine may comprise a key step in the production of various alkaloid products from a precursor. In some examples, the precursor is an L-amino acid or a sugar (e.g., glucose). The various alkaloid products may include, without limitation, medicinal TAs, non-medicinal TAs, and non-natural TAs. Any suitable carbon source can be used as a precursor for an alcohol-aldehyde interconversion of TA. Suitable precursors may include, without limitation, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or a combination thereof. . In some examples, unpurified mixtures of renewable feedstocks (e.g., corn liquor, sugar beet molasses, barley malt, biomass hydrolyzate) may be used. In still other embodiments, the carbon precursor may 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 can be used, for example, methylamine, glucosamine and amino acids (for example, Larginine and L-phenylalanine). In some examples, a TA or a precursor of a TA possessing an alcohol and / or aldehyde functional group can be added directly to a designed host cell of the invention, including, for example, tropine, pseudotropin, ecgonine, methylecgonine, littorin, hyoscyamine aldehyde, hyoscyamine, anisodamine and scopolamine. In some embodiments, the substrate used to carry out the alcohol-aldehyde interconversion of TA may comprise one or more alcohol and / or aldehyde functional groups, wherein only one of said functional groups is oxidized or reduced to the corresponding aldehyde or alcohol group. . Methods to increase intracellular and extracellular metabolite transport Some methods, processes and systems provided herein describe the use of proteins (hereinafter referred to as transporters) to translocate metabolites across lipid membranes (hereinafter referred to as transmembrane transport). Some of these methods, processes and systems may comprise an engineered host cell. In some examples, transmembrane transport is a key step in the conversion of a substrate to a wide range of alkaloids. In certain embodiments, the host cell includes one or more heterologous coding sequences for one or more transporters or active fragments thereof that localize to a lipid membrane and translocate a TA or a TA precursor across the same lipid membrane. In some examples, the lipid membrane is the membrane of the vacuole. In other examples, the lipid membrane is the ER membrane. In some examples, the lipid membrane is the peroxisome membrane. In other examples, the lipid membrane is the cell plasma membrane. In some examples, TAs and TA precursors transported in this manner include, but are not limited to, putrescine, N-methylputrescine, 4-methylaminobutanal, N-methylpyrrolinium, tropinone, tropine, phenyl lactic acid, Ι-Ο-β-phenyl lactoylglucose , littorin, hyoscyamine, anisodamine and scopolamine. The accumulation of such TAs or TA precursors in specific subcellular compartments may prevent the access of operatively linked blosynthetic enzymes in different compartments; therefore, the use of transporters that translocate TAs or TA precursors from one compartment to another may mitigate such transport limitations. In certain cases, the expression of heterologous coding sequences for one or more transporters within a host cell can increase the production of a TA or a TA precursor. In some embodiments, the transporter or active fragment thereof is a multidrug and toxin extrusion (MATE) transporter. Any convenient MATE transporter that transports one or more of the above-mentioned TAs or TA precursors finds use in target host cells. Carrier proteins of interest include, but are not limited to, enzymes such as Nicotiana tabacum jasmonate-inducible alkaloid transporter 1 (NtJATI), N. tabacum MATE1, N. tabacum MATE2, or any others as described in Table 1 and Table 4. In certain embodiments, the transporter or active fragment thereof is a nitrate / peptide family (NPF) transporter. Any suitable NPF transporter that transports one or more of the TAs or TA precursors mentioned above finds use in the target host cells. In other embodiments, the transporter or active fragment thereof is an ATP-binding cassette (ABC) transporter. Any suitable NPF transporter that transports one or more of the TAs or TA precursors mentioned above finds use in the target host cells. In some embodiments, the transporter or active fragment thereof is a pleiotropic drug resistance (PDR) transporter. Any convenient PDR carrier that transports one or more of the above-mentioned TAs or TA precursors finds use in target host cells. In certain embodiments, the host cell includes a heterologous coding sequence for a transporter or an active fragment thereof. In some embodiments of the invention, the amino acid sequence of a transporter is subject to one or more modifications that alter the subcellular localization, direction of substrate translocation, and / or topological orientation of the enzyme. Examples of such modifications include, but are not limited to: addition, deletion or replacement of N-terminal, C-terminal or internal signal sequences; addition, deletion, replacement or rearrangement of transmembrane helices; and fusion of protein domains to the N- and / or C-terminus of the transporter. Examples of transporter amino acid sequences that can be used to mitigate substrate transport limitations and / or to increase the accumulation of TAs or TA precursors in specific cellular compartments are provided in Table 4. An amino acid sequence for a transporter used in this way in non-plant cells may be 50% or more identical to a given amino acid sequence, as listed in Table 4. For example, an amino acid sequence for such a transporter may comprising 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, % or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, % or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, % or more, or 99% or more identical to an amino acid sequence as provided herein. Furthermore, in certain embodiments, an identical amino acid sequence contains at least 80%-99% identity at the amino acid level with the specific amino acid sequence. In some cases, an identical amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, and more in certain cases, at least 95%, 96%, 97%, 98% and 99% identity, at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence is altered to optimize codon usage for the host organism, for example. An engineered non-plant host cell may be provided that produces a transporter that translocates one or more TA or TA precursors from one cellular compartment to another, wherein the transporter comprises an amino acid sequence selected from the group consisting of those sequences in Table 4. In some examples, the methods provide engineered non-plant host cells that produce an alkaloid product, wherein transmembrane transport of TA may comprise a key step in the production of an alkaloid product. In some examples, the alkaloid produced is a medicinal TA. In still other embodiments, the alkaloid produced is derived from a medicinal TA, including, for example, non-natural TA. In still other embodiments, the alkaloid product is selected from the group consisting of medicinal TA, non-medicinal TA and non-natural TA. Heterologous coding sequences In some cases, host cells are cells that harbor one or more heterologous coding sequences (such as two or more, three or more, four or more, five or more, or even more) that encode activity(s) that allow the host cells produce the desired TAs of interest, for example, as described herein. As used herein, the term "heterologous coding sequence" is used to indicate any polynucleotide that encodes, or ultimately encodes, a peptide or protein or its equivalent amino acid sequence, for example, an enzyme, that is not normally present in the host organism and that can be expressed in the host cell under suitable conditions. As such, heterologous coding sequences include multiple copies of coding sequences that are normally present in the host cell, such that the cell is expressing additional copies of a coding sequence that are not normally present in the cells. Heterologous coding sequences may be RNA or any type thereof, for example, mRNA, DNA or any type thereof, for example, cDNA, or an RNA / DNA hybrid. Coding sequences of interest include, but are not limited to, full-length transcription units that include such features as the coding sequence, introns, promoter regions, 3'-UTR, and enhancer regions. In the examples, the designed host cell comprises a plurality of heterologous coding sequences, each of which encodes an enzyme. In some examples, the plurality of enzymes encoded by the plurality of heterologous coding sequences may be distinct from each other. In some examples, some of the plurality of enzymes encoded by the plurality of heterologous coding sequences may be different from each other and some of the plurality of enzymes encoded by the plurality of heterologous coding sequences may be duplicate copies. In some examples, the heterologous coding sequences may be operably connected. Heterologous coding sequences that are operably connected may be within the same pathway of production of a particular tropane alkaloid product. In some examples, the operably connected heterologous coding sequences may be directly sequential along the production pathway of a particular tropane alkaloid product. In some examples, the operably connected heterologous coding sequences may have one or more native enzymes among one or more of the enzymes encoded by the plurality of heterologous coding sequences. In some examples, the heterologous coding sequences may have one or more heterologous enzymes among one or more of the enzymes encoded by the plurality of heterologous coding sequences. In some examples, the heterologous coding sequences may have one or more non-native enzymes among one or more of the enzymes encoded by the plurality of heterologous coding sequences. In some embodiments, the host cell includes putrescine N-methyltransferase (PMT) activity. Any suitable PMT enzyme finds use in the target host cells. PMT enzymes of interest include, but are not limited to, enzymes such as EC 2.1.1.53, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for a PMT or an active fragment of the same. In some cases, the host cell includes one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert NMP to 4MAB. In certain cases, one or more enzymes are selected from plant methylputrescine oxidases (MPOs) and eukaryotic MPOs (e.g., EC 1.4.3.22). In certain embodiments, the cell includes one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert NMPy to MPOB. In certain cases, the one or more enzymes is a type III polyketide synthase (for example, EC 2.3.1.-). The one or more heterologous coding sequences may be derived from any convenient species (e.g., as described herein). In some cases, one or more heterologous coding sequences may be derived from a species described in Table 1. In some cases, one or more heterologous coding sequences are present in a gene or enzyme selected from those described in Table 1. In certain embodiments, the host cell includes tropinone synthase activity. Any convenient tropinone synthase enzyme (e.g., CYP82M3) finds use in target host cells. Tropinone synthase enzymes of interest include, but are not limited to, enzymes such as EC 1.14.14.-, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for a tropinone synthase or a active fragment of it. In certain embodiments, the host cell includes tropinone reductase activity. Any suitable tropinone reductase enzyme finds use in the target host cells. Tropinone reductase enzymes of interest include, but are not limited to, enzymes such as EC 1.1.1.206, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for a tropinone reductase or an active fragment. Of the same. In some cases, the host cell includes phenylpyruvate reductase (PPR) activity. Any suitable PPR enzyme finds use in the target host cells. Some PPR enzymes of interest include, but are not limited to, enzymes such as EC 1.1.1.237, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for a PPR or an active fragment thereof. . In certain embodiments, the host cell includes phenyl lactate glycosyltransferase activity. Any suitable phenyl lactate glycosyltransferase enzyme finds use in the target host cells. Glycosyltransferase enzymes include, but are not limited to, enzymes such as 2.4.1.-, which transfer a glucose moiety from UDP-glucose to phenyl lactate via a glycosidic ester bond, as described in Table 1. In In certain embodiments, the host cell includes a heterologous coding sequence for a phenyl lactate glycosyltransferase or an active fragment thereof. In certain embodiments, the cell includes one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert tropine and 1-Ο-β-phenyl lactoylglucose to littorin. In some embodiments, the host cell includes littorin synthase activity. Any suitable littorin synthase enzyme or enzyme comprising active fragments of littorin synthase finds use in the target host cells. Littorin synthase enzymes of interest include, but are not limited to, enzymes such as EC 2.3.1.-, as described in Table 1, and enzymes comprising littorin synthase enzymes whose N-terminal ends are fused to soluble protein domains. described in Table 3. In certain embodiments, the host cell includes a heterologous coding sequence for a littorin synthase or an active fragment thereof. In certain cases, the host cell includes littorin mutase activity. Any suitable littorin mutase enzyme finds use in the target host cells. Littorin mutase enzymes of interest include, but are not limited to, enzymes such as EC 1.14.19.-, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for a littorin mutase or a active fragment of it. In some embodiments, the host cell includes hyoscyamine dehydrogenase (HDH) activity. Any suitable HDH enzyme finds use in the target host cells. Some HDH enzymes of interest include, but are not limited to, the sequences described in Table 2. In certain embodiments, the host cell includes a heterologous coding sequence for an HDH or an active fragment thereof. In certain embodiments, the host cell includes hyoscyamine 6βhydroxylase / dioxygenase (H6H) activity. Any suitable H6H enzyme finds use in the target host cells. Some H6H enzymes of interest include, but are not limited to, enzymes such as EC 1.14.11.11, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for an H6H or an active fragment of the same. In certain examples, the engineered host cell comprises a plurality of heterologous coding sequences, each of which encodes a transmembrane metabolite transporter. In some examples, the plurality of transporters encoded by the plurality of heterologous coding sequences may be distinct from each other. In some examples, some of the plurality of transporters encoded by the plurality of heterologous coding sequences may be different from each other and some of the plurality of transporters encoded by the plurality of heterologous coding sequences may be duplicate copies. As used herein, the term heterologous coding sequences also includes the coding portion of the peptide or enzyme, that is, the cDNA or mRNA sequence, of the peptide or enzyme, as well as the coding portion of the full-length transcriptional unit, that is, the gene that includes introns and exons, as well as sequences with optimized codons, truncated sequences or other forms of altered sequences that encode the enzyme or encode its equivalent amino acid sequence, provided that the equivalent amino acid sequence produces a functional protein. Said equivalent amino acid sequences may have a deletion of one or more amino acids, the deletion being N-terminal, C-terminal or internal. Truncated forms are contemplated as long as they have the catalytic capacity indicated herein. Fusions of two or more enzymes are also envisioned to facilitate the transfer of metabolites in the pathway, as long as catalytic activities are maintained. Also included are fusions of one or more enzyme or catalytic protein domains with one or more non-catalytic protein domains in a manner in which the non-catalytic protein domain facilitates solubilization, folding, maturation and / or activity of the fused catalytic domain. . Operable fragments, mutants or truncated forms can be identified by modeling and / or selection. This is possible by adding or removing, for example, N-terminal, C-terminal or internal regions of the protein in a stepwise manner, followed by analysis of the resulting derivative with respect to its activity for the desired reaction in comparison to the original sequence. If the derivative in question operates in this capacity, it is considered to constitute an equivalent derivative of the enzyme itself. Aspects of the present invention also relate to heterologous coding sequences that encode amino acid sequences that are equivalent to the native amino acid sequences for the various enzymes. An amino acid sequence that is equivalent is defined as an amino acid sequence that is not identical to the specific amino acid sequence, but rather contains at least some amino acid changes (deletions, substitutions, inversions, insertions, etc.) that do not essentially affect to the biological activity of the protein compared to a similar activity of the specific amino acid sequence, when used for a desired purpose. Biological activity refers, in the example of a decarboxylase, to its catalytic activity. Equivalent sequences are also intended to include those that have been designed and / or evolved to have properties different from the original amino acid sequence. Mutable properties of interest include catalytic activity, substrate specificity, selectivity, stability, solubility, localization, etc. In certain embodiments, an equivalent amino acid sequence contains at least 80%-99% identity at the amino acid level with the specific amino acid sequence, in some cases at least about 85%, 86%, 87%, 88%, 89%. , 90%, 91%, 92%, 93%, 94% and more in certain cases, at least 95%, 96%, 97%, 98% and 99% identity, at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence is altered to optimize codon usage for the host organism, for example. Host cells can also be modified to possess one or more genetic alterations to accommodate heterologous coding sequences. Alterations of the native host genome include, but are not limited to, modifying the genome to reduce or eliminate the expression of a specific protein that may interfere with the desired pathway. The presence of such native proteins can rapidly convert one of the intermediate or final products of the pathway into a metabolite or other compound that cannot be used in the desired pathway. Therefore, if the activity of the native enzyme were reduced or completely disappeared, the intermediates produced would be more readily available for incorporation into the desired product. In some cases, where ablation of protein expression may be of interest, alteration occurs in proteins involved in the pleiotropic response to the drug, including, but not limited to, ATP-binding cassette (ABC) transporters. , multidrug resistance (MDR) pumps and associated transcription factors. These proteins are involved in the export of TA molecules and TA precursors to the culture medium, so the deletion controls the export of the compounds to the medium, making them more available for incorporation into the desired product. In some embodiments, deletions of host cell genes of interest include genes associated with the unfolded protein response and endoplasmic reticulum (ER) proliferation. Such gene deletions may lead to enhanced TA production. Expression of cytochrome P450s can induce the unfolded protein response and can lead to ER proliferation. Deleting genes associated with these stress responses may control or reduce the overall load on the host cell and improve the performance of the pathway. Genetic alterations may also include modifying the promoters of endogenous genes to increase expression and / or introduce additional copies of endogenous genes. Examples of this include the construction / use of strains that overexpress the endogenous yeast NADPH-P450 reductase Ncplp to increase the activity of heterologous P450 enzymes. Furthermore, endogenous enzymes such as Spelp, Fmslp, Carlp, Arg2p, Aro8p, Aro9p, Pha2p, Ugplp, and Leu2p that are directly involved in the synthesis of intermediate metabolites may also be overexpressed. Heterologous coding sequences of interest include, but are not limited to, sequences that encode enzymes, whether wild type or equivalent sequences, that are typically responsible for the production of TAy precursors in plants. In some cases, the enzymes encoded by the heterologous sequences may be any of the enzymes of the TA pathway, and may be from any convenient source. The choice and number of enzymes encoded by the heterologous coding sequences for the particular synthesis pathway can be selected based on the desired product. In certain embodiments, the host cells of the present invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more heterologous coding sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 heterologous coding sequences. In some cases, the polypeptide sequences encoded by the heterologous coding sequences are as reported in GENBANK. Enzymes of interest include, but are not limited to, the enzymes described herein and those shown in Table 1. Host cells may include any combination of the listed enzymes, from any source. Unless otherwise indicated, accession numbers in Table 1 refer to GenBank. Some accession numbers refer to the Saccharomyces Genome Database (SGD), which is available on the global web at yeastgenome.org. In some embodiments, the host cell (e.g., a yeast strain) is engineered for selective production of a TA of interest by localizing one or more enzymes to a compartment of the cell. In some cases, an enzyme may be located in the host cell so that the compound produced by this enzyme is spontaneously rearranged or converted by another enzyme to a desirable metabolite before reaching a localized enzyme that can convert the compound to an undesirable metabolite. . The spatial distance between two enzymes can be selected to prevent one of the enzymes from acting directly on a compound to produce an undesirable metabolite and to restrict the production of undesirable end products (e.g., an undesirable opioid byproduct). In some other cases, an enzyme may be located 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 parameter for its activity. than the compartment in which the enzyme is naturally found. In certain cases, an enzyme can be located in a specific compartment within the host cell so that the intracellular trafficking pathway by which the enzyme is transported to said compartment provides 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 multienzyme complex formation. In certain embodiments, any of the enzymes described herein, either individually or together with a second enzyme, can be located in any convenient compartment in the host cell, including, but not limited to, an organelle, endoplasmic reticulum, golgi, vacuole. , nucleus, plasma membrane, mitochondria, peroxisome, periplasm, the lumen of any of the organelles mentioned above, or the membrane enclosing or associated with any of the organelles mentioned above. In cases where one or more enzymes are located in a membrane associated with any of the organelles mentioned above, the enzyme can be oriented so that the catalytic domain of the enzyme faces the cytosol, the lumen of the organelle and / or any another intracellular space. In some embodiments, the host cell includes one or more of the enzymes that include a localization tag. Any convenient label can be used. In some cases, the localization tag is a peptide sequence that binds to the N-terminus and / or C-terminus of the enzyme. Any convenient method of attaching a tag to the enzyme can be used. In some cases, the localization tag is derived from an endogenous yeast protein. Such tags may provide a route to a variety of yeast organelles including, but not limited to, the endoplasmic reticulum (ER), Golgi apparatus (GA), mitochondria (MT), plasma membrane (PM), peroxisome (POX). ) and vacuole (V). In certain embodiments, the label is a routing label to the RE (eg, ER1). In certain embodiments, the tag is a vacuole tag (eg, V1). In certain embodiments, the tag is a plasma membrane tag (eg, P1). In certain embodiments, the tag is a peroxisome-targeted sequence (eg, PTS1). In certain instances, the tag includes or is derived from a transmembrane domain within the class of tail-anchored proteins. In some embodiments, the localization tag locates the enzyme outside of an organelle. In certain embodiments, the localization tag locates the enzyme within an organelle. In some embodiments, the localization tag localizes the enzyme such that one or more portions of the enzyme are located both inside and outside an organelle. In some embodiments of the invention, the host cell is modified by the expression of one or more coding sequences that encode one or more enzymes comprising a localization tag described above. In certain embodiments, the host cell is 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 decarboxylases, putrescine N-methyltransferases, pyrrolidine ketide synthases, tropinone reductases, MA / 1 1 phenylpyruvate reductases, UDP-glucosyltransferases and 2-oxoglutarate-dependent dioxygenases, such as hyoscyamine 6p-hydroxylase / dioxygenase. In certain embodiments, the host cell is modified by the expression of one or more heterologous coding sequences such that one or more enzymes are expressed on the ER membrane. Examples of such enzymes include, but are not limited to, cytochromes P450 such as tropinone synthase (CYP82M3) and littorin mutase (CYP80F1), and NADP+cytochrome P450 reductases. In certain embodiments, the host cell is modified by the 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 synthases. In other embodiments, the host cell is modified by the 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 the expression of one or more heterologous coding sequences such that one or more enzymes are expressed in the lumen of the vacuole. Examples of such enzymes include, but are not limited to, serine carboxypeptidase-type acyltransferases such as littorin synthase and engineered variants thereof. In other embodiments, the host cell is modified by the expression of one or more heterologous coding sequences such that one or more enzymes or proteins are expressed in the membrane of the vacuole. Examples of such proteins include, but are not limited to, multidrug and toxin extrusion transporters, nitrate / peptide family transporters, and ATP-binding cassette transporters. In other embodiments, the host cell is modified by the expression of one or more heterologous coding sequences such that one or more enzymes or proteins are expressed on the plasma membrane. Examples of such proteins include, but are not limited to, ATP-binding cassette transporters, pleiotropic drug resistance transporters, and multidrug resistance transporters. In some cases, the expression of each type of enzyme is increased through additional gene copies (i.e., multiple copies), increasing intermediate accumulation and / or production of TAs of interest. Embodiments of the present invention include increased production of TA of interest in a host cell by simultaneously expressing multiple species variants of one or more enzymes. In some cases, additional gene copies of one or more enzymes are included in the host cell. Any convenient method can be used, including multiple copies of a heterologous coding sequence for an enzyme in the host cell. In some embodiments, the host cell includes multiple copies of a heterologous coding sequence for an enzyme, such as 2 or more, 3 or more, 4 or more, 5 or more, or even 10 or more copies. In certain embodiments, the host cell includes multiple copies of heterologous coding sequences for one or more enzymes, such as multiple copies of two or more, three or more, four or more, etc. In some cases, multiple copies of the heterologous coding sequence of an enzyme are derived from two or more different source organisms compared to the host cell. For example, the host cell may include multiple copies of a heterologous coding sequence, each of the copies being derived from a different source organism. As such, each copy may include some explicit sequence variations based on differences between species of the enzyme of interest that is encoded by the heterologous coding sequence. In some embodiments of the host cell, the heterologous coding sequence is from a source organism selected from the group consisting of Escherichia coli, Bacillus coagulans, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus spp, Wickerhamia fluorescens, Aequoria spp, Discosoma spp, Arabidopsis thaliana, Avena sativa, Solanum lycopersicum, Solanum tuberosum, Nicotiana tabacum, Nicotiana benthamiana, Atropa belladonna, Hyoscyamus niger, Hyoscyamus muticus, Datura stramonium, Datura metel, Datura innoxia, Duboisia myoporoides, Anisodus luridus, Anisodus tanguticus, Anisodus acutangulus, Brugmansia ar borea, Brugmansia x candida, Brugmansia sanguine, Erythroxylum coca, Cochlearia officinalis, Solanum spp, Nicotiana spp, Atropa spp, Hyoscyamus spp, Datura spp, Duboisia spp, Anisodus spp, Brugmansia spp, Erythroxylum spp, or Cochlearia spp. In certain cases, the heterologous coding sequence is from a source organism selected from A. belladonna, H. niger, and D. stramonium. In some embodiments, the host cell includes a heterologous coding sequence from one or more of the source organisms described in Table 1. The engineered host cell medium can be sampled and monitored for production of the TAs of interest. The TAs 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 fragmentation patterns and MS / MS, and quantification or measurement of the compound can be achieved by trace LC peaks of known retention time and / or MS peak analysis. EIC by reference to the corresponding LC-MS analysis of a known quantity of a compound standard. Methods Process steps As summarized above, aspects of the invention include methods for preparing a tropane alkaloid (TA) of interest. As such, aspects of the invention include culturing a host cell under conditions in which one or more modifications of the host cell (e.g., as described herein) are functionally expressed such that the cell converts the starting compounds. of interest in TA products of interest or precursors thereof (for example, pre-esterification TAs). Also provided are methods that include culturing a host cell under conditions suitable for protein production, such that one or more heterologous coding sequences are functionally expressed and convert starting compounds of interest into TA products of interest. In some cases, the method is a method of preparing a tropane alkaloid (TA), which includes culturing a host cell (e.g., as described herein); adding a starting compound to the cell culture; and recover the TA from the cell culture. In some embodiments of the method, the starting compound, the TA product and the host cell are described by one of the entries in Table 1. The fermentation media may contain suitable carbon substrates. The carbon source suitable for performing the methods of this disclosure can encompass a wide variety of carbon-containing substrates. Suitable substrates may include, without limitation, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or a combination thereof. . In some cases, unpurified mixtures of renewable raw materials can be used (e.g., corn 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 can be used, for example, methylamine, glucosamine and amino acids. Any convenient host cell culture method can be employed to produce the TA precursors and downstream TAs of interest. The particular protocol that is employed may vary, for example, depending on the host cell, heterologous coding sequences, desired TA precursors and downstream TAs of interest, etc. The cells may be present in any convenient environment, such as an environment in which the cells are capable of expressing one or more functional heterologous enzymes. In vitro, as used herein, simply means outside a living cell, regardless of the location of the cell. As used herein, the term in vivo indicates the interior of a living cell, regardless of the location of the cell. In some embodiments, cells are cultured under conditions conducive to expression of the enzyme and with appropriate substrates available to allow production of TA and downstream TA precursors of interest in vivo. In some embodiments, functional enzymes are extracted from the host for the production of TAs under in vitro conditions. In some cases, the host cells are reattached to a multicellular host organism. Host cells are in any growth phase, including, but not limited to, stationary phase and logarithmic growth phase, etc. Additionally, the crops themselves may be continuous crops or they may be batch crops. The cells can be cultured in an appropriate fermentation medium at a temperature between 2040 °C. Cells can be grown with shaking at any convenient speed (eg, 200 rpm). Cells can be cultured at a suitable pH. Suitable pH ranges for fermentation can be between pH 5-9. Fermentations can be carried out under aerobic, anaerobic or microaerobic conditions. Any suitable growth medium can be used. Suitable growth media may include, without limitation, common commercially prepared media such as synthetically defined minimal media (SD) or rich yeast extract peptone dextrose (YEPD) medium. Any other rich, defined or synthetic growth medium appropriate for the microorganism can be used. Cells can be grown in a container of essentially any size and shape. Examples of vessels suitable for performing the methods of this disclosure may include, without limitation, multi-well stir plates, test tubes, flasks (baffled and unbaffled), and bioreactors. Culture volume can range from 10 microliters to over 10,000 liters. This may include the addition of agents to the growth medium that are known to modulate metabolism in a manner desirable for alkaloid production. In a non-limiting example, adenosine 2'3'-cyclic monophosphate can be added to the growth medium to modulate catabolite repression. Any cell culture condition suitable for a particular cell type can be used. In certain embodiments, host cells that include one or more modifications are cultured under standard or easily optimized conditions, with standard cell culture media and supplements. As an example, standard growth media when selective pressure is not required for plasmid maintenance may contain 20 g / L yeast extract, 10 g / L peptone, and 20 g / L dextrose (YPD). Plasmid-containing host cells are grown in complete synthetic (SC) medium containing 1.7 g / L yeast nitrogen base, 5 g / L ammonium sulfate and 20 g / L dextrose, supplemented with the appropriate amino acids necessary for growth and selection. Alternative carbon sources that may be useful for the expression of inducible enzymes include, but are not limited to, sucrose, raffinose, and galactose. The cells are grown at any convenient temperature (e.g. 30°C) with shaking at any convenient speed (e.g. 200 rpm) in a container, e.g. in test tubes or flasks in volumes ranging from 1-1000. mL or more in the laboratory. Culture volumes can be scaled for growth in larger fermentation vessels, for example as part of an industrial process. The industrial fermentation process 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. A batch fermentation is a closed system, in which the composition of the medium is established at the beginning of the fermentation and is not altered during the fermentation process. The desired organisms are inoculated into the medium at the beginning of fermentation. In some cases, batch fermentation is done with modifications to the system to control factors such as pH and oxygen (but not carbon) concentration. In this type of fermentation system, the biomass and metabolite compositions of the system change continuously during the course of fermentation. Cells typically go through a dormancy phase, then a logarithmic phase (high growth rate), then a stationary phase (reduced or arrested growth rate), and finally a death phase (if left untreated). . A fed-batch fermentation is similar to a batch fermentation, except that the substrate is added at intervals to the system during the course of the fermentation process. Feed-batch systems are used to reduce the impact of catabolite repression on host cell metabolism and in other circumstances where it is desired to have limited amounts of substrate in the growth medium. A continuous fermentation is an open system, in which a defined fermentation medium is continuously added to the 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 cell loss due to medium removal must be balanced with the growth rate in the fermentation. Continuous fermentations are generally operated under conditions where the cells have a constant, high cell density. Continuous fermentations allow the modulation of one or more factors that affect the concentration of the white product and / or cell growth. The liquid medium may include, but is not limited to, a defined rich or synthetic medium having an additive component described above. Media components can be dissolved in water and sterilized by heat, pressure, filtration, radiation, chemicals, or any combination thereof. Various components of the medium can be prepared separately and sterilized, and then combined in the fermentation vessel. The culture medium can be buffered to help maintain a constant pH during fermentation. Process parameters including temperature, dissolved oxygen, pH, agitation, aeration rate, and cell density can be monitored or controlled during the course of fermentation. For example, the temperature of a fermentation process can be monitored using a temperature probe immersed in the culture medium. Growing temperature can be controlled at the set point by regulating the jacket temperature. The water can be cooled in an external chiller and then flow to the bioreactor control tower and circulate to the jacket at the temperature required to maintain the set point temperature in the vessel. Additionally, a gas flow parameter can be monitored in a fermentation process. For example, gases can flow into the medium through a disperser. Gases suitable for the methods of this disclosure may include compressed air, oxygen and nitrogen. Gas flow can be at a fixed rate or regulated to maintain a dissolved oxygen set point. The pH of a culture medium can also be monitored. In the examples, the pH can be monitored using a pH probe that is immersed in the culture medium inside the container. If pH control is in effect, the pH can be adjusted by acid and base pumps that add each solution to the medium at the required ratio. Acid solutions used to control pH can be sulfuric acid or hydrochloric acid. The basic solutions used to control pH can be sodium hydroxide, potassium hydroxide or ammonium hydroxide. Additionally, dissolved oxygen can be monitored in a culture medium using a dissolved oxygen probe immersed in the culture medium. If dissolved oxygen regulation is in effect, the oxygen level can be adjusted by increasing or decreasing the stirring 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. The stirring speed can also be monitored in a fermentation process. In examples, the stirrer motor may drive a stirrer. The agitator speed can be set at a constant rpm during fermentation or can be dynamically regulated to maintain a set dissolved oxygen level. Additionally, turbidity can be monitored in a fermentation process. In the examples, cell density can be measured using a turbidity probe. Alternatively, cell density can be measured by taking samples from the bioreactor and analyzing them in a spectrophotometer. Furthermore, samples can be removed from the bioreactor at time intervals through a sterile sampling apparatus. Samples can be analyzed for alkaloids produced by host cells. Samples can also be analyzed for other metabolites and sugars, depletion of culture medium components, or cell density. In another example, a raw material parameter can be monitored during a fermentation process. In particular, raw materials that include sugars and other carbon sources, nutrients and cofactors that can be added to the fermentation by an external pump. Other components may also be added during fermentation including, without limitation, antifoams, salts, chelating agents, surfactants and organic liquids. Any suitable codon optimization technique for optimizing the expression of heterologous polynucleotides in host cells can be adapted for use in target host cells and methods, see, for example, Gustafsson, C. etal. (2004) Trends Biotechnol, 22, 346-353, which is incorporated by reference in its entirety. The objective method may also include the addition of a starting compound to the cell culture. Any convenient addition method can be adapted for use in the target methods. The cell culture may be supplemented with a sufficient amount of the starting materials of interest (e.g., as described herein), for example, an amount of mM to μΜ such as between about 1-5 mM of a starting compound. . It is understood that the amount of starting material added, the time and rate of addition, the shape of the added material, etc., may vary according to a variety of factors. The starting material can be added pure or previously dissolved in a suitable solvent (for example, cell culture medium, water or an organic solvent). The starting material can be added in concentrated form (e.g., 10 times 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., hours or days). Methods for isolating products from the fermentation medium Target methods may also include recovering the TA of interest from the cell culture. Any convenient separation and isolation method (e.g., chromatography methods or precipitation methods) can be adapted for use in the target methods to recover the TA of interest from cell culture. Filtration methods can be used to separate soluble from insoluble fractions of the cell culture. In some cases, liquid chromatography methods (e.g., reverse phase HPLC, size exclusion, normal phase chromatography) 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. The alkaloids produced can be isolated from the fermentation medium using methods known in the art. Various recovery steps can be performed immediately after (or in some cases, during) fermentation for initial recovery of the desired product. Through these steps, alkaloids (e.g., TA) can be separated from cells, cellular residues and debris, and other nutrients, sugars, and organic molecules can remain in the spent culture medium. This process can be used to produce a TA-enriched product. In one example, a product stream having a tropane alkaloid (TA) product is formed by providing engineered yeast cells and a feedstock including nutrients and water to a batch reactor. Engineered yeast cells may have at least one modification selected from the group consisting of: a mutation that mitigates feedback inhibition in a biosynthetic enzyme gene native to the cell; a transcriptional modulation modification of a cell native biosynthetic enzyme gene; and an inactivating mutation in an enzyme native to the cell. When the engineered yeast cells are inside the batch reactor, the engineered yeast cells can undergo fermentation. In particular, the engineered yeast cells can be subjected to fermentation by incubating the engineered yeast cells for a period of time of at least about 5 minutes to produce a solution comprising the TA product and the cellular material. Once the engineered yeast cells have undergone fermentation, at least one separation unit can be used to separate the TA product from the cellular material to provide the product stream comprising the TA product. In particular, the product stream may include the TA product, as well as additional components, such as a clarified yeast culture medium. Additionally, a TA product may comprise one or more TAs of interest, such as one or more TA compounds. Different methods can be used to remove cells from a bioreactor medium that includes a TA of interest. In the examples, the cells may be removed by sedimentation over time. This sedimentation process can be accelerated by cooling or adding fining agents 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 base of the reactor. Alternatively, the 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 using a continuous extractor. If some valuable TAs of interest are present inside the cells, the cells can be permeabilized or lysed and cellular debris can be removed by any of the methods described above. Agents used to permeabilize cells may include, without limitation, organic solvents (e.g., DMSO) or salts (e.g., lithium acetate). Methods to lyse cells may include the addition of surfactants such as sodium dodecyl sulfate, or mechanical disruption by bead milling or sonication. MA / t / ZUZ1 1 The TAs of interest can be extracted from the clarified spent culture medium by liquid-liquid extraction by the addition of an organic liquid that is not miscible 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, oleic alcohol, ethyl butyrate. The organic liquid can be added up to 10% or up to 100% of the volume of the aqueous medium. In some cases, the organic liquid can be added at the beginning of fermentation or at any time during fermentation. This extractive fermentation process can increase the yield of TAs of interest from host cells by continuously removing TA precursors or TAs to the organic phase. Agitation can cause the organic phase to form an emulsion with the aqueous culture medium. Methods to promote separation of the two phases into distinct layers may include, without limitation, the addition of a demulsifier or nucleating agent, or adjustment of pH. The emulsion can also be centrifuged to separate the two phases, for example by continuous conical plate centrifugation. Alternatively, the organic phase can be isolated from the aqueous culture medium so that it can be physically removed after extraction. For example, the solvent can be encapsulated in a membrane. In the examples, TAs of interest can be extracted from a fermentation medium using adsorption methods. In particular, TAs of interest can be extracted from the clarified spent culture medium by adding a resin such as Amberlite® XAD4 or another agent that removes TAs by adsorption. The TAs 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. TAs of interest can also be extracted from a fermentation medium using filtration. At high pH, ​​the TAs of interest can form a crystalline precipitate in the bioreactor. This precipitate can be removed directly by filtration through a filter, membrane or other porous material. The precipitate can also be collected by centrifugation and / or decantation. The extraction methods described above can be carried out in situ (in the bioreactor) or ex situ (for example, in an external loop through which the media flows out of the bioreactor and contacts the extraction agent, then is recirculated back to the container). Alternatively, extraction methods can be performed after the fermentation is completed using the clarified medium extracted from the bioreactor vessel. Methods for purifying products from solutions enriched with alkaloids Subsequent purification steps may involve treating the post-fermentation TA precursor or TA-enriched product using methods known in the art to recover individual product species of interest to high purity. In one example, TA precursors or TAs extracted in an organic phase 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 suitable pH. In a further example, the TA precursors or TAs can be extracted from the organic phase by addition of an aqueous solution at a suitable pH that promotes extraction of the TA precursors or TAs into the aqueous phase. The aqueous phase can then be removed by decantation, centrifugation, or another method. The TA or TA precursor-containing solution can be further treated to remove metals, for example by treating it with a suitable chelating agent. The solution containing TA precursor or TA can be further treated to remove other impurities, such as proteins and DNA, by precipitation. In one example, the TA or TA precursor-containing solution is treated with an appropriate precipitation agent such as ethanol, methanol, acetone or isopropanol. In an alternative example, DNA and protein can be removed by dialysis or by other size exclusion methods that separate smaller alkaloids from contaminating biological macromolecules. In other examples, the solution containing TA precursor, TA or modified TA can be extracted to high purity by continuous cross-flow filtration using methods known in the art. If the solution contains a mixture of TA precursors or TAs, it can be subjected to an acid-base treatment to produce individual TAs of the species of interest using methods known in the art. In this process, the pH of the aqueous solution is adjusted to precipitate individual TA precursors or the TAs at their respective pKa. For small-scale high-purity preparations, TA precursors or TAs can be purified in a single step by liquid chromatography. Yeast-derived alkaloid APIs versus plant-derived APIs Clarified yeast culture medium (CYCM) can contain a variety of impurities. The clarified yeast culture medium can be dehydrated by vacuum and / or heat to produce an alkaloid-rich powder. This product is analogous to solanaceous leaf concentrate (CNL), which is used by active pharmaceutical ingredient (API) manufacturers for the extraction of tropane alkaloids to be subjected to subsequent 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 the impurities in these two products that may be specific to CYCM or CNL or may be present in both. By testing a product of unknown origin for a subset of these impurities, one skilled in the art could determine if the product is from a yeast-producing host or from a plant. API grade pharmaceutical ingredients are highly purified molecules. As such, impurities that could indicate the plant or yeast origin of an API (such as those listed in Tables 2 and 3) may not be present in that API stage of the product. In fact, many of the yeast strain-derived API products of the present invention may be largely indistinguishable from traditional plant-derived APIs. In some cases, however, conventional alkaloid compounds can undergo chemical modifications using chemical synthesis approaches that may appear as chemical impurities in plant-based products requiring such chemical modifications. For example, chemical derivatization can often result in a pool of impurities related to chemical synthesis processes. In certain situations, these modifications can be made biologically in the yeast production platform, thereby preventing some of the impurities associated with chemical derivatization from being present in the yeast-derived product. In particular, these chemically derived product impurities may be present in an API product that is produced using chemical synthesis processes, but may be absent in an API product that is produced using a yeast derived product. Alternatively, if a yeast-derived product is mixed with a chemically derived product, the resulting impurities may be present, but in a smaller amount than would be expected in an API that only or primarily contains chemically derived products. In this example, by analyzing the API product for a subset of these impurities, a person skilled in the art could determine whether the product originated from a yeast production host or from the traditional chemical derivatization route. Non-limiting examples of impurities that may be present in chemically derivatized tropane alkaloid APIs, but not in biosynthesized APIs, include hydrogen halides such as hydrogen chloride, hydrogen iodide and hydrogen bromide formed by N-alkylaclone chemistry, such as N-methylation and N-butylation of hyoscyamine and scopolamine. However, in the case where the yeast-derived compound and the plant-derived compound are subjected to chemical modification by chemical synthesis approaches, the same impurities associated with the chemical synthesis process can be expected in the products. In such a situation, the starting material (eg CYCM or CNL) can be analyzed as described above. Host cell design methods Also included are methods for designing host cells in order to produce the TAs of interest or precursors thereof. Insertion of DNA into host cells can be accomplished using any convenient method. The methods are used to insert the heterologous coding sequences into host cells so that the host cells functionally express the enzymes and convert the starting compounds of interest into TA products of interest. Any suitable promoter can be used in the host cells and target methods. Promoters that drive expression of heterologous coding sequences can be constitutive or inducible promoters, as long as the promoters are active in the host cells. Heterologous coding sequences can be expressed from their native promoters or non-native promoters can be used. These promoters can be of low to high resistance in the host in which they are used. Promoters can be regulated or constitutive. In certain embodiments, promoters are used that are not repressed by glucose, or only slightly repressed by the presence of glucose in the culture medium. Promoters of interest include, but are not limited to, promoters of glycolytic genes such as the promoter of the B. subtilis tsr gene (which encodes the promoter region of the fructose bisphosphate aldolase gene) or the promoter of the yeast S gene. cerevisiae encoding glyceraldehyde 3-phosphate dehydrogenase (GPD, GAPDH or TDH3), the baker's yeast ADH1 promoter, promoters induced by phosphate starvation, such as the yeast PH05 promoter, the alkaline phosphatase promoter of B. licheniformis, yeast inducible promoters such as Gal1-10, Gal1, GalL, GalS, the Met25 repressible promoter, teto, and constitutive promoters such as the glyceraldehyde 3phosphate dehydrogenase (GPD) promoter, alcohol dehydrogenase (ADH) promoter, translation elongation factor-1-α (TEF) promoter, cytochrome c-oxidase (CYC1) promoter, MRP7 promoter, phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI), etc. Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, spheroids and thyroid hormones can also be used and include, but are not limited to, the glucorticoid-responsive element (GRE) and the hormone-responsive element. thyroid (TRE). These and other examples are described in US Patent No. 7,045,290, which is incorporated by reference, including the references cited therein. Additional vectors containing constitutive or inducible promoters, such as a factor, alcohol oxidase and PGH, may be used. Furthermore, any promoter / enhancer combination (according to the EPDB eukaryotic promoter database) could also be used to drive gene expression. Any appropriate promoter suitable for the host cell can be selected, for example, E. coli. Promoter selection can also be used to optimize transcription and therefore enzyme levels to maximize production while minimizing energy resources. Any suitable vector can be used in the host cells and target methods. Vectors of interest include vectors for use in yeast and other cells. Yeast vector types can be divided into 4 general categories: integrating vectors (Ylp), high copy number autonomously replicating vectors (YEp or 2p plasmids), low copy number autonomously replicating vectors (YCp or 2p plasmids). centromeric) and vectors for the cloning of large fragments (YAC). The vector DNA is introduced into prokaryotic or eukaryotic cells by any convenient transformation or transfection technique. Utility The host cells and methods of the invention, for example, as described above, find use in a variety of applications. Applications of interest include, but are not limited to: research applications and therapeutic applications. The methods of the invention find use in a variety of different applications, including any suitable application in which the production of TAs is of interest. Host cells and targeting methods find use in a variety of therapeutic applications. Therapeutic applications of interest include those applications in which the preparation of pharmaceutical products that include TA is of interest. The host cells described herein produce tropane alkaloid precursors (TA precursors) and the TAs of interest. Tropinone and tropine are the main branch point intermediates of interest in the synthesis of TAs, including design efforts to produce end products such as medicinal TA products. Target host cells can be used to produce TA precursors from simple and inexpensive starting materials that can find use in the production of TAs of interest, including tropinone, tropine, and TA end products. As such, target host cells find use in delivering therapeutically active TAs or precursors thereof. In some cases, host cells and methods find use in the production of commercial-scale quantities of TAs or precursors thereof when chemical synthesis of these compounds is low-yield and is not a viable means for large-scale production. scale. In certain cases, the host cells and methods are used in a fermentation facility that would include bioreactors (fermenters) of, for example, 5,000-200,000 liters capacity that allow rapid production of the TAs of interest or precursors thereof for therapeutic products. Such applications may include industrial scale production of TAs of interest from fermentable carbon sources such as cellulose, starch and free sugars. Host cells and targeting methods find use in a variety of research applications. Host cell and targeting methods can be used to analyze the effects of a variety of enzymes on the biosynthetic pathways of a variety of TAs of interest or precursors thereof. Furthermore, host cells can be engineered to produce TAs or precursors thereof that find use in bioactivity tests of interest in yet unproven therapeutic functions. In some cases, engineering host cells to include a variety of heterologous coding sequences encoding a variety of enzymes elucidates high-throughput biosynthetic pathways toward TAs of interest or precursors thereof. In certain cases, research applications include the production of precursors for therapeutic molecules of interest that can then be chemically modified or derivatized to obtain the desired products or for the evaluation of further therapeutic activities of interest. In some cases, host cell strains are used to detect enzymatic activities that are of interest in such pathways, which may lead to the discovery of enzymes by converting TA metabolites produced in these strains. Host cells and targeting methods can be used as a production platform for specialized metabolites in plants. Host cells and targeting methods can be used as a platform for drug library development as well as plant enzyme discovery. For example, host cell and targeting methods may find use in developing drug libraries based on natural products, taking yeast strains that produce interesting scaffold molecules, such as hyoscyamine and scopolamine, and further functionalizing the compound structure. by combinatorial biosynthesis or by chemical means. By producing drug libraries in this way, any potential drug success is already associated with a production host that is amenable to large-scale cultivation and production. As another example, these host cells and targeting methods may find use in the discovery of plant enzymes. Target host cells provide a clean environment of defined metabolites to express expressed sequence tag (EST) libraries in plants to identify novel enzymatic activities. Host cells and targeting methods provide expression methods and culture conditions for functional expression and increased activity of plant enzymes in yeast. Kits and systems Aspects of the invention further include kits and systems, where the kits and systems may include one or more components used in the methods of the invention, for example, host cells, starting compounds, heterologous coding sequences, vectors, culture medium, etc., as described herein. In some embodiments, the target kit includes a host cell (e.g., as described herein) and one or more components selected from the following: starting compounds, a heterologous coding sequence, and / or a vector that includes the same vectors. , growth raw materials, components suitable for use in expression systems (e.g. cells, cloning vectors, multiple cloning sites (MCS), bidirectional promoters, an internal ribosome entry site (IRES), etc.) and a culture medium. Any of the components described herein may be provided in the kits, for example, host cells including one or more modifications, starting compounds, culture medium, etc. A variety of components suitable for use in the manufacture and use of heterologous coding sequences, cloning vectors and expression systems may find use in the target kits. Kits may also include tubing, buffer solutions, etc. and instructions for use. The various reagent components of the kits may be present in separate containers, or some or all of them may be pre-combined into a single container reagent mixture, as desired. Systems for producing a TA of interest are also provided, where the systems may include engineered host cells that include one or more modifications (e.g., as described herein), starting compounds, culture medium, a fermenter, and production equipment. fermentation, for example, an apparatus suitable for maintaining the growth conditions of host cells, sampling and control equipment and components, and the like. A variety of components suitable for use in large-scale fermentation of yeast cells may find use in the target systems. In some cases, the system includes components for large-scale fermentation of engineered host cells and monitoring and purification of TA compounds produced by host cell fermentations. In certain embodiments, one or more starting compounds (e.g., as described herein) are added to the system, under conditions whereby the engineered host cells in the fermenter produce one or more desired TA products or precursors thereof. themselves. In some cases, host cells produce a TA of interest (e.g., as described herein). In some cases, the TA products of interest are medicinal TA products, such as hyoscyamine, N-methylhyoscyamine, anisodamine, scopolamine, N-methylscopolamine, and N-butylscopolamine. In some cases, the system includes means for monitoring and / or analyzing one or more TA compounds or precursors thereof produced by the target host cells. For example, an LC-MS analysis system as described herein, a chromatography system or any convenient system in which the sample can be analyzed and compared to a standard, for example, as described herein. The fermentation medium can be monitored at any convenient time before and during fermentation by sampling and analysis. When the conversion of the starting compounds to TA products or precursors of interest is complete, fermentation can be stopped and purification of the TA products can be performed. As such, in some cases, the target system includes a purification component suitable for purifying the TA products or precursors of interest from the host cell medium in which it is produced. The purification component may include any convenient means that can be used to purify the TA products or fermentation precursors, 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 target system provides for the production and isolation of TA fermentation products of interest after input of one or more starting compounds to the system. The following examples are set forth in order to provide those skilled in the art with a full disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors consider their invention nor are they intended to represent that the experiments The following are all or the only experiments carried out. Efforts have been made to ensure accuracy with respect to numbers used (e.g. quantities, temperature, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Examples of methods The following section provides examples of methods and procedures that can be used to construct, grow and test microbial strains, such as yeast strains, for the production of TA precursors and TAs, as well as to perform fermentations of such strains to produce precursors. of TA and the TAs. Also included are examples of methods, procedures and materials that can be used to generate the DNA sequences necessary for the modification of microbial hosts and to introduce desired DNA sequences into microbial hosts. Standards and chemical compounds. Chemical standards of TA precursors and TAs to verify the identity and quantify the metabolites produced by the engineered host cells can be purchased from commercial suppliers. For example, putrescine dihydrochloride, Nmethylputrescine, hygrin, tropinone, and tropin can be purchased from Santa Cruz Biotechnology (Dallas, TX). 4-(methylamino)butyric acid hydrochloride can be purchased from Sigma (St. Louis, MO). γmethylaminobutyraldehyde (4MAB) diethyl acetal and littorin can be purchased from Toronto Research Chemicals (Toronto, ON). A chemical standard for NMPy can be synthesized by deprotecting one volume of the diethyl acetal with five volumes of HCl2 M at 60 °C for 30 min as described previously (see Feth, F., Wray, V. and Wagner, K. G. Determination of methylputrescine oxidase by high performance liquid chromatography. Phytochemistry 24, 1653-1655 (1985)), incubating overnight at room temperature and then washing the resulting concentrate twice with three volumes of diethyl ether to remove residual organic impurities. Construction of plasmids. Oligonucleotides used for generation of novel DNA sequences by polymerase chain reaction (PCR) and for DNA sequencing can be obtained from a DNA synthesis company, such as IDT DNA, Twist Bioscience, or Stanford Protein and Nucleic Acid Facility (Stanford, CA). Native yeast genes can be amplified from S. cerevisíae genomic DNA by colony PCR (see Kwiatkowski, T. J., Zoghbi, H. Y., Ledbetter, S. A., Ellison, K. A. and Chinault, A. C. Rapid identification of yeast artificial chromosome clones by matrix pooling and crude lysate PCR. Nucleic Acids Res. 18, 7191 (1990)). Gene sequences for heterologous enzymes can be codon optimized to improve expression in S. cerevisíae using suitable codon optimization software, such as GeneArt GeneOptimizer software (Thermo Fisher Scientific). Heterologous genetic sequences can be synthesized as double-stranded linear DNA fragments by a commercial DNA synthesis company. Two types of plasmids can be used for gene expression in yeast: direct expression (DE) plasmids to test biosynthetic genes of interest and yeast integration (Yl) plasmids to provide a template for genomic integration of promoter-cassettes. selected gene-terminator. DE plasmids comprise a gene of interest flanked by a constitutive promoter and terminator, a low-copy CEN6 / ARS4 yeast origin of replication, and an auxotrophic selection marker. DE plasmids can be constructed by PCR amplification of the genes of interest to add 5' and 3' restriction sites using overhang primers, digesting the PCR products or the synthesized gene fragments with the appropriate restriction enzyme pairs ( for example, Spel, BamHI, EcoRI, Pstl or -ccdB, or pAG416GPD-ccdB (see Alberti, S., Gitler, A. D. and Lindquist, S. A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisíae. Yeast 24, 913-9 (2007)) using the T4 DNA ligase. Yl plasmids comprise a gene of interest flanked by a constitutive promoter and terminator, but lack a yeast origin of replication or an auxotrophic selection marker. Yl plasmids can be constructed by linearizing empty retention vectors with suitable promoters and terminators using around-the-horn PCR with primers designed to join at the 3' and 5' ends of the promoter and terminator, respectively. Genes of interest can also be amplified by PCR to add 5' and 3' overhangs with 35-40 bp of homology to the ends of the linearized vector backbones. Gene assembly can then be performed on Yl vectors using Gibson assembly. DE plasmids expressing GFP fusions of biosynthetic enzymes can be prepared by first assembling PCR-amplified DNA fragments that separately encode GFP, the target enzyme, and a Yl vector backbone using Gibson assembly, and subsequently subcloning the GFP fusion constructs. Yl plasmids into DE vectors using restriction enzymes and ligation cloning as described. PCR amplification can be performed using any high-fidelity recombinant DNA polymerase available from commercial suppliers and the linear DNA can be purified using a suitable DNA column purification kit. The assembled plasmids can be propagated in any chemically competent E. coli strain by heat shock transformation and selection in Luria-Bertani (LB) broth or on LB agar plates with carbenicillin (100 pg / mL), kanamycin (50 pg / mL ) or another selection antibiotic. Plasmid DNA can be isolated by alkaline lysis of overnight E. coli cultures at 37°C and 250 rpm in selective LB medium using plasmid purification columns according to the manufacturer's protocol. Plasmid sequences should be verified by Sanger sequencing. Construction of yeast strains. Any suitable laboratory yeast strain can be used as the host organism. The yeast strains described in the examples of the Experimental Section are derived from the parental strain CEN.PK2-1 D (see Entian, K. D. and Kótter, P. 25 Yeast Genetic Strain and Plasmid Collections. Methods Microbiol. 36, 629-666 (2007)), called CEN.PK2. Strains can be grown non-selectively in yeast-peptone medium supplemented with 2% w / v dextrose (YPD medium), yeast nitrogen-based defined medium (YNB) supplemented with a complete mixture of synthetic amino acids (YNB -SC) 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 in YNB medium supplemented with 2% w / v dextrose and the appropriate removal solution (YNB-DO) or in YNB-DO agar plates. Yeast strains that are deficient in acetate metabolism can be grown in the aforementioned media supplemented with 0.1% w / v potassium acetate (i.e., YPAD or YNBA). Genomic modifications can be made to yeast using the CRISPRm method (see Ryan, O. W. et al. Selection of chromosomal DNA libraries using a multiplex CRISPR system. Elife 3, 1-15 (2014)). CRISPRm plasmids express Cas9 from Streptococcus pyogenes and a single guide RNA (guRNA) targeting a locus of interest in the yeast genome, and can be constructed by assembly PCR and Gibson assembly of DNA fragments encoding SpCas9, promoter of tRNA and HDV ribozyme, a 20 nt guide RNA sequence, and a traceRNA and a terminator. For gene insertions, integration fragments comprising one or more genes of interest flanked by unique promoters and terminators can be constructed using PCR amplification and cloned into retention vectors by Gibson assembly. Integration fragments are amplified by PCR using an appropriate high-fidelity DNA polymerase with 40 bp microhomology regions flanking adjacent fragments and / or the yeast genome at the integration site. In the case of gene interruptions, the integration fragments comprise 6 to 8 stop codons in the three reading frames flanked by 40 bp of microhomology to the interruption site, which is located within the first half of the reading frame. open reading. For full gene deletions, the integration fragments comprise an auxotrophic marker gene flanked by 40 bp of microhomology at the deletion site. Each integration fragment is co-transformed with the CRISPRm plasmid directed to the desired genomic site. Positive integrants can be identified by yeast colony PCR, Sanger sequencing and / or functional selection by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Yeast transformations. Yeast strains can be transformed using any suitable method, including heat shock, electroporation and chemical transformation. For example, the yeast strains described in the examples 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. Saturated cultures are rediluted 1:10 to 1:50 in YP(A)D medium and grown for an additional 5-7 hours to reach the exponential phase. Cultures are pelleted by centrifugation at 500xg for 4 min and then washed twice by resuspending the pellet in 50 mM Tris-HCI buffer, pH 8.5. The washed pellets are resuspended in 20 pL of EZ2 solution per transformation and then mixed with 100-600 ng of total DNA and 200 pL of EZ3 solution. The yeast suspensions are incubated at 30 °C with gentle rotation for one hour. For plasmid transformations, transformed yeast is plated directly onto YNB(A)-DO agar plates. For Cas9-mediated chromosome modifications, yeast suspensions are mixed with 1 mL of YP(A)D medium, pelleted by centrifugation at 500xg for 4 min, and then resuspended in 250 pL of fresh YP(A)D medium. The suspensions are then incubated at 30 °C with gentle rotation for an additional two hours to allow the production of G418 resistance proteins and then spread on YP(A)D plates containing 400 mg / L G418 sulfate (geneticin ). The plates are then incubated at 30°C for 48-60 hours to allow colony formation. Point dilution tests. 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 500xg for 4 min and resuspended in sterile Tris-HCI buffer, pH 8.0 at a concentration of 107 cells / mL based on DOeoo. Ten-fold serial dilutions of each strain are then prepared in Tris-HCI buffer and 10 pL of each dilution is plated on pre-warmed YNB(A)-DO plates. Plates are incubated at 30°C and imaged after 48 hours. Growth conditions for metabolite assays. Small-scale metabolite production tests can be performed in YNB(A)-SC or YNB(A)-DO media. Yeast colonies can be inoculated into 300-500 pL of medium and grown in 2 mL deep-well 96 plates covered with gas permeable film for 48-72 hours at 30°C, 460 rpm, and 80% relative humidity. in a shaker. Analysis of metabolite production. Metabolite profiles and titers can be analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). To separate cells from the medium for analysis, fermentation cultures can be pelleted by centrifugation at 3,500xg for 5 min at 12°C and then 100-200 pL aliquots can be removed from the supernatant for direct analysis. Metabolite production can be analyzed by LC-MS / MS using any suitable HPLC device paired with a triple quadrupole mass spectrometer, such as the Agilent 1260 Infinity Binary HPLC and Agilent 6420 Triple Quadrupole mass spectrometer. Chromatography can be performed using a C18 reverse phase column, such as a Zorbax EclipsePlus C18 column (2.1 x 50 mm, 1.8 pm; Agilent Technologies), with 0.1% v / v formic acid in water as mobile phase Solvent A and 0.1% v / v formic acid in acetonitrile as solvent B. The column operates with a constant flow rate of 0.4 mL / min at 40 °C and a sample injection volume of 5 pL. Separation of compounds can be performed using the following gradient: 0.00-0.75 min, 1% B; 0.751.33 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; 3,714.33 min, 95% B; 4.33-4.34 min, 95-1% B; 4.34-5.00 min, equilibrated with 1% B. LC eluent directed to MS 0.01-5 min operating with electrospray ionization (ESI) in positive mode, source gas temperature 350 °C, gas flow 11 L / min , and nebulizer pressure 40 psi. Metabolites can be quantified by integrated peak area based on multiple reaction monitoring (MRM) parameters and standard curves. Fluorescence microscopy. Single 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. Overnight cultures are pelleted by centrifugation at 500xg for 4 min and resuspended in 2 mL of YNB-DO medium with 2% w / v dextrose and then cultured at 30°C and 250 rpm for an additional 4-6 hours. to reach the exponential phase and allow the expressed fluorescent proteins to fully fold. Next, approximately 5-10 pL of culture is placed on a glass microscope slide and covered with a glass coverslip and then imaged using a suitable inverted fluorescence microscope 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 with a CCD camera, and subsequent image analysis can be performed in any suitable scientific image analysis software, such as ImageJ (NIH). Identification of novel gene variants from transcriptome databases. Innovative genes and variants thereof can be identified using sequence alignment-based searches of transcriptome and genome databases. For example, N. tabacum N-methylputrescine oxidase orthologs (NtMPOI) were identified by a tBLASTn search of the D. metel and A. belladonna transcriptomes in the 1000 Plants Project database (see Matasci, N. etal. Data access for the 1,000 Plants (1KP) project. Gigascience 3, 17 (2014)). The coding sequences of the putative genes identified using these search strategies can then be optimized for expression in yeast and then cloned into expression vectors as described above. Structural analysis of enzymes. 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 any suitable homology modeling or de novo structure prediction software. like RaptorX or Rosetta. The resulting protein models can be visualized with any three-dimensional molecular visualization software, such as PyMOL (Schrodinger) or UCSF Chimera. The affinity of enzymes for specific substrates can be analyzed using any suitable ligand docking simulation software, such as AutoDock, SwissDock, GOLD or Glide. Analysis of protein expression in yeast by Western blot. For immunoblot analysis of proteins expressed by yeast, a suitable strain is transformed with an expression vector harboring an epitope-tagged protein of interest. Three days after transformation, transformed colonies are inoculated into 2 mL of YNB-DO medium and grown overnight (-16-20 h) to stationary phase at 30 °C and 460 rpm. Cells are pelleted by centrifugation at 3,000 x g for 5 minutes, resuspended in 200 pL H2O, mixed with 200 pL 0.2 M NaOH, and incubated at room temperature for 5 minutes to allow hydrolysis of cell wall glycoproteins. . Cells are pelleted again at 3,000 x g for 5 minutes, resuspended in 75 pL H2O, mixed with 25 pL 4X NuPAGE LDS sample buffer (Thermo Fisher), and boiled at 95 °C for 3 minutes to lyse the cells. cells. The suspensions are pelleted by centrifugation at 16,000 x g for 5 minutes to remove insoluble remains and the supernatants are transferred to previously refrigerated tubes. For analysis under reducing conditions, protein lysates are mixed with β-mercaptoethanol (10% final concentration) and incubated at 70°C for 10 min. Approximately 20-40 pg of total protein is loaded on NuPAGE Bis-Tris 4-12% acrylamide gels (Thermo Fisher) with Precision Plus Dual Color protein molecular weight marker (BioRad). Electrophoresis is performed in 1X NuPAGE MOPS SDS running buffer at 150 V for 90 minutes. Protein transfer to a nitrocellulose membrane is performed at 15 V for 15 min using a Trans-Blot Semi-Dry apparatus (BioRad) and NuPAGE transfer buffer (Thermo Fisher) according to the manufacturer's instructions. For reducing conditions, the antioxidant NuPAGE (Thermo Fisher) is added at a final concentration of 1X to both the running and transfer buffers. Membranes with transferred protein are washed for 5 minutes in Tris-buffered saline and Tween (TBS-T; 137 mM NaCl, 2.7 mM KCl, 19 mM Tris base, 0.1% Tween 20, pH 7.4) and then blocked. with 5% skim milk in TBS-T for 1 hour at room temperature. Membranes are incubated overnight at 4°C with an appropriate dilution of an HRP-conjugated antibody in TBS-T with 5% milk, washed three times for 5 minutes each with TBS-T, and then visualized using the Western Pico PLUS HRP substrate (Thermo Fisher) and a suitable imager. experimental part A series of specific genetic modifications provide a biosynthetic process in Saccharomyces cerevisiae for the production of TAs from simple and inexpensive raw materials or precursor molecules. Methods are described to construct innovative strains capable of producing the first TA molecules putrescine, N-methylputrescine, 4-methylaminobutanal, N-methylpyrrolinium (NMPy), tropinone, tropin, phenyllactic acid (PLA) and Ι-Ο-β-phenyl lactoylglucose (PLA glucoside) from non-TA precursors or simple raw materials. NMPy is the natural precursor of all known TA molecules. Methods for manipulating the regulation of yeast biosynthetic pathways and for optimizing the production of amino acid-derived TA precursors are also described. Methods for constructing novel strains capable of producing non-medicinal TAs such as pseudotropin alkaloids and callistegins from simple raw materials are described. In addition, methods for constructing novel strains capable of producing medicinal TAs such as hyoscyamine, anisodamine and scopolamine from TA precursors or simple raw materials are described. In addition, methods are described for constructing novel strains capable of producing unnatural TAs, such as cinnamoyltropin, from non-TA precursors or simple starting materials. Example 1. Design of a yeast strain platform for high levels of putrescine production The tropine portion of TAs is derived from the amino acid arginine via the polyamine molecule putrescine. S. cerevisiae strains with enhanced flux through the arginine and polyamine biosynthesis pathways are developed in order to increase intracellular concentrations of TA precursor molecules, including putrescine, NMP, 4MAB, and NMPy. These strains combine genetic modifications to increase the flux of carbon and nitrogen from central metabolism to arginine and polyamine biosynthesis in general, and include the introduction of key heterologous enzymes for further production of the TA precursor, putrescine. Genetic modifications are employed, including introducing mutations that mitigate feedback inhibition in genes encoding native biosynthetic enzymes and regulatory proteins, adjusting the transcriptional regulation of native biosynthetic enzymes, deleting or disrupting genes that they encode enzymes that divert precursor molecules away from the intended pathway, and the introduction of heterologous enzymes for the conversion of endogenous molecules to TA precursor molecules. 1.1) The biosynthetic pathway in the designed strain incorporates the overexpression of native yeast genes involved in arginine metabolism and polyamine biosynthesis (Figure 4). 1.1.1) Examples of native genes overexpressed in yeast include, but are not limited to: glutamate N-acetyltransferase (Arg2p), which catalyzes the first step in the biosynthesis of arginine from glutamate; arginase (Carlp), which removes the guanidinium group from arginine to produce ornithine in the mitochondrial matrix; a mitochondrial membrane transporter (Ort1 p), which exports ornithine from the mitochondrial matrix to the cytosol; ornithine decarboxylase (Spelp), which decarboxylates cytosolic ornithine to putrescine; and a polyamine oxidase (Fmslp), which dealkylates spermine and spermidine MA / 1 1 to turn them into putrescine. 1.1.2) The impact of overexpression of these native enzymes on putrescine production was examined by co-transforming a yeast strain with different combinations of three low-copy plasmids, each of which expressed one of SPE1, ORT1, CAR1, ARG2, FMS1, or blue fluorescent protein (BFP) as a negative control. The putrescine titer accumulated in the extracellular medium of the co-transformed cells after 48 hours of growth in selective media was quantified by LC-MS / MS (Figure 5). Overexpression of SPE1 alone resulted in a 13.4-fold increase in putrescine titer to 23 mg / L. While co-overexpression of CAR1 or ARG2 with SPE1 resulted in 27% and 12% increases in putrescine production relative to SPE1 alone, overexpression of ORT1 with SPE1 caused a 35% decrease in putrescine titer. compared to SPE1. Overexpression of three of SPE1, CARI, ARG2, and FMS1 collectively increased extracellular putrescine titers to 34–35 mg / L. 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). 1.2.1) In addition to the ornithine-dependent pathway found in most plants, animals and fungi, by which putrescine is synthesized via deguanidation of arginine followed by decarboxylation of ornithine, many Bacteria and plants also express an alternative pathway through which arginine is first decarboxylated by arginine decarboxylase (ADC) to produce agmatine. In plants, the guanidine group of agmatine is converted to urea by an iminohydrolase (AIH) to produce N-carbamoylputrescine (NCP), from which the amide group is removed by an amidase (CPA) to produce putrescine (see Patel, J. et al. Dual functioning of plant arginases provides a third route for putrescine synthesis. Plant Sel. 262, 62-73 (2017)). Some bacteria have evolved an enzyme agmatine ureohydrolase (AUH) that allows the direct removal of the guanidine group from agmatine to produce putrescine without an N-carbamoylate intermediate (see Klein, R. D. et al. Reconstitution of a bacterial / plant polyamine biosynthesis pathway in Saccharomyces cerevisiae. Microbiology 145 (Pt 2, 3017(1999)). 1.2.2) To reconstruct the heterologous putrescine biosynthetic pathways in yeast, the following enzymes can be used: ADC, AIH, CPA and AUH. As an example of an engineered strain that possesses these enzymatic activities, an ADC from oats {Avena sativa; AsADC) with activity previously demonstrated in S. cerevisiae (see Klein, R. D. et al. Reconstitution of a bacterial / plant polyamine biosynthesis pathway in Saccharomyces cerevisiae. Microbiology 145 Pt2, 301-7 (1999)), an AIH from Arabidopsis thaliana was selected (AtAIH), two orthologs of CPA from tomato (Solanum lycopersicum; SICPA) and A. thaliana (AtCPA), and two AUH from E. coli (speB) and A. thaliana (AtARGAH2) for expression in yeast. 1.2.3) To establish the functionality of each heterologous enzyme in yeast, the three-step (arginine —> agmatine —» NCP —» putrescine) or two-step (arginine —> agmatine putrescine) putrescine pathways were reconstituted. in a stepwise manner by co-transforming the wild-type yeast strain with low-copy plasmids expressing AsADC, AtAIH and SICPA or AtCPA} or AsADC and speB or AÍARGAH2. To eliminate the effects on cell growth and metabolite production derived from different levels of auxotrophy, all transformations were performed with three low-copy plasmids harboring different auxotrophic markers, using BFP as a negative control instead of a blank plasmid or absent. The relative accumulation of agmatine, NCP and putrescine in the extracellular medium of transformed cells after 48 hours of growth in selective media was analyzed by LC-MS / MS, which indicated that all enzymes, except SICPA and AtARGAH2, retained their activity. in yeast (Figures 6, 7). Reconstitution of the plant-specific pathway comprising AsADC, AtAIH, and AtCPA enabled production of putrescine at titers of 23 mg / L, a 22-fold improvement over wild-type titers. The tomato orthologous CPA (SICPA) enabled putrescine production at titers of 4.5 mg / L when combined with AsADC and AtAIH, similar to putrescine levels in cells expressing AsADC and AtAIH. Reconstitution of the bacterial direct access pathway through AsADC and E. coli ureohydrolase (speB) allowed the production of putrescine at titers of 34 mg / L, 32 times higher than the wild type. 1.3) The biosynthetic pathway of the designed strain incorporates the overexpression of native yeast genes involved in the biosynthesis of arginine and polyamines and the expression of heterologous biosynthetic enzymes from polyamine production pathways found in organisms other than yeast. to further increase putrescine production. 1.3.1) The highest yielding overexpressed native gene triad for putrescine biosynthesis (SPE1, ARG2, CAR1; 1.1.2) was combined with the highest yielding heterologous putrescine pathway (AsADC, speB; 1.2.3) by co-transforming the wild-type yeast strain with a low-copy plasmid encoding SPE1, AsADC and speB and low-copy plasmids encoding ARG2 and CAR1. Putrescine titers in the culture medium of transformed cells were measured by LC-MS / MS analysis after 48 hours. The resulting strain produced putrescine at titers of 47 mg / L (Figure 10). 1.4) Polyamine biosynthesis in yeast is regulated by several mechanisms (Figure 8). The biosynthetic pathway of the engineered strain incorporates disruptions of one or more of these regulatory mechanisms to reduce feedback inhibition of putrescine production. 1.4.1) Native yeast genes that are involved in the regulation of polyamine biosynthesis and that can therefore be disrupted to enhance intracellular accumulation of putrescine, include, but are not limited to, the following examples ( Figure 8). Methylthioadenosine phosphorylase (Meulp) catalyzes the driving step in the decarboxylated S-adenosylmethionine (dcSAM) recycling pathway, which constitutes the donor alkyl group for the conversion of putrescine to spermidine and spermine catalyzed by spermidine synthase (Spe3p) and spermine synthase (Spe4p) (see Chattopadhyay, Μ. K., Tabor, C. W. and Tabor, H. Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1A mutant. Biochem. Biophys. Res. Commun. 343, 203-207 (2006)) . Methylthioadenosine is known to inhibit spermidine synthase activity (see Chattopadhyay, Μ. K., Tabor, C. W. and Tabor, H. Studies on the regulation of ornithine decarboxylase in yeast: Effect of deletion in the MEU1 gene. Proc. Nati Acad. Sci. 102, 16158-16163 (2005). Polyamine biosynthesis is regulated by an antizyme-mediated negative feedback loop that is conserved in fungi and metazoans (see Pegg, A. E. Regulation of ornithine decarboxylase. Journal of Biological Chemistry 281, 14529-14532 (2006)) . In yeast, the OAZ1 gene comprises two exons separated by a single nucleotide that collectively encode antizyme-1, a competitive inhibitor of ornithine decarboxylase (Spelp). A polyamine-induced ribosomal frameshift mechanism allows translation of the full-length antizyme only at high levels of polyamines, thus imposing feedback inhibition of its 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 Skylp, a protein kinase that is believed to interact with Agp2p. 1.4.2) Single gene disruption strains for each of MEU1, OAZI, SPE4, SKY1 and AGP2 were constructed by inserting a series of tandem nonsense mutations within the first third of each open reading frame in yeast. wild type. To characterize the effects of each regulatory alteration in the context of the native and heterologous putrescine production pathways, ODC was overexpressed in yeast (SPE1), 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 by LC-MS / MS after 72 hours of growth (Figure 9). Disruption of MEU1 enhanced putrescine titers by 68% when the native putrescine production pathway via SPE1 was overexpressed. Similarly, disruption of OAZI markedly enhanced putrescine production by 174% when combined with SPE1 overexpression. Disruption of OAZ1 resulted in a 21-fold increase in putrescine titer in nontransformed cells without overexpressing native or heterologous putrescine pathways. Disruption of SKY1 and AGP2 resulted in increases of 29% and 14% respectively in putrescine titer when overexpressed with SPE1. Disruption of SKY1 resulted in a 41% decrease in putrescine titer when combined with heterologous expression of AsADC and speB. 1.5) The biosynthetic pathway in the engineered strain combines deletion of the regulatory genes MEU1 and OAZ1 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 a meu1 / oaz1 double knockout strain. This strain was transformed with a low-copy plasmid expressing SPE1, AsADC, and speB. LC-MS / MS analysis of the extracellular medium of this transformed strain indicated that putrescine titers reached 86 mg / L after 48 hours of growth in selective media (Figure 10). Example 2. Design 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 combine genetic modifications in order to increase the flux of carbon and nitrogen from putrescine towards NMPy biosynthesis, and include the introduction of key heterologous enzymes for the production of the ΤΑ NMP precursors, 4MAB and NMPy. Genetic modifications are used that include the modification of the N- and / or C-terminal domains of the enzymes of interest to improve activity in a heterologous host, and the deletion or alteration of the genes that encode the enzymes that divert the precursor molecules. off the planned path. 2.1) The biosynthetic pathway of the designed strain allows the production of NMPy from endogenous putrescine. Putrescine is first converted to N-methylputrescine (NMP) by a SAM-dependent Nmethyltransferase (PMT), which is subsequently oxidized to 4-methylaminobutanal (4MAB) by a copper-dependent diamine oxidase (MPO). 4MAB, like many aldehyde compounds, is unstable in aqueous solution and cyclizes spontaneously by a base-catalyzed nucleophilic attack to form NMPy (Figure 11). 2.1.1) The putrescine-overproducing strain of Example 1.5, harboring a low-copy plasmid expressing SPE1, AsADC, and speB for putrescine overproduction, was cotransformed with additional low-copy plasmids expressing a PMT from A. belladonna (AbPMTI) and a downstream MPO enzyme from Nicotiana tabacum (NtMPOI). The accumulation of intermediates in the extracellular medium of transformed cells expressing each successive enzyme between putrescine and NMPy was compared by LC-MS / MS analysis after 48 hours of growth. The immediate product of NtMPOI (4MAB) as well as its spontaneous cyclization product (NMPy) were produced with the expression of AbPMTI and NtMPOI (Figure 11), as well as their precursors, NMP and putrescine (Figure 12). 2.1.2) The accumulation of NMP in the growth medium of putrescine-overproducing yeast strains with and without alteration of the MEU1 gene (described in Example 1.4.2) was measured by LC-MS / MS analysis. This analysis indicated that prior disruption of MEU1 in the putrescine-overproducing strain and its concomitant impact on SAM recycling did not inhibit the N-methylation of putrescine by AbPMTI (Figure 13). 2.2) Enzymes can localize to different subcellular compartments when expressed heterologously than in their original host organism, resulting in reduced function. The biosynthetic pathway in the designed strain can incorporate modifications in the polypeptide sequences of the native and heterologous enzymes to induce the localization of these modified enzymes in sub-cellular compartments other than those where they are located naturally. For example, previous studies have shown that while NtPMT is expressed in the cytosol of tobacco cells, NtMPOI is localized in the lumen of the peroxisome (see Naconsie, M., Kato, K., Shoji, T. and Hashimoto, T. Molecular evolution of n-methylputrescine oxidase in Tobacco. Plant Cell Physiol. 55, 436-444 (2014). 2.2.1) The sub-cellular localization of NtMPOI was examined by performing an in silico prediction of the subcellular localization of the enzyme using the SherLoc2 utility for signal peptide detection (see Briesemeister, S. et al. SherLoc2: A high-accuracy hybrid method for predicting subcellular localization of proteins. J. Proteome Res. 8, 5363-5366 (2009)). This analysis indicated that NtMPOI harbors a strong yeast peroxisome targeting consensus sequence (PTS) at its C-terminus (Ala-Lys-Leu, denoted PTS1), suggesting that NtMPOI may localize to peroxisomes when heterologously expressed. in yeast (Figure 14). 2.2.2) Fluorescence microscopy of wild-type yeast cells expressing N- or C-terminal GFP-tagged AbPMTI and NtMPOI from low-copy plasmids indicated that while AbPMT 1 is primarily found in the cytosol, the Localization of NtMPOI in peroxisomes depends on an exposed C-terminal PTS (Figures 15 panel A, Figure 16). 2.2.3) The cytosolic expression of NtMPOI achieved by masking the Cterminal PTS with a GFP fusion did not have a significant impact on the extracellular levels of 4MAB or NMPy (Figure 15 panel B). 2.3) The biosynthetic pathway of the engineered strain may incorporate orthologs of biosynthetic enzymes other than those listed in Table 1. Different orthologs of an enzyme may present significant differences in their activity when expressed in heterologous hosts. Therefore, the orthologs of the biosynthetic enzymes provided as examples herein and listed in Table 1 can also be used in non-plant cells engineered to perform the same biochemical conversions. 2.3.1) A tBLASTn search of the transcriptomes of A. belladonna and Datura metel was performed in the 1000 Plants Project database (see Matase!, N. et al. Data access for the 1,000 Plants (1KP) project. Gigascience 3 , 17 (2014)) was performed using the amino acid sequence of NtMPOI as a query and an E-value threshold of 10150. Two full-length orthologous sequences named AbMPOI and DmMPOI were identified, each of which shared a sequence identity of the 91% with NtMPOI (Figure 17 panel A). 2.3.2) Yeast codon-optimized sequences for AbMPOI and DmMPOI were obtained and cloned into low-copy expression plasmids. To evaluate their activity, each of the three MPO variants was co-expressed with AbPMTI from low-copy plasmids in the putrescine-overproducing strain of Example 1.5, and the accumulation of 4MAB and NMPy in the extracellular medium was measured by LC-MS / MS after 48 hours of growth on selective media. DmMPOI showed levels of 4MAB and NMPy production comparable to those of the original NtMPOI variant (Figure 17 panel B). 2.3.3) Differences in activity between orthologous enzymes can often be attributed, at least in part, to structural differences in their active sites. Template-based homology models of NtMPOI, AbMPOI and DmMPOI were constructed from the crystal structure of a copper-containing amino oxidase from Pisum sativum (PDB: 1KSI) using the RaptorX web server (see Kállberg, M. et al. Template -based protein structure modeling using the RaptorX web server. Nat. Protoc. 7, 1511-22 (2012)). Homology models indicated that the orthologs possess long, unstructured N- and C-terminal tail regions (Figure 17 panel C). 2.3.4) The activity of truncations of the two active orthologs, NtMPOI and DmMPOI, was tested 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 where the unstructured tail was removed, but the PTS was retained (termed ac-ptsij each of the MPO truncations was co-expressed with AbPMTI from low-copy plasmids in the putrescine-overproducing strain of the Example 1.5, and the 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 NtMPOI truncations (Figure 18). Deletion of the C-terminal unstructured region of DmMPOI, while retaining the C-terminal PTS tripeptide, resulted in a 31% increase in extracellular 4MAB levels relative to the wild-type DmMPOI enzyme. 2.4) The biosynthetic pathway of the designed strain incorporates one or more genetic modifications to reduce or eliminate the metabolic flow of undesirable secondary reactions. Biosynthetic enzymes expressed in heterologous hosts can participate in undesirable side reactions that divert the flow of metabolites from the biosynthesis of desired compounds. For example, yeast aldehyde dehydrogenases can oxidize heterologous aldehyde molecules, such as 4MAB, to their cognate carboxylic acids. Based on LC-MS / MS analysis, accumulation of 4MAB acid was observed in the growth medium of the putrescine-overproducing strain of Example 1.5 when AbPMTI and DmMP01ÚC~PTS1 were coexpressed from low-copy plasmids, but not in the absence of the MPO enzyme (Figure 11). 2.4.1) It has been shown in the literature that six yeast genes (ALD2-ALD6 and HFD1) encode enzymes with aldehyde dehydrogenase activity (see Datta, S., Annapure, U. S. and Timson, D. J. Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii. Biosci. Rep. 37, BSR20160529 (2017); and also Nakahara, K. et al. The Sjógren-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., and Toyn, J. H. Specialization of Function Among Aldehyde Dehydrogenases: Genetics 163, 69-77 (2003)). The ALD4, ALD5 and ALD6 genes respectively encode two mitochondrial and a cytosolic acetaldehyde dehydrogenase that, in addition to oxidizing acetaldehyde to acetate during fermentative growth on glucose and ethanol (see Saint-Prix, F., Bónquist, L. and 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 shown that they oxidize a matrix from various aliphatic and aromatic aldehydes to carboxylic acids (see Datta, S., Annapure, U. S. and Timson, D. J. Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii. Biosci. Rep. 37, BSR20160529 (2017)). Individual knockout strains for these four target genes were constructed by inserting a series of tandem nonsense mutations within the first third of their open reading frames into the putrescine-overproducing strain of Example 1.5. The contribution of each of the four dehydrogenases to 4MAB oxidation was assessed by co-expressing AbPMTI and DmMP01¿CPTS1a from low-copy plasmids in each single disruption strain and measuring the accumulation of 4MAB acid in the medium by LC-MS / MS after 48 hours of growth. Marginal decreases in 4MAB acid levels were observed with individual alterations of HFD1 and ALD46 (Figure 19). 2.4.2) Although the ALD4-6 genes are considered essential due to their role in the production of acetate and acetyl-CoA, previous studies have shown that all three genes are at least partially redundant and that the lethal phenotype of doubles and triples knockouts can be rescued by supplementing the medium with acetate (see Saint-Pnx, F., Bónquist, L. and Dequin, 8. 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. and Van Vuuren, H. J. J. Functional improvement of Saccharomyces cerevisiae to reduce volatile acidity in wine. FEMS Yeast Res. 13, 485-494 (2013)). A quadruple knockout yeast strain with alterations in the open reading frames of HFD1 and ALD4-6, and expressing both AbPMTI and DmMPO1AC-PTS1, was constructed from low-copy plasmids. This strain showed a 45% reduction in 4MAB acid levels (Figure 20 panel A) and a concomitant 46% increase in NMPy production compared to the uninterrupted strain (Figure 20 panel B). 2.4.3) An ALD-null strain was constructed by deleting the ALD2-ALD3 genes in tandem from the genome of the quadruple knockout strain of example 2.4.2 and co-expressing AbPMTI and DmMP01ACPTS1 from low-copy plasmids. After 48 hours of growth, LC-MS / MS analysis indicated that deletion of ALD2 and ALD3 completely eliminated the acid side product 4MAB and increased the production of 4MAB and NMPy by 83% and 75%, respectively, in comparison with the strain with all six ALD genes intact (Figure 20 panels A and B). 2.4.4) An NMPy-producing yeast strain was constructed by integrating a putrescine overproduction gene cassette (SPE1, AsADC, speB) previously carried by a plasmid into the genome of the ALD-null strain of Example 2.4.3 , and the additional integration of AbPMTI and DmMP01AC PTS1. LC-MS / MS analysis confirmed that the production of NMPy in this strain after 48 hours of growth in non-selective medium was comparable to that of the ADL-null strain of example 2.4.3 that expressed the genes necessary for production of putrescine, AbPMTI and DmMPO1AC-pTS1, from low-copy plasmids and cultured in selective medium (Figure 21). Example 3. Yeast strains designed for the production of tropine from simple sugars and nutrients A type III polyketide synthase (PKS) and a cytochrome P450 allow the conversion of NMPy to tropinone via the ΤΑ precursor MPOB. Tropinone can be reduced by a stereospecific reductase, called tropinone reductase 1 (TR1), to produce tropin (see Kim, N., Estrada, O., Chavez, B., Stewart, C. and D'Auria, J. C. Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis. Molecules 21, (2016)) (Figure 22). 3.1) The biosynthetic pathway of the engineered strain incorporates a pyrrolidine ketide synthase, a CYP82M3 tropinone synthase, one or more cytochrome P450 reductases and a tropinone reductase 1 to convert NMPy to tropin. 3.1.1) Yeast DNA sequences with optimized codons encoding A. belladonna pyrrolidine ketide synthase (AbPYKS), tropinone synthase (AbCYP82M3) and Datura stramonium tropinone reductase 1 (DsTR1) were obtained. Codon-optimized sequences from yeast were also obtained for a panel of four different CPRs, including three plant CPRs from A. thaliana, Eschscholzia californica (California poppy), and Papaver somniferum (opium poppy), and the native yeast CPR. (NCP1), for its expression in yeast, since P450 enzymes require the collaboration of NADP+- cytochrome P450 reductase (CPR) for the continuous exchange of electrons. A yeast strain was constructed by integrating DsTR1 into the genome of the NMPy-producing strain of Example 2.4.4, and expressing AbPYKS, AbCYP82M3, and each of the four CPRs from low-copy plasmids. To validate the enzymatic activity and identify possible bottlenecks, the accumulation of NMPy, MPOB, tropinone and tropin was monitored by LC-MS / MS in the media of the transformed strains after 48 hours of growth (Figure 23). Comparable levels of de novo tropin production (175-210 pg / L) were observed with all four CPR collaborators under the trial conditions. 3.2) The presence of metabolic bottlenecks, defined as biosynthetic enzymes or spontaneous steps whose low activity limits flux through a part of a biosynthetic pathway, can lead to suboptimal production of desired TAs and precursors. 3.2.1) For example, analysis of the accumulation of TA intermediates in the media of the engineered strains of Example 3.1.1 indicated that, although the accumulation of tropinone, the product AbCYP82M3, was minimal, a substantial portion of MPOB produced by AbPYKS remained without being consumed by AbCYP82M3 (Figure 24). 3.2.2) Integration of tropin biosynthesis genes into the yeast genome may improve tropin production by allowing more stable expression of AbCYP82M3. A tropin-producing platform strain was constructed by integrating AtATRI with AbPYKS and AbCYP82M3 into the genome of the NMPy-producing strain of Example 3.1.1. Tropine and hygrin accumulation for the integrated strain was compared to plasmid-based expression of the same genes by LC-MS / MS analysis after 48 hours (Figure 28). Genomic expression of AbPYKS, AbCYP82M3, and AtATRI increased tropin titers by almost threefold (565 pg / L) relative to plasmid-based expression (189 pg / L). The engineered strain also showed a 2.6-fold increase in hygrin accumulation. 3.3) The accumulation of secondary products in the biosynthetic pathway of the engineered strain can lead to suboptimal production of the desired TAs and precursors. 3.3.1) For example, analysis of the accumulation of TA intermediates in the medium of the engineered strains of Example 3.1.1 indicated substantial accumulation of hygrin, a derivative of NMPy, at titers almost four times higher than tropine (775 -900 pg / L). In the relevant literature, hygrin has been observed to accumulate through the spontaneous decarboxylation of MPOB (see Bedewitz, M. A., Jones, A. D., D'Auria, J. C. and Barry, C. S. Tropinone synthesis via an atypical polyketide synthase and P450mediated cyclization . Nat. Commun. 9, 5281 (2018)) (Figure 22). As another example, LCMS / MS analysis of the growth medium of the engineered strains of Example 3.1.1 indicated that hygrin also accumulated in the negative control strain lacking AbPYKS and AbCYP82M3 due to decarboxylative condensation with NMPy ( Figure 22). 3.3.2) Modulation of growth temperature can be used to reduce the accumulation of side products in the biosynthetic pathway of the engineered strain to increase flux to desired TAs and precursors. In one example, the impact of temperature on spontaneous hygrin production was evaluated by taking advantage of a kinetic principle that the rates of enzymatic and spontaneous reactions decrease at lower temperatures. Since A. belladonna and other TA-producing Solanaceae are adapted for optimal growth in colder climates, growth of yeast strains expressing Solanaceae genes at 25°C may improve folding and / or enzyme activity, thereby allows comparable production of enzymatically generated tropin for growth at 30 °C while reducing the rate of spontaneous hygrin production. Cultures of the tropin-producing strain of Example 3.2.2 were grown in non-selective defined medium at 30°C and 25°C and the accumulation of tropine and hygrin was compared by LC-MS / MS analysis of the growth medium. after 48 hours. Tropine titers were minimally affected by decreasing temperature. Hygrin accumulation was reduced by 42% at 25°C compared to 30°C, resulting in a 60% increase in the ratio of tropine to hygrin produced (Figure 25). 3.3.3) Reduction or elimination of undesirable side reactions can be used to improve the flux of metabolites toward desirable TAs and TA precursors in the biosynthetic pathway of the engineered strain. In one example, flux to the TA precursor tropine can be enhanced by reducing hygrin production resulting from spontaneous decarboxylative condensation with acetate. The impact of removing the fed acetate from the medium of the NMPy-producing strain of Example 2.4.4 on the production of hygrin and tropine was evaluated. The effect of abolishing acetate auxotrophy in the engineered strain of Example 2.4.4 was evaluated by expressing functional copies of ALD4 and ALD6 on low-copy plasmids and then monitoring the accumulation of hygrin and 4MAB acid by LC-MS / analysis. MS after 48 hours of growth. While reconstitution of ALD4 or ALD6 allowed growth on selective media in the absence of fed acetate (Figure 26 panel A), addition of ALD4 caused a five-fold increase in 4MAB acid accumulation while ALD6 did not produce a significant increase. (Figure 26 panel B). Furthermore, removal of acetate feed with ALD4 or ALD6 resulted in a 38% and 59% decrease in hygrin accumulation, respectively (Figure 26 panel B). 3.3.4) A functional copy of the ALD6 gene was reinstated in the tropine-producing strain of Example 3.2.2 at the previously altered ald6 locus. The impact of this integration on the accumulation of all metabolites between NMPy and tropine was measured by LC-MS / MS analysis after 48 h of growth in non-selective medium. Restoration of acetate metabolism through Ald6p resulted in a 2.7-fold increase in tropin titers as well as a 1.6-fold increase in hygrin accumulation (Figure 28). Furthermore, ALD6 integration resulted in substantial increases in NMPy and tropinone production, as well as increased MPOB consumption (Figure 27). 3.3.5) An additional copy of each putrescine-tropin biosynthetic enzyme gene (i.e., AbPMTI, DmMP01ÚC-pTS1, AbPYKS and AbCYP82M3) was expressed from a low-copy plasmid in the engineered strain of Example 3.3.4 and the 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 tropin production (Figure 29). Expression of an additional copy of AbPMTI resulted in significant improvements in the production of all TA precursors between NMP and tropinone, as well as a 2.4-fold increase in tropin production (Figure 29). Therefore, additional copies of PMT (AbPMTI and DsPMTI) and PYKS (AbPYKS) were integrated into the genome of the tropin-producing strain of Example 3.3.4 (CSY1249) at the PAD1 locus. The resulting engineered strain (CSY1251) was grown at 25 °C in non-selective medium for 48 hours, resulting in tropin production at titers of 3.4 mg / L, 2.2 times higher than the tropin-producing strain ( CSY1249) in Example 3.3.4 (Figure 30). Example 4: Yeast designed for the production of pseudotropin alkaloids from Larginine. Yeast strains can be engineered for the production of non-medicinal TAs from early amino acid precursors such as L-arginine. As an example, the platform yeast strains described in Example 3 can be further engineered to produce pseudotropine alkaloids from L-arginine (Figure 1). The platform yeast strain that produces tropinone from L-arginine (see descriptions in Example 3) can be further engineered to incorporate a stereospecific reductase, for example, tropinone reductase 2 (TR2; EC 1.1.1.236), to convert tropinone biosynthesized into pseudotropin. An expression cassette harboring a strong constitutive promoter such as TDH3 and a coding sequence for a TR2 variant, for example, Datura stramonium TR2 (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 pseudotropin, for example, callistegins, by integrating one or more expression cassettes harboring a strong constitutive promoter such as PGK1 and a hydroxylating enzyme such as a cytochrome P450 that acts on the pseudotropin scaffold. . By incorporating multiple P450 enzymes, each acting at a different position on the pseudotropin backbone, a variety of callistegins and derivatives thereof can be biosynthesized. Engineered strains can be grown in nonselective synthetic complete medium at 30°C or 25°C for 48 to 96 hours, after which the accumulation of pseudotropin alkaloids in the culture medium can be analyzed by LC-MS / MS. Example 5: Yeast designed for the overproduction of phenylpyruvate and associated TA precursors. Yeast strains can be engineered for overproduction of phenylpyruvate, which represents the precursor of acyl donor molecules necessary for the production of medicinal TAs (Figure 2), in order to increase the flux of carbon and nitrogen from central metabolism. towards the desired TAs and TA precursors. Yeast strains can be engineered for overproduction of phenylpyruvate by incorporating genetic modifications, including, but not limited to, fine-tuning the transcriptional regulation of native biosynthetic enzymes, deletion or alteration of genes encoding enzymes that divert precursor molecules. of the desired pathway and introduction of heterologous enzymes for the conversion of endogenous molecules into TA precursor molecules. In one example, a yeast strain 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 core metabolites. These extra copies can be controlled by strong constitutive promoters, such as GPD, TEF1 or PGK1. Examples of native gene targets include, but are not limited to, ARO8 and ARO9 aromatic acid aminotransferases, and PHA2 dehydratase. In one case, one or more additional copies of ARO8 can be incorporated into the engineered strain under the control of a strong constitutive promoter. In one case, one or more additional copies of ARO9 can be incorporated into the engineered strain under the control of a strong constitutive promoter. In another case, one or more additional copies of PHA2 can be incorporated into the engineered strain under the control of a strong constitutive promoter. In one embodiment of the invention, one or more additional copies of one or more genes selected from the group including ARO8, ARO9 and PHA2 can be incorporated into the engineered strain under the control of strong and unique constitutive promoters. Example 6: Yeast designed 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 various acyl-donating phenylpropanoid compounds from L-phenylalanine and L-tyrosine, including PLA, cinnamic acid, coumaric acid, ferulic acid, benzoic acid, and glucoside and thioester derivatives of coenzyme A. of these compounds, which can undergo esterification with tropine, pseudotropin or their derivatives to biosynthesize medicinal TAs, non-medicinal TAs and non-natural TAs (Figures 1 to 3). 6.1) As wild-type yeast produces only trace levels of PLA, production of this TA precursor must be increased to allow sufficient TA accumulation downstream. To enhance PLA production, heterologous phenylpyruvate reductases (PPRs) can be expressed in engineered host cells. PPR orthologues from E. coli, Lactobacillus, A. belladonna and Wickerhamia fluorescens, as well as lactate dehydrogenases (LDH) from Bacillus and Lactobacillus with reported activity on 3-phenylpyruvate (Table 1), were screened for their activity in yeast expressing each enzyme starting from a low-copy plasmid in CSY1251 and measuring PLA production by LC-MS / MS after 72 h of growth in selective medium. All LDH candidates as well as the PPRs from L. plantarum, E. coli, and A. belladonna produced modest improvements (1.3- to 3.5-fold) in PLA production relative to the control, while PPR expression of W. fluorescens resulted in a nearly 80-fold increase in PLA production at -250 mg / L (Figure 31). As such, WfPPR was selected for integration into CSY1251 to produce strain CSY1287. 6.2) As another example, yeast strains can be engineered for the production of cinnamic acid and coumaric acid, which are phenylpropanoids that can be used as acyl donor compounds for esterification with tropine or pseudotropin to form the unnatural TAs, from Lphenylalanine. and L-tyrosine, respectively. Yeast can be engineered for the production of cinnamic acid from L-phenylalanine by incorporating an ammonium lyase such as phenylalanine ammonium lyase (PAL; EC 4.3.1.24). Similarly, yeast can be engineered for the production of coumaric acid from L-tyrosine by incorporating an ammonia lyase such as a tyrosine ammonia lyase (TAL; EC 4.3.1.23). A yeast strain was engineered to produce cinnamic acid from L-phenylalanine by transforming it with a low-copy CEN / ARS plasmid with a TRP1 selective marker, TEF1 promoter, and a coding sequence for a PAL variant of Arabidopsis thaliana (AtPALI). The resulting strain harboring the low-copy plasmid was grown in complete synthetic medium with the appropriate amino acid removal solution (-Ura) at 30°C. After 48 hours of growth, the cinnamic acid content in the medium was analyzed by LC-MS / MS analysis (Figure 32). 6.3) In A. belladonna, PLA is activated for acyl-to-tropin transfer via glycosylation by UDP-glucosyltransferase 84A27 (AbUGT) (see Qiu, F. et al., Functional genomics analysis reveáis two novel genes required for littorine biosynthesis. New PhytoL, nph.16317 (2019)). As plants, UGTs participate in the biosynthesis of various phenylpropanoids and often exhibit a wide 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 a UGT with sufficiently high activity on a desired acyl donor. 6.3.1) As an example, AbUGT activity on different acyl-donating phenylpropanoids, including the canonical substrate, PLA, was evaluated by expressing AbUGT from a low-copy plasmid at CSY1251 and measuring the conversion of three acyl-donating phenylpropanoids ( PLA, cinnamic acid, ferulic acid) to their respective glycosides. While AbUGT glycosylated ~60% and 90% of cinnamic acid and ferulic acid, respectively, PLA glycosylation was the lowest of the substrates tested with <3% conversion (Figure 33). 6.3.2) The activity of AbUGT orthologs from other TA-producing solanaceae on PLA and other phenylpropanoids can be evaluated. In this example, transcripts encoding UGT84A27 from the Brugmansia sanguineous (BsUGT) and D. metel (DmUGT) transcriptomes in the 1000Plants database using a tBLASTn search. Yeast codon-optimized sequences encoding these orthologous UGTs were screened for activity expressing AbUGT, BsUGT, DmUGT or a BFP negative control of low copy plasmids on CSY1251. Glycoside production was measured in cultures of the transformed strains by LC-MS / MS after 72 h of growth in selective medium supplemented with 500 μΜ PLA, cinnamic acid (CA) or ferulic acid (FA) as glucose acceptors. All three UGT orthologs showed substantial glycation of CA (34-65% conversion) and FA (85-90% conversion) and only minimal activity on PLA (<3% conversion), with AbUGT showing the highest activity. conversion of PLA (2.7%) (Figures 33, 34). 6.3.3) Given the disproportionate variation in AbUGT activity on the structurally similar substrates cinnamate, ferulate, and PLA, a structure-guided rational mutagenesis approach can be implemented to engineer the AbUGT active site to enhance activity in PLA. In this example, a homology model of AbUGT bound to UDP-glucose was first constructed based on the crystal structure of salicylate UDP-glucosyltransferase UGT74F2 from Arabidopsis thaliana (PDB: MA / 1 1 5V2K) using the RaptorX web server (Figure 35). Docking of D-PLA to the active site was then simulated using the Maestro / GlideXP software package. Based on the energy-minimizing binding mode, the aryl ring of D-PLA is likely stabilized by p¡ stacking interactions with F130, while its α-hydroxyl and carboxylate groups are respectively stabilized by hydrogen bonds with Q151 and H24. , such that the oxygen of the nucleophilic carboxylate is within 4 A of the electrophilic C1 carbon of UDP-glucose (Figure 35). D-PLA is additionally adjacent to residues L205 and I292, neither of which appear to interact with either substrate. This suggests that mutation of (i) F130 to tyrosine could preserve p¡ stacking with the aryl ring of D-PLA while providing an additional hydrogen bond to stabilize the α-hydroxyl oxygen of D-PLA, which is absent from cinnamate. and ferulate; (ii) L205 to phenylalanine could increase the stabilization of the pi stacking of D-PLA with F130Y; and (i¡) I292 to glutamine would generate two additional stabilizing hydrogen bonds with D-PLA and UDP-glucose (Figure 35). The activity of AbUGT point mutants F130Y, L205F and I292Q was examined by expressing each mutant, wild-type AbUGT or a BFP control from a low-copy plasmid in CSY1251 and glycoside production was measured by LCMS / MS after 72 h. growth in selective media supplemented with 500 μM of PLA, CA or FA. All three mutants exhibited comparatively low (and statistically indistinguishable) activity in PLA relative to wild-type AbUGT (<3% conversion), although the F130Y and I292Q mutations significantly reduced UGT activity in CA (Figure 36). 6.3.4) Based on the results described in sections 6.1 and 6.3, strain CSY1288 was constructed by integrating WfPPR and AbUGT with optimized yeast codons into the CSY1251 genome, validated by verifying PLA production (66 mg / L) and minimal accumulation of PLA glycosides (Figure 37). 6.4) As the poor activity of AbUGT on PLA is likely to limit the flux of TA precursors to downstream TAs, the flux of phenylalanine to the PLA glucoside can be increased by incorporating genetic modifications that promote the accumulation of UDP-glucose and decrease the glycoside degradation. 6.4.1) UDP-glucose is essential for the formation of storage polysaccharides, cell wall glycans and glycoproteins and therefore its biosynthesis is strictly regulated (see Nishizawa, M., Tanabe, M., Yabuki, N., Kitada, K., Toh-e, A. Pho85 kinase, a yeast cyclin-dependent kinase, regulates the expression of UGP1 encoding UDP-glucose pyrophosphorylase. Yeast. 18, 239-249 (2001)). During growth on glucose, yeast directs glucose-6-phosphate along two main metabolic pathways, glycolysis and starch biosynthesis. Since citrate is an allosteric inhibitor of the rate-limiting glycolytic enzyme phosphofructokinase (see L, Y. et al., Production of Rebaudioside A from Stevioside Catalyzed by the Engineered Saccharomyces cerevísiae. Appl. Biochem. Biotechnol. 178, 1586 -1598 (2016)), partial suppression of glycolysis by citrate supplementation could increase UDP-glucose availability and glycoside production (Figure 38). Strain CSY1288, which encodes genomic WfPPR and AbUGT for endogenous PLA glycoside production, was grown in medium supplemented with 2% citrate and 500 μΜ CA or FA, and glycoside production was compared by LC-MS / MS after 72 hours of growth. Citrate supplementation reduced PLA, CA, and FA glycosylation by 83%, 56%, and 78%, respectively (Figure 39). 6.4.2) Overexpression of PGM2 and UGP1, whose gene products respectively catalyze the isomerization of glucose-6-phosphate to glucose-1-phosphate and the conversion of glucose-1-phosphate to UDPglucose, can be used to increase the supply of UDP-glucose. 6.4.2.1) Additional copies of PGM2 and UGP1 were expressed from low-copy plasmids in CSY1288 and the production of PLA glycosides was measured after 72 h of growth in selective medium. While overexpression of PGM2 did not produce any improvement relative to the control, overexpression of UGP1 resulted in a ~1.8-fold increase in PLA glycoside production (Figure 40), supporting that increased UDP-pool Glucose enhances PLA utilization by AbUGT. 6.4.2.2) Native glycosidases can act on PLA and other TA precursor glycosides to reduce accumulation, as other heterologous glycosides have been shown to be hydrolyzed in this way in yeast (see Schmidt, S., Rainieri, S. , Witte, S., Matern, U., Martens, S., Identification of a Saccharomyces cerevisiae glucosidase that hydrolyzes flavonoid glucosides. Appl. Environ. Microbiol. 77, 1751-1757 (2011); see also Wang, H.etal. , Engineering Saccharomyces cerevisiae with the deletion of endogenous glucosidases for the production of flavonoid glucosides. Microb. Cell Fací 15, 1-12 (2016)). In this example, three native deglucosidase genes—EXG1, SPR1, and EGH1—were disrupted in CSY1288 and PLA glycoside production was measured after 72 h of growth of disruption mutants on nonselective media. Disruption of EGH1 more than doubled PLA glycoside production (Figure 41), indicating that hydrolysis by Eghlp constitutes a substantial loss of TA precursor flux. Example 7: Yeast designed for the conversion of littorin to hyoscyamine aldehyde Yeast strains can be engineered for the conversion of littorin to hyoscyamine aldehyde (Figure 2). For example, the tropine and PLA glycoside-producing yeast strain described in Example 6 can be further engineered to express a cytochrome P450 CYP80F1 (EC 1.14.19.-) to catalyze the rearrangement of littorin to hyoscyamine aldehyde, and a cytochrome P450 reductase (CPR; EC 1.6.2.4) to support the activity of the P450 enzyme. A yeast strain was engineered to convert fed littorin to hyoscyamine aldehyde by transforming it with a low-copy CEN / ARS plasmid with a LEU2 selection marker, TDH3 promoter, and coding sequence for an A. belladonna CYP80F1 variant (AbCYP80F1); and with a low-copy CEN / ARS plasmid with a TRP1 selection marker, TEF1 promoter and a coding sequence for a cytochrome P450 reductase (CPR) from S. cerevisiae (NCP1) or from A. thaliana (AtATRI). The resulting strain harboring the low-copy plasmids was grown in complete synthetic medium with the appropriate amino acid removal solution (-Leu -Trp) supplemented with 1 mM littorin at 30 °C. After 48 hours of growth, the hyoscyamine aldehyde content in the medium was analyzed by LC-MS / MS analysis (Figure 42). Example 8: Yeast designed for the conversion of hyoscyamine to scopolamine. Yeast strains can be engineered for the conversion of hyoscyamine to scopolamine (Figure 2). For example, the yeast strain described in Example 7 can be further engineered to incorporate enzymes possessing hydroxylase activity at the 6β position of hyoscyamine to form anisodamine and enzymes possessing dioxygenase activity at the 6p-hydroxyl position of anisodamine to form scopolamine, or enzymes that possess both activities (EC 1.14.11.11). Yeast strains were engineered to convert fed hyoscyamine to scopolamine by transforming them with a low-copy CEN / ARS plasmid with a LEU2 selection marker, TDH3 promoter, and coding sequence for a D. stramonium hyoscyamine 6p-hydroxylase / dioxygenase (H6H). (DsH6H), Anisodus acutangulus (AaH6H), Brugmansia arborea (BaH6H) or Datura metel (DmH6H). The resulting strains harboring the low-copy plasmids were grown in complete synthetic medium with the appropriate amino acid removal solution (-Leu) and supplemented with 1 mM hyoscyamine at 30°C. After 72 hours of growth, the scopolamine content in the medium was analyzed by LC-MS / MS analysis (Figure 43). The strain expressing the H6H variant of D. stramonium exhibited the highest conversion of fed hyoscyamine to scopolamine, although all variants tested showed H6H activity in vivo. Further optimization of cofactor requirements was performed by supplementing different cofactors into the culture media of this engineered yeast strain and analyzing the media by LCMS / MS after 72 hours of growth. This analysis identified that ferrous iron supplementation increases the conversion of hyoscyamine to scopolamine (Figure 44). Example 9. Identification of hyoscyamine dehydrogenase enzyme candidates and reduction of hyoscyamine aldehyde to hyoscyamine in engineered non-plant cells To identify a dehydrogenase enzyme suitable for performing the TA alcohol-aldehyde interconversions of the methods described herein, and in particular for reducing the aldehyde from hyoscyamine to hyoscyamine, a hyoscyamine dehydrogenase (HDH) open reading frame was identified. from publicly available plant RNA sequencing data. 9.1) Tissue-specific abundances (fragments per kilobase of contiguous sequence per million mapped reads, FPKM) and structural and functional annotations of putative proteins were obtained for each of the 43,861 unique transcripts identified from the Michigan State University A. belladonna transcriptome. Medicinal Plant Genomics Resource. Transcripts encoding hyoscyamine dehydrogenase candidates were identified based on clustering of tissue-specific expression profiles with those of the bait genes CYP80F1 (littorin mutase) and H6H (hyoscyamine 6p-hydroxylase / dioxygenase), which precede and follow respectively to the dehydrogenase step in the TA biosynthetic pathway, using the following computational filtering algorithm. First, the complete list of 43,861 transcripts was filtered for those annotated with any of the following protein family IDs (PFAM): PF00106, PF13561, PF08659, PF08240, PF00107, PF00248, PF00465, PF13685, PF13823, PF13602, PF 16884 , PF00248; or any of the following functional annotation keywords: alcohol dehydrogenase, aldehyde reductase, short chain, aldo / keto. Additionally, all functionally annotated transcripts containing the keywords putrescine, tropinone, and tropin were included in the filter as positive control TA-associated genes to validate clustering with bait genes. Tissue-specific mean expression profiles were then generated for the bait genes CYP80F1 and H6H. For each of the two bait genes, linear regression models were constructed to express the bait gene expression profile (in FPKM) as a linear function of each candidate gene profile, and correlation p values ​​were calculated for each candidate. Candidates identified using each of the two bait genes were clustered and duplicates were removed. The combined p values ​​for each candidate were calculated as the sum of the Iog10 p values ​​of the correlations with each of the two bait genes. Transcripts matching known dehydrogenases in the TA biosynthetic pathway (i.e., tropinone reductases I and II) were removed, and the remaining candidates were ranked by combined p-value and by distance from bait genes using hierarchical clustering. tissue-specific expression profiles (Figure 45). 9.2) Almost all candidates identified in Example 9.1 exhibited the same secondary root-specific expression pattern observed for known TA biosynthetic genes. A BLASTp search of the resulting ~30 candidates against the UniPROT / SwissPROT database revealed that many transcripts were missing terminal or internal sequence regions. To address this, de novo assembly of the transcriptome was repeated from deposited raw RNAseq reads using the Trinity software package (see Haas, B. J. et aL, De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 8, 1494-512 (2013)), and all missing sequence fragments for twelve of the HDH candidates were reconstituted by performing BLAST alignments of regions of incomplete sequence against the newly assembled transcriptome (Table 2 ). 9.3) The failing HDH activity was identified by selecting the candidates generated in Examples 9.1 and 9.2 in yeast. 9.3.1) The lack of an authentic commercial standard for hyoscyamine aldehyde and the insufficient yield of chemical syntheses required the co-expression of HDH candidates with the upstream biosynthetic enzyme—cytochrome P450 littorin mutase (CYP80F1)—to detection of in vivo activity through fed littorin (see Example 7). As littorin exhibits chromatographic and mass spectrometric properties similar to those of the HDH hyoscyamine product, an HDH selection strain (CSY1292) was constructed by integrating yeast codon-optimized AbCYP80F1 and DsH6H (see Example 8) into the CSY1251 genome, which allows selection of HDH candidates through detection of scopolamine (m / z+304) produced from fed littorin (m / z+290) via a three-step biosynthetic pathway (Figure 2). 9.3.2) Yeast codon-optimized sequences encoding each of the twelve HDH candidates were expressed from a low-copy plasmid in strain CSY1292, and scopolamine production was measured after 72 h of growth in medium supplemented with 1 mM littorin. One of the twelve candidates, HDH2 (termed AbHDH), showed a 35% decrease in hyoscyamine aldehyde levels and a medium accumulation of scopolamine (7.2 pg / L), indicating that it encoded failing HDH activity (Figure 46). 9.4) Structural and phylogenetic analyzes provided more information on the catalytic mechanism and evolutionary history of HDH. 9.4.1) A model of AbHDH homology was constructed based on the crystal structure of the Populus tremuloides sinapyl alcohol dehydrogenase (PtSAD; PDB: 1YQD) (Figure 47). AbHDH is a member of the zinc-dependent alcohol dehydrogenase (ZADH) family within the medium chain dehydrogenase / reductase (MDR) superfamily. Typical of this family, AbHDH exhibits a bi-lobular structure with a well conserved nucleotide binding domain and a more variable substrate binding domain. Alignment of residues S216, T217, S218, and K221 within the nucleotide-binding domain of AbHDH with phosphate-stabilizing residues S214, T215, S216, and K219 in PtSAD suggests that AbHDH is a NADPH-dependent oxidoreductase. Also typical of ZADH, AbHDH appears to bind to a structural Zn2+ using a tetrad of cysteine ​​residues near the protein surface (C105, C108, C111, and C119) and a catalytic Zn2+ within the active site. 9.4.2) The catalytic mechanism of AbHDH was elucidated by molecular coupling of the substrate, hyoscyamine aldehyde, to the active site using the Maestro / Glide software package (Figure 47). The most favorable binding mode positions the aldehyde group of the substrate at a distance of ~5 A from both the catalytic Zn2+ and the NADPH hydride donor. The result of the coupling and the general mechanism of ZADHs (see Bomati, E.K., Noel, J.P., Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase. Plant CelL 17, 1598-1611 (2005)) suggests the following mechanism catalytic for AbHDH. In the absence of substrate, the catalytic Zn2+ within the active site is stabilized with C52, H74, C168 and a water molecule, which is placed by polar interaction with S54 and displaced upon hyoscyamine aldehyde binding. The nucleophilic attack of the carbonyl of the aldehyde by a dihydronicotinamide hydride forms an oxyanion intermediate stabilized by the interaction with the catalytic Zn2+, and which is probably protonated through a proton shuttle between the ribose group of NADP+ and S54. 9.5) To confirm whether orthologous oxidoreductases catalyze hyoscyamine biosynthesis in other TA-producing Solanaceae, AbHDH coding sequence variants were identified from Datura innoxia and Datura stramonium transcriptomes by tBLASTx search (Figure 48). The HDH activity of the two identified orthologs (DiHDH, DsHDH) was validated by co-expression of codon-optimized yeast sequences with an additional copy of the flow-limiting DsH6H from low-copy plasmids at CSY1292 and measuring scopolamine production. in media supplemented with 1 mM littorin. DsHDH showed the highest substrate depletion and product accumulation of the tested variants (Figure 49). 9.6) The biosynthetic branch of medicinal TA comprising optimal enzyme variants and overexpression of a flow-limiting enzyme was integrated into a platform yeast strain. Strain CSY1294 was constructed by integrating yeast codon-optimized WfPPR and AbUGT, DsHDH, and a second copy of DsH6H into CSY1292. The production of scopolamine from fed littorin was verified in CSY1294 (Figure 50). Example 10: Yeast designed for the esterification of acyl donors and acceptors for the production of TA scaffolds. Yeast strains can be engineered to express enzymes that catalyze the esterification of activated acyl donor compounds and acyl acceptor compounds to produce various TA scaffolds (Figures 2, 3). Activation of the acyl donor group can be achieved by engineering an acyl donor-producing yeast strain to incorporate an enzyme that adds a chemical moiety with high group transfer potential, such as coenzyme A (CoA) or glucose ( glucoside), to the carboxyl group of the acyl donor, as described in Example 6. Examples of acyl donor activating enzymes that can be used in this capacity include CoA ligases and UDPglycosyltransferases. Examples of esterifying enzymes that can be used to catalyze the esterification of activated acyl donor and acyl acceptor compounds such as tropine and pseudotropin are acyltransferases, including serine carboxypeptidase type acyltransferases (SCPL-AT) and BAHD type acyltransferases. The coding sequence of such acyltransferases can be modified to improve their activity when expressed in a heterologous host such as yeast. 10.1) In plants, where SCPL-AT typically occur naturally, the SCPL-AT coding sequence includes N-terminal signal peptides that direct the nascent polypeptide to the endoplasmic reticulum (ER). Once localized to the ER, the SCPL-AT polypeptide is transported via the secretory trafficking pathway through the Golgi to the lumen of the vacuole, where it is found to exhibit activity. During this process of trafficking from the ER to the vacuole, they undergo several post-translational modifications (Figure 51), including, but not limited to, signal peptide cleavage, Nglycosylation, removal of internal propeptide sequences, and formation. of disulfide bonds (see Stehle, F., Stubbs, M.T., Strack, D. and Milkowski, C. Heterologous expression of a serine carboxypeptidaselike acyltransferase and characterization of the kinetic mechanism, FEBS Journal, 275, (2008)). However, as intracellular trafficking pathways and patterns of post-translational modifications differ between organisms, expression of SCPL-AT in heterologous hosts may result in incorrect subcellular localization and / or incorrect post-translational modification of the activity. As an example, the coding sequence of a SCPL-AT such as littorin synthase (LS) (Table 1) can be modified to improve activity when expressed in yeast. 10.1.1) Signal peptide sequences can affect the processing and localization of SCPL-AT in yeast. 10.1.1.1) The presence of a putative N-terminal signal peptide in AbLS suggests that it follows the expected ER-to-vacuole trafficking pathway of SCPL in planta. AbLS localization in yeast was examined by expressing AbLS N- and C-terminal GFP fusions from low-copy plasmids in CSY1294. Fluorescence microscopy revealed that the N-terminal fusion (GFP-AbLS) co-localized with the vacuolar membrane stain FM4-64 (Figure 52). No fluorescence was detected for the C-terminal fusion (AbLS-GFP), consistent with reports that a native C-terminus is crucial for the stability of SCPL acyltransferases (see Stehle, F., Stubbs, M.T., Strack , D. and Milkowski, C. Heterologous expression of a serine carboxypeptidase-like acyltransferase and characterization of the kinetic mechanism, FEBS Journal, 275, (2008)). 10.1.1.2) Vacuolar sequestration of SCPL-AT in yeast could prevent access to cytosolic substrate pools, since yeast probably lacks the necessary tonoplastic transporters present in plants for the exchange of secondary metabolites with the cytosol. To determine whether forced localization of AbLS to other yeast compartments—presumably, with better access to cytosolic metabolites—would enable activity, the wild-type N-terminal SP sequence was replaced with a panel of N-terminal signal sequences. taken from yeast proteins targeting the lumen of the vacuole (Prclp and Pep4p), the lumen-oriented vacuole membrane (Dap2p), the trans-Golgi network (Och1p), the lumen-oriented ER membrane (Mnslp ) and to the mitochondrial matrix (Cit1 p) (Figure 53). The wild-type SP was also completely deleted, and for another variant, a canonical peroxisome-targeting sequence (PTS1) was added to the C-terminus. These chimeric AbLS variants were expressed from high-copy plasmids in CSY1294 and the transformants were analyzed for activity by LC-MS / MS after 96 h of growth on selective media. No production of littorin or downstream intermediates was observed with any of the variants (Figure 53). 10.1.2) Incorrect post-translational processing of SCPL-AT in yeast could prevent expression of the active enzyme. 10.1.2.1) Protein N-glycosylation patterns differ between yeast and plants, and previous reports have suggested that correct N-glycosylation of various plant enzymes is important for their folding, stability and / or activity (see Kar, B ., Verma, P., den Haan, R., Sharma, A.K., Effect of N-linked glycosylation on the activity and stability of a β-glucosidase from Putranjiva roxburghii. Int. J. Biol. Macromol. 112, 490-498 (2018); see also Podzimek, T. etal., N-glycosylation of tomato nuclease TBN1 produced in N. benthamiana and its effect on the enzyme activity. Plant Sci. 276, 152-161 (2018); see also Strasser, R ., Plant protein glycosylation. Glycobiology. 26, 926-939 (2016)). In silico analysis of the AbLS polypeptide predicted four N-glycosylation sites (N152, N320, N376, N416) and no O-glycosylation of this protein was detected in N. benthamiana (Figure 54). Expressed in CSY1294 and wild-type N. benthamiana AbLS tagged with HA C-terminally, each of the four N^Q mutants (where the N to Q mutation abolishes N-glycosylation (23)), or one mutant quadruple N—>Q, and the glycosylation profiles were compared by Western blot. While wild-type AbLS, N^Q single mutants, and the quadruple mutant appeared as single bands in N. benthamiana, indicating a single glycosylation state, only the N—>Q quadruple mutant produced a single band in yeast; all other variants appeared as double or triple bands, indicating a combination of multiple glycosylation states (Figure 55). However, as the denser of the two bands of wild-type AbLS in yeast showed partial overlap with that of wild-type AbLS in tobacco, at least a fraction of AbLS expressed in yeast must be in a correct glycosylation state, and erroneous glycosylation is unlikely to explain the complete lack of AbLS activity in yeast. (Figures 54-55). 10.1.2.2) A subset of SCPL acyltransferases, including the sinapoylglucose:choline sinapoyltransferase from Arabidopsis thaliana (AtSCT) and an Avenacin synthase from Avena strigosa (AsSCPLI), contain an internal propeptide linker that is proteolytically removed to produce a heterodimer. active linked by disulfide bonds (see Shirley, A.M., Chapple, C., Biochemical characterization of sinapoylglucose:choline sinapoyltransferase, a serine carboxypeptidase like protein that functions as an acyltransferase in plant secondary metabolism. J. Biol. Chem. 278, 1987019877 (2003 ); see also Mugford, S.T. et al., A serine carboxypeptidase-like acyltransferase is required for synthesis of antimicrobial compounds and disease resistance in oats. Plant Cel1. 21,2473-2484 (2009)). Comparison of the amino acid sequence of AbLS with those of previously characterized plant serine carboxypeptidases and SCPL acyltransferases revealed the presence of an internal sequence of 25 to 30 residues that aligns with the highly variable propeptide of AtSCT, AsSCPLI, and carboxypeptidase 2. wheat (TaCBP2), suggesting that AbLS also undergoes endoproteolytic cleavage to form a heterodimer (Figure 56). Furthermore, an AbLS homology model suggested that the predicted internal propeptide blocks the active site, making it necessary to remove it for activity (Figure 57). However, wild-type AbLS expressed in N. benthamiana does not appear to undergo proteolytic cleavage, as no expected -20-25 kDa C-terminal fragment was detected by Western blotting under disulfide-reducing conditions (Figure 54, Figure 55). As the putative propeptide does not appear to be cleaved or eliminated in planta, AbLS could adopt a native conformation in plants that moves the propeptide away from the active site, but differences in the biochemical environment of the yeast secretory pathway and / or the vacuole prevent this change. , blocking the activity. To address this failure mode, split AbLS controls were constructed in which the N- ...

Claims

1. An engineered non-plant cell that produces a tropane alkaloid precursor, a tropane alkaloid product, or a tropane alkaloid derivative, wherein the engineered non-plant cell comprises a plurality of heterologous coding sequences encoding a plurality of enzymes within a pathway for producing the tropane alkaloid precursor, the tropane alkaloid product, or the tropane alkaloid derivative; wherein the cell comprises one or more alterations of one or more endogenous metabolic pathways or regulatory mechanisms selected from the group of endogenous arginine metabolism, endogenous phenylalanine and phenylpropanoid metabolism, endogenous polyamine regulatory mechanisms and metabolism, endogenous acetate metabolism, and endogenous glycoside metabolism.

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

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

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

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

6. The cell according to claims 1 to 5, wherein the engineered cell comprises one or more heterologous coding sequences for one or more enzymes, wherein at least one of the enzymes is selected from the group consisting of arginine decarboxylase, agmatine ureohydrolase, agmatinase, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidine ketide synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylalanine ammonia-lyase, tyrosine ammonia-lyase, phenylpyruvate reductase, 4-coumarate-CoA ligase, 3-phenyllactic acid UDP-glucosyltransferase 84A27, littorine synthase, littorine mutase, hyoscyamine dehydrogenase, hyoscyamine 63-hydroxylase / dioxygenase, and cocaine synthase.

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

8. The cell according to any one of claims 1 to 7, wherein the metabolism of endogenous phenylalanine and phenylpropanoids is altered in the cell by modifications in one or more coding sequences of one or more endogenous enzymes, wherein at least one of the enzymes is selected from the group consisting of pentafunctional AROM polypeptide, chorismate synthase, chorismate MA / 1 1 109 mutase, prephenate dehydratase, aromatic aminotransferase, and phenylacrylic acid decarboxylase.

9. The cell according to any one of claims 1 to 8, wherein the regulatory mechanisms of endogenous polyamines are altered in the cell by modifications in one or more coding sequences of one or more endogenous proteins, 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. The cell according to any of claims 1 to 9, wherein the metabolism of endogenous acetate is altered in the cell by modifications in one or more coding sequences of one or more endogenous enzymes, wherein at least one of the enzymes is selected from the group consisting of alcohol dehydrogenase and aldehyde dehydrogenase.

11. The cell according to any of claims 1 to 10, wherein the metabolism of endogenous glycosides is altered in the cell by modifications in one or more coding sequences of one or more endogenous enzymes, wherein at least one of the enzymes is selected from the group consisting of glucan 1,3-3-glucosidase and steryl-p-glucosidase.

12. The cell according to any one of claims 6 to 11, wherein the modifications of one or more coding sequences are selected from the group consisting of a feedback inhibition mitigating mutation in a cell-native biosynthetic enzyme or regulatory protein gene, a transcriptional modulating modification of a cell-native biosynthetic enzyme gene, and an inactivating mutation in a cell-native enzyme or protein.

13. The cell according to any of claims 1 to 12, wherein the engineered cell comprises one or more heterologous coding sequences encoding one or more enzymes comprising one or more soluble protein domains fused to the N-terminus of a serine carboxypeptidase-type acyltransferase domain for the purpose of enabling functional expression of the acyltransferase domain in a subcellular compartment of the engineered cell.

14. The cell according to any one of claims 1 to 13, wherein the cell produces a precursor of a tropane alkaloid product selected from the group consisting of an agmatine, N-carbamoylputrescine, N-methylputrescine, 4-methylaminobutanal, N-methylpyrrolinium, 4-(1-methyl-2-pyrrodynyl)-3-oxobutanoic acid, tropinone, tropine, pseudotropine, ecgonine, methylecgonine, coenzyme A covalently linked to phenyllactic acid by means of a thioester bond, or a sugar covalently linked to cinnamic acid, ferulic acid, coumaric acid, or phenyllactic acid by means of a glycosidic bond.

15. The cell according to any of claims 1 to 14, wherein the cell produces a tropane alkaloid product selected from the group consisting of hyoscyamine, atropine, anisodamine, scopolamine, calistegine, cocaine, or a non-natural tropane alkaloid.

16. The cell according to any of claims 1 to 15, 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.

17. The cell according to claim 15, wherein the cell produces a tropane alkaloid product selected from the group consisting of hyoscyamine, atropine, or scopolamine.

18. The cell according to any of claims 1 to 17, wherein the transport of one or more of the TA, one or more TA precursors and / or one or more TA derivatives across the intracellular membranes or across the plasma membrane is modified in the cell.

19. The cell according to claim 18, wherein the modified transport is enabled by one or more heterologous coding sequences encoding one or more transporters, wherein at least one of the transporters is selected from the group consisting of a multidrug and toxin extrusion transporter, a nitrate / peptide family transporter, an ATP-binding cassette transporter, and a pleiotropic drug resistance transporter.

20. An engineered non-plant cell that produces a tropane alkaloid product or a derivative of a tropane alkaloid product, wherein the engineered non-plant cell comprises a plurality of heterologous coding sequences encoding a plurality of enzymes within a pathway for producing the tropane alkaloid product or the derivative of a tropane alkaloid product.

21. The cell according to claim 20, wherein the cell is a microbial cell.

22. The cell according to claim 21, wherein the cell is a fungal cell.

23. The cell according to claims 20 to 22, wherein the engineered cell comprises one or more heterologous coding sequences for one or more enzymes, wherein at least one of the enzymes is selected from the group consisting of arginine decarboxylase, agmatine ureohydrolase, agmatinase, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidine ketide synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylalanine ammonia-lyase, tyrosine ammonia-lyase, phenylpyruvate reductase, 4-coumarate-CoA ligase, 3-phenyllactic acid UDPglucosyltransferase 84A27, littorine synthase, littorine mutase, hyoscyamine dehydrogenase, hyoscyamine 6β-hydroxylase / dioxygenase, and cocaine synthase.

24. The cell according to any of claims 20 to 23, wherein the engineered cell comprises one or more heterologous coding sequences encoding one or more enzymes comprising one or more soluble protein domains fused to the N-terminus of a serine carboxypeptidase-type acyltransferase domain for the purpose of enabling functional expression of the acyltransferase domain in a subcellular compartment of the engineered cell.

25. The cell according to any of claims 20 to 24, wherein the cell produces a tropane alkaloid product selected from the group consisting of hyoscyamine, atropine, anisodamine, scopolamine, calistegine, cocaine, or a non-natural tropane alkaloid.

26. The cell according to any of claims 20 to 25, 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-chloropescopolamine, p-bromoscopolamine, N-methylhyoscyamine, N-butylhyoscyamine, N-methylscopolamine, N-butylscopolamine, N-acetylhyoscyamine, and N-acetylscopolamine.

27. The cell according to claim 25, wherein the cell produces a tropane alkaloid product selected from the group consisting of hyoscyamine, atropine, or scopolamine.

28. The cell according to any of claims 20 to 27, wherein the transport of one or more of the TA, one or more TA precursors and / or one or more TA derivatives across intracellular membranes or across the plasma membrane is modified in the cell.

29. The cell according to claim 28, wherein the modified transport is enabled by one or more heterologous coding sequences encoding one or more transporters, wherein at least one of the transporters is selected from the group consisting of a multidrug and toxin extrusion transporter, a nitrate / peptide family transporter, an ATP-binding cassette transporter, and a pleiotropic drug resistance transporter.

30. 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 a cell according to any one of claims 1 to 29 under conditions suitable for protein production; (b) adding a starting compound to the cell culture; and (c) recovering the tropane alkaloid product, the precursor of a tropane alkaloid product, or the derivative of a tropane alkaloid product from the culture.

31. The method according to claim 30, wherein the cells are grown in a batch or fed-batch fermentation.

32. The method according to claims 30 and 31, wherein the starting compound added to the cell culture is a sugar or a substrate containing one or more sugars, or which is converted into one or more sugars during microbial fermentation.

33. The method according to claims 30 and 31, 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.

34. The method according to claims 30 and 31, wherein the starting compound added to the cell culture is a precursor of a tropane alkaloid product.

35. The method according to claims 30 to 34, wherein the precursor of a tropane alkaloid product, the tropane alkaloid product or the derivative of a tropane alkaloid product is recovered by a process comprising liquid-liquid extraction, chromatographic separation, distillation or recrystallization.