Non-plant host cells producing tropane alkaloids (TA) and methods of making and using the same

Abstract

In particular, provided herein is 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. Also described is a method for using the cell to produce a tropane alkaloid, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application Serial Nos. 62 / 815,709, filed March 8, 2019, 62 / 848,419, filed May 15, 2019, and 62 / 891,771, filed August 26, 2019, which are incorporated herein by reference.

[0003] Government Rights

[0004] This invention was made with government support under Contracts GM110699 and AT007886 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Background Art

[0005] Tropane alkaloids (TAs) are a class of anticholinergic secondary metabolites produced by plants of the nightshade family (Solanaceae). Several TAs, including atropine, hyoscyamine and scopolamine, are listed 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, and for the management of symptoms of Parkinson's disease. Therefore, it is of great significance to have an adequate and stable supply of these TA molecules so that researchers and doctors can use them. The current supply chain for pharmaceutical TAs relies on extraction from unsustainable and geographically restricted monocultures, in which TAs account for only 0.2-4% of the dry weight, and the plants are susceptible to pests, land use changes and climate. Due to the difficulties posed by TA stereochemistry, the total chemical synthesis of TAs from simple raw materials has not yet proven to be economical enough for industrial use. In addition, to date, poor economies of scale and long generation times have made engineering transgenic plants or plant cultures with increased TA production an unfeasible strategy for obtaining these compounds. Therefore, methods for preparing TA are of interest. Summary of the invention

[0006] The present invention includes non-plant organisms engineered for producing various tropane alkaloids (TA) from precursors and sugars. For example, the present invention includes engineered microbial strains for producing pharmaceutical TAs, which are defined herein as naturally occurring TAs with established uses in current medical practice, including hyoscyamine, atropine, anisodamine, and scopolamine, as well as precursors and derivatives thereof. The present invention also includes engineered microbial strains for producing non-pharmaceutical TAs, which are defined herein as naturally occurring TAs that have no established uses in current medical practice but may have biological activities of pharmaceutical interest, including calystegine, cocaine, and precursors and derivatives thereof. The present invention also includes engineered microbial strains for producing non-natural TAs, which are defined herein as TAs that are not produced by unmodified organisms, such as TAs produced by esterification of acyl donor and acyl acceptor compounds that are not esterified in naturally occurring organisms, including derivatives of pharmaceutical TAs and derivatives of non-pharmaceutical TAs. Examples of the solutions included in the present invention are Figure 1-3 Described in detail.

[0007] The present invention includes methods of producing pseudotropines and alkaloids derived from pseudotropines (e.g., caltropin) using microorganisms engineered to express at least one heterologous enzyme as a microbial catalyst. The present invention also includes methods of 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 a microbial catalyst. The present invention also includes methods of esterifying acyl donors and acceptors for the production of TA scaffolds using microorganisms engineered to express at least one heterologous enzyme as a microbial catalyst. The present invention also includes methods of modifying and culturing engineered microbial strains for the production of medicinal TAs such as hyoscyamine and scopolamine, non-medicinal TAs such as caltropin, and non-natural TAs such as those derived from the esterification of tropine with acyl donor compounds other than 3-phenyllactic acid (PLA).

[0008] Provided are host cells engineered to produce tropane alkaloids (TA) of interest, such as hyoscyamine and scopolamine. The TA of interest may include TA precursors, TA, and modifications of TA, including derivatives of TA. The host cell may have one or more modifications selected from the following: feedback inhibition relief mutations in enzyme genes; transcriptional regulatory modifications of biosynthetic enzyme genes; inactivation mutations in enzymes; and heterologous coding sequences. Also provided are methods for producing TA of interest using host cells and compositions that can be used in the methods of the invention, such as kits, systems, and the like.

[0009] One aspect of the present invention provides a method for forming a product stream having a tropane alkaloid (TA) product. The method includes providing an engineered non-plant cell and a raw material including nutrients and water to a batch reactor, wherein the engineered non-plant cell has at least one modification selected from the group consisting of: a feedback inhibition relief mutation in a cell-intrinsic biosynthetic enzyme gene; a transcriptional regulatory modification of a cell-intrinsic biosynthetic enzyme gene; and an inactivation mutation in a cell-intrinsic enzyme. In addition, the method includes fermenting the engineered non-plant cell in a batch reactor by incubating the engineered non-plant cell for a period of at least about 5 minutes to produce a solution comprising a TA product and a cell material. The method also includes separating the TA product from the cell material using at least one separation unit to provide the product stream comprising the TA product.

[0010] In another aspect, the present invention provides a method for forming a product stream having a TA product. The method includes providing engineered non-plant cells and a feedstock including nutrients and water to a reactor. The method also includes fermenting the engineered non-plant cells in the reactor by incubating the engineered yeast cells for a period of at least about 5 minutes (e.g., 5 minutes or longer) to produce a solution including cell material and the TA product. In addition, the method includes separating the TA product from the cell material using at least one separation unit to provide a product stream comprising the TA product.

[0011] Another aspect of the present invention provides an engineered non-plant cell that produces a tropane alkaloid (TA) product, wherein the engineered non-plant cell has at least one modification selected from the group consisting of: a feedback inhibition relief mutation in a cell-intrinsic biosynthetic enzyme gene; a transcriptional regulation modification of a cell-intrinsic biosynthetic enzyme gene; and an inactivating mutation in a cell-intrinsic enzyme. The engineered non-plant cell comprises at least one heterologous coding sequence encoding at least one enzyme selected from the group consisting of arginine decarboxylase, agmatine ureahydrolase, agmatinease, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidine ketide synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylpyruvate reductase, 3-phenyllactate UDP-glucosyltransferase 84A27, littorine synthase, littorine mutase, scopolamine dehydrogenase, scopolamine 6β-hydroxylase / dioxygenase, and cocaine synthase. In some examples, the engineered non-plant cell comprises a plurality of heterologous coding sequences encoding enzymes selected from the group consisting of arginine decarboxylase, agmatine ureahydrolase, agmatinease, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidone synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylpyruvate reductase, 3-phenyllactate UDP-glucosyltransferase 84A27, ritolin synthase, ritolin mutase, scopolamine dehydrogenase, scopolamine 6β-hydroxylase / dioxygenase, and cocaine synthase. In some examples, the heterologous coding sequences may be operably linked. The operably linked heterologous coding sequences may be in the same pathway for producing a specific tropane alkaloid product. In some examples, the engineered non-plant cell comprises one or more improvements to intracellular compartmentalization selected from the group including, but not limited to, improved intracellular trafficking of an enzyme, improved intracellular localization of an enzyme, and improved intracellular transport of a metabolite.

[0012] In another aspect of the present invention, a therapeutic agent is provided, wherein the therapeutic agent comprises a tropane alkaloid product. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings. It should be emphasized that, according to common practice, the various features of the drawings are not drawn to scale. Instead, the dimensions of the various features are arbitrarily expanded or reduced for clarity. The accompanying drawings include the following illustrations.

[0014] Figure 1An exemplary biosynthetic scheme for converting L-arginine to a non-pharmaceutical TA is shown. ADC, arginine decarboxylase; ARG, arginase; AUH, agmatine ureahydrolase; ODC, ornithine decarboxylase; PAO, polyamine oxidase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; spont., spontaneous (non-enzymatic) step; PYKS, pyrrolidone synthase; CYP82M3, tropinone synthase; CPR, cytochrome P450-NADP + Reductase; TR2, tropinone reductase 2; P450, cytochrome P450. Arginine, ornithine, spermine, spermidine, and putrescine are naturally synthesized in yeast. All other metabolites shown are not naturally produced in yeast. The end products indicated inside the boxes are examples of non-pharmaceutical TAs.

[0015] Figure 2 Exemplary biosynthetic pathways by which amino acids can be converted into pharmaceutical TA molecules of interest and their precursor molecules are shown. This example shows the conversion of L-arginine and L-phenylalanine to pharmaceutical TA. ADC, arginine decarboxylase; ARG, arginase; AUH, agmatine ureahydrolase; ODC, ornithine decarboxylase; PAO, polyamine oxidase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; spont., spontaneous (nonenzymatic) step; PYKS, pyrrolidone synthase; CYP82M3, tropinone synthase; CPR, cytochrome P450-NADP+ reductase; TR1, tropinone reductase 1; ArAT, aromatic aminotransferase; PPR, phenylpyruvate reductase; UGT84A27, 3-phenyllactate UDP-glucosyltransferase; LS, ritolin synthase; CYP80F1, ritolin mutase; HDH, (S)-hyoscine dehydrogenase; H6H, (S)-hyoscine 6β-hydroxylase / dioxygenase. Arginine, ornithine, spermine, spermidine, putrescine, phenylalanine, 3-phenylpyruvic acid and trace amounts of 3-phenyllactic acid are naturally synthesized in yeast. All other metabolites shown are not naturally produced in yeast. The end products indicated inside the boxes are examples of pharmaceutical TAs.

[0016] Figure 3Exemplary biosynthetic pathways by which amino acids can be converted to non-pharmaceutical TAs and their precursor molecules are shown. In this example, L-arginine and L-phenylalanine are converted to non-natural TAs. ADC, arginine decarboxylase; ARG, arginase; AUH, agmatine ureahydrolase; ODC, ornithine decarboxylase; PAO, polyamine oxidase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; spont., spontaneous (non-enzymatic) step; PYKS, pyrrolidone synthase; CYP82M3, tropinone synthase; CPR, cytochrome P450-NADP + Reductase; TR1, tropinone reductase 1; PAL, phenylalanine ammonia lyase; 4CL, 4-coumarate-CoA ligase; CS, cocaine synthase. Arginine, ornithine, spermine, spermidine, putrescine, and phenylalanine are naturally synthesized in yeast. All other metabolites shown are not naturally produced in yeast. The end products indicated inside the boxes are examples of non-natural TAs.

[0017] Figure 4 Exemplary biosynthetic pathways for producing putrescine from amino acids and other polyamine molecules are shown. The figure shows how putrescine can be made from central metabolites using endogenous yeast and heterologous biosynthetic pathways.

[0018] Figure 5 Yeast strains engineered to overexpress endogenous biosynthetic enzymes involved in arginine and polyamine metabolism are shown to produce putrescine in liquid culture. Additional copies of native genes were expressed by low-copy plasmids in wild-type yeast (CEN.PK2). Before LC-MS / MS analysis, the transformed strains were cultured at 30°C for 48h in selective medium with 2% dextrose. All data represent the mean of at least three biological replicates, and error bars show standard deviations. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001. Unless otherwise noted, statistical significance relative to the corresponding control (i.e., CEN.PK2) is shown.

[0019] Figure 6Yeast strains engineered to heterologously express biosynthetic enzymes from organisms other than yeast that participate in arginine and polyamine metabolism can produce putrescine in liquid culture. In this example, yeast strains are engineered to express heterologous biosynthetic pathways from plants and bacteria. Heterologous enzymes are expressed by low copy plasmids in wild-type yeast. Before LC-MS / MS analysis, the transformed strains were cultured at 30°C in selective medium with 2% dextrose for 48h. All data represent the mean of at least three biological replicates, and error bars show standard deviations. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001. Unless otherwise noted, statistical significance relative to corresponding controls (i.e., CEN.PK2) is shown.

[0020] Figure 7 Yeast strains engineered to heterologously express biosynthetic enzymes from organisms other than yeast that participate in arginine and polyamine metabolism can produce TA precursors and intermediates agmatine, N-carbamoyl putrescine and putrescine in liquid culture. The figure shows the functional verification of agmatine / putrescine biosynthetic pathway genes in yeast. Wild-type yeast strain CEN.PK2 was transformed with three low-copy plasmids to co-express between zero (negative control) and three specified biosynthetic genes. The plasmid expressing blue fluorescent protein (BFP) was used as a negative control for each of three auxotrophic selection markers URA3, TRP1 and LEU2. Before LC-MS / MS analysis was performed on metabolite yield, the transformed strain was cultured at 30°C in a selective medium with 2% dextrose for 48h. All data show the titer measured by LC-MS / MS peak area relative to a negative control (CEN.PK2). Data represent the mean value of three biological replicates, and error bars show standard deviation.

[0021] Figure 8 Shown is an endogenous regulatory pathway that tightly controls intracellular putrescine levels during normal yeast growth.

[0022] Fig. 9 Heat map of putrescine production in yeast strains is shown, in which endogenous polyamine biosynthesis regulatory mechanisms are disrupted. For the overexpression of natural or heterologous putrescine pathways, the specified gene is expressed by low copy plasmid in wild-type yeast (WT) or each single-disruption strain. Before LC-MS / MS analysis, the strain was cultured at 30°C for 72h in the selective (YNB-DO) medium with 2% dextrose. All data represent the mean value of at least three biological replicates. The figure shows that yeast strains with single destruction of polyamine metabolic genes and overexpressed endogenous or heterologous putrescine biosynthetic pathways can produce putrescine in liquid culture.

[0023] Fig.10A summary of engineering efforts to increase putrescine production in yeast is provided. The '+' symbol indicates expression of at least one gene from the pathway, while '-' indicates no gene expression from the pathway. Prior to LC-MS / MS analysis, the strain was cultured at 30°C for 48h in selective medium with 2% dextrose. All data represent the mean of at least three biological replicates, and error bars show standard deviations. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001. Unless otherwise indicated, statistical significance relative to the corresponding control (i.e., CEN.PK2) is shown.

[0024] Fig.11 LC-MS / MS chromatograms are shown, which show that putrescine is gradually converted to TA intermediate NMPy and byproduct 4MAB acid through intermediates NMP and 4MAB in engineered yeast according to an embodiment of the present invention. A proposed mechanism for the formation of 4MAB acid byproducts through the activity of an endogenous yeast enzyme (ALD) is shown. Extracted ion chromatogram MRM traces of each metabolite along the pathway and authentic standards using the highest precursor ion / product ion transition of each metabolite are shown. The control represents strain CSY1235 expressing SPE1, AsADC and speB on a low copy plasmid (see Example 1.5). The chromatogram traces represent three biological replicates. Enzyme symbols: PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; ALD, aldehyde dehydrogenase.

[0025] Fig.12LC-MS / MS chromatograms are shown showing the relative yields of TA precursors (A) putrescine, (B) NMP, (C, E) 4MAB, and (D, F) NMPy in liquid cultures of engineered yeast expressing AbPMT1 and MPO enzymes according to embodiments of the present invention. (A) MRM chromatogram of putrescine (m / z+89→72) of CSY1235 containing pCS4239 to overproduce putrescine. (B) MRM chromatogram of NMP (m / z+103→72) of CSY1235 containing pCS4239 and expressing AbPMT1 from a low copy plasmid. (C, D) Corresponding MRM chromatograms of 4MAB (m / z+102→71) and NMPy (m / z+84→57) of CSY1235 containing pCS4239 and expressing AbPMT1 and NtMPO1 from a low copy plasmid. (E, F) Corresponding MRM chromatograms of 4MAB (m / z+102→71) and NMPy (m / z+84→57) of CSY1235 containing pCS4239 and expressing AbPMT1 and DmMPO1ΔC-PTS1 from low copy plasmids. The Y axis of the traces is the raw MRM ion counts. All chromatograms were generated by LC-MS / MS analysis of the extracellular medium after growth for 48 hours at 30°C in selective medium with 2% dextrose. The traces represent at least three biological replicates.

[0026] Fig.13 The effect of MEU1 disruption on SAM-dependent putrescine N-methylation by AbPMT1 is shown. Wild-type strain CEN.PK2 or meu1 disruption strain CSY1229 was co-transformed with low-copy plasmids expressing SPE1, AsADC, and speB and AbPMT1. Data indicate average NMP titers relative to CEN.PK2 controls, as quantified by LC-MS / MS peak areas of 3 biological replicates after 48 hours of growth at 30°C in selective medium with 2% dextrose. Error bars show standard deviations. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001.

[0027] Fig.14 In silico predictions of the subcellular localization of NMPy biosynthetic genes in plant and yeast / fungal cells using the SherLoc2 web server are shown. The values ​​and colors represent the probability scores (0 to 1) of localization to each of the following compartments: CYT, cytosol; NUC, nucleus; VAC, vacuole; CHL, chloroplast; MIT, mitochondria; POX, peroxisome.

[0028] Fig.15Shown are (A) colocalization of NtMPO1 with N-terminal and C-terminal GFP tags and the PEX3 peroxisomal marker according to an embodiment of the present invention, and (B) the effect of N-terminal and C-terminal GFP tagging of NtMPO1 on the production of TA precursors 4MAB and NMPy in liquid culture of engineered yeast. The figure shows experimental verification of the subcellular localization of NtMPO1. (A) Fluorescence microscopy of NtMPO1 N-terminal and C-terminal GFP fusions coexpressed with the peroxisomal marker mCherry-PEX3 in wild-type yeast (CEN.PK2). White arrows indicate the colocalization of NtMPO1 with GFP tags and peroxisomes. Scale bar, 10 μm. (B) The effect of forced cytosol localization of NtMPO1 on the production of 4MAB or NMPy. Wild-type yeast (CEN.PK2) was co-transformed with low-copy plasmids expressing wild-type NtMPO1 or N-terminal or C-terminal GFP fusions and low-copy plasmids expressing SPE1, AsADC, and speB and AbPMT1. LC-MS / MS analysis was performed after 48 hours of growth at 30°C in selective medium with 2% dextrose. Data represent the mean of three biological replicates; error bars show standard deviation. The most likely subcellular compartments are indicated based on the microscopy data in (a).

[0029] Fig.16 Provides fluorescence microscopy data depicting the subcellular localization of AbPMT1 and NtMPO1 when heterologously expressed in yeast. Microscopy was performed on wild-type yeast expressing AbPMT1 or NtMPO1 with N-terminal or C-terminal GFP tags from low-copy plasmids. Scale bar, 10 μm.

[0030] Fig.17Shown are (A) sequence alignments of NtMPO1 and putative MPO enzymes AbMPO1 and DmMPO1 identified from plant transcriptome data (from top to bottom: SEQ ID NOs: 27-29), (B) comparison of production of TA precursors 4MAB and NMPy in liquid cultures of engineered yeast strains expressing NtMPO1, AbMPO1 or DmMPO1, and (C) comparison of predicted three-dimensional structures of NtMPO1, AbMPO1 and DmMPO1 determined by homology modeling according to embodiments of the present invention. (A) Alignment of query NtMPO1 sequence with AbMPO1 and DmMPO1 candidates from the 1000Plants Project database. Blue indicates conservation of amino acid structure; red indicates mismatch. (B) Comparison of relative activities of MPO orthologs. Putrescine overproducing strain CSY1235 (see Example 1.5) was co-transformed with low-copy plasmids expressing SPE1, AsADC and speB, AbPMT1, and one of the three MPO variants. LC-MS / MS analysis was performed after 48 h of growth at 30°C in selective medium. Data represent the mean of three biological replicates; error bars show standard deviation. (C) Homology model of the MPO enzyme (pink) constructed based on the crystal structure of the copper-containing amino oxidase of pea (Pisum sativum) (PDB: 1KSI, blue) using the RaptorX web server. Top: NtMPO1; Center: AbMPO1; Bottom: DmMPO1.

[0031] Fig.18 4MAB production in liquid cultures of engineered yeast strains that overproduce putrescine and express AbPMT1 and N- and C-terminal truncations of NtMPO1 and DmMPO1 is shown. The figure shows the effect of N- and C-terminal truncations of methylputrescine oxidase on 4MAB production in engineered yeast. The wild-type (WT) enzyme and the indicated truncations were expressed from low copy plasmids in the putrescine-overproducing strain CSY1235 (see Example 1.5). The strains were cultured in selective medium with 2% dextrose at 30°C for 48h prior to LC-MS / MS analysis. All data represent the mean of at least three biological replicates, and the error bars show standard deviations. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001.

[0032] Fig.19The production of TA precursors 4MAB and NMPy and the byproduct 4MAB acid in liquid cultures of engineered yeast strains containing a single disruption of one of the four native aldehyde dehydrogenase genes is shown. The figure shows the effect of disruption of a single aldehyde dehydrogenase on the accumulation of 4MAB acid. Putrescine overproducing strain CSY1235 (control) or substrains with nonsense mutation disruptions of hfd1, ald4, ald5, or ald6 were cultured with strains expressing SPE1, AsADC and speB, AbPMT1, and 4MAB acid titer as measured by LC-MS / MS peak area normalized to CSY1235 (no ALD disruption) after 48 h of growth at 30°C in selective medium. Data represent the mean of three biological replicates, and error bars show standard deviation. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001.

[0033] Fig. 20 The production of (A) 4MAB acid byproducts and (B) TA precursors 4MAB and NMPy in liquid cultures of engineered yeast strains containing one or more disruptions of native aldehyde dehydrogenases is shown. The figure shows the effect of aldehyde dehydrogenase gene disruption on the production of (A) 4MAB acid byproducts and (B) 4MAB and NMPy in engineered yeast. The '+' and '-' symbols indicate the presence or absence of functional enzymes, respectively. Prior to LC-MS / MS analysis, the strains were cultured at 30°C in selective (YNB-DO) medium with 2% dextrose for 48h. All data represent the mean of at least three biological replicates, and error bars show standard deviations. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001. Unless otherwise indicated, statistical significance relative to the corresponding control (CSY1235) is shown.

[0034] Fig.21 1 shows a comparison of the production of the TA precursor NMPy in liquid culture of engineered yeast strains in the presence of low-copy plasmid-based expression or genomic expression of putrescine overproducing genes AbPMT1 and DmMPO1 truncations according to embodiments of the present invention. The figure provides a comparison of 4MAB and NMPy production in the presence of plasmid-based expression (CSY1241) and genomic expression (CSY1243) of NMPy biosynthetic genes. Strain CSY1241 was engineered to express putrescine overproducing genes (SPE1, AsADC, speB), AbPMT1 and The strain CSY1243 was transformed with a low copy plasmid. All the above genes were expressed by genomic integrated copies. After 48 h of growth at 30 ° C in selective (CSY1241) or non-selective (CSY1243) medium, NMPy levels were quantified by LC-MS / MS. Data represent the average of at least two biological replicates, and error bars indicate standard deviations.

[0035] Fig. 22 The biosynthetic pathway for producing the byproduct scorbutine from NMPy and MPOB according to an embodiment of the present invention is shown. The putative major and minor side reactions in yeast are indicated by bold and dashed arrows, respectively.

[0036] Fig.23 Comparison of the yields of TA precursors tropinone and tropine and byproduct scopolamine in liquid cultures of engineered yeast strains expressing low-copy plasmid-based AbPYKS, AbCYP82M3, DsTR1, and one of four different CPRs is shown. The figure shows the yields of tropine and related intermediates in the case of expressing AbPYKS, AbCYP82M3, and DsTR1 in engineered yeast. The designated genes were expressed from low-copy plasmids in CSY1246; the '+' and '–' symbols indicate the presence or absence of enzymes. Prior to LC-MS / MS analysis, the strains were cultured at 30°C in selective medium with 2% dextrose for 48 h. The data represent the mean of three biological replicates, and the error bars show the standard deviation. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001.

[0037] Fig.24(A) LC-MS / MS chromatograms are shown, which show the characteristic triplet of the TA precursor MPOB produced in liquid culture of engineered yeast strains, and (B) the production of TA precursors NMPy and MPOB in liquid culture of yeast strains engineered to express AbPYKS, AbCYP82M3 and one of the four CPRs by plasmids. The figure shows the accumulation of NMPy and MPOB in the culture medium of the engineered strain expressing AbPYKS. (A) Representative LC-MS / MS multiple reaction monitoring (MRM) chromatograms for detecting MPOB in the extracellular medium of CSY1246 expressing only AbPYKS by a low copy plasmid. The three characteristic MPOB isoform peaks are labeled with (I), (II) and (III). LC-MS / MS analysis was performed after 48 h of growth at 30°C in selective medium. (B) Relative abundance of NMPy and MPOB in the extracellular medium of CSY1246 expressing AbPYKS, AbCYP82M3 and one of the four CPRs from low-copy plasmids after growth in selective medium at 30°C for 48 h (all 3 peaks). '+' and '-' symbols indicate the presence or absence of the gene. Data represent the mean of three biological replicates; error bars indicate standard deviation.

[0038] Fig.25 The effect of growth temperature on the production of TA precursor tropine and byproduct scopolamine in liquid culture of engineered yeast is shown. The figure shows the effect of growth temperature on spontaneous scopolamine production in a tropine-producing yeast strain (CSY1248). Relative selectivity represents the ratio of relative tropine titer to relative scopolamine titer. Prior to LC-MS / MS analysis, the strains were cultured at 30°C or 25°C in non-selective medium with 2% dextrose for 48h. The data represent the mean of three biological replicates, and the error bars show standard deviations. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001.

[0039] Fig.26Shown are the effects of (A) ALD4 and ALD6 reconstruction on the growth of engineered yeast strains producing tropin on medium supplemented or not supplemented with acetate, and (B) the effect of eliminating acetate auxotrophy on the production of byproducts 4MAB acid and scopolamine in liquid culture of engineered yeast strains producing tropin according to embodiments of the present invention. The figure shows the effect of eliminating acetate auxotrophy in engineered tropin-producing yeast strains. (A) The effect of reconstructing functional ALD4 or ALD6 genes on the growth of a yeast strain (CSY1246) producing NMPy with and without acetate supplementation. ALD4 and ALD6 are expressed by low copy plasmids. 'WT' indicates CSY1246 with a control (BFP) plasmid. The adjacent columns show ten-fold dilutions. (B) The production of 4MAB acid and scopolamine byproducts obtained by reconstructed acetate metabolism in engineered yeast. '+' and '–' symbols indicate the presence or absence of feed metabolites (acetate) or ALD4 and ALD6 genes expressed from low-copy plasmids. Strains were cultured in selective (YNB-DO) medium with 2% dextrose at 30 °C for 48 h prior to LC-MS / MS analysis. Data represent the mean of three biological replicates, and error bars show standard deviations. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001.

[0040] Fig. 27 Shown are (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 tropine, and (B) representative LC-MS / MS chromatograms of the TA precursor MPOB produced in liquid cultures of yeast strains engineered to produce tropine with and without acetate auxotrophy according to embodiments of the present invention. The figure shows the effect of reconstructing ALD6 activity on the flux of metabolites toward tropine through NMPy in engineered yeast. (A) The yield of intermediates between NMPy and tropinone in engineered strains with and without functional Ald6p. Intermediate abundance was measured by LC-MS / MS MRM in the extracellular medium of a tropigenin-producing integrated strain (CSY1248) grown in non-selective medium supplemented with 0.1% w / v potassium acetate (grey) at 25°C for 48 h or a tropigenin-producing strain (CSY1249) with reconstituted ALD6 grown in non-selective medium not supplemented with acetate (pink) at 25°C for 48 h. Data represent the mean of three biological replicates; error bars indicate standard deviation. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001. (B) Representative MRM chromatograms of MPOB production from CSY1248 (grey) and CSY1249 (red) cultured as described in (a).

[0041] Fig.28 Progress in improving the production of the TA precursor tropine and the byproduct scopolamine in liquid cultures of engineered yeast strains is shown. The figure provides a summary of strains engineered to increase tropine production in yeast. The '-' symbol indicates the absence of a gene; 'p' and 'i' indicate gene expression from a low copy plasmid or genomic integration, respectively. Prior to LC-MS / MS analysis, the strains were cultured at 30°C or 25°C in selective or non-selective medium with 2% dextrose for 48h. The data represent the mean of three biological replicates, and the error bars show standard deviations. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001.

[0042] Fig.29 1 shows the effect of expressing additional copies of the heterologous biosynthetic enzymes PMT, MPO, PYKS, and CYP82M3 on the yield of each TA precursor between putrescine and tropine in liquid culture of engineered yeast according to embodiments of the present invention. The figure identifies the metabolic bottleneck in the optimized tropine-producing strain (CSY1249). The strain CSY1249 was transformed with a low copy plasmid of an additional copy of AbPYKS or AbCYP82M3. After 48 h of growth at 25°C in selective medium, the intermediate levels in the extracellular medium were quantified by LC-MS / MS. The data indicate the mean of three biological replicates, and the error bars show the standard deviation.

[0043] Fig.30 The effect of additional copies of the bottleneck enzymes PMT and PYKS on tropine production in engineered yeast is shown. The figure shows the alleviation of metabolic bottlenecks by genomic integration of additional copies of the PMT and PYKS enzymes. Tropine-producing strains CSY1249 and CSY1251 were cultured in non-selective medium at 25°C for 48h prior to LC-MS / MS analysis of the growth medium. Data represent the mean of three biological replicates, and error bars show standard deviations. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001.

[0044] Fig.31The output of TA precursor acyl donor compound PLA in the liquid culture of the engineered yeast strain expressing heterologous lactate dehydrogenase and phenylpyruvate reductase is shown.The figure illustrates the LC-MS / MS analysis of the yeast strain engineered to convert L-phenylalanine into 3-phenyllactic acid.The yeast strain is engineered to have a low copy CEN / ARS plasmid containing a LEU2 selection marker, a TDH3 promoter and the coding sequence of the following: BFP as a negative control; LDH variants from Bacillus coagulans (B.coagulans) (BcLLDH), Lactobacillus casei (L.casei) (LcLLDH), Lactobacillus plantarum (L.plantarum) (LpLLDH); or PPR variants from A.belladonna (A.belladonna) (AbPPR), Lactobacillus plantarum (LpPPR), Escherichia coli (Escherichia coli) (hcxB) or W.fluorescens (WfPPR). Yeast from freshly transformed colonies were grown in 300 μL of selective medium (-Leu) in 96-well deep-well microtiter plates. After 72 hours of growth in a shaking incubator at 25°C and 460 rpm, yeast were pelleted and the culture supernatant analyzed by LC-MS / MS. Data are shown based on extracted ion chromatograms (ammonium adducts, EIC m / z + =184) Relative 3-phenyllactic acid titer normalized to trace levels present in negative control. Data represent the mean of three biological replicates, and error bars show standard deviation. Student's two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001.

[0045] Fig.32 LC-MS / MS chromatograms are shown, which illustrate the production of TA precursor acyl donor compound cinnamic acid in the liquid culture of the engineered yeast strain expressing phenylalanine ammonia lyase. The figure shows the LC-MS / MS analysis of the yeast strain engineered to convert L-phenylalanine into cinnamic acid. The yeast strain is engineered to have a low copy CEN / ARS plasmid containing a TRP1 selective marker, a TEF1 promoter, and (i) BFP or (ii) the coding sequence of Arabidopsis thaliana (A.thaliana) phenylalanine ammonia lyase (AtPAL1). The yeast from the freshly transformed bacterium colony is grown in 300 μL selective culture medium (-Trp) in a 96-well deep well microtiter plate. After growing for 48 hours in a shaking incubator at 30°C and 460rpm, the yeast is precipitated and the culture supernatant is analyzed by LC-MS / MS. The chromatogram traces show cinnamic acid produced by these strains based on the most abundant multiple reaction monitoring (MRM) transition of cinnamic acid (m / z +149→131). Each trace represents three samples.

[0046] Fig.33 Shown are substrate specificities of orthologs of UDP-glucosyltransferase 84A27 (UGT84A27) from TA-producing Solanaceae plants expressed in engineered yeast. The 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-phenyllactic acid (PLA); middle, trans-cinnamic acid (CA); bottom, trans-ferulic acid (FA). (B) Heat map of the percentage of conversion of fed phenylpropanoids to glucosides achieved by yeast engineered to express UGT84A27. UGT84A27 orthologs or BFP negative controls were expressed from low copy plasmids in CSY1251. Transformed cells were cultured in selective medium supplemented with 500 μM PLA, CA or FA for 72 h prior to LC-MS / MS analysis. Data represent mean ± SD of n = 3 biologically independent samples.

[0047] Fig.34 An example of chromatographic and mass spectrometric analysis of UGT84A27 activity is shown. The figure shows a representative LC-MS / MS trace, which is shown in FIG. Fig.33 In CSY1251 cultured for 120 h as in Figure B, PLA, CA, and FA were converted to homologous glucosides by AbUGT to achieve more complete glucosylation. For PLA, the acid (top trace in each panel) and glucosides (bottom trace in each panel) were converted to homologous glucosides by AbUGT to achieve more complete glucosylation. + The adducts were distinguished by their parent masses and different retention times. For CA and FA, rapid fragmentation was required to detect the glucosides based on the lower retained peaks produced by their phenylpropane fragments.

[0048] Fig.35Figure 1 shows the structure-guided active site engineering of AbUGT to change substrate specificity. The figure shows the structural analysis of AbUGT 3D structure to identify potential mutations that increase activity to PLA. (A) Homology model of AbUGT84A27 constructed based on the crystal structure (PDB: 5V2K) of Arabidopsis salicylic acid UDP-glucosyltransferase UGT74F2 with bound UDP. Based on docking simulation, PLA (orange) is shown with the preferred binding posture of UDP-glucose (pink). (B) A magnified view of the AbUGT active site with docked D-PLA and UDP-glucose. Potential mutations identified to improve PLA selectivity (F130Y, L205F, I292Q) are shown; dotted lines indicate putative polarity / hydrogen bond interactions.

[0049] Fig.36 The substrate specificity of AbUGT84A27 active site mutant is shown. This figure shows the heat map of the percentage of feed phenylpropanoids to glucoside conversion achieved by yeast engineered to express AbUGT mutants. AbUGT wild type, active site mutants or BFP negative controls were expressed by low copy plasmids in CSY1251. Before LC-MS / MS analysis, transformed cells were cultivated for 72h in a selective medium supplemented with 500 μM PLA, CA or FA. Data represent the mean ± standard deviation of n=3 biological independent samples.

[0050] Fig.37 LC-MS / MS chromatograms are shown that verify the stepwise biosynthesis of PLA glucosides in yeast engineered to produce tropine. The figure shows multiple reaction monitoring (MRM) and extracted ion chromatogram (EIC) traces of the culture medium from a yeast strain engineered to stepwise reconstruct PLA glucosides. The strains were grown in non-selective medium for 72 h prior to LC-MS / MS analysis of the culture supernatant. The chromatogram traces represent three biological replicates.

[0051] Fig.38 Schematic diagram of the biosynthetic pathways showing the dual metabolic fate of glucose in yeast. This figure shows the effect of citrate on glucoside production by inhibiting glycolysis. Abbreviations: HXK, hexokinase; GPI, glucose-6-phosphate isomerase; PFK, phosphofructokinase; PGM, phosphoglucomutase; UGP, UDP-glucose pyrophosphorylase.

[0052] Fig.39The effect of supplementing citrate on the production of heterologous glucosides in engineered yeast is shown. This figure shows the effect of supplementing 2% citrate on the conversion of phenylpropanoid acid to glucosides achieved by yeast engineered to express AbUGT. Strain CSY1288 was cultured in a non-selective medium with or without 2% citrate and without additional supplementation (to evaluate the glucosylation of endogenously produced PLA) or supplemented with 500 μM trans-cinnamic acid (CA) or trans-ferulic acid (FA). The cultures were grown for 72 h before LC-MS / MS analysis. The data represent the mean values ​​(open circles) of n=3 biologically independent samples and the error bars show standard deviations. Student's two-tailed t-test: *P<0.05, **P<0.01, ***P<0.001.

[0053] Fig.40 Relative PLA glucoside production in yeast strains engineered to overexpress UDP-glucose biosynthetic enzymes is shown. This figure shows the effect of overexpressing natural enzymes involved in the biosynthesis of the glucoside precursor UDP-glucose on the production of PLA glucosides in engineered yeast. Enzymes or negative controls (BFP) were expressed by low copy plasmids in strain CSY1288. The strains were cultured in selective medium for 72 h prior to LC-MS / MS analysis of metabolites in the culture supernatant. Data represent the mean values ​​(open circles) of n=3 biologically independent samples and error bars show standard deviations. Student's two-tailed t-test: *P<0.05, **P<0.01, ***P<0.001. Statistical significance is shown relative to the corresponding controls.

[0054] Fig.41 Relative PLA glucoside production in CSY1288 with disruption of endogenous glucosidase is shown. This figure shows the effect of disrupting each of the three native glucosidase genes on PLA glucoside accumulation in engineered yeast. The strains were cultured in non-selective medium for 72 h prior to LC-MS / MS analysis of the culture supernatant. Data represent the mean values ​​(open circles) of n=3 biologically independent samples and error bars show standard deviations. Student's two-tailed t-test: *P<0.05, **P<0.01, ***P<0.001. Statistical significance is shown relative to the corresponding controls.

[0055] Fig.42LC-MS / MS chromatogram is shown, and it is shown that in the liquid culture of the engineered yeast cell expressing AbCYP80F1, medicinal TA precursor scopolamine is produced by ritolin.This figure illustrates the LC-MS / MS analysis of the yeast strain engineered to convert (R)-ritolin into scopolamine.Yeast strain is engineered to have low copy CEN / ARS plasmid, and the plasmid contains LEU2 selective marker, TDH3 promotor and the coding sequence of ritolin mutase CYP80F1 (AbCYP80F1) from belladonna.Bacterial strain also has the second low copy plasmid, and the plasmid contains TRP1 selective marker, TDH3 promotor and the coding sequence of the following: (i) BFP as negative control, (ii) saccharomyces cerevisiae CPR (NCP1), or (iii) Arabidopsis thaliana CPR (AtATR1). Yeast from freshly transformed colonies were grown in 300 μL selective medium (-Leu–-Trp) supplemented with 1 mM ritolin in 96-well deep-well microtiter plates. After 48 hours of growth in a shaking incubator at 30°C and 460 rpm, the yeast were pelleted and the culture supernatant was analyzed by LC-MS / MS. The chromatogram traces show scopolaldehyde produced by these strains based on the most abundant MRM transition (m / z+288→124). Arrows indicate putative scopolaldehyde peaks. Each trace represents three samples.

[0056] Fig.43 It is shown that the medicinal TA scopolamine is produced by the medicinal TA scopolamine in the liquid culture of the engineered yeast cells expressing the orthologs of scopolamine 6β-hydroxylase / dioxygenase (H6H). This figure shows that (S)-scopolamine is converted to (S)-scopolamine by the engineered yeast strain expressing the orthologs of H6H. The yeast strain is engineered to have a low copy CEN / ARS plasmid, and the plasmid contains LEU2 selection markers, TDH3 promoter and BFP as a negative control or the coding sequence of the H6H variant from Datura stramonium (D.stramonium) (DsH6H), A.acutangulus (AaH6H), B.arborea (B.arborea) (BaH6H) or D.metel (D.metel) (DmH6H). Yeast from freshly transformed colonies were grown in 300 μL selective medium (-Leu) supplemented with 1 mM scopolamine in 96-well deep-well microtiter plates. After 48 hours of growth in a shaking incubator at 30°C and 460rpm, yeast was precipitated and the culture supernatant was analyzed by LC-MS / MS. Data represent the mean of three biological replicates and are normalized for the amount of scopolamine contaminants in the feed scopolamine. Error bars represent standard deviations. Relative scopolamine titers were quantified based on the peak area of ​​the m / z+304→138MRM transition.

[0057] Fig.44 The availability of cofactor in culture medium and the impact of supplementing the conversion of hyoscyamine to scopolamine in the liquid culture of the engineered yeast cells expressing DsH6H are shown. This figure shows the impact of supplementing cofactor on the conversion of (S)-hyoscyamine to (S)-scopolamine in the yeast of through engineering approaches. Yeast strains are engineered to have low copy CEN / ARS plasmids, and the plasmid contains LEU2 selection marker, TDH3 promoter and the following coding sequences: (i) BFP as negative control, or (iii) hyoscyamine 6β-hydroxylase / dioxygenase (DsH6H) from Datura stramonium. Yeast from freshly converted bacterium colonies is grown in 300 μ L selective medium (-Leu) supplemented with specified substrate and / or cofactor in 96-well deep well microtiter plates. After 48 h of growth in a shaking incubator at 30 °C and 460 rpm, yeasts were pelleted and culture supernatants were analyzed by LC-MS / MS. Relative (S)-scopolamine titers were quantified based on the integrated peak area of ​​the m / z+304→138 MRM transition and normalized to strains expressing DsH6H and with all supplemented cofactors and substrates. Data represent the mean of three biological replicates and error bars indicate standard deviations. Hyo, (S)-scopolamine; 2-OG, 2-oxoglutaric acid; L-AA, L-ascorbic acid.

[0058] Fig.45 Heatmap showing hierarchical clustering of scopolamine dehydrogenase gene candidates identified from the Atropaeolum solanum transcriptome by analysis of tissue co-expression data. This figure shows clustering of tissue-specific expression profiles of transcripts in the Atropaeolum solanum transcriptome that potentially encode enzymes with scopolamine dehydrogenase activity. Transcript expression for each candidate was scaled by row using a normal distribution. The dendrogram indicates the hierarchical clustering of candidates obtained by tissue-specific expression profiling. Known TA pathway genes are identified by name; putative HDH candidates are indicated with locus ID. Black triangles indicate candidates screened for activity; double black triangles indicate candidates with experimentally validated HDH activity.

[0059] Fig.46The production of the medicinal TA scopolamine from ritolin in liquid culture of engineered yeast cells expressing scopolamine dehydrogenase (HDH) candidates is shown. This figure shows an experimental screening for activity of HDH candidates identified from the Atropa belladonna transcriptome in engineered yeast. The yeast strain was engineered to express Atropa belladonna ritolin mutase (AbCYP80F1) and Datura scopolamine 6β-hydroxylase / dioxygenase (DsH6H) from constitutive promoters within expression cassettes integrated into the genome, and each of the 13 HDH candidates was expressed from a low copy CEN / ARS plasmid containing the TRP1 selection marker and the TDH3 promoter. Yeast from freshly transformed colonies was grown in 300 μL selective medium (-Trp) supplemented with 1 mM ritolin in 96-well deep well microtiter plates. After 72 h of growth in a shaking incubator at 30 °C and 460 rpm, the yeast were pelleted and the culture supernatant was analyzed by LC-MS / MS. Relative scopolaldehyde titer was based on m / z + The integrated peak area of ​​the 288→124 MRM transition was quantified and normalized to an engineered strain expressing BFP instead of the HDH candidate. (S)-Scopamine titers were based on m / z + The integrated peak area of ​​the 304→138 MRM transition and the standard curve of authentic scopolamine standards were used for quantification. The data represent the mean of three biological replicates, and the error bars indicate the standard deviation.

[0060] Fig.47 The three-dimensional structure of scopolamine dehydrogenase from Atropa belladonna is shown. This figure shows a cartoon representation of the structure of AbHDH, which is a homology model built based on the crystal structure of sinapyl alcohol dehydrogenase (PtSAD; PDB: 1YQD) from Populus tremuloides as a template. NADPH and Zn 2+ Shown in the active site. The inset box shows a magnified view of the AbHDH active site with NADPH and docked scopolaldehyde. The dashed lines indicate interactions that are important for catalysis.

[0061] Fig.48Phylogenetic tree showing three identified HDH orthologs (AbHDH, DiHDH, DsHDH) and the closest protein hits in the UniProt / SwissProt database. This figure shows the clustering of three identified HDH enzyme orthologs with closely related protein sequences based on a BLAST search of the UniProt / SwissProt database. The sequences shown include the top 50 BLASTp hits based on E-values, as well as 10 additional hits selected from the next 100 rankings. The phylogenetic relationships were inferred by bootstrap neighbor-joining in ClustalX2 using n=1000 trials, and the resulting tree was visualized using 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.

[0062] Fig.49 The production of the pharmaceutical TA scopolamine from ritolin in liquid culture of engineered yeast cells expressing scopolamine dehydrogenase orthologs is shown. This figure shows the activity comparison between the identified HDH enzyme orthologs expressed in engineered yeast. The yeast strain was engineered to express the belladonna ritolin mutase (AbCYP80F1) and the Datura stramonium scopolamine 6β-hydroxylase / dioxygenase (DsH6H) from constitutive promoters within expression cassettes integrated into the genome, each of the 3 HDH orthologs (AbHDH, DiHDH, DsHDH) was expressed from a low copy CEN / ARS plasmid containing the TRP1 selection marker and the TDH3 promoter, and an additional copy of DsH6H was expressed from a low copy CEN / ARS plasmid containing the LEU2 selection marker and the TDH3 promoter. Yeast from freshly transformed colonies were grown in 300 μL selective medium (-Leu-Trp) supplemented with 1 mM ritolin in 96-well deep-well microtiter plates. After 72 hours of growth in a shaking incubator at 30°C and 460 rpm, yeast were pelleted and the culture supernatant analyzed by LC-MS / MS. Relative scopolaldehyde titers were based on m / z + The integrated peak area of ​​the 288→124 MRM transition was quantified and normalized to an engineered strain expressing AbHDH and BFP instead of DsH6H. (S)-Scopamine titers were based on m / z+ The integrated peak area of ​​the 304→138 MRM transition and the standard curve of authentic scopolamine standards were used for quantification. Data represent the mean of three biological replicates, and error bars indicate standard deviation.

[0063] Fig.50 The experimental verification of converting feed ritolin into scopolamine by yeast engineered to express CYP80F1, HDH and H6H is shown. This figure shows the multiple reaction monitoring (MRM) LC-MS / MS traces from the culture medium of the yeast strain engineered to convert ritolin into scopolamine. Before the metabolites in the culture supernatant were analyzed by LC-MS / MS, the strain was cultivated in a non-selective medium supplemented with 1mM ritolin for 72h. The dark trace (CSY1294, scopolamine) in the lower right figure represents the scopolamine standard of 125nM (38μg / L). The chromatogram trace represents three biological replicates.

[0064] Fig.51 The classical plant ER to vacuolar trafficking and maturation pathway of SCPL acyltransferase (SCPL-AT) is shown. This figure shows a schematic diagram of the typical ER to vacuolar protein trafficking pathway in plants followed by SCPL-AT, with Atropurpura belladonna ritolin synthase (AbLS) shown as an example. The circled numbers indicate the major steps in SCPL-AT expression and activity, including maturation in the (1) ER lumen and (2) Golgi apparatus, (3) trafficking to the vacuole, and vacuolar (4) substrate inward transport and (5) product efflux transport.

[0065] Fig.52 Co-localization of wild-type ritolin synthase from Atropa belladonna expressed in engineered yeast is shown. This figure shows epifluorescence microscopy of yeast engineered to express AbLS with an N-terminal GFP tag (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 μm.

[0066] Fig.53 The strategy for forcing the localization of ritolin synthase to different yeast subcellular compartments by signal sequence replacement is shown. This figure illustrates a protein engineering method for improving the subcellular localization of AbLS to solve the potential restriction of substrate availability in different compartments. (A) is used for the schematic diagram of the yeast subcellular compartment of AbLS by signal sequence exchange. The signal sequence source protein of each compartment is indicated. (B) The end and residue selected for the replacement of the AbLS signal sequence. The residues comprising each signal sequence domain are selected based on the structural annotation in the UniProt / SwissProt database.

[0067] Fig.54 A protein blot of wild-type AbLS expressed in tobacco and treated with a deglycosylase is shown. This figure shows the identification of the glycosylation modification type of AbLS expressed in plants. AbLS with a C-terminal HA tag was transiently expressed in N. benthamiana leaves by Agrobacterium infiltration. Crude leaf extracts were 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 NuPAGE 4-12% Bis-Tris gels and then transferred to nitrocellulose membranes for immunodetection using a chimeric rabbit IgGκ anti-HA HRP-conjugated antibody. All electrophoresis and blotting steps were performed under disulfide reducing conditions (see Online Methods). Lane 'L', Bio-Rad Precision Plus Dual Color protein ladder.

[0068] Fig.55 The protein blotting of AbLS glycosylation site mutants expressed in yeast and tobacco is shown. This figure illustrates the comparison of the N-glycosylation pattern for AbLS expressed in yeast and tobacco. Wild-type AbLS, monoglycosylation site point mutants (N→Q) or quadruple mutants with C-terminal HA tags were infiltrated in Nicotiana benthamiana ('Nb') (A) or transiently expressed by low copy plasmids in CSY1294 ('yeast') (B). The preparation of tobacco and yeast crude extracts was carried out under denatured disulfide reduction conditions (see online method). Crude extracts were separated by electrophoresis on NuPAGE 4-12% Bis-Tris gel, and then transferred to nitrocellulose membranes to use chimeric rabbit IgG κ anti-HA HRP conjugated antibodies for immunodetection. All electrophoresis and blotting steps were carried out under disulfide reduction conditions (see online method). For (A) and (B), the control of corresponding yeast and tobacco expression was included for comparison. Lane 'L', Bio-Rad Precision Plus Dual Color protein ladder.

[0069] Fig.56Phylogenetic identification of putative internal proteolytic propeptide removal in ritolin synthase is shown. This figure shows a sequence alignment of AbLS with characterized serine carboxypeptidases and SCPL acyltransferases known to have (AtSCT, AsSCPL1, TaCBP2) or lack (AtSMT, yPRC1) an internal propeptide linker (bold, gray) that is proteolytically removed. The putative N-terminal signal peptide is in bold (black); disulfide bonds are represented as connecting lines. AtSCT, Arabidopsis thaliana sinapinic acid glucose:choline sinapinic acid transferase; AtSMT, Arabidopsis thaliana sinapinic acid glucose:malate sinapinic acid transferase; AbLS, belladonna ritolin synthase; AsSCPL1, Avena strigosa avenin synthase; TaCBP2, Triticum aestivum carboxypeptidase 2; yPRC1, yeast carboxypeptidase Y. From top to bottom: SEQ ID NOs: 30-35.

[0070] Fig.57 Structural identification showing putative internal proteolytic propeptide removal in ritolin synthase. This figure shows a comparison of the three-dimensional structures of two SCPL-ATs, one of which is known to contain an internal propeptide sequence that is removed by proteolytic means. Left: (top) cartoon and (bottom) surface representation of the crystal structure of TaCBP2 (PDB: 1WHT), showing disulfide bonds and internal propeptide removal sites. Right: (top) cartoon and (bottom) surface representation of the homology model of AbLS based on the crystal structure of TaCBP2, showing the N-terminal signal peptide, disulfide bonds, and a putative internal propeptide that appears to block access to the active site.

[0071] Fig.58The analysis of the proteolytic cleavage pattern of AbLS split control and presumed propeptide exchange variants in yeast is shown. This figure shows the protein fragment size produced by the AbLS split control and propeptide variants expressed in engineered yeast by Western blot analysis. The AbLS variant with C-terminal HA tag is expressed by low copy plasmid in CSY1294 (lanes 1-6); the wild-type AbLS with HA tag expressed in Nicotiana benthamiana (Nb) is shown as another control (lane 7). Gel electrophoresis and blotting are carried out under disulfide reduction conditions, and anti-HA antibodies are used to detect (referring to online methods). Lane symbols: L, protein molecular weight ladder; WT, wild-type AbLS; SPL, AbLS cleaved at the putative propeptide with signal peptides on both fragments; SPL-T, AbLS cleaved at the putative propeptide without signal peptides on either fragment; GS, AbLS variant with the wild-type propeptide exchanged for a flexible Gly-Ser linker; SCT, AbLS variant with the wild-type propeptide exchanged for the AtSCT propeptide sequence; CUT, AbLS variant with the wild-type propeptide exchanged for a synthetic poly-arginine site recognized and cleaved by the Kex2p protease.

[0072] Fig.59 De novo hyoscine and scopolamine production in yeast strains engineered to express AbLS N-terminal fusions are shown. This figure shows a comparison of de novo hyoscine and scopolamine production in yeast strains expressing AbLS, which have different soluble protein domains fused to the N-terminus. Wild type (control) or AbLS fusions were expressed by low copy plasmids in CSY1294. Transformed strains were cultured in selective media for 96 h before LC-MS / MS analysis of metabolites in the culture supernatant. Data represent the mean (open circles) of n=3 biological independent samples and error bars show standard deviations. Student's two-tailed t-test: *P<0.05, **P<0.01, ***P<0.001.

[0073] Fig.60 Fluorescence microscopy of tobacco alkaloid transporters expressed in CSY1296 to alleviate vacuolar TA transport restrictions is shown. This figure shows fluorescence microscopy images of engineered yeast expressing tobacco alkaloid transporters fused to GFP at their C-termini to enable identification of their subcellular localization. (A) C-terminal GFP fusions of NtJAT1 and (B) NtMATE2 were expressed from low copy plasmids in CSY1296. Scale bar, 5 μm.

[0074] Fig.61The output of tropine, hyoscyamine and scopolamine in CSY1296 engineered to express heterologous alkaloid transporters is shown. This figure shows the practicality of different plant alkaloid transporters in relieving intracellular substrate transport restrictions in yeast engineered for TA production. Common tobacco jasmonate-inducible alkaloid transporter 1 (NtJAT1), multidrug and toxin efflux (MATE) transporter 1 or 2, or negative control (BFP) are expressed by low copy plasmids in CSY1296. Before LC-MS / MS analysis of metabolites in culture supernatant, transformed strains are cultivated in selective medium for 96h. Data represent the mean value (open circles) of n=3 biological independent samples and error bars show standard deviation. Student's two-tailed t test: *P<0.05, **P<0.01, ***P<0.001.

[0075] Fig.62LC-MS / MS chromatograms in (A) product ion mode and (B) multiple reaction monitoring mode are shown, which show the de novo production of the non-natural TA cinnamoyl tropine in engineered yeast. This figure shows the LC-MS / MS analysis of engineered yeast strains that produce the non-natural TA cinnamoyl tropine. (A) Tandem MS / MS spectra of the extracellular medium of the following: (i) tropine-producing strain CSY1251; (ii) CSY1251 expressing phenylalanine ammonia lyase (AtPAL1), 4-coumarate-CoA ligase 5 (At4CL5) and cocaine synthase (EcCS), denoted as CSY1282; or (iii) an authentic cinnamoyl tropine standard with a parent mass of m / z+=272. The blue diamond represents the parent compound peak. (B) The activity of EcCS acyltransferase on cinnamic acid and α-tropine was verified by substrate feeding. Strains were transformed with plasmid combinations expressing AtPAL1 (low-copy plasmid pCS4252) and / or At4CL5 and EcCS (high-copy plasmid pCS4207) and then cultured 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 + AtPAL1 AL1+At4CL5+EcCS;(iv) CEN.PK2+AtPAL1+At4CL5+EcCS+0.5mM α-tropine;(v) CSY1251+At4CL5+EcCS;(vi) CSY1251+At4CL5+EcCS+0.2mM trans-cinnamic acid;(vii) CSY1251+AtPAL1+At4CL5+EcCS;(viii) 25nM cinnamoyl tropine standard. For (A) and (B), yeast strains were cultured in selective medium (YNB-DO+2% dextrose+5% glycerol) at 25°C for 72h before LC-MS / MS analysis.

[0076] Fig.63The effect of different carbon sources fed with (A) dextrose alone or (B) on the yield of tropine and related TA precursors in liquid culture of engineered yeast is shown. This figure shows the optimization of carbon sources to improve the tropine yield in engineered yeast. Overnight cultures of tropine-producing strain CSY1249 (see Example 3.3.4) were grown in non-selective enrichment medium (YPD). Overnight cultures were precipitated and resuspended in non-selective defined medium (YNB-SC), which had all amino acids and (A) 2% of each carbon source or (B) 2% of dextrose and 2% of each other carbon source, including dextrose. Before analyzing the growth medium by LC-MS / MS, the culture was grown at 25°C for 48h. The data show the relative titer of each metabolite normalized for (A) 2% dextrose or (B) 2%+2% dextrose. The data represent the mean value of three biological replicates, and the error bars indicate the standard deviation.

[0077] Fig.64 Metabolic bottleneck analysis of scopolamine-producing strain CSY1296 is shown. This figure shows the effect of expressing additional copies of flux-limiting enzymes on the yield of TA and TA precursors in engineered yeast. Additional copies of each biosynthetic enzyme between tropine and scopolamine are expressed in strain CSY1296 by the following low-copy plasmids: (A) WfPPR, pCS4436; (B) AbUGT, pCS4440; (C) DsRed-AbLS, pCS4526; (D) AbCYP80F1, pCS4438; (E) DsHDH, pCS4478; (F) DsH6H, pCS4439; or BFP control (pCS4208, pCS4212 or pCS4213), corresponding to the same auxotrophic markers as each biosynthetic gene plasmid. Transformed strains were cultured in appropriate selective media at 25°C for 96 hours prior to quantification of metabolites in the growth medium by LC-MS / MS. Data represent the mean of n=3 biologically independent samples (open circles) and error bars show standard deviation. Student's two-tailed t-test: *P<0.05, **P<0.01, ***P<0.001.

[0078] Fig.65Show the influence of hyoscine and scopolamine production in the alleviation flux and transport restriction in engineered yeast.This figure shows the comparison of de novo hyoscine and scopolamine production in yeast strains CSY1296 and CSY1297, wherein the latter has the other genome copy of flux restriction enzyme (WfPPR and DsH6H) and tobacco vacuolar alkaloid inward transporter (NtJAT1).Before the metabolites in the culture supernatant are carried out LC-MS / MS analysis, the strain is cultivated in non-selective medium for 96h.Data represent the mean value (open circles) of n=3 biological independent samples and error bars show standard deviation.Student's two-tailed t test: *P<0.05, **P<0.01, ***P<0.001.

[0079] definition

[0080] Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the specification.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd Edition, John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the general meaning of many of the terms used herein to the skilled person. Nevertheless, for clarity and ease of reference, certain terms are defined below.

[0082] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "primer" refers to one or more primers, i.e., a single primer and multiple primers. It should also be noted that the claims may be drafted to exclude any optional elements. Therefore, the statement is intended to serve as antecedent basis for using exclusive terminology such as "solely," "only," etc., or using a "negative" limitation in conjunction with the recitation of claim elements.

[0083] As used herein, the terms "determine," "measure," "assess," and "assay" are used interchangeably and include both quantitative and qualitative determinations.

[0084] As used herein, the term "polypeptide" refers to a polymeric form of amino acids of any length, including peptides with a length in the range of 2-50 amino acids and polypeptides with a length greater than 50 amino acids. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" includes polymers of coding and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides with modified peptide backbones, wherein conventional backbones have been replaced by non-naturally occurring or synthetic backbones. Polypeptides can 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." Peptide" can 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 length of the peptide is between 5 and 30 amino acids.

[0085] As used herein, the term "isolated" means that the fraction of interest is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components associated with the fraction prior to purification.

[0086] As used herein, the term "encoded by" refers to a nucleic acid sequence encoding a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of 3 or more amino acids, such as 5 or more, 8 or more, 10 or more, 15 or more or 20 or more amino acids from the polypeptide encoded by the nucleic acid sequence. The term also encompasses a polypeptide sequence that is immunologically identifiable using a polypeptide encoded by the sequence.

[0087] "Vector" is capable of transferring a gene sequence to a target cell. As used herein, the terms "vector construct," "expression vector," and "gene transfer vector" are used interchangeably to refer to any nucleic acid construct capable of directing the expression of a gene of interest, and which can transfer a gene sequence to a target cell, either by genome integration of all or a portion of the vector, or by transient or heritable maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning and expression vehicles as well as integration vectors.

[0088] An "expression cassette" includes any nucleic acid construct capable of directing the expression of a gene / coding sequence of interest operably linked to a promoter of the expression cassette. Such cassettes are constructed as "vectors," "vector constructs," "expression vectors," or "gene transfer vectors" to facilitate transfer of the expression cassette into a target cell. Thus, the term includes cloning and expression vehicles as well as viral vectors.

[0089] A "plurality" comprises at least 2 members. In some 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 embodiment, a plurality may have 2-20 members.

[0090] The term "tropane alkaloid product" is intended to refer to any molecule whose backbone contains an 8-azabicyclo[3.2.1]octane core group comprising a cycloheptane ring and a nitrogen bridge connecting carbon atoms 1 and 5, wherein the 8-azabicyclo[3.2.1]octane group is covalently bonded to an acyl group via an ester bond at position 3, and / or wherein the 8-azabicyclo[3.2.1]octane group is functionalized with a hydroxyl group at position 3 and with one or more hydroxyl groups at positions 2, 4, 5, 6 and / or 7. Tropane alkaloid products include, but are not limited to, ritolin, hyoscyamine, atropine, anisodamine, scopolamine, cocaine, and any other similar tropine / pseudotropine+acyl natural or unnatural tropane alkaloids (e.g., caltropin).

[0091] 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 can be converted to a tropane alkaloid product in one or more (e.g., one or two) biosynthetic steps; wherein the carbon source is a carbohydrate, a non-carbohydrate sugar, a sugar alcohol, a lipid, a fatty acid, or a substrate that is converted to one or more of the above carbon sources by a metabolic pathway; and wherein the nitrogen source is ammonia, urea, nitrate, nitrite, any amino acid other than glutamate, arginine, ornithine and citrulline, a peptide, a protein, or any substrate that is converted to one or more of the above nitrogen sources by a metabolic pathway.

[0092] The term "derivative of a tropane alkaloid product" is intended to refer to any molecule that is not naturally produced by an unmodified organism, wherein the backbone of the molecule comprises a tropane alkaloid product and differs from the tropane alkaloid product by the attachment of functional groups without modification of the backbone 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.

[0093] Numeric ranges are inclusive of the numbers defining the range.

[0094] Methods described herein include multiple steps. As required, each step can be performed after a predetermined amount of time between steps. Therefore, the time between performing each step can be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, and include 5 hours or more. In certain embodiments, each subsequent step is performed immediately after the previous step is completed. In other embodiments, step can be performed after incubation or waiting time (e.g., a few minutes to overnight waiting time) after completing the previous step.

[0095] Additional definitions for terms may appear throughout the specification. DETAILED DESCRIPTION

[0096] Provided are host cells engineered to produce tropane alkaloids (TA) of interest, such as hyoscyamine and scopolamine. The host cell may have one or more engineered modifications selected from the following: feedback inhibition relief mutations in enzyme genes; transcriptional regulatory modifications of biosynthetic enzyme genes; inactivation mutations in enzymes; and heterologous coding sequences. Also provided are methods for producing TAs of interest using host cells and compositions, such as kits, systems, etc., that can be used in the methods of the invention.

[0097] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, as such may vary. It should also be understood that the terminology used herein is only used for the purpose of describing specific embodiments, and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

[0098] Where a range of values ​​is provided, it is to be understood that each intermediate value between the upper and lower limits of the range (to one tenth of the unit of the lower limit, unless the context clearly indicates otherwise) and any other stated or intermediate value in the range are encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in smaller ranges and are also encompassed within the present invention, subject to any specific exclusions in the stated ranges. When a range is specified to include one or two limits, ranges excluding any one or both of those included limits are also encompassed within the present invention.

[0099] Certain ranges are presented herein with numerical values ​​preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows it, as well as for numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number may be a number that, in the context in which it appears, provides a substantial equivalent to the specifically recited number.

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used to practice or test the present invention, representative illustrative methods and materials are now described.

[0101] 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 associated with the cited publications. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the publication dates provided may be different from the actual publication dates and may need to be independently confirmed.

[0102] As will be apparent to those skilled in the art after reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the invention. Any recited method is carried out in the order of events recited or in any other order that is logically possible.

[0103] In further describing the present invention, the TA precursors, TA and modifications of TA, including derivatives of TA, of interest are first described in more detail, followed by host cells for producing them. Next, methods of interest in which the host cells can be used are reviewed. Kits useful for practicing the methods of the present invention are also described.

[0104] Tropane alkaloid (TA) precursor

[0105] As described above, a host cell for producing a tropane alkaloid precursor (TA precursor) is provided. A TA precursor can be any intermediate or precursor compound in a synthetic pathway (e.g., as described herein) that leads to the production of a TA of interest (e.g., as described herein). In some cases, a TA precursor has a structure that can be characterized as a TA or a derivative thereof. In some cases, a TA precursor has a structure that can be characterized as a fragment of a TA. In some cases, a TA precursor is an early TA. As used herein, "early TA" refers to an early intermediate when a TA of interest is synthesized in a cell, wherein the early TA is produced by a host cell from a host cell raw material or a simple starting compound. In some cases, an early TA is a TA intermediate produced by a subject host cell only from a host cell raw material (e.g., a carbon and nutrient source) without the need to add a starting compound to the cell. The term early TA can refer to a precursor of a TA end product of interest, regardless of whether the early TA itself can be characterized as a tropane alkaloid.

[0106] In some cases, the TA precursor is an early TA, such as a pre-tropic tropane alkaloid or a pre-ritolinic tropane alkaloid. Thus, host cells are provided that produce pre-tropic tropane alkaloids (pre-tropic TAs) and pre-ritolinic tropane alkaloids (pre-ritolinic TAs). Tropine is a major branch point intermediate of interest in downstream TA synthesis through cell engineering efforts to produce end products such as pharmaceutical TA products derived from ritolinic ( Figure 2 The subject host cells can produce TA precursors from simple and inexpensive starting materials that can be used to produce tropine, ritolin, and downstream TA end products.

[0107] As used herein, the terms "pre-esterified tropane alkaloid," "pre-esterified TA," and "pre-esterified TA precursor" are used interchangeably and refer to a biosynthetic precursor of ritolin, cinnamoyl tropine, or other products of the esterification of an acyl donor and an acyl acceptor, regardless of whether the structure of the esterified precursor itself is characterized as a tropane alkaloid. The term pre-esterified TA is intended to include biosynthetic precursors, intermediates, and metabolites thereof of any convenient member of the host cell biosynthetic pathway that can produce an esterified product such as ritolin. In some cases, the pre-esterified TA includes a tropane alkaloid fragment, such as a tropine fragment, a phenylpropanoid fragment, or a precursor or derivative thereof. In some cases, the pre-esterified TA has a structure that can be characterized as a tropane alkaloid or a derivative thereof.

[0108] TA precursors of interest include, but are not limited to, tropine and phenyllactic acid (PLA), and tropine and PLA precursors such as arginine, ornithine, agmatine, N-carbamoylputrescine (NCP), putrescine, N-methylputrescine (NMP), 4-methylaminobutyraldehyde, N-methylpyrrolinium (NMPy), 4-(1-methyl-2-pyrrolidinyl)-3-oxobutyric acid (MPOB), tropinone, phenylalanine, prephenic acid, and phenylpyruvic acid (PPA). In some embodiments, one or more TA precursors are tropine and PLA. In some cases, one or more TA precursors are tropine and a phenylpropanecarboxylic acid other than PLA, such as cinnamic acid. Figure 1 , 2 3 and 4 show the biosynthesis of non-drug, drug and non-natural TA from various TA and non-TA precursor molecules, respectively.

[0109] Synthetic pathways for TA precursors can be generated in host cells and can begin with any convenient starting compounds or materials. Figure 1-4 Interesting synthetic pathways of TA precursors starting from amino acids are shown. The starting material may be non-naturally occurring, or the starting material may be naturally occurring in the host cell. Based on the synthetic pathways present in the host cell, any convenient compounds and materials may be used as starting materials. The source of the starting material may be from the host cell itself, such as arginine or phenylalanine, or the starting material may be added or supplemented to the host cell from an external source. Thus, in some cases, the starting compound refers to a compound in the cell's synthetic pathway that is added to the host cell from an external source that is not part of the growth feedstock or cell growth medium. Interesting starting compounds include, but are not limited to, N-methylputrescine, 4-methylaminobutyraldehyde, tropinone, tropine, PLA, cinnamic acid, and Figure 1-4 Any compound shown in. For example, if the host cell is grown in a liquid culture medium, starting materials can be supplemented in the cell culture medium, and the starting materials are transported into the cell and converted into the desired product by the cell. The starting materials of interest include, but are not limited to, cheap raw materials and simple precursor molecules. In some cases, the host cell utilizes raw materials including simple carbon sources as starting materials, and the host cell utilizes the raw materials to produce compounds of the synthetic pathways of the cell. The host cell growth raw materials may include one or more components, such as carbon sources such as cellulose, starch, free sugars and nitrogen sources, such as ammonium salts or cheap amino acids. In some cases, the growth raw materials that can be used as starting materials can be derived from sustainable sources, such as biomass grown on marginal land, including switchgrass and algae, or biomass waste products from other industrial or agricultural activities.

[0110] Tropane alkaloids (TA)

[0111] As described above, a host cell producing a tropane alkaloid (TA) of interest is provided. In some embodiments, the engineered strains of the present invention will provide a platform for producing a tropane alkaloid of interest and modifications thereof across several categories, including but not limited to medicinal TAs, such as those derived from tropine and PLA; non-medicinal TAs, such as those derived from tropine, pseudotropine or norpseudotropine; and non-natural TAs, such as those derived from TA precursors (e.g., acyl donors and acyl acceptor compounds) rather than esterification of tropine and PLA. Each of these categories is intended to include biosynthetic precursors, intermediates and metabolites of any convenient member of the host cell biosynthetic pathway that can produce members of the category. Non-limiting examples of compounds are given below for each of these categories. In some embodiments, the structure of a given example may or may not be characterized as a tropane alkaloid itself. The chemical entity of the present invention is intended to include all possible isomers, including single enantiomers, racemic mixtures, optically pure forms, mixtures of diastereomers and intermediate mixtures.

[0112] Medicinal TAs may include, but are not limited to, ritolin, hyoscyamine, atropine, anisodamine, scopolamine, and their derivatives that are naturally produced by plants.

[0113] Non-medicinal TAs may include, but are not limited to, cupulein, cocaine and its derivatives produced naturally from plants.

[0114] Non-natural TAs may include, but are not limited to, cinnamoyl tropine, cinnamoyl-3β-tropine, coumaroyl tropine, coumaroyl-3β-tropine, benzoyl tropine, benzoyl-3β-tropine, caffeoyl tropine, caffeoyl-3β-tropine, feruloyl tropine, and feruloyl-3β-tropine.

[0115] Modifications of TA including derivatives

[0116] As described above, host cells are provided that produce modified derivatives of tropane alkaloids (TAs) of interest. In some embodiments, the engineered strains of the invention will provide a platform for derivatizing TAs of interest, including TA precursors, pharmaceutical TAs, non-pharmaceutical TAs, and non-natural TAs produced by the engineered host cells or fed to the growth medium of the engineered host cells.

[0117] As used herein, the terms "derivatization", "functionalization", "modification by derivatization", and "modification by functionalization" refer to the modification of TA or TA precursors by the attachment of functional groups, without modifying the TA backbone 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.

[0118] In some embodiments of the invention, derivatization of the TA of interest can be achieved enzymatically by feeding a pre-functionalized TA precursor (e.g., a halogenated or alkylated amino acid) to a host cell that is engineered to take up the fed TA precursor and then convert it to the TA of interest. In other embodiments of the invention, in addition to the enzyme and cell modifications required to produce unmodified TA, derivatization of the TA of interest can be achieved enzymatically by engineering the host cell to express an enzyme with the desired activity to attach a functional group to the target TA. In other embodiments of the invention, derivatization of the TA of interest can be achieved enzymatically by treating the unmodified TA produced by the engineered host cell with a purified enzyme capable of attaching the desired functional group or with a crude lysate of a host cell engineered to express an enzyme with the desired derivatization activity. In other embodiments of the invention, derivatization of the TA of interest can be achieved non-enzymatically by treating the unmodified TA produced by the engineered host cell with a chemical agent having the desired functional group.

[0119] Modified derivatives of TA include, but are not limited to, p-hydroxyatropine, p-hydroxyscopolamine, p-fluscopolamine, p-chloroscopolamine, p-bromoscopolamine, p-fluoroscopolamine, p-chloroscopolamine, p-bromoscopolamine, N-methylscopolamine, N-butylscopolamine, N-methylscopolamine, N-butylscopolamine, N-acetylscopolamine, and N-acetylscopolamine.

[0120] Host cells

[0121] As described above, one aspect of the invention is a host cell that produces one or more TAs of interest. Any convenient cell can be used in the subject host cells and methods. In some cases, the host cell is a non-plant cell. In some cases, the host cell can be characterized as a microbial cell. In some cases, the host cell is an insect cell, a mammalian cell, a bacterial cell, or a fungal cell. Any convenient type of host cell can be used to produce a subject TA-producing cell, see, for example, US2008 / 0176754, US2014 / 0273109, and WO2014 / 143744, now published as U.S. Patent No. 8,975,063; its disclosure is incorporated by reference in its entirety. Host cells of interest include, but are not limited to, bacterial cells, such as Bacillus subtilis (Bacillus subtilis), Escherichia coli, Streptomyces, Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas Halomonas, Klebsiella, Kocuria, Lactobacillus, Leucononstoc, Macrococcus, Methylomonas, Methylobacter, Methylocella, Methylococcus, Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus, Propionibacterium, Proteus,Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, Zymomonas, and Salmonella typhimuium cells, insect cells such as Drosophila melanogaster S2 and Spodoptera frugiperda frugiperda) Sf9 cells, and yeast cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Yarrowia lipolytica, Candida albicans, Aspergillus spp., Rhizopus spp., Penicillium spp., and Trichoderma reesei cells. In some embodiments, the host cell is a yeast cell or an Escherichia coli cell. In some cases, the host cell is a yeast cell. In some cases, the host cell is from a yeast strain engineered to produce a TA of interest. Any host cell described in US2008 / 0176754, US2014 / 0273109, and WO2014 / 143744, now published as U.S. Pat. No. 8,975,063, may be suitable for use in the subject cells and methods. In certain embodiments, the yeast cell may belong to the species S. cerevisiae. In certain embodiments, the yeast cell may belong to the species Schizosaccharomyces pombe. In certain embodiments, the yeast cell may belong to the species Pichia pastoris. Yeast is of interest as a host cell because cytochrome P450 proteins involved in some biosynthetic pathways of interest can be properly folded into the endoplasmic reticulum membrane, thereby maintaining their activity.

[0122] Yeast strains of interest that can be used in the present invention include, but are not limited to, CEN.PK (genotype: MATa / αura3-52 / ura3-52trp1-289 / trp1-289leu2-3_112 / leu2-3_112his3Δ1 / his3Δ1MAL2-8C / MAL2-8C SUC2 / SUC2), S288C, W303, D273-10B, X2180, A364A, ∑1278B, AB972, SK1, and FL100. In some cases, the yeast strain is any one of the following: S288C (MATα; SUC2 mal mel gal2 CUP1 flo1 flo8-1hap1), BY4741 (MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0), BY4742 (MATα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0), BY4743 (MATa / MATα; his3Δ1 / his3Δ1; leu2Δ0 / leu2Δ0; met15Δ0 / MET15; LYS2 / lys2Δ0; ura3Δ0 / ura3Δ0), and derivatives of WAT11 or W(R) and W303-B strains (MATa; ade2-1; his3-11, -15; leu2-3, -112; ura3-1; canR; cyr+), which express Arabidopsis NADPH-P450 reductase ATR1 and yeast NADPH-P450 reductase CPR1, respectively. In another embodiment, the yeast cell is W303α (MATα; his3-11, 15trp1-1 leu2-3 ura3-1 ade2-1). The identities and genotypes of other yeast strains of interest can be found at EUROSCARF (web.uni-frankfurt.de / fb15 / mikro / euroscarf / col_index.html).

[0123] In some cases, the host cell is a fungal cell. In certain embodiments, the fungal cell may belong to Aspergillus species and strains include Aspergillus niger (ATCC 1015, ATCC 9029, CBS 513.88), Aspergillus oryzae (ATCC 56747, RIB40), Aspergillus terreus (NIH2624, ATCC 20542) and Aspergillus nidulans (Aspergillus nidulans) (FGSC A4).

[0124] In certain embodiments, the heterologous coding sequence can be codon optimized for expression in Aspergillus species and expressed by an appropriate promoter. In certain embodiments, the promoter can be selected from phosphoglycerate kinase promoter (PGK), MbfA promoter, cytochrome c oxidase subunit promoter (CoxA), SrpB promoter, TvdA promoter, malate dehydrogenase promoter (MdhA), β-mannosidase promoter (ManB). In certain embodiments, the terminator can be selected from glucoamylase terminator (GlaA) or TrpC terminator. In certain embodiments, the expression cassette consisting of promoter, heterologous coding sequence and terminator can be expressed by plasmid or integrated into the host's genome. In certain embodiments, the selection of cells maintaining plasmids or integration boxes can be performed by antibiotic selection such as hygromycin or nitrogen source utilization such as using acetamide as the sole nitrogen source. In certain embodiments, established transformation methods such as protoplast transformation, lithium acetate or electroporation can be used to introduce DNA constructs into host cells. In certain embodiments, cells can be cultured in liquid ME or solid MEA (3% malt extract, 0.5% peptone, and ±1.5% agar) with or without selection, or in Vogel's minimal medium.

[0125] In some cases, the host cell is a bacterial cell. The bacterial cell can be selected from any bacterial species. Examples of species from which the bacterial cell can be derived include Anabaena, Arthrobacter, Acetobacter, Acetobacter, Bacillus, Bifidobacterium, Brevibacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconoacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Coxsackie, Lactobacillus, Leuconostoc, Megacoccus, Methylomonas, Methylobacterium, Methylobacterium, Methylococcus, Methylococcus, Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus, Propionibacterium, Proteus, Pseudoalteromonas, Pseudomonas, Psychrophiles, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella and Zymomonas. Examples of bacterial species that can be used with the methods of the present disclosure include Arthrobacter nicotianae, Acetobacter aceti, Arthrobacter arilaitensis, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus pumilus, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium adolescentis, Brachybacterium tyrofermentans, Brevibacterium linens, Carnobacterium divergens, Corynebacterium flavescens), Enterococcus faecium, Gluconacetobacter europaeus, Gluconacetobacter johannae, Gluconobacter oxydans, Hafnia alvei, Halomonas elongatuselongata), Kocuria rhizophila, Lactobacillus acidifarinae, Lactobacillus jensenii, Lactococcus lactis, Lactobacillus yamanashiensis, Leuconostoc citreum, Macrococcus caseolyticus, Microbacterium foliorum, Micrococcus lylae, Oenococcus oeni, Pediococcus acidilactici, Propionibacterium acidipropionici, Proteus vulgaris, Pseudomonas fluorescens, Psychrobacter celer), Staphylococcus condimenti, Streptococcus thermophilus, Streptomyces griseus, Tetragenococcus halophilus, Weissella cibaria, Weissella koreensis, Zymomonas mobilis, Corynebacterium glutamicum, Bifidobacterium bifidum / breve / longum, Streptomyces lividans, Streptomyces coelicolor, Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus casei, Pseudoalteromonas citrinum citrea), Pseudomonas putida, Young's / Acetic acid / Acetobutanol / Beijing's / Clostridium butyricumljungdahlii / aceticum / acetobutylicum / beijerinckii / butyricum) and Moorella themocellum / thermoacetica.

[0126] In certain embodiments, bacterial cells may belong to Escherichia coli strains. In certain embodiments, Escherichia coli strains may be selected from BL21, DH5α, XL1-Blue, HB101, BL21 and K12. In certain embodiments, heterologous coding sequences may be codon optimized for expression in Escherichia coli and expressed by appropriate promoters. In certain embodiments, promoters may be selected from T7 promoters, tac promoters, trc promoters, tetracycline inducible promoters (tet), lac operon promoters (lac), lacO1 promoters. In certain embodiments, the expression cassette consisting of promoter, heterologous coding sequence and terminator may be expressed by plasmid or integrated into the genome. In certain embodiments, plasmids are selected from pUC19 or pBAD. In certain embodiments, the selection of cells maintaining plasmids or integration cassettes may be performed by antibiotic selection, such as kanamycin, chloramphenicol, streptomycin, spectinomycin, gentamicin, erythromycin or ampicillin. In certain embodiments, established transformation methods such as conjugation, heat shock chemical transformation or electroporation may be used to introduce DNA constructs into host cells. In certain embodiments, cells can be cultured in liquid Luria-Bertani (LB) medium at about 37°C, with or without antibiotics.

[0127] In certain embodiments, the bacterial cell can be a strain of bacillus subtilis. In certain embodiments, the strain of bacillus subtilis can be selected from 1779, GP25, RO-NN-1, 168, BSn5, BEST195, 1A382 and 62178. In certain embodiments, the heterologous coding sequence can be codon optimized for expression in Bacillus species and expressed by a suitable promoter. In certain embodiments, the promoter can be selected from grac promoter, p43 promoter or trnQ promoter. In certain embodiments, the expression cassette consisting of promoter, heterologous coding sequence and terminator can be expressed by plasmid or integrated into the genome. In certain embodiments, plasmid is selected from pHP13 pE194, pC194, pHT01 or pHT43. In certain embodiments, integration vectors such as pDG364 or pDG1730 can be used for expression cassette integrated into the genome. In certain embodiments, the selection of cells maintaining plasmids or integration boxes can be carried out by antibiotic selection, such as erythromycin, kanamycin, tetracycline and spectinomycin. In certain embodiments, established transformation methods such as natural competence, heat shock or chemical transformation can be used to introduce DNA constructs into host cells. In certain embodiments, cells can be cultured in liquid Luria-Bertani (LB) medium at 37°C or in M9 medium with glucose and tryptophan.

[0128] Genetic modification of host cells

[0129] Host cells can be engineered to include one or more modifications (such as two or more, three or more, four or more, five or more, or even more modifications) that provide the production of the TA of interest. In some cases, modification refers to genetic modification, such as mutation, addition or deletion of a gene or its fragment, or transcriptional regulation of a gene or its fragment. In some cases, one or more (such as two or more, three or more, or four or more) modifications are selected from: feedback inhibition relief mutations in cell-intrinsic biosynthetic enzyme genes; transcriptional regulation modifications of cell-intrinsic biosynthetic enzyme genes; inactivation mutations in cell-intrinsic enzymes; heterologous coding sequences encoding enzymes; and heterologous coding sequences encoding proteins that improve subcellular transport and / or localization of enzymes or metabolites. Cells comprising one or more modifications may be referred to as modified cells.

[0130] The modified cells may overproduce one or more precursor TAs, TAs, or modified TA molecules. Overproduction means that the cell has improved or increased production of the TA molecule of interest relative to a control cell (e.g., an unmodified cell). Improved or increased production means that a certain amount of the TA of interest is produced when the control has no TA precursor production, and 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, when the control has some TA of interest production.

[0131] In some cases, the host cell is capable of producing an increased amount of putrescine relative to a control host cell lacking one or more modifications (e.g., as described herein). In some cases, the increased amount of putrescine is an increase of about 10% or more relative to a control host cell, such as an increase of 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 a control host cell.

[0132] In some cases, the host cell is capable of producing an increased amount of N-methylpyrrolinium relative to a control host cell lacking one or more modifications (e.g., as described herein). In some cases, the increased amount of N-methylpyrrolinium is an increase of about 10% or more relative to the control host cell, such as an increase of 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.

[0133] In some cases, the host cell is capable of producing an increased amount of a tropine relative to a control host cell lacking one or more modifications (e.g., as described herein). In some cases, the increased amount of a tropine is an increase of about 10% or more relative to a control host cell, such as an increase of 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 a control host cell.

[0134] In some cases, the host cell is capable of producing an increased amount of phenylpyruvate relative to a control host cell lacking one or more modifications (e.g., as described herein). In some cases, the increased amount of phenylpyruvate is an increase of about 10% or more relative to a control host cell, such as an increase of 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 a control host cell.

[0135] In some cases, the host cell is capable of producing an increased amount of phenyllactic acid relative to a control host cell lacking one or more modifications (e.g., as described herein). In some cases, the increased amount of phenyllactic acid is an increase of about 10% or more relative to the control host cell, such as an increase of 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.

[0136] In some cases, the host cell is capable of producing an increased amount of ritolin relative to a control host cell lacking one or more modifications (e.g., as described herein). In some cases, the increased amount of ritolin is an increase of about 10% or more relative to a control host cell, such as an increase of 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 a control host cell.

[0137] In some cases, relative to a control host cell lacking one or more modifications (e.g., as described herein), the host cell is capable of producing an increased amount of scopolamine. In some cases, the increased amount of scopolamine is an increase of about 10% or more relative to the control host cell, such as an increase of 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.

[0138] In some cases, relative to a control host cell lacking one or more modifications (e.g., as described herein), the host cell is capable of producing the scopolamine of the amount increased. In some cases, the scopolamine of the amount increased is an increase of about 10% or more relative to the control host cell, such as an increase of 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.

[0139] In some embodiments, the host cell is capable of producing a 10% or greater yield of tropine from a starting compound, such as arginine, such as 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or even 90% or greater yield of tropine from a starting compound.

[0140] In some embodiments, the host cell is capable of producing a 10% or greater yield of phenyllactate from a starting compound such as phenylalanine, such as 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or even 90% or greater yield of phenyllactate from a starting compound.

[0141] In some embodiments, the host cell is capable of producing a 10% or greater yield of scopolamine from a starting compound such as arginine or phenylalanine, such as 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or even 90% or greater yield of scopolamine from the starting compound.

[0142] In some embodiments, the host cell is capable of producing a 10% or greater yield of scopolamine from a starting compound such as arginine or phenylalanine, such as 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or even 90% or greater yield of scopolamine from a starting compound.

[0143] 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-methylaminobutyraldehyde, N-methylpyrrolinium, 4-(1-methyl-2-pyrrolidinyl)-3-oxobutyric acid, tropinone, tropine, phenylalanine, prephenic acid, phenylpyruvate, phenyllactic acid, glucose-1-O-phenyllactate, ritolin, hyoscyamine, scopolamine, and scopolamine.

[0144] Any convenient combination of one or more modifications may be included in the subject host cell. In some cases, two or more (such as two or more, three or more, or four or more) different types of modifications are included. In some cases, two or more (such as three or more, four or more, five or more, or even more) different modifications of the same modification type are included in the subject cell.

[0145] In some embodiments of the host cell, when the cell includes one or more heterologous coding sequences encoding one or more enzymes, it includes at least one additional modification selected from the group consisting of: feedback inhibition relief mutations in cell-intrinsic biosynthetic enzyme genes; transcriptional regulatory modifications of cell-intrinsic biosynthetic enzyme genes; and inactivating mutations in cell-intrinsic enzymes. In certain embodiments of the host cell, when the cell includes one or more feedback inhibition relief mutations in cell-intrinsic biosynthetic enzyme genes, it includes at least one additional modification selected from the group consisting of: transcriptional regulatory modifications of cell-intrinsic biosynthetic enzyme genes; inactivating mutations in cell-intrinsic enzymes; and heterologous coding sequences encoding enzymes. In some embodiments of the host cell, when the cell includes one or more transcriptional regulatory modifications of cell-intrinsic biosynthetic enzyme genes, it includes at least one additional modification selected from the group consisting of: feedback inhibition relief mutations in cell-intrinsic biosynthetic enzyme genes; inactivating mutations in cell-intrinsic enzymes; heterologous coding sequences encoding enzymes; and heterologous coding sequences encoding proteins that improve subcellular transport and / or localization of enzymes or metabolites. In certain cases of the host cell, when the cell includes one or more inactivating mutations in one or more enzymes intrinsic to the cell, it includes at least one additional modification selected from the group consisting of: a feedback inhibition relief mutation in a cell-intrinsic biosynthetic enzyme gene; a transcriptional regulation modification of a cell-intrinsic biosynthetic enzyme gene; a heterologous coding sequence encoding an enzyme; and a heterologous coding sequence encoding a protein that improves subcellular trafficking and / or localization of an enzyme or metabolite.

[0146] In certain embodiments of the host cell, the cell includes one or more feedback inhibition relief mutations in one or more biosynthetic enzyme genes inherent to the cell; and one or more transcriptional regulatory modifications of one or more biosynthetic enzyme genes inherent to the cell. In certain embodiments of the host cell, the cell includes one or more feedback inhibition relief mutations in one or more biosynthetic enzyme genes inherent to the cell; and one or more inactivation mutations in the enzyme inherent to the cell. In certain embodiments of the host cell, the cell includes one or more feedback inhibition relief mutations in one or more biosynthetic enzyme genes inherent 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) including one or more genes of interest described in Table 1.

[0147] Feedback inhibition alleviates mutations

[0148] In some cases, the host cell is a cell that includes one or more feedback inhibition relief mutations (such as two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell. In some cases, one or more biosynthetic enzyme genes are intrinsic to the cell (e.g., present in unmodified cells). As used herein, the term "feedback inhibition relief mutation" refers to a mutation that relieves the feedback inhibition control mechanism of the host cell. Feedback inhibition is a control mechanism of the cell in which an enzyme in the synthetic pathway of a regulated compound is inhibited when the compound accumulates to a certain level, thereby balancing the amount of the compound in the cell. In some cases, one or more feedback inhibition relief mutations are in Figure 1-4 biosynthetic pathway or Figure 8 The enzyme depicted in the schematic diagram of FIG. A mutation that relieves feedback inhibition reduces inhibition of the regulated enzyme in the cell of interest relative to control cells and provides increased levels of the regulated compound or its downstream biosynthetic product. In some cases, relieving inhibition of the regulated enzyme refers to an inhibition of the IC 50 Increase 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. Increased levels refer to 110% or more of the level of the regulated compound or its downstream product in the control cell, 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 of the regulated compound or its downstream product in the host cell, such as at least 3 times or more, at least 5 times or more, at least 10 times or more, or even more.

[0149] A variety of feedback inhibition control mechanisms and biosynthetic enzymes inherent to host cells intended to regulate the level of TA precursors can be targeted in host cells for mitigation. The host cell may include one or more feedback inhibition mitigation mutations in one or more biosynthetic enzyme genes inherent to the cell. The mutation may be located in any convenient biosynthetic enzyme gene inherent to the host cell in which the biosynthetic enzyme is regulated. In some embodiments, one or more biosynthetic enzyme genes encode one or more enzymes selected from the following: ornithine decarboxylase (ODC), ornithine decarboxylase antizyme (antizyme) and putrescine N-methyltransferase. In some embodiments, one or more biosynthetic enzyme genes encode ornithine decarboxylase. In some cases, one or more biosynthetic enzyme genes encode ornithine decarboxylase antizyme. In some embodiments, one or more biosynthetic enzyme genes encode putrescine N-methyltransferase. In some cases, one or more feedback inhibition mitigation mutations are present in a biosynthetic enzyme gene selected from SPE1, OAZ1 and PMT. In some cases, one or more feedback inhibition mitigation mutations are present in a biosynthetic enzyme gene that is SPE1. In some cases, one or more feedback inhibition mitigation mutations are present in a biosynthetic enzyme gene that is OAZ1. In some cases, one or more feedback inhibition relieving mutations are present in a biosynthetic enzyme gene that is a PMT.In some embodiments, the host cell comprises one or more feedback inhibition relieving mutations in one or more biosynthetic enzyme genes, such as one of those genes described in Table 1.

[0150] The mutation of any convenient number and type can be utilized to alleviate the feedback inhibition control mechanism.As used herein, the term "mutation" refers to the disappearance, insertion or replacement of one or more amino acid residues or one or more nucleotide residues relative to a reference sequence or motif.The mutation can be incorporated as the directed mutation of the native gene at the original locus.In some cases, the mutation can be incorporated as an additional copy of a gene (introduced as the genetic integration at a separate locus), or as an additional copy on an additional vector (such as 2μ or a centromeric plasmid).In some cases, the feedback inhibition copy of the enzyme is under the transcriptional regulation of the native cell.In some cases, by being placed under the control of a synthetic promoter, the feedback inhibition copy of the enzyme is introduced, with the constitutive or dynamic regulation of the engineering of protein expression.

[0151] In certain embodiments, the host cells of the invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more feedback inhibition relief mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 feedback inhibition relief mutations in one or more biosynthetic enzyme genes native to the host cell.

[0152] Transcriptional regulatory modifications

[0153] Host cells can include one or more transcriptional regulation 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 cells. In some cases, one or more biosynthetic enzyme genes are intrinsic to cells. Any convenient biosynthetic enzyme gene of cells can be targeted for transcriptional regulation. Transcriptional regulation refers to regulating the expression of a gene of interest in modified cells, for example, increasing or decreasing, enhancing or suppressing relative to a control cell (for example, an unmodified cell). In some cases, the transcriptional regulation of a gene of interest includes increasing or enhancing expression. Increasing or enhancing expression refers to increasing 2 times or more, such as 5 times or more, and sometimes increasing 25, 50 or 100 times or more, and 300 times or more or higher (for example, by using any convenient gene expression assay) compared to a control (that is, expression in unmodified same cells), in certain embodiments. Alternatively, in the case of low to undetectable expression of a gene of interest in a cell, if expression is increased to a level that can be easily detected, it is considered that the expression level of a gene of interest increases. In some cases, the transcriptional regulation of a gene of interest includes reducing or suppressing expression. Reduction or suppression expression refers to that compared with the control, the expression level of the gene of interest is reduced by 2 times or more, such as 5 times or more, and sometimes reduced by 25, 50 or 100 times or more, and in certain embodiments 300 times or more or higher. In some cases, expression is reduced to undetectable levels. The improvement of the host cell process of interest applicable to the subject host cell is described in the U.S. Publication No. 20140273109 (14 / 211,611) of Smolke et al., and its publication is incorporated herein by reference in its entirety.

[0154] Any convenient biosynthetic enzyme gene may be transcriptionally regulated, and includes but is not limited to Figure 1-3In some embodiments, the host cell comprises one or more transcriptional regulatory modifications to one or more genes, such as those described in Table 1. In some embodiments, the host cell comprises one or more transcriptional regulatory modifications to one or more genes, such as those described in Table 1. In some embodiments, the host cell comprises one or more transcriptional regulatory modifications to one or more genes, such as those described in Table 1. Figure 1-4 A biosynthetic pathway or Figure 8 One or more transcriptional regulatory modifications of one of the genes described in the schematic diagram of .

[0155] In some embodiments, the transcriptional regulation modification includes replacing the natural promoter of one or more biosynthetic enzyme genes with a strong promoter or expressing one or more additional copies of one or more genes under the control of a strong promoter. The promoter driving the expression of the gene of interest can be a constitutive promoter or an inducible promoter, provided that the promoter can be active in the host cell. The gene of interest can be expressed by its natural promoter, or a non-natural promoter can be used. Although not necessary, such promoters should have medium to high intensity in the host using them. The promoter can be regulated or constitutive. In some embodiments, a promoter that is not suppressed by glucose or is only slightly suppressed by the presence of glucose in the culture medium is used. There are many suitable promoters, examples of which include promoters of glycolytic genes, such as promoters of Bacillus subtilis tsr genes (encoding fructose bisphosphate aldolase) or GAPDH promoters from Saccharomyces cerevisiae (encoding glyceraldehyde-phosphate dehydrogenase) (Bitter GA, Meth. Enzymol. 152: 673 684 (1987)). Other strong promoters of interest include, but are not limited to, the ADHI promoter of baker's yeast (Ruohonen L. et al., J. Biotechnol. 39:193 203 (1995)), phosphate starvation-inducible promoters such as the yeast PHO5 promoter (Hinnen, A. et al., in Yeast Genetic Engineering, Barr, PJ et al., eds., Butterworths (1989), the alkaline phosphatase promoter from B. licheniformis (Lee. JWK et al., J. Gen. Microbiol. 137:1127-1134), the promotor of yeast (Lee, J. W. K. ... 1133 (1991)), GPD1 and TEF1. Yeast promoters of interest include, but are not limited to, inducible promoters such as Gal1-10, Gal1, GalL, GalS, repressible promoters Met25, tetO, and constitutive promoters such as glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation elongation factor 1-α promoter (TEF), cytochrome c-oxidase promoter (CYC1), MRP7 promoter, phosphoglycerate kinase (PGK), triosephosphate isomerase (TPI), etc. In some cases, the strong promoter is GPD1. In some cases, the strong promoter is TEF1. Self-replicating yeast expression vectors containing hormone-inducible promoters such as glucocorticoids, steroids, and thyroid hormones are also known and include, but are not limited to, glucocorticoid response elements (GRE) and thyroid hormone response elements (TRE), see, for example, those described in U.S. Pat. No. 7,045,290.Can use the carrier containing constitutive or inducible promoter such as alpha factor, alcohol oxidase and PGH.In addition, any promoter / enhancer combination (according to the eukaryotic promoter database (Eukaryotic Promoter Data Base, EPDB)) can also be used to drive the expression of the gene of interest.It should be understood that any convenient promoter specific to the host cell (e.g., Escherichia coli) can be selected.In some cases, promoter selection can be used to optimize transcription, and therefore optimize enzyme levels, so that the yield is maximized while minimizing energy resources.

[0156] Inactivating mutation

[0157] The host cell may include one or more inactivating mutations (such as two or more, three or more, four or more, five or more, or even more) of an enzyme of the cell. Inclusion of one or more inactivating mutations may improve the flux of a synthetic pathway of the host cell to increase the level of a TA of interest or a desired enzyme or precursor that produces the TA. In some cases, the one or more inactivating mutations are for an enzyme intrinsic to the cell. Figure 8 Demonstrating a natural regulatory mechanism acting on the polyamine production pathway in yeast, and Fig. 9 The impact of the destruction of these natural regulatory systems on putrescine production is shown. As used herein, "inactivation mutation" refers to one or more mutations of a cell's gene or regulatory DNA sequence, wherein one or more mutations inactivate the biological activity of the protein expressed by the gene of interest. In some cases, the gene is intrinsic to the cell. In some cases, the gene encodes an enzyme that is inactivated and is a part of or is connected to the synthetic pathway of the TA of interest produced by the host cell. In some cases, the inactivation mutation is located in the regulatory DNA sequence controlling the gene of interest. In some cases, the inactivation mutation is for the promoter of the gene. Any convenient mutation (for example, as described herein) can be used to inactivate the gene of interest or the regulatory DNA sequence. "Inactivation" or "inactivation..." refers to a reduction of 10% or more in the biological activity of the protein expressed by the mutant gene relative to the control protein expressed by the non-mutated control gene, 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. In some cases, the protein is an enzyme and the inactivating mutation reduces the activity of the enzyme.

[0158] In some embodiments, the cell includes an inactivating mutation in an enzyme that is intrinsic to the cell. Any convenient enzyme can be targeted for inactivation. Enzymes of interest include, but are not limited to Figure 1-4, 8, 11, 22 and 41, whose role in the biosynthetic pathway of the host cell tends to reduce the level of the TA of interest. In some cases, the enzyme has methylthioadenosine phosphorylase activity. In certain embodiments, the enzyme comprising the inactivating mutation is MEU1 (see, e.g., Figure 8 , 9and 13). In some cases, the enzyme has ornithine decarboxylase antienzyme activity. In certain embodiments, the enzyme comprising an inactivating mutation is OAZ1. In some cases, the enzyme has spermidine synthase activity. In certain embodiments, the enzyme comprising an inactivating mutation is SPE3. In some cases, the enzyme has spermine synthase activity. In some embodiments, the enzyme comprising an inactivating mutation is SPE4. In some cases, the enzyme is a membrane transporter with polyamine efflux activity (export activity). In certain embodiments, the enzyme or protein comprising an inactivating mutation is TPO5. In some cases, the enzyme has phenylacrylic acid decarboxylase activity. In certain embodiments, the enzyme comprising an inactivating mutation is PAD1. In some cases, the enzyme has alcohol dehydrogenase activity. In some embodiments, the enzyme comprising an inactivating mutation is selected from ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and SFA1. In certain embodiments, the enzyme comprising one or more inactivating mutations is ADH2. In certain embodiments, the enzyme comprising one or more inactivating mutations is ADH3. In certain embodiments, the enzyme comprising one or more inactivating mutations is ADH4. In certain embodiments, the enzyme comprising one or more inactivating mutations is ADH5. In certain embodiments, the enzyme comprising one or more inactivating mutations is ADH6. In certain embodiments, the enzyme comprising one or more inactivating mutations is ADH7. In some cases, the enzyme has aldehyde oxidoreductase activity. In certain embodiments, the enzyme comprising an inactivating mutation is selected from HFD1, ALD2, ALD3, ALD4, ALD5 and ALD6. In certain embodiments, the enzyme comprising one or more inactivating mutations is HFD1. In certain embodiments, the enzyme comprising one or more inactivating mutations is ALD2. In certain embodiments, the enzyme comprising one or more inactivating mutations is ALD3. In certain embodiments, the enzyme comprising one or more inactivating mutations is ALD4. In certain embodiments, the enzyme comprising one or more inactivating mutations is ALD5. In certain embodiments, the enzyme comprising one or more inactivating mutations is ALD6. In some cases, the enzyme has glucosidase activity. In certain embodiments, the enzyme comprising an inactivating mutation is selected from EXG1, SPR1, and EGH1. In certain embodiments, the enzyme comprising one or more inactivating mutations is EXG1. In certain embodiments, the enzyme comprising one or more inactivating mutations is SPR1. In certain embodiments, the enzyme comprising one or more inactivating mutations is EGH1. In some embodiments, the host cell comprises one or more inactivating mutations to one or more genes described in Table 1.

[0159] Methods for TA acyl transfer reactions using functional expression of acyltransferases in non-plant hosts

[0160] Some methods, processes and systems provided herein describe the synergistic reaction of one or more TA precursors comprising an acyl donor group with one or more TA precursors comprising an acyl acceptor group in a non-plant cell to produce one or more TAs (hereinafter referred to as TA acyl transfer reactions). Some of these methods, processes and systems may include engineered host cells. In some instances, the TA acyl transfer reaction is a key step in converting a substrate into a variety of alkaloids. In some instances, the TA acyl transfer reaction includes a condensation reaction.

[0161] In some instances, TA acyl transfer may involve at least one condensation reaction. In some cases, at least one of the condensation reactions is performed 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 may be used to catalyze the condensation reaction.

[0162] In some methods, processes and systems described herein, the condensation reaction can be carried out in the presence of an enzyme. In some examples, the enzyme can be an acyltransferase. The acyltransferase can use TA with an alcohol or carboxylate functional group as a substrate. The acyltransferase can use TA containing a carboxylate group activated by a 1-O-β glycosidic bond with a sugar (hereinafter referred to as a glycoside) as a substrate. The acyltransferase can convert TA alcohol and carboxylate / glycoside functional groups into corresponding ester derivatives. Non-limiting examples of enzymes suitable for condensation of TA precursors in the present disclosure include serine carboxypeptidase-like acyltransferases (SCPL-AT). For example, ritolin synthase (EC 2.3.1.-) can condense tropine and other TA precursors containing alcohol functional groups with 1-O-β-phenyllactyl-glucose and other TA glycoside precursors into ritolin and other corresponding ester products. In some examples, a protein comprising the SCPL-AT domain of any of the aforementioned examples can be condensed. In some examples, SCPL-AT can catalyze condensation reactions within a host cell (such as an engineered host cell), as described herein. In other examples, SCPL-AT can catalyze condensation reactions within a subcellular compartment within a host cell (such as an engineered host cell), as described herein.

[0163] In some embodiments of the invention, the amino acid sequence of an acyltransferase (such as a SCPL-AT enzyme) for performing a TA acyl transfer reaction is subjected to one or more modifications that alter the post-translational processing, trafficking, folding, oligomerization, and / or subcellular localization of the enzyme. Since some acyltransferases, including SCPL-AT enzymes, have never been shown to exhibit catalytic activity in living non-plant cells, such modifications may prove useful or may be necessary for activity in non-plant host cells. Examples of such modifications include, but are not limited to, adding, removing, or replacing an N-terminal signal peptide sequence; adding, removing, or replacing an internal propeptide sequence; adding or removing an asparagine-linked N-glycosylation site; adding or removing a serine-linked O-glycosylation site; and fusion of a protein domain to the N- and / or C-terminus of an acyltransferase domain.

[0164] In one embodiment of the invention, the SCPL-AT enzyme domain is modified at its N-terminus by the fusion of a soluble protein domain. This soluble domain shields any internal signal sequence in the acyltransferase domain, thereby improving the transport and / or subcellular localization of the fused SCPL-AT domain. In some instances, the domain fused at the N-terminus induces the SCPL-AT domain to be transported to subcellular compartments, including but not limited to ER membranes, ER chambers, cis-Golgi bodies, trans-Golgi bodies, lysosomes, vacuolar membranes, and vacuolar chambers. The soluble domain fused at the N-terminus can also change the oligomeric state of the SCPL-AT domain from its native state (monomer) to any state, including but not limited to homodimers, heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, homohexamers, heterohexamers, homooctamers, heterooctamers, or higher degrees of oligomerization.

[0165] In one example, the soluble protein domain fused at the N-terminus is a fluorescent protein selected from the group including but not limited to: a fluorescent protein derived from a species of Aequoria and a fluorescent protein derived from a species of Discosoma. In one example, the soluble protein domain fused at the N-terminus is a red fluorescent protein (DsRed) from a species of Discosoma. In other examples, the soluble protein domain fused at the N-terminus is another enzyme in the TA biosynthetic pathway, including but not limited to ornithine decarboxylase, putrescine N-methyltransferase, pyrrolidone synthase, tropinone reductase, phenylpyruvate reductase, phenyllactate UDP-glucosyltransferase 84A and scopolamine dehydrogenase.

[0166] Examples of amino acid sequences of soluble protein domains that can be fused to the N-terminus of the SCPL-AT domain (which can then be used to perform TA acyl transfer reactions in non-plant cells) are provided in Table 3. The amino acid sequence of the SCPL-AT enzyme comprising the fused N-terminal domain and used for TA acyl transfer reactions in non-plant cells can have 50% or more identity with a given amino acid sequence listed in Table 3. For example, the amino acid sequence of such acyltransferases can comprise an amino acid sequence having 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 identity to an amino acid sequence as provided herein. In addition, in certain embodiments, an "identical" amino acid sequence comprises at least 80%-99% identity to a particular amino acid sequence at the amino acid level. In some cases, "identical" amino acid sequences comprise at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more, in some cases at least 95%, 96%, 97%, 98% and 99% identity at the amino acid level. In some cases, the amino acid sequences may be identical, but the DNA sequence is altered, such as to optimize codon usage for, for example, a host organism.

[0167] An engineered non-plant host cell that produces an acyltransferase that catalyzes a TA acyl transfer reaction can be provided, wherein the acyltransferase comprises an amino acid sequence fused at its N-terminus to an amino acid sequence of a soluble protein domain selected from the group consisting of those sequences in Table 3. The acyltransferase produced within the engineered host cell can be recovered and purified to form a biocatalyst. One or more enzymes recovered from the engineered host cell that produces the acyltransferase can be used in a process for performing a TA acyltransfer reaction. The process may include contacting a TA precursor having an alcohol and / or carboxylate / glycoside functional group with an acyltransferase in an amount sufficient to convert the alcohol and / or carboxylate / glycoside group into a corresponding ester group. In an example, a TA precursor having an alcohol and / or carboxylate / glycoside functional group can be contacted with a sufficient amount of one or more enzymes such that at least 5% of the TA precursor is converted into a corresponding ester. In further examples, a TA having alcohol and / or carboxylate / glycoside functional groups can be contacted with a sufficient amount of one or more enzymes such that at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or 100% of the TA precursor is converted to the corresponding ester.

[0168] One or more enzymes useful for performing a TA acyl transfer reaction may contact a TA precursor in vitro. Additionally or alternatively, one or more enzymes useful for performing a TA acyl transfer reaction may contact a TA precursor in vivo. Furthermore, one or more enzymes useful for performing a TA acyl transfer reaction may be provided to a cell having a TA precursor therein, or the enzymes may be produced in an engineered non-plant host cell.

[0169] In some examples, the method provides an engineered non-plant host cell that produces an alkaloid product, wherein the TA acyl transfer reaction can constitute a key step in the production of the alkaloid product. In some examples, the alkaloid produced is a medicinal TA. In other embodiments, the alkaloid produced is derived from a medicinal TA, including, for example, a non-natural TA. In other embodiments, the alkaloid product is selected from the group consisting of a medicinal TA, a non-pharmaceutical TA, and a non-natural TA.

[0170] In some examples, the substrate is a TA precursor selected from the group consisting of tropine, pseudotropine, ecgonine, methylecgonine, phenyllactic acid, cinnamic acid, ferulic acid, coumaric acid, and glycosides of the listed compounds.

[0171] In some instances, the method provides an engineered non-plant host cell that produces an alkaloid product by tropine and 1-O-β-phenyl lactyl glucose. Tropine and 1-O-β-phenyl lactyl glucose are condensed into ritolin, which can constitute a key step in producing various alkaloid products by a precursor. In some instances, the precursor is L-amino acid or sugar (e.g., glucose). Various alkaloid products can include but are not limited to medicinal TA, non-medicinal TA and non-natural TA.

[0172] Any suitable carbon source can be used as a precursor for the TA acyl transfer reaction. Suitable precursors may include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or combinations thereof. In some instances, unpurified mixtures from renewable raw materials (e.g., corn syrup, beet molasses, barley malt, biomass hydrolysate) may be used. In 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 other embodiments, other carbon-containing compounds such as methylamine, glucosamine, and amino acids (e.g., L-arginine and L-phenylalanine) may be used. In some instances, TA or TA precursors having alcohol and / or carboxylate / glycoside functional groups may be directly added to the engineered host cells of the present invention, including, for example, tropine, pseudotropine, ecgonine, methylecgonine, phenyllactic acid, cinnamic acid, ferulic acid, coumaric acid, and glycosides of the listed compounds.

[0173] In some embodiments, substrates for performing vacuolar TA acyl transfer reactions may contain one or more alcohol and / or carboxylate / glycoside functional groups, wherein only one of the functional groups is condensed with the corresponding ester.

[0174] TA alcohol-aldehyde interconversion

[0175] Some methods, processes and systems provided herein describe the conversion of TAs having aldehyde functional groups to TAs having alcohol (hydroxyl) functional groups, and the conversion of TAs having alcohol functional groups to TAs having aldehyde functional groups (hereinafter referred to as TA alcohol-aldehyde interconversion). Some of these methods, processes and systems may include engineered host cells. In some instances, TA alcohol-aldehyde interconversion is a key step in converting substrates into various alkaloids. In some instances, the conversion of TA aldehyde groups to TA alcohol groups includes a reduction reaction. In some cases, the reduction of the substrate TA aldehyde to the alcohol can be carried out by reducing the aldehyde substrate to the corresponding tetrahedral oxyanion intermediate and then protonating the intermediate to the hydroxyl group, such as Figure 2 As provided in and as generally represented in Scheme 1. As provided in Scheme 1, R 1 It can be H, CH3 or a higher alkyl group; R 2 and R3 It can be H, OH or OCH3; R 4 can be H; and R 5 It can be H, OH, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 acyl, F, Cl or Br.

[0176]

[0177] Solution 1

[0178] In some examples, the TA alcohol-aldehyde interconversion may involve at least one oxidation reaction or at least one reduction reaction. In some cases, at least one of the oxidation or reduction reactions is carried out in the presence of an enzyme. In some cases, at least one of the oxidation or reduction reactions is catalyzed by an enzyme. In some cases, both the oxidation and reduction reactions are carried out in the presence of at least one enzyme. In some cases, at least one enzyme can be used to catalyze the oxidation and reduction reactions. The oxidation and reduction reactions can be catalyzed by the same enzyme.

[0179] In some methods, processes and systems described herein, the oxidation or reduction reaction may be carried out in the presence of an enzyme. In some instances, the enzyme may be a dehydrogenase. The dehydrogenase may use a TA with an alcohol or aldehyde functional group as a substrate. The dehydrogenase may convert the TA alcohol or aldehyde functional group into a corresponding aldehyde or alcohol derivative. The dehydrogenase may be referred to as hyoscyamine dehydrogenase (HDH). Non-limiting examples of enzymes suitable for oxidation and / or reduction of TA in the present disclosure include cytochrome P450 oxidase, 2-oxoglutarate-dependent oxidase, flavoprotein oxidase, short-chain dehydrogenase-reductase (SDR), medium-chain dehydrogenase-reductase (MDR), cinnamyl alcohol dehydrogenase (CAD) and aldehyde-ketone reductase (AKR). For example, tropinone reductase 1 (EC 1.1.1.206) may oxidize tropinone and other TA precursors with a ketone functional group into tropine (3α-tropine alcohol) and other corresponding alcohol products. In some examples, a protein comprising a dehydrogenase domain of any of the foregoing examples can be oxidized or reduced. In some examples, a dehydrogenase can catalyze oxidation and / or reduction reactions within a host cell (such as an engineered host cell), as described herein.

[0180] Examples of amino acid sequences of dehydrogenases that can be used to perform TA alcohol-aldehyde interconversions are provided in Table 2. The amino acid sequences of dehydrogenases for TA alcohol-aldehyde interconversions can have 50% or more identity with a given amino acid sequence as listed in Table 2. For example, the amino acid sequence of such a dehydrogenase can comprise an amino acid sequence that has 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 identity with an amino acid sequence as provided herein. In addition, in certain embodiments, an "identical" amino acid sequence comprises at least 80%-99% identity with a particular amino acid sequence at the amino acid level. In some cases, "identical" amino acid sequences comprise at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more, in some cases at least 95%, 96%, 97%, 98% and 99% identity at the amino acid level. In some cases, the amino acid sequences may be identical, but the DNA sequence is altered, such as to optimize codon usage for, for example, a host organism.

[0181] An engineered host cell can be provided that produces a dehydrogenase that catalyzes a TA alcohol-aldehyde interconversion, wherein the dehydrogenase comprises an amino acid sequence selected from the group consisting of those sequences in Table 2. The dehydrogenase produced within the engineered host cell can be recovered and purified to form a biocatalyst. One or more enzymes recovered from the engineered host cell that produces the dehydrogenase can be used in a process for performing a TA alcohol-aldehyde interconversion. The process can include contacting a TA having an alcohol and / or aldehyde functional group with an amount of the dehydrogenase sufficient to convert the alcohol and / or aldehyde groups of the TA to the corresponding aldehyde and / or alcohol groups. In an example, a TA having an alcohol and / or aldehyde functional group can be contacted with a sufficient amount of one or more enzymes such that at least 5% of the TA is converted to its corresponding aldehyde and / or alcohol group. In further examples, a TA having an alcohol and / or aldehyde functional group can be contacted with a sufficient amount of one or more enzymes such that at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or 100% of the TA is converted to its corresponding aldehyde and / or alcohol group.

[0182] One or more enzymes useful for performing alcohol-aldehyde interconversion of TA can contact TA in vitro. Additionally or alternatively, one or more enzymes useful for performing alcohol-aldehyde interconversion of TA can contact TA in vivo. Furthermore, one or more enzymes useful for performing alcohol-aldehyde interconversion of TA can be provided to a cell having TA therein, or the enzymes can be produced within an engineered host cell.

[0183] In some examples, the method provides an engineered host cell that produces an alkaloid product, wherein the TA alcohol-aldehyde interconversion can constitute a key step in the production of the alkaloid product. In some examples, the alkaloid produced is a pharmaceutical TA. In other embodiments, the alkaloid produced is derived from a pharmaceutical TA, including, for example, a non-natural TA. In another embodiment, a TA with an alcohol and / or aldehyde functional group is an intermediate of a product of the engineered host cell. In other embodiments, the alkaloid product is selected from the group consisting of a pharmaceutical TA, a non-pharmaceutical TA, and a non-natural TA.

[0184] In some examples, the substrate is TA or a precursor of TA selected from the group consisting of ritolin, hyoscyaldehyde, hyoscyamine, anisodamine, and scopolamine.

[0185] In some instances, the method provides an engineered host cell that produces an alkaloid product from scopolamine. Scopolamine is reduced to scopolamine, which can constitute a key step in producing various alkaloid products from a precursor. In some instances, the precursor is an L-amino acid or a sugar (e.g., glucose). Various alkaloid products may include, but are not limited to, medicinal TA, non-medicinal TA, and non-natural TA.

[0186] Any suitable carbon source can be used as a precursor for TA alcohol-aldehyde interconversion. Suitable precursors may include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or combinations thereof. In some instances, unpurified mixtures from renewable raw materials (e.g., corn syrup, beet molasses, barley malt, biomass hydrolysate) may be used. In 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 other embodiments, other carbon-containing compounds such as methylamine, glucosamine, and amino acids (e.g., L-arginine and L-phenylalanine) may be used. In some examples, TA or a TA precursor having an alcohol and / or aldehyde functional group can be added directly to the engineered host cells of the present invention, including, for example, tropine, pseudotropine, ecgonine, methylecgonine, ritolin, hyoscyamine, scopolamine, anisodamine, and scopolamine.

[0187] In some embodiments, substrates for performing TA alcohol-aldehyde interconversions may contain one or more alcohol and / or aldehyde functional groups, wherein only one of the functional groups is oxidized or reduced to the corresponding aldehyde or alcohol group.

[0188] Methods for increasing intracellular and extracellular metabolite transport

[0189] 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 include engineered host cells. In some instances, transmembrane transport is a key step in converting substrates into a variety of alkaloids.

[0190] In certain embodiments, the host cell includes one or more heterologous coding sequences of one or more transporters or active fragments thereof that localize to a lipid membrane and translocate TA or TA precursor across the same lipid membrane. In some instances, the lipid membrane is a vacuole membrane. In other instances, the lipid membrane is an ER membrane. In some instances, the lipid membrane is a peroxisomal membrane. In other instances, the lipid membrane is a cytoplasmic membrane.

[0191] In some examples, TA and TA precursors transported in this manner include, but are not limited to, putrescine, N-methylputrescine, 4-methylaminobutyraldehyde, N-methylpyrrolinium, tropinone, tropine, phenyllactic acid, 1-O-β-phenyllactyl glucose, ritolin, hyoscyamine, anisodamine, and scopolamine. The accumulation of such TA or TA precursors in specific subcellular compartments can prevent the entry of biosynthetic enzymes operably linked in different compartments; therefore, the use of transporters that translocate TA or TA precursors from one compartment to another can alleviate such transport limitations. In some cases, the expression of heterologous coding sequences for one or more transporters in host cells can increase the production of TA or TA precursors.

[0192] In some embodiments, the transporter or its active fragment is a multidrug and toxin efflux (MATE) transporter. Any convenient MATE transporter for transporting one or more of the above-mentioned TA or TA precursors can be used in the subject host cell. The transporter of interest includes but is not limited to enzymes such as the inducible alkaloid transporter 1 (NtJAT1) of common tobacco jasmonate, common tobacco MATE1, common tobacco MATE2 or any other protein as described in Table 1 and Table 4.

[0193] In certain embodiments, the transporter or its active fragment is a nitrate / peptide family (NPF) transporter. Any convenient NPF transporter that transports one or more of the above-mentioned TAs or TA precursors can be used in the subject host cell. In other embodiments, the transporter or its active fragment is an ATP binding cassette (ABC) transporter. Any convenient NPF transporter that transports one or more of the above-mentioned TAs or TA precursors can be used in the subject host cell. In some embodiments, the transporter or its active fragment is a pleiotropic drug resistance (PDR) transporter. Any convenient PDR transporter that transports one or more of the above-mentioned TAs or TA precursors can be used in the subject host cell.

[0194] In certain embodiments, the host cell comprises a heterologous coding sequence of a transporter or an active fragment thereof. In some embodiments of the invention, the amino acid sequence of the transporter is subjected to one or more modifications that change the subcellular localization, the direction of substrate translocation, and / or the topological orientation of the enzyme. Examples of such modifications include, but are not limited to: adding, removing, or replacing an N-terminal, C-terminal, or internal signal sequence; adding, removing, replacing, or rearranging transmembrane helices; and fusion of a protein domain to the N and / or C-terminal of the transporter.

[0195] Examples of amino acid sequences of transporters that can be used to alleviate substrate transport limitations and / or increase accumulation of TA or TA precursors in specific cellular compartments are provided in Table 4. The amino acid sequence of the transporter used in this manner in non-plant cells can have 50% or more identity to a given amino acid sequence as listed in Table 4. For example, the amino acid sequence of such a transporter can comprise an amino acid sequence that is at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to an amino acid sequence as provided herein. In addition, in certain embodiments, an "identical" amino acid sequence comprises at least 80%-99% identity at the amino acid level to a particular amino acid sequence. In some cases, an "identical" amino acid sequence comprises at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more, and in some cases at least 95%, 96%, 97%, 98% and 99% identity at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence is altered, such as to optimize, for example, codon usage of a host organism.

[0196] An engineered non-plant host cell can be provided that produces a transporter that translocates one or more TAs or TA precursors from one cell compartment to another, wherein the transporter comprises an amino acid sequence selected from the group consisting of those sequences in Table 4. In some examples, the method provides an engineered non-plant host cell that produces an alkaloid product, wherein TA transmembrane transport can constitute a key step in the production of the alkaloid product. In some examples, the alkaloid produced is a pharmaceutical TA. In other embodiments, the alkaloid produced is derived from a pharmaceutical TA, including, for example, a non-natural TA. In other embodiments, the alkaloid product is selected from the group consisting of a pharmaceutical TA, a non-pharmaceutical TA, and a non-natural TA.

[0197] Heterologous coding sequence

[0198] In some cases, the host cell is a cell containing 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 one or more activities that enable the host cell to produce, for example, a desired TA of interest 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 (e.g., an enzyme) that is not normally present in the host organism and can be expressed in the host cell under appropriate conditions. Thus, a "heterologous coding sequence" includes multiple copies of a coding sequence that is normally present in a host cell, such that the cell expresses additional copies of a coding sequence that is not normally present in the cell. A heterologous coding sequence can be RNA or any type thereof (e.g., mRNA), DNA or any type thereof (e.g., cDNA), or a hybrid of RNA / DNA. Coding sequences of interest include, but are not limited to, full-length transcription units including features such as coding sequences, introns, promoter regions, 3'-UTRs, and enhancer regions.

[0199] In an example, the engineered host cell comprises a plurality of heterologous coding sequences that each encodes an enzyme. In some examples, the plurality of enzymes encoded by the plurality of heterologous coding sequences may be different 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.

[0200] In some instances, the heterologous coding sequence can be operably connected. The heterologous coding sequence that is operably connected can be in the same approach of producing a specific tropane alkaloid product. In some instances, the heterologous coding sequence that is operably connected can be directly continuous along the approach that produces a specific tropane alkaloid product. In some instances, the heterologous coding sequence that is operably connected can have one or more natural enzymes between one or more enzymes encoded by a plurality of heterologous coding sequences. In some instances, the heterologous coding sequence can have one or more heterologous enzymes between one or more enzymes encoded by a plurality of heterologous coding sequences. In some instances, the heterologous coding sequence can have one or more non-natural enzymes between one or more enzymes encoded by a plurality of heterologous coding sequences.

[0201] In some embodiments, the host cell includes putrescine N-methyltransferase (PMT) activity. Any convenient PMT enzyme can be used in the subject host cell. Interested PMT enzymes include, but are not limited to, enzymes such as EC 2.1.1.53, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for a PMT or an active fragment thereof.

[0202] In some cases, the host cell includes one or more heterologous coding sequences of one or more enzymes or active fragments thereof that convert NMP to 4MAB. In some cases, the one or more enzymes are selected from plant methylputrescine oxidase (MPO) and eukaryotic MPO (e.g., EC 1.4.3.22).

[0203] In certain embodiments, the cell includes one or more heterologous coding sequences of one or more enzymes or active fragments thereof that convert NMPy into MPOB. In some cases, the one or more enzymes are type III polyketide synthases (e.g., EC 2.3.1.-). The one or more heterologous coding sequences can be derived from any convenient species (e.g., as described herein). In some cases, the one or more heterologous coding sequences can be derived from the species described in Table 1. In some cases, the one or more heterologous coding sequences are present in a gene or enzyme selected from those described in Table 1.

[0204] In certain embodiments, the host cell includes tropinone synthase activity. Any convenient tropinone synthase (e.g., CYP82M3) can be used in the subject host cell. Interested tropinone synthase includes but is not limited to enzymes such as EC1.14.14.-, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence of tropinone synthase or its active fragment.

[0205] In certain embodiments, the host cell comprises tropinone reductase activity. Any convenient tropinone reductase can be used in the subject host cell. Interested tropinone reductases 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 comprises a heterologous coding sequence of tropinone reductase or an active fragment thereof.

[0206] In some cases, the host cell includes phenylpyruvate reductase (PPR) activity. Any convenient PPR enzyme can be used in the subject host cell. 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.

[0207] In certain embodiments, the host cell comprises phenyllactate glycosyltransferase activity. Any convenient phenyllactate glycosyltransferase can be used in the subject host cell. Glycosyltransferases include, but are not limited to, enzymes such as 2.4.1.-, which transfer the glucose moiety from UDP-glucose to phenyllactate via a glycosidic ester bond, as described in Table 1. In certain embodiments, the host cell comprises a heterologous coding sequence for a phenyllactate glycosyltransferase or an active fragment thereof.

[0208] In certain embodiments, the cell includes one or more heterologous coding sequences of one or more enzymes or active fragments thereof that convert tropine and 1-O-β-phenyllactyl glucose into ritolin. In some embodiments, the host cell includes ritolin synthase activity. Any convenient ritolin synthase or enzyme comprising an active fragment of ritolin synthase can be used in the subject host cell. The ritolin synthase of interest includes but is not limited to enzymes such as EC 2.3.1.-, as described in Table 1, and enzymes of ritolin synthases comprising soluble protein domains fused to their N-terminals as described in Table 3. In certain embodiments, the host cell includes a heterologous coding sequence of a ritolin synthase or an active fragment thereof.

[0209] In some cases, the host cell includes a ritolin mutase activity. Any convenient ritolin mutase can be used in the subject host cell. Interested ritolin mutases 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 ritolin mutase or an active fragment thereof.

[0210] In some embodiments, the host cell includes scopolamine dehydrogenase (HDH) activity. Any convenient HDH enzyme can be used in the subject host cell. Some HDH enzymes of interest include, but are not limited to, those sequences described in Table 2. In certain embodiments, the host cell includes a heterologous coding sequence for HDH or an active fragment thereof.

[0211] In certain embodiments, the host cell comprises scopolamine 6β-hydroxylase / dioxygenase (H6H) activity. Any convenient H6H enzyme can be used in the subject host cell. 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 comprises a heterologous coding sequence for H6H or an active fragment thereof.

[0212] In some instances, the host cell of through engineering approaches comprises a plurality of heterologous coding sequences that encode transmembrane metabolite transporters, respectively. In some instances, the plurality of transporters encoded by the plurality of heterologous coding sequences may be different from each other. In some instances, 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.

[0213] As used herein, the term "heterologous coding sequence" also includes the coding portion of a peptide or enzyme, i.e., the cDNA or mRNA sequence of the peptide or enzyme, and the coding portion of the full-length transcription unit, i.e., genes including introns and exons, and "codon-optimized" sequences, truncated sequences or other forms of altered sequences encoding enzymes or encoding equivalent amino acid sequences thereof, provided that the equivalent amino acid sequence produces a functional protein. Such equivalent amino acid sequences may have one or more amino acid deletions, wherein the deletion is N-terminal, C-terminal or internal. Truncated forms are envisioned, as long as they have the catalytic ability indicated herein. Fusions of two or more enzymes are also envisioned to promote the transfer of metabolites in the pathway, provided that the catalytic activity is maintained. Fusions of one or more enzymes or catalytic protein domains with one or more non-catalytic protein domains are also included, and the fusion mode thereof makes the non-catalytic protein domains conducive to the dissolution, folding, maturation and / or activity of the fused catalytic domains.

[0214] Operable fragments, mutants or truncated forms can be identified by modeling and / or screening. This is made possible by adding or deleting, for example, the N-terminus, C-terminus or internal regions of the protein in a stepwise manner and then analyzing the activity of the resulting derivatives with respect to the desired reaction compared to the original sequence. If the derivative in question operates in this capacity, it is considered to constitute a fully equivalent derivative of the enzyme.

[0215] Aspects of the present invention also relate to heterologous coded sequences of amino acid sequences equivalent to the natural amino acid sequences of various enzymes. The amino acid sequence that is "equivalent" is defined as such an amino acid sequence: it is not identical with the specific amino acid sequence, but comprises at least some amino acid changes (deletion, substitution, inversion, insertion, etc.), and when used for the desired purpose, compared with the similar activity of the specific amino acid sequence, the change does not affect the biological activity of the protein substantially. In the example of decarboxylase, biological activity refers to its catalytic activity. Equivalent sequences are also intended to include those that have been engineered and / or evolved to have characteristics that are different from the original amino acid sequence. Interested variable properties include catalytic activity, substrate specificity, selectivity, stability, solubility, positioning, etc. In certain embodiments, an "equivalent" amino acid sequence comprises at least 80%-99% identity at the amino acid level to a particular amino acid, in some cases at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more, and in some cases at least 95%, 96%, 97%, 98% and 99% identity at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence is altered, such as to optimize, for example, codon usage for a host organism.

[0216] Host cells may also be modified to have one or more genetic changes to adapt to heterologous coding sequences. Changes in the natural host genome include, but are not limited to, modifying the genome to reduce or eliminate the expression of specific proteins that may interfere with the desired pathway. The presence of such natural proteins may rapidly convert one of the intermediates or final products of the pathway into metabolites or other compounds that are unavailable in the desired pathway. Therefore, if the activity of the natural enzyme is reduced or completely absent, the intermediate produced will be more easily available for incorporation into the desired product.

[0217] In some cases, when the ablation of protein expression may be of interest, the changes are in proteins involved in pleiotropic drug responses, including but not limited to ATP binding cassette (ABC) transporters, multidrug resistance (MDR) pumps, and related transcription factors. These proteins are involved in the outward transport of TA molecules and TA precursors into the culture medium, so the outward transport of the control compound into the culture medium is deleted, making them more available for incorporation into the desired product. In some embodiments, the host cell gene deletion of interest includes genes associated with the unfolded protein response and endoplasmic reticulum (ER) proliferation. Such gene deletions can lead to an increase in TA production. The expression of cytochrome P450 can induce the unfolded protein response and can lead to ER proliferation. The deletion of genes associated with these stress responses can control or reduce the overall burden of the host cell and improve pathway performance. Genetic changes can also include modifying the promoter of an endogenous gene to increase expression and / or introducing additional copies of an endogenous gene. Examples of this include constructing / using strains that overexpress endogenous yeast NADPH-P450 reductase Ncp1p to increase the activity of heterologous P450 enzymes. In addition, endogenous enzymes directly involved in the synthesis of intermediate metabolites, such as Spelp, Fmslp, Car1p, Arg2p, Aro8p, Aro9p, Pha2p, Ugplp, and Leu2p, can also be overexpressed.

[0218] Interested heterologous coding sequences include, but are not limited to, sequences encoding enzymes, which are wild type or equivalent sequences, and the enzymes are generally responsible for the production of TA and precursors in plants. In some cases, the enzyme encoded by the heterologous sequence can be any enzyme in the TA pathway, and can be from any convenient source. The selection and quantity of the enzyme encoded by the heterologous coding sequence for a specific synthetic pathway can be selected based on the desired product. In certain embodiments, the host cell of the present invention can 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.

[0219] In some cases, the polypeptide sequence encoded by the heterologous coding sequence is reported in GENBANK. Enzymes of interest include but are not limited to those described herein and those shown in Table 1. Host cells may include any combination of listed enzymes from any source. Unless otherwise indicated, the accession numbers in Table 1 refer to GenBank. Some accession numbers refer to the Saccharomyces genome database (SGD), which can be obtained on the world wide web at yeastgenome.org.

[0220] In some embodiments, host cells (e.g., yeast strains) are engineered to selectively produce TAs of interest by locating one or more enzymes to a compartment in the cell. In some cases, the enzyme may be located in the host cell so that the compound produced by the enzyme is spontaneously rearranged, or is converted into a desired metabolite by another enzyme before reaching a localized enzyme (localized enzyme) that can convert the compound into an undesirable metabolite. The spatial distance between the two enzymes can be selected to prevent one of the enzymes from acting directly on the compound to produce an undesirable metabolite, and to limit the production of undesirable end products (e.g., undesirable opioid byproducts). In some other cases, the enzyme may be located in the host cell so that the subcellular compartment in which it is located provides a better pH, cofactor concentration, redox potential, substrate concentration, and / or other biochemical parameters for its activity than the compartment in which the enzyme naturally exists. In some cases, the enzyme may be located in a specific compartment in the host cell so that the intracellular transport pathway that transports the enzyme to the compartment provides the necessary post-translational modification to allow the enzyme to exhibit activity. Such post-translational modifications include but are not limited to acetylation, acetylated glycosylation, amidation, carboxylation, methylation, glutathionylation, hydroxylation, glycosylation, phosphorylation, sulfonation, disulfide bond formation, cutting of signal sequence and multi-enzyme complex formation. In certain embodiments, any enzyme as described herein, alone or together with the second enzyme, can be positioned in any convenient compartment in the host cell, including but not limited to the cavity of organelle, endoplasmic reticulum, Golgi body, vacuole, nucleus, plasma membrane, mitochondria, peroxisomes, periplasm, any above-mentioned organelle, or surround any above-mentioned organelle or the film associated therewith. In the case where one or more enzymes are positioned in the film associated with any above-mentioned organelle, the enzyme can be directed to make the catalytic domain of the enzyme face the cavity of cytosol, organelle and / or any other intracellular space. In some embodiments, the host cell includes one or more enzymes including positioning tags. Any convenient label can be used. In some cases, the positioning tag is a peptide sequence connected to the N-terminal and / or C-terminal of the enzyme.

[0221] Any convenient method can be used to connect the label to the enzyme. In some cases, the positioning label is derived from endogenous yeast protein. Such labels can provide paths leading to a variety of yeast organelles, including but not limited to endoplasmic reticulum (ER), Golgi body (GA), mitochondria (MT), plasma membrane (PM), peroxisome (POX) and vacuole (V). In certain embodiments, the label is an ER routing label (for example, ER1). In certain embodiments, the label is a vacuole label (for example, V1). In certain embodiments, the label is a plasma membrane label (for example, P1). In certain embodiments, the label is a peroxisome targeting sequence (for example, PTS1). In some cases, the label includes or is derived from a transmembrane domain in a protein of a tail anchoring category. In some embodiments, the positioning label positions the enzyme outside the organelle. In certain embodiments, the positioning label positions the enzyme inside the organelle. In some embodiments, the positioning label positions the enzyme so that one or more parts of the enzyme are found inside and outside the organelle.

[0222] In some embodiments of the present invention, the host cell is modified by expressing one or more coding sequences encoding one or more enzymes comprising the above-mentioned positioning tags. In certain embodiments, the host cell is modified by expressing one or more heterologous coding sequences so that one or more enzymes are expressed in the cytosol. Examples of such enzymes include, but are not limited to, arginine decarboxylase, putrescine N-methyltransferase, pyrrolidone synthase, tropinone reductase, phenylpyruvate reductase, UDP-glucosyltransferase, and 2-oxoglutarate-dependent dioxygenases, such as scopolamine 6β-hydroxylase / dioxygenase. In certain embodiments, the host cell is modified by expressing one or more heterologous coding sequences so that one or more enzymes are expressed in the ER membrane. Examples of such enzymes include, but are not limited to, cytochrome P450, such as tropinone synthase (CYP82M3) and ritolin mutase (CYP80F1), and NADP +-Cytochrome P450 reductase. In certain embodiments, the host cell is modified by expressing one or more heterologous coding sequences so that one or more enzymes are expressed in the mitochondria. Examples of such enzymes include, but are not limited to, N-acetylglutamate synthase. In other embodiments, the host cell is modified by expressing one or more heterologous coding sequences so 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 expressing one or more heterologous coding sequences so that one or more enzymes are expressed in the vacuole cavity. Examples of such enzymes include, but are not limited to, serine carboxypeptidase-like acyltransferases, such as ritolin synthase, and engineered variants thereof. In other embodiments, the host cell is modified by expressing one or more heterologous coding sequences so that one or more enzymes or proteins are expressed in the vacuole membrane. Examples of such proteins include, but are not limited to, multidrug and toxin efflux transporters, nitrate / peptide family transporters, and ATP binding cassette transporters. In other embodiments, the host cell is modified by expressing one or more heterologous coding sequences so that one or more enzymes or proteins are expressed in the plasma membrane. Examples of such proteins include, but are not limited to, ATP-binding cassette transporters, pleiotropic resistance transporters, and multidrug resistance transporters.

[0223] In some cases, expression of each type of enzyme is increased by additional gene copies (i.e., multiple copies), which increases intermediate accumulation and / or the production of the TA of interest. Embodiments of the present invention include increasing the production of the TA of interest in a host cell by simultaneously expressing multiple species variants of a single or multiple enzymes. In some cases, additional gene copies of a single or multiple enzymes are included in the host cell. Any convenient method can be used, including multiple copies of the heterologous coding sequence of the enzyme in the host cell.

[0224] In some embodiments, host cell comprises multiple copies of the heterologous coding sequence of enzyme, such as 2 or more, 3 or more, 4 or more, 5 or more, or even 10 or more copies.In certain embodiments, host cell comprises multiple copies of the heterologous coding sequence of 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 enzyme derive from two or more source organisms different from host cell.For example, host cell can comprise multiple copies of a heterologous coding sequence, wherein each copy derives from different source organisms.Therefore, based on the interspecies difference of the interested enzyme encoded by the heterologous coding sequence, each copy can comprise some variations in exogenous sequence.

[0225] 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 species, Wicki fluorescens, Aequorea species, Lentinula species, Arabidopsis thaliana, Avena, Solanum lycopersicum, Solanum tuberosum, Nicotiana tabacum, Nicotiana benthamiana, Solanum belladonna, Hyoscyamus niger, Hyoscyamus muticus, Datura stramonium, Datura metel, Datura innoxia, Duboisia myoporoides, Anisodus luridus, Anisodus tanguticus, Brugmansia truncatula, In some cases, the heterologous coding sequence is from a source organism selected from Solanum spp., Nicotiana spp., Atropa spp., Hyoscyamus spp., Datura spp., Duboisia spp., Anisodus spp., Brugmansia spp., Erythroxylum spp., or Cochlearia spp. In some embodiments, the host cell includes a heterologous coding sequence from one or more source organisms described in Table 1.

[0226] The engineered host cell culture medium can be sampled and the production of the TA of interest can be monitored. Any convenient method can be used to observe and measure the TA of interest. Methods of interest include, but are not limited to, LC-MS methods (e.g., as described herein), in which samples of interest are analyzed by comparison with known amounts of standard compounds. Identity can be confirmed, for example, by m / z and MS / MS fragmentation patterns, and quantification or measurement of compounds can be achieved by LC trace peaks of known retention times and / or EIC MS peak analysis with reference to corresponding LC-MS analysis of known amounts of compound standards.

[0227] method

[0228] Process Steps

[0229] As described above, aspects of the invention include methods for preparing a tropane alkaloid (TA) of interest. Accordingly, aspects of the invention include culturing a host cell under conditions under which one or more host cell modifications (e.g., as described herein) are functionally expressed such that the cell converts a starting compound of interest into a product TA or a precursor thereof (e.g., pre-esterified TA). Methods are also provided, which include culturing a host cell under conditions suitable for protein production such that one or more heterologous coding sequences are functionally expressed and convert the starting compound of interest into a product TA of interest. In some cases, the method is a method for 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 recovering TA from the cell culture. In some embodiments of the method, the starting compound, TA product, and host cell are described by one of the entries in Table 1.

[0230] The fermentation medium may include a suitable carbon substrate. Carbon sources suitable for performing the disclosed method may include a wide variety of carbon-containing substrates. Suitable substrates may include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose) or combinations thereof. In some cases, unpurified mixtures (e.g., corn syrup, beet molasses, barley malt) from renewable raw materials may be used. In some cases, the carbon substrate may be a one-carbon substrate (e.g., methanol, carbon dioxide) or a two-carbon substrate (e.g., ethanol). In other cases, other carbon-containing compounds may be used, such as methylamine, glucosamine and amino acids.

[0231] Any convenient method of culturing host cells can be used to produce TA precursors and downstream TAs of interest. The specific scheme adopted may vary, for example, depending on the host cell, the heterologous coding sequence, the desired TA precursor and the downstream TA of interest, etc. The cell may be present in any convenient environment, such as an environment in which the cell is capable of expressing one or more functional heterologous enzymes. In vitro, as used herein, refers only to the outside of living cells, regardless of the location of the cell. As used herein, the term in vivo means inside living cells, regardless of the location of the cell. In some embodiments, the cell is cultured under conditions conducive to enzyme expression and with appropriate substrates that are available to allow the production of TA precursors and downstream TAs of interest in vivo. In some embodiments, functional enzymes are extracted from the host to produce TA under in vitro conditions. In some cases, the host cell is placed back into a multicellular host organism. The host cell is in any growth phase, including but not limited to the stationary phase and the logarithmic growth phase, etc. In addition, the culture itself can be a continuous culture or they can be a batch culture.

[0232] Cells can be grown at a temperature between 20-40° C. in an appropriate fermentation medium. Cells can be grown under shaking at any convenient speed (e.g., 200 rpm). Cells can be grown at a suitable pH. The pH range suitable for fermentation can be between pH 5-9. Fermentation can be carried out under aerobic, anaerobic or microaerobic conditions. Any suitable growth medium can be used. Suitable growth medium can include, but is not limited to, common commercial preparation mediums, such as synthetic defined (SD) minimal medium or yeast extract peptone dextrose (YEPD) enriched medium. Any other enriched, defined or synthetic growth medium suitable for microorganisms can be used.

[0233] Cells can be cultured in containers of substantially any size and shape. Examples of containers suitable for performing the methods of the present disclosure may include, but are not limited to, multi-well shake plates, test tubes, flasks (with and without baffles), and bioreactors. The volume of the culture medium may range from 10 microliters to greater than 10,000 liters.

[0234] This may include adding to the growth medium an agent known to modulate metabolism in a manner desired for the production of alkaloids. In one non-limiting example, cyclic adenosine 2'3'-monophosphate may be added to the growth medium to modulate catabolite repression.

[0235] Any convenient cell culture conditions for a particular cell type can be utilized. In certain embodiments, host cells comprising one or more modifications are cultivated with standard cell culture media and supplements under standard or easily optimized conditions. For example, when the selective pressure for plasmid maintenance is not required, standard growth medium can contain 20g / L yeast extract, 10g / L peptone and 20g / L dextrose (YPD). Host cells containing plasmids grow in synthetic complete (SC) culture media, and the culture media contains 1.7g / L yeast nitrogen base, 5g / L ammonium sulfate and 20g / L dextrose, and are supplemented with growth and the appropriate amino acids required for selection. Alternative carbon sources that can be used for inducible enzyme expression include but are not limited to sucrose, raffinose and galactose. In the laboratory, cells are grown in containers at any convenient temperature (for example, 30°C) under the vibration of any convenient rate (for example, 200rpm), and the container, for example, a volume range of 1-1000mL or a larger test tube or flask.

[0236] For example, as part of an industrial process, the culture volume can be expanded to grow in a larger fermentation vessel. Industrial fermentation processes can be carried out under closed batch, fed-batch or continuous chemostat conditions or any suitable fermentation mode. In some cases, cells can be fixed on a substrate as a whole cell catalyst and subjected to fermentation conditions to produce alkaloids.

[0237] Batch fermentation is a closed system, wherein the composition of the substratum is set at the beginning of fermentation and does not change during the fermentation process. At the beginning of fermentation, one or more desired organisms are inoculated into the substratum. In some cases, batch fermentation is operated under the situation that changes are made to the system to control factors such as pH and oxygen concentration (but not carbon). In the fermentation system of this type, the biomass and metabolite composition of the system are constantly changing during the fermentation process. Cells usually experience a hysteresis phase, then enter the logarithmic phase (high growth rate), then enter the stationary phase (growth rate reduces or stops), and finally enter the death phase (if not treated).

[0238] Fed-batch fermentation is similar to batch fermentation, except that substrate is added to the system at intervals during the fermentation process. Fed-batch systems are used to reduce the effects of catabolite repression on host cell metabolism, and in other situations where it is desirable to have limited amounts of substrate in the growth medium.

[0239] Continuous fermentation is an open system in which a defined fermentation medium is continuously added to a bioreactor and an equal amount of fermentation medium is continuously removed from the container for processing. Continuous fermentation systems are usually operated to maintain steady-state growth conditions so that cell losses due to removal of the medium must be balanced by the growth rate in the fermentation. Continuous fermentation is usually operated under conditions where the cells are at a constant high cell density. Continuous fermentation allows for the regulation of one or more factors that affect target product concentration and / or cell growth.

[0240] Liquid culture media may include, but are not limited to, enriched or synthetic defined media with the above-mentioned additive components. The culture media components may be dissolved in water and sterilized by heating, pressurization, filtration, radiation, chemicals, or any combination thereof. Several culture media components may be prepared and sterilized separately and then compounded in a fermentation vessel. The culture media may be buffered to help maintain a constant pH throughout the fermentation process.

[0241] Process parameters including temperature, dissolved oxygen, pH, agitation, aeration rate, and cell density can be monitored or controlled during the fermentation process. For example, the temperature of the fermentation process can be monitored by a temperature probe immersed in the culture medium. The culture temperature can be controlled at a set point by adjusting the jacket temperature. Water can be cooled in an external cooler and then flowed into the bioreactor control tower and circulated to the jacket at a temperature required to maintain the set point temperature in the vessel.

[0242] In addition, gas flow parameters can be monitored during the fermentation process. For example, the gas can flow into the culture medium through a sparger. Gases suitable for the method of the present disclosure may include compressed air, oxygen and nitrogen. The gas flow may be at a fixed rate or adjusted to maintain a dissolved oxygen set point.

[0243] The pH of the culture medium can also be monitored. In an example, the pH can be monitored by a pH probe immersed in the culture medium inside the container. If the pH control is effective, the pH can be adjusted by an acid pump and an alkali pump, which add each solution to the culture medium at a desired rate. The acid solution used to control the pH can be sulfuric acid or hydrochloric acid. The alkali solution used to control the pH can be sodium hydroxide, potassium hydroxide or ammonium hydroxide.

[0244] In addition, the dissolved oxygen in the culture medium can be monitored by a dissolved oxygen probe immersed in the culture medium. If dissolved oxygen regulation is effective, the oxygen content can be adjusted by increasing or decreasing the stirring speed. The dissolved oxygen content can also be adjusted by increasing or decreasing the gas flow rate. The gas can be compressed air, oxygen or nitrogen.

[0245] The agitation speed can also be monitored during the fermentation process. In an example, a stirrer motor can drive an agitator. The agitator speed can be set to a consistent rpm throughout the fermentation process, or can be dynamically adjusted to maintain a set dissolved oxygen content.

[0246] In addition, turbidity can be monitored during fermentation. In an example, a turbidity probe can be used to measure cell density. Alternatively, cell density can be measured by sampling from a bioreactor and analyzing it in a spectrophotometer. In addition, samples can be taken out from a bioreactor at time intervals by a sterile sampling device. The alkaloids produced by the host cells in the sample can be analyzed. The consumption of other metabolites and sugars, culture medium components, or the density of the cell can also be analyzed.

[0247] In another example, feedstock parameters can be monitored during the fermentation process. Specifically, feedstocks including sugars and other carbon sources, nutrients, and cofactors can be added to the fermentation using an external pump. Other components can also be added during the fermentation process, including but not limited to defoamers, salts, chelating agents, surfactants, and organic liquids.

[0248] Any convenient codon optimization technique for optimizing expression of a heterologous polynucleotide in a host cell may be adapted for use with the subject host cells and methods, see, for example, Gustafsson, C. et al. (2004) Trends Biotechnol, 22, 346-353, the entire contents of which are incorporated by reference.

[0249] The subject method can also include adding a starting compound to the cell culture. Any convenient addition method can be applicable to the subject method. The cell culture can be supplemented with a sufficient amount of the starting material of interest (e.g., as described herein), such as mM to μM amount, such as the starting compound between about 1-5mM. It should be understood that the amount of the starting material added, the time and rate of addition, the form of the material added, etc. can vary according to various factors. The starting material can be added purely or dissolved in a suitable solvent (e.g., cell culture medium, water or organic solvent) in advance. The starting material can be added in a concentrated form (e.g., more than 10x of the desired concentration) to minimize the dilution of the cell culture medium after addition. The starting material can be added in one or more batches or by continuously adding over an extended period of time (e.g., hours or days).

[0250] Method for separating products from fermentation medium

[0251] The subject method may also include recovering the TA of interest from the cell culture. Any convenient separation and isolation methods (e.g., chromatography or precipitation) may be applicable to the subject method to recover the TA of interest from the cell culture. Filtration methods can be used to separate the soluble fraction of the cell culture from the insoluble fraction. 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.

[0252] The alkaloid produced can be separated from the fermentation medium using methods known in the art. Multiple recovery steps can be performed immediately after fermentation (or in some cases, during fermentation) to initially recover the desired product. Through these steps, the alkaloid (e.g., TA) can be separated from cells, cell debris and waste, and other nutrients, sugars and organic molecules can be retained in the spent medium. This process can be used to obtain a product rich in TA.

[0253] In one example, a product stream having a tropane alkaloid (TA) product is formed by providing engineered yeast cells and raw materials including nutrients and water to a batch reactor. The engineered yeast cells may have at least one modification selected from the group consisting of: a feedback inhibition relief mutation in a cell-intrinsic biosynthetic enzyme gene; a transcriptional regulation modification of a cell-intrinsic biosynthetic enzyme gene; and an inactivation mutation in a cell-intrinsic enzyme. When the engineered yeast cells are in a batch reactor, the engineered yeast cells may be fermented. Specifically, the engineered yeast cells may be fermented by incubating the engineered yeast cells for a period of at least about 5 minutes to produce a solution comprising a TA product and a cell material. Once the engineered yeast cells have been fermented, at least one separation unit may be used to separate the TA product from the cell material to provide a product stream comprising a TA product. Specifically, the product stream may include a TA product and additional components, such as a clarified yeast culture medium. In addition, the TA product may include one or more TAs of interest, such as one or more TA compounds.

[0254] Different methods can be used to remove cells from a bioreactor culture medium including a TA of interest. In an example, cells can be removed by sedimentation over time. This sedimentation process can be accelerated by cooling or by adding a fining agent (finingagent) (such as silicon dioxide). The spent culture medium can then be siphoned from the top of the reactor, or the cells can be decanted from the bottom of the reactor. Alternatively, cells can be removed by filtering using a filter, membrane or other porous material. Cells can also be removed by centrifugation, for example, by continuous flow centrifugation or by using a continuous extractor.

[0255] If some valuable TA of interest is present inside the cell, the cell may be permeabilized or lysed, and cell debris may be removed by any of the above methods. Agents used to permeabilize the cell may include, but are not limited to, organic solvents (e.g., DMSO) or salts (e.g., lithium acetate). Methods for lysing the cell may include the addition of a surfactant such as sodium dodecyl sulfate, or mechanical disruption by bead milling or ultrasound.

[0256] The TA of interest can be extracted from the clarified spent culture medium by liquid-liquid extraction by adding an organic liquid that is immiscible with the aqueous culture medium. Examples of suitable organic liquids include, but are not limited to, isopropyl myristate, ethyl acetate, chloroform, butyl acetate, methyl isobutyl ketone, methyl oleate, toluene, oleyl alcohol, ethyl butyrate. The organic liquid can be added to as little as 10% or as much as 100% of the volume of the aqueous culture medium.

[0257] In some cases, the organic liquid can be added at the beginning of the fermentation or at any time during the fermentation. This extractive fermentation process can increase the yield of the TA of interest from the host cells by continuously removing TA precursors or TA into the organic phase.

[0258] Agitation may cause the organic phase to form an emulsion with the aqueous medium. Methods for promoting the separation of the two phases into different layers may include, but are not limited to, adding demulsifiers or nucleating agents, or adjusting pH. The emulsion may also be centrifuged to separate the two phases, for example, by continuous conical plate centrifugation.

[0259] Alternatively, the organic phase may be separated from the aqueous medium for physical removal following extraction. For example, the solvent may be encapsulated in a membrane.

[0260] In an example, an adsorption method can be used to extract the TA of interest from the fermentation medium. Specifically, the TA of interest can be extracted by adding a resin such as XAD4 or other agents that remove TAs by adsorption extract the TA of interest from the clarified spent culture medium. An organic solvent may then be used to release the TA of interest from the resin. Examples of suitable organic solvents include, but are not limited to, methanol, ethanol, ethyl acetate, or acetone.

[0261] Filtration can also be used to extract the TA of interest from the fermentation medium. At high pH, ​​the TA of interest may form a crystalline precipitate in the bioreactor. This precipitate can be directly removed by filtration using a filter, membrane or other porous material. The precipitate can also be collected by centrifugation and / or decantation.

[0262] The above extraction methods can be performed in situ (in the bioreactor) or ex situ (e.g., in an external loop through which the culture medium flows out of the bioreactor and contacts the extractant and then is recirculated back into the vessel). Alternatively, the extraction method can be performed using clarified culture medium removed from the bioreactor vessel after fermentation has terminated.

[0263] Process for purifying products from alkaloid-rich solutions

[0264] Subsequent purification steps may involve treating the post-fermentation TA precursor or TA-rich product using methods known in the art to recover the respective product species of interest in high purity.

[0265] In one example, the TA precursor or TA extracted in the organic phase can be transferred to an aqueous solution. In some cases, the organic solvent can be evaporated by heating and / or vacuum, and the resulting powder can be dissolved in an aqueous solution of a suitable pH. In another example, the TA precursor or TA can be extracted from the organic phase by adding an aqueous solution of a suitable pH that promotes the extraction of the TA precursor or TA into the aqueous phase. The aqueous phase can then be removed by decantation, centrifugation, or another method.

[0266] The solution containing the TA precursor or TA can be further treated to remove metals, for example, by treatment with a suitable chelating agent. The solution containing the TA precursor or TA can be further treated by precipitation to remove other impurities, such as proteins and DNA. In one example, the solution containing the TA precursor or TA is treated with a suitable precipitant such as ethanol, methanol, acetone or isopropanol. In an alternative example, DNA and proteins can be removed by dialysis or by other size exclusion methods that separate smaller alkaloids from contaminating biomacromolecules.

[0267] In further examples, a solution containing a TA precursor, TA or modified TA can be extracted to high purity by continuous cross-flow filtration using methods known in the art.

[0268] If the solution contains a mixture of TA precursors or TAs, it can be subjected to acid-base treatment using methods known in the art to obtain a single TA species of interest. In this process, the pH of the aqueous solution is adjusted to precipitate a single TA precursor or TA at its respective pKa.

[0269] For high purity, small-scale preparations, TA precursor or TA can be purified in a single step by liquid chromatography.

[0270] Yeast-derived alkaloid APIs versus plant-derived APIs

[0271] Clarified yeast culture medium (CYCM) may contain a variety of impurities. Clarified yeast culture medium can be dehydrated by vacuum and / or heating to produce an alkaloid-rich powder. This product is similar to the concentrate of Solanum leaf (CNL), which is used by active pharmaceutical ingredient (API) manufacturers to extract tropane alkaloids to be further chemically processed and purified. For the purposes of the present invention, CNL is a representative example of any type of purified plant extract, from which one or more desired alkaloid products can be further purified. Table 5 highlights the impurities in these two products, which can be unique to CYCM or CNL, or can be present in both. By analyzing products of unknown origin for a subset of these impurities, one skilled in the art can determine whether the product is derived from yeast or a plant production host.

[0272] API-grade pharmaceutical ingredients are highly purified molecules. Therefore, impurities (such as those listed in Tables 2 and 3) of plant or yeast origin that can indicate API may not exist at the API stage of the product. In fact, many API products derived from yeast strains of the present invention may be indistinguishable from traditional plant-derived APIs to a large extent. However, in some cases, conventional alkaloid compounds can be chemically modified using chemical synthesis methods, which can be displayed as chemical impurities in plant-based products that require such chemical modifications. For example, chemical derivatization can generally lead to a group of impurities associated with chemical synthesis processes. In some cases, these modifications can be carried out biologically in yeast production platforms, thereby avoiding some impurities associated with chemical derivatization from being present in yeast-derived products. Specifically, these impurities from chemically derived products may be present in API products produced using chemical synthesis processes, but may not be present in API products produced using yeast-derived products. Alternatively, if yeast-derived products are mixed with chemically derived products, the resulting impurities may be present, but the amount is lower than the amount expected in the API that only or mainly comprises chemically derived products. In this example, by analyzing the API product for a subset of these impurities, one skilled in the art can determine whether the product is derived from a yeast production host or a traditional chemical derivatization route.

[0273] Non-limiting examples of impurities that may be present in chemically derived tropane alkaloid APIs but not in biosynthesized APIs include hydrogen halides such as hydrogen chloride, hydrogen iodide, and hydrogen bromide formed by chemical N-alkylation (such as N-methylation and N-butylation) of hyoscyamine and scopolamine.

[0274] However, in the case where both the yeast-derived compound and the plant-derived compound are chemically modified by chemical synthesis methods, the presence of the same impurities associated with the chemical synthesis process can be expected in the product. In this case, the starting material (e.g., CYCM or CNL) can be analyzed as described above.

[0275] Methods for engineering host cells

[0276] Also included are methods for engineering host cells for producing a TA of interest or a precursor thereof. Insertion of DNA into a host cell can be accomplished using any convenient method. The method is used to insert a heterologous coding sequence into a host cell such that the host cell functionally expresses the enzyme and converts the starting compound of interest into the product TA of interest.

[0277] Any convenient promoter can be used in the subject host cells and methods. The promoter driving the expression of the heterologous coding sequence can be a constitutive promoter or an inducible promoter, provided that the promoter is active in the host cell. The heterologous coding sequence can be expressed by its native promoter, or a non-natural promoter can be used. Such promoters can have low to high strength in the host using them. The promoter can be regulated or constitutive. In certain embodiments, a promoter that is not inhibited by glucose or is only slightly inhibited by the presence of glucose in the culture medium is used. Promoters of interest include, but are not limited to, promoters of glycolytic genes, such as the promoter of the Bacillus subtilis tsr gene (the promoter region of the fructose bisphosphate aldolase gene encoding) or the promoter from the Saccharomyces cerevisiae gene encoding glyceraldehyde 3-phosphate dehydrogenase (GPD, GAPDH or TDH3), the ADH1 promoter of baker's yeast, phosphate starvation-inducible promoters such as the PHO5 promoter of yeast, the alkaline phosphatase promoter from Bacillus licheniformis, yeast inducible promoters such as Gal1-10, Gal1, GalL, GalS, the repressible promoters Met25, tetO, and constitutive promoters such as the glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), the alcohol dehydrogenase promoter (ADH), the translation elongation factor-1-alpha promoter (TEF), the cytochrome c-oxidase promoter (CYC1), the MRP7 promoter, phosphoglycerate kinase (PGK), triosephosphate isomerase (TPI), and the like. Also can use the self-replicating yeast expression vector containing the promoter that can be induced by hormones such as glucocorticoid, steroid and thyroid hormone and include but not limited to glucocorticoid response element (GRE) and thyroid hormone response element (TRE).These and other examples are described in U.S. Patent No. 7,045,290, which is incorporated by reference, including the references cited therein.Other carriers containing constitutive or inducible promoters such as α factor, alcohol oxidase and PGH can be used.In addition, any promoter / enhancer combination (according to the eukaryotic promoter database EPDB can also be used to drive the expression of gene.Any convenient appropriate promoter can be selected for host cell, such as Escherichia coli.Promoter selection can also be used to optimize transcription, and therefore optimize enzyme level, so that output is maximized, while minimizing energy resources.

[0278] Any convenient vector can be used in the subject host cells and methods. Interested vectors include vectors for yeast and other cells. The types of yeast vectors can be divided into 4 major categories: integrating vectors (YIp), autonomously replicating high copy number vectors (YEp or 2μ plasmids), autonomously replicating low copy number vectors (YCp or centromeric plasmids) and vectors for cloning large fragments (YAC). The vector DNA is introduced into prokaryotic or eukaryotic cells by any convenient transformation or transfection technique.

[0279] Practicality

[0280] The host cells and methods of the invention, for example, as described above, can be used in a variety of applications. Applications of interest include, but are not limited to, research applications and therapeutic applications. The methods of the invention can be used in a variety of different applications, including any convenient application in which the production of TA is of interest.

[0281] The subject host cells and methods can be used for a variety of therapeutic applications. Therapeutic applications of interest include those applications of interest in preparing pharmaceutical products including TAs. The host cells described herein produce tropane alkaloid precursors (TA precursors) and TAs of interest. Tropinone and tropine are major branch point intermediates of interest in TA synthesis, including engineering efforts to produce end products such as pharmaceutical TA products. The subject host cells can be used to produce TA precursors from simple and inexpensive starting materials, which can be used to produce TAs of interest, including tropinone, tropine, and TA end products. Therefore, the subject host cells can be used to supply therapeutically active TAs or their precursors.

[0282] In some cases, the host cells and methods can be used to produce commercial-scale quantities of TAs or their precursors, where chemical synthesis of these compounds has low yields and is not a feasible means of large-scale production. In some cases, the host cells and methods are used in fermentation facilities that will include, for example, bioreactors (fermentors) of 5,000-200,000 liter capacity, allowing rapid production of the TA of interest or its precursor for therapeutic products. Such applications may include industrial-scale production of the TA of interest from fermentable carbon sources such as cellulose, starch, and free sugars.

[0283] The subject host cells and methods can be used for a variety of research applications. The subject host cells and methods can be used to analyze the effects of various enzymes on the biosynthetic pathways of various TAs or their precursors of interest. In addition, the host cells can be engineered to produce TAs or their precursors, which can be used to test the biological activity of interest in terms of unproven therapeutic functions. In some cases, engineering the host cells to include a variety of heterologous coding sequences encoding a variety of enzymes illustrates a high-yield biosynthetic pathway toward the TA of interest or its precursor. In some cases, research applications include the production of precursors of therapeutic molecules of interest, which can then be further chemically modified or derivatized to a desired product, or used to screen for increased therapeutic activities of interest. In some cases, host cell strains are used to screen for enzyme activities of interest in such pathways, which can lead to the discovery of enzymes through the conversion of TA metabolites produced in these strains.

[0284] The subject host cell and method can be used as a production platform for plant-specific metabolites. The subject host cell and method can be used as a platform for drug library development and plant enzyme discovery. For example, the subject host cell and method can be used for obtaining a yeast strain that produces a scaffold molecule of interest (such as hyoscyamine and scopolamine), and further functionalizing the compound structure by combinatorial biosynthesis or chemical means to develop a drug library based on natural products. By producing a drug library in this way, any potential drug hit has been associated with a production host suitable for large-scale cultivation and production. As another example, these subject host cells and methods can be used for plant enzyme discovery. The subject host cell provides a clean background for limiting metabolites, to express a plant-expressed sequence tag (EST) library, to identify new enzyme activity. The subject host cell and method provide expression methods and culture conditions for functional expression and activity increase of plant enzymes in yeast.

[0285] Kits and Systems

[0286] Aspects of the invention also include kits and systems, wherein the kits and systems may include one or more components used in the methods of the invention, such as host cells, starting compounds, heterologous coding sequences, vectors, culture media, etc., as described herein. In some embodiments, the subject kits include host cells (e.g., as described herein) and one or more components selected from the following: starting compounds, heterologous coding sequences and / or vectors comprising said heterologous coding sequences, vectors, growth materials, components suitable for expression systems (e.g., cells, cloning vectors, multiple cloning sites (MCS), bidirectional promoters, internal ribosome entry sites (IRES), etc.) and culture media.

[0287] Any component described herein can be provided in a kit, for example, including one or more modified host cells, starting compounds, culture media, etc. Various components suitable for preparing and using heterologous coding sequences, cloning vectors, and expression systems can be used in the subject kit. The kit can also include tubes, buffers, etc., and instructions for use. As needed, the various reagent components of the kit can be present in separate containers, or some or all of them can be pre-mixed into a reagent mixture in a single container.

[0288] Also provided are systems for producing a TA of interest, wherein the systems may include engineered host cells comprising one or more modifications (e.g., as described herein), starting compounds, culture media, fermentors, and fermentation equipment, such as devices suitable for maintaining growth conditions for the host cells, sampling and monitoring equipment and components, etc. A variety of components suitable for large-scale fermentation of yeast cells can be used in the subject systems.

[0289] In some cases, the system includes large-scale fermentation of engineered host cells, and monitoring and purification components of TA compounds produced by fermented host cells. In certain embodiments, the engineered host cells in the fermentor are added to the system under conditions where one or more desired TA products or precursors thereof are produced. In some cases, the host cells produce TAs of interest (e.g., as described herein). In some cases, the TA products of interest are pharmaceutical TA products, such as hyoscyamine, N-methylhyoscyamine, anisodamine, scopolamine, N-methylscopolamine, and N-butylscopolamine.

[0290] In some cases, the system includes a device for monitoring and or analyzing one or more TA compounds or precursors thereof produced by the subject host cell. For example, an LC-MS analysis system, a chromatography system, or any convenient system in which samples can be analyzed and compared with, for example, a standard as described herein. The fermentation medium can be monitored by sampling and analysis at any convenient time before and during fermentation. When the conversion of the starting compound to the TA product or precursor of interest is completed, the fermentation can be stopped and the purification of the TA product can be performed. Therefore, in some cases, the subject system includes a purification component suitable for purifying the TA product or precursor of interest from the host cell culture medium that produces the TA product or precursor of interest. The purification component may include any convenient means that can be used to purify the fermented TA product or precursor, 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, after one or more starting compounds are input into the system, the subject system provides the production and separation of the TA fermentation product of interest.

[0291] The following examples are given to provide a complete disclosure and description of how to prepare and use the present invention for those of ordinary skill in the art, and are neither intended to limit the scope of their invention as the inventors believe, nor to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numerals (e.g., amounts, temperatures, etc.) used, but some experimental errors and deviations should be considered. Unless otherwise stated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0292] Exemplary Methods

[0293] The following section provides examples of methods and procedures that can be used to construct, culture and test microbial strains (such as yeast strains) for producing TA precursors and TAs, and to perform fermentation of such strains to produce TA precursors and TAs. Also included are examples of methods, procedures and materials that can be used to generate the DNA sequences required for modification of microbial hosts and to introduce the desired DNA sequences into microbial hosts.

[0294] Chemical compounds and standards. Chemical standards for TA precursors and TA used to verify the identity and quantify metabolites produced by engineered host cells can be purchased from commercial suppliers. For example, putrescine dihydrochloride, N-methylputrescine, scopolamine, tropinone, and tropine can be purchased from Santa Cruz Biotechnology (Dallas, TX). 4-(Methylamino)butyric acid hydrochloride can be purchased from Sigma (St. Louis, MO). γ-Methylaminobutyraldehyde (4MAB) diethyl acetal and ritolin can be purchased from Toronto Research Chemicals (Toronto, ON). Chemical standards of NMPy can be synthesized by deprotecting one volume of diethyl acetal with five volumes of 2 M HCl at 60°C for 30 minutes as described previously (see Feth, F., Wray, V., and Wagner, KG Determination of methylputrescine oxidase by high performance liquid chromatography. Phytochemistry 24, 1653–1655 (1985)), incubating at room temperature overnight, and then washing the resulting concentrate twice with three volumes of diethyl ether to remove residual organic impurities.

[0295] Plasmid construction. Oligonucleotides for generating new DNA sequences and DNA sequencing by polymerase chain reaction (PCR) can be obtained from DNA synthesis companies, such as IDT DNA, Twist Bioscience or Stanford Protein and Nucleic Acid Facility (Stanford, CA). Natural yeast genes can be amplified by colony PCR from Saccharomyces cerevisiae genomic DNA (see Kwiatkowski, TJ, Zoghbi, HY, Ledbetter, SA, Ellison, KA and Chinault, AC Rapid identification of yeast artificial chromosome clones by matrix pooling and crude iysate PCR. Nucleic Acids Res. 18, 7191 (1990)). Suitable codon optimization software, such as GeneArt GeneOptimizer software (Thermo Fisher Scientific), can be used to codon optimize the gene sequence of heterologous enzymes to improve expression in Saccharomyces cerevisiae. The heterologous gene sequence can then be synthesized into linear double-stranded DNA fragments by commercial DNA synthesis companies. Two types of plasmids can be used for gene expression in yeast: direct expression (DE) plasmids for testing biosynthetic genes of interest and yeast integration (YI) maintenance plasmids that provide a template for genomic integration of a selected promoter-gene-terminator cassette.

[0296] DE plasmids contain a gene of interest flanked by a constitutive promoter and terminator, a low copy CEN6 / ARS4 yeast origin of replication, and an auxotrophic selection marker. DE plasmids can be constructed by PCR amplifying the gene of interest to attach 5' and 3' restriction sites using primer overhangs, digesting the PCR product or synthetic gene fragment with an appropriate restriction enzyme pair (e.g., SpeI, BamHI, EcoRI, PstI, or XhoI), and then ligating the gene fragment into a similarly digested vector with a suitable yeast promoter, terminator, and replication sequence using T4 DNA ligase, such as plasmids pAG414GPD-ccdB, pAG415GPD-ccdB, or pAG416GPD-ccdB (see Alberti, S., Gitler, AD, and Lindquist, SA suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast 24, 913–9 (2007)).

[0297] The YI plasmid comprises a gene of interest flanked by a constitutive promoter and terminator, but lacks a yeast origin of replication or an auxotrophic selection marker. The YI plasmid can be constructed by utilizing a primer designed to be incorporated into the 3' and 5' ends of the promoter and terminator, using 'around-the-horn' PCR to linearize the empty carrier with a suitable promoter and terminator. PCR amplification can also be performed on the gene of interest, with 5' and 3' overhangs with 35-40bp homology attached to the end of the linearized carrier backbone. The Gibson assembly can then be used to assemble the gene into the YI vector. The DE plasmid expressing the GFP fusion of a biosynthetic enzyme can be prepared in the following manner: first, the PCR amplified DNA fragments encoding GFP, target enzyme and the YI carrier backbone are assembled, and then the fusion construct from the YI plasmid is subcloned into the DE vector using restriction enzymes and ligation cloning as described.

[0298] PCR amplification can be performed using any high-fidelity recombinant DNA polymerase available from commercial suppliers, and linear DNA can be purified using a suitable DNA column purification kit. In Luria-Bertani (LB) liquid medium or LB agar plates with carbenicillin (100 μg / mL), kanamycin (50 μg / mL) or another antibiotic selection, heat shock transformation and selection can be used to breed assembled plasmids in any chemically competent E. coli strain. Plasmid DNA can be isolated from overnight E. coli cultures grown at 37 ° C and 250 rpm in selective LB medium using plasmid purification columns by alkaline lysis according to the manufacturer's protocol. The plasmid sequence should be verified by Sanger sequencing.

[0299] Yeast strain construction. Any suitable laboratory yeast strain can be used as a host organism. The yeast strains described in the examples of the experimental section are derived from the parent strain CEN.PK2-1D (see Entian, KD and P.25Yeast Genetic Strain and Plasmid Collections.Methods Microbiol.36,629–666(2007)), referred to as CEN.PK2. The strain can be grown non-selectively in yeast-peptone medium (YPD medium) supplemented with 2% w / v dextrose, yeast nitrogen base (YNB) defined medium supplemented with synthetic complete amino acid mixture (YNB-SC) and 2% w / v dextrose, or on agar plates of the above medium. 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 appropriate dropout solutions (YNB-DO) or on YNB-DO agar plates. Yeast strains deficient in acetic acid metabolism can grow on the above medium (i.e., YPAD or YNBA) supplemented with 0.1% w / v potassium acetate.

[0300] Yeast genome modification can be performed using the CRISPRm method (see Ryan, OW et al. Selection of chromosomal DNA libraries using a multiplex CRISPR system. Elife 3, 1–15 (2014)). The CRISPRm plasmid expresses Streptococcus pyogenes Cas9 and a single guide RNA (sgRNA) targeting a locus of interest in the yeast genome, and can be constructed by assembly PCR and Gibson assembly of DNA fragments encoding Sp Cas9, tRNA promoters and HDV ribozymes (20-nt guide RNA sequences), as well as tracrRNA and terminators. For gene insertion, PCR amplification can be used to construct an integration fragment of a gene of interest containing one or more flanks with unique promoters and terminators, and cloned into a maintenance vector by Gibson assembly. The integration fragment is PCR amplified using a suitable high-fidelity DNA polymerase at the integration site using a 40bp microhomology region flanking adjacent fragments and / or the yeast genome. For gene disruption, the integration fragment contains 6-8 stop codons in all three reading frames, and its flank has 40bp microhomology with the disruption site, which is located in the first half of the open reading frame. For complete gene deletion, the integration fragment contains an auxotrophic marker gene, and its flank has 40bp microhomology with the deletion site. Each integration fragment is co-transformed with the CRISPRm plasmid targeting the desired genomic site. Positive integrants can be identified by yeast colony PCR, Sanger sequencing and / or by functional screening performed by liquid chromatography and tandem mass spectrometry (LC-MS / MS).

[0301] Yeast transformation. Yeast strains can be transformed using any suitable method, including heat shock, electroporation and chemical transformation. For example, the yeast strains described in the examples of the experimental section are 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 250rpm. Saturated cultures are back-diluted to between 1:10 and 1:50 in YP(A)D medium and grown for another 5-7 hours to reach the exponential phase. The culture is precipitated by centrifugation at 500 × g for 4 minutes, then washed twice by resuspending the precipitate in 50mM Tris-HCl buffer at pH 8.5. Each transformation resuspends the washed precipitate in 20 μL of EZ2 solution, then mixed with 100-600ng of total DNA and 200 μL of EZ3 solution. The yeast suspension is gently rotated and incubated at 30°C for 1 hour. For plasmid transformation, the transformed yeast was plated directly onto YNB(A)-DO agar plates. For Cas9-mediated chromosome modification, the yeast suspension was alternatively mixed with 1 mL of YP(A)D medium, precipitated by centrifugation at 500 × g for 4 minutes, and then resuspended in 250 μL of fresh YP(A)D medium. The suspension was then incubated at 30°C with gentle rotation for another two hours to produce G418 resistance protein, which was then plated on YP(A)D plates containing 400 mg / L G418 (Geneticin) sulfate. The plates were then incubated at 30°C for 48-60 hours to allow colonies to form.

[0302] Spot dilution assay. The strain was inoculated into YNB(A)-DO medium and grown overnight at 30°C and 250 rpm. The saturated overnight culture was pelleted by centrifugation at 500×g for 4 minutes and resuspended in sterile Tris-HCl buffer (pH 8.0) to an OD based on the OD value. 600 For 10 7 The concentration of cells / mL was then prepared. 10-fold serial dilutions of each strain were then prepared in Tris-HCl buffer and 10 μL of each dilution was spotted on pre-warmed YNB(A)-DO plates. The plates were incubated at 30°C and imaged after 48 hours.

[0303] Growth conditions for metabolite assays. Small-scale metabolite production tests can be performed in YNB(A)-SC or YNB(A)-DO medium. Yeast colonies can be inoculated into 300-500 μL of medium and grown in a shaker at 30°C, 460 rpm, and 80% relative humidity for 48-72 hours in a 2 mL deep-well 96-well plate covered with a gas-permeable membrane.

[0304] Metabolite production analysis. Liquid chromatography and tandem mass spectrometry (LC-MS / MS) can be used to analyze metabolite profiles and titers. In order to separate cells from the culture medium for analysis, the fermentation culture can be precipitated by centrifugation at 3,500 × g for 5 minutes at 12 ° C, and then aliquots of 100-200 μL supernatant can be taken out for direct analysis. The production of metabolites can be analyzed by LC-MS / MS using any suitable HPLC device (such as Agilent 1260 Infinity Binary HPLC and Agilent 6420 triple quadrupole mass spectrometer) paired with a triple quadrupole mass spectrometer. A C18 reverse phase column such as Zorbax Eclipse Plus C18 column (2.1x 50mm, 1.8μm; Agilent Technologies) can be used for chromatography, wherein 0.1% v / v formic acid aqueous solution is used as mobile phase solvent A and 0.1% v / v formic acid / acetonitrile is used as solvent B. The column was operated at 40°C with a constant flow rate of 0.4 mL / min and a sample injection volume of 5 μL. Compound separation was performed using the following gradient: 0.00-0.75 min, 1% B; 0.75-1.33 min, 1-25% B; 1.33-2.70 min, 25-40% B; 2.70-3.70 min, 40-60% B; 3.70-3.71 min, 60-95% B; 3.71-4.33 min, 95% B; 4.33-4.34 min, 95-1% B; 4.34-5.00 min, equilibrated with 1% B. The LC eluent was directed to the MS at 0.01-5 min, operated with electrospray ionization (ESI) in positive mode, source gas temperature 350°C, gas flow rate 11 L / min, and nebulizer pressure 40 psi. Metabolites can be quantified by integrating peak areas based on multiple reaction monitoring (MRM) parameters and standard curves.

[0305] Fluorescence microscopy. Individual colonies of yeast strains transformed with plasmids encoding biosynthetic enzymes fused to fluorescent protein reporter genes were inoculated into 1 mL of YNB-DO medium and allowed to grow overnight at 30°C and 250 rpm. Overnight cultures were pelleted by centrifugation at 500 × g for 4 minutes and resuspended in 2 mL of YNB-DO medium with 2% w / v dextrose, then grown for another 4-6 hours at 30°C and 250 rpm to reach exponential phase and allow the expressed fluorescent protein to fully fold. Approximately 5-10 μL of the culture was then spotted onto a glass microscope slide and covered with a glass coverslip and 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 by a CCD camera and subsequent image analysis can be performed in any suitable scientific image analysis software, such as ImageJ (NIH).

[0306] Identify new gene variants from the transcriptome database.Sequence alignment-based searches of transcriptome and genomic databases can be used to identify new genes and their variants.For example, the ortholog of N-methylputrescine oxidase (NtMPO1) of common tobacco was identified in the 1000Plants Project database using tBLASTn searches of the transcriptomes of datura and belladonna (see Matasci, N. et al. Data access for the 1,000Plants (1KP) project. Gigascience 3, 17 (2014)).Then the coding sequences of the putative genes identified using these search strategies can be optimized for yeast expression, and then cloned into expression vectors as previously described.

[0307] Enzyme structure analysis. By examining the homology model constructed using any suitable homology modeling or ab initio structure prediction software (such as RaptorX or Rosetta), the structural features of heterologous enzymes can be analyzed, which may be problematic during expression in yeast, such as large unstructured regions. The resulting protein model can be visualized using any three-dimensional molecular viewing software, such as PyMOL (Schrodinger) or UCSF Chimera. Any suitable ligand docking simulation software, such as AutoDock, SwissDock, GOLD or Glide, can be used to analyze the affinity of the enzyme for a specific substrate.

[0308] Protein expression in yeast was analyzed by Western blotting. For immunoblot analysis of yeast-expressed proteins, suitable strains were transformed with expression vectors containing proteins of interest with epitope tags. Three days after transformation, the transformed colonies were inoculated into 2 mL of YNB-DO medium and grown to stationary phase overnight (~16-20 h) at 30 ° C and 460 rpm. The cells were pelleted by centrifugation at 3,000 × g for 5 minutes, resuspended in 200 μL H2O, mixed with 200 μL of 0.2M NaOH, and incubated at room temperature for 5 minutes to allow cell wall glycoprotein hydrolysis. The cells were reprecipitated at 3,000 × g for 5 minutes, resuspended in 75 μL H2O, mixed with 25 μL of 4X NuPAGE LDS sample buffer (Thermo Fisher), and then boiled at 95 ° C for 3 minutes to lyse the cells. The suspension was precipitated by centrifugation at 16,000 × g for 5 minutes to remove insoluble debris, and the supernatant was transferred to a pre-cooled tube. For analysis under reducing conditions, protein lysates were mixed with β-mercaptoethanol (final concentration 10%) and incubated at 70°C for 10 minutes. Approximately 20-40 μg of total protein was loaded onto NuPAGE Bis-Tris 4-12% acrylamide gels (ThermoFisher) with Precision Plus DualColor protein molecular weight markers (BioRad). Electrophoresis was performed at 150V for 90 minutes in 1X NuPAGE MOPS SDS running buffer. Proteins were transferred to nitrocellulose membranes at 15V for 15 minutes using a Trans-Blot Semi-Dry device (BioRad) and NuPAGE transfer buffer (Thermo Fisher) according to the manufacturer's instructions. For reducing conditions, NuPAGE antioxidant (Thermo Fisher) was added to the running buffer and transfer buffer to a final concentration of 1X. The membrane with the transferred protein was washed in Tris-buffered saline with Tween (TBS-T; 137 mM NaCl, 2.7 mM KCl, 19 mM Tris base, 0.1% Tween 20, pH 7.4) for 5 minutes and then blocked with 5% skim milk in TBS-T for 1 h at room temperature. The membrane was incubated overnight at 4°C with appropriate dilutions of HRP-conjugated antibodies in TBS-T with 5% milk, washed 3 times with TBS-T for 5 minutes each, and then visualized using Western Pico PLUS HRP substrate (Thermo Fisher) and an appropriate imager.

[0309] experiment

[0310] A series of specific genetic modifications provide biosynthetic processes in Saccharomyces cerevisiae for producing TAs from simple, inexpensive raw materials or precursor molecules. Methods for constructing novel strains capable of producing early TA molecules putrescine, N-methylputrescine, 4-methylaminobutyraldehyde, N-methylpyrrolinium (NMPy), tropinone, tropine, phenyllactic acid (PLA) and 1-O-β-phenyllactyl glucose (PLA glucoside) from non-TA precursors or simple raw materials are described. NMPy is the natural precursor of all known TA molecules. Methods for manipulating the regulation of yeast biosynthetic pathways and optimizing the production of amino acid-derived TA precursors are also described. Methods for constructing novel strains capable of producing non-pharmaceutical TAs such as pseudotropine alkaloids and hyoscyamine 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 non-TA precursors or simple raw materials are also described. Furthermore, methods for constructing novel strains capable of producing non-natural TAs such as cinnamoyl tropine from non-TA precursors or simple feedstocks are described.

[0311] Example 1. Engineering a platform yeast strain for high-level putrescine production

[0312] The tropine part of TA is derived from the amino acid arginine through the polyamine molecule putrescine. For the purpose of increasing the intracellular concentration of TA precursor molecules including putrescine, NMP, 4MAB and NMPy, a Saccharomyces cerevisiae strain with improved flux through the arginine and polyamine biosynthetic pathways has been developed. These strains combine genetic modifications to increase the carbon and nitrogen flux from central metabolism to arginine and polyamine biosynthesis in general, and include the introduction of key heterologous enzymes to additionally produce the TA precursor putrescine. Genetic modifications are used, including the introduction of feedback inhibition relief mutations of genes encoding natural biosynthetic enzymes and regulatory proteins, fine-tuning of transcriptional regulation of natural biosynthetic enzymes, deletion or destruction of genes encoding enzymes that deviate from the expected pathway for precursor molecules, and the introduction of heterologous enzymes for converting endogenous molecules into TA precursor molecules.

[0313] 1.1) Biosynthetic pathways in engineered strains incorporate overexpression of native yeast genes involved in arginine metabolism and polyamine biosynthesis ( Figure 4 ).

[0314] 1.1.1) Examples of overexpressed native genes in yeast include, but are not limited to: glutamate N-acetyltransferase (Arg2p), which catalyzes the first step in the biosynthesis of arginine from glutamate; arginase (Car1p), which removes the guanidinium group of arginine to produce ornithine in the mitochondrial matrix; mitochondrial membrane transporter (Ort1p), which transports ornithine out of the mitochondrial matrix to the cytosol; ornithine decarboxylase (Spe1p), which decarboxylates cytosolic ornithine to putrescine; and polyamine oxidase (Fms1p), which dealkylates spermine and spermidine to putrescine.

[0315] 1.1.2) The effect of overexpression of these native enzymes on putrescine production was examined by co-transforming yeast strains with different combinations of three low-copy plasmids, each expressing one of SPE1, ORT1, CAR1, ARG2, FMS1 or blue fluorescent protein (BFP) as a negative control. After 48 h of growth in selective medium, the titer of putrescine accumulated in the extracellular medium of the co-transformed cells 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 a 27% and 12% increase in putrescine production relative to SPE1 alone, overexpression of ORT1 with SPE1 resulted in a 35% decrease in putrescine titer compared to SPE1. Overexpression of any three of SPE1, CAR1, ARG2, and FMS1 together increased extracellular putrescine titer to 34-35 mg / L.

[0316] 1.2) Biosynthetic pathways in engineered strains incorporate expression of heterologous enzymes from polyamine production pathways found in organisms other than yeast to further increase putrescine production ( Figure 4 ).

[0317] 1.2.1) In addition to the ornithine-dependent pathway found in most plants, animals and fungi (whereby putrescine is synthesized by deguanidine of arginine and subsequent decarboxylation of ornithine), many bacteria and plants also express alternative pathways by which arginine is first decarboxylated by arginine decarboxylase (ADC) to generate agmatine. In plants, the guanidine group of agmatine is then converted to urea by iminohydrolase (AIH) to produce N-carbamoylputrescine (NCP), from which the amide group is then removed by amidase (CPA) to give putrescine (see Patel, J. et al. Dual functioning of plant arginases provides a third route for putrescine synthesis. Plant Sci. 262, 62–73 (2017)). Some bacteria have evolved agmatine urea hydrolases (AUHs) that are able to remove the guanidine group directly from agmatine to produce putrescine without an N-carbamylated intermediate (see Klein, RD et al. Reconstitution of abacterial / plant polyamine biosynthesis pathway in Saccharomyces cerevisiae. Microbiology 145 (Pt 2, 301–7 (1999)).

[0318] 1.2.2) To reconstruct the heterologous putrescine biosynthesis pathway in yeast, the following enzymes can be used: ADC, AIH, CPA and AUH. As an example of an engineered strain with these enzymatic activities, ADC from oats (Avena sativa; AsADC) previously shown to be active in Saccharomyces cerevisiae (see Klein, RD et al. Reconstitution of abacterial / plant polyamine biosynthesis pathway in Saccharomyces cerevisiae. Microbiology 145 (Pt 2, 301–7 (1999)), AIH from Arabidopsis thaliana (AtAIH), two CPA orthologs from tomato (Solanum lycopersicum; SlCPA) and Arabidopsis thaliana (AtCPA), and two AUHs from Escherichia coli (speB) and Arabidopsis thaliana (AtARGAH2) were selected for expression in yeast.

[0319] 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 pathway was reconstructed in a stepwise manner by co-transforming wild-type yeast strains with low-copy plasmids expressing AsADC, AtAIH and SlCPA or AtCPA; or AsADC and speB or AtARGAH2. To eliminate the effects on cell growth and metabolite production caused by different auxotrophic levels, all transformations were performed using three low-copy plasmids containing different auxotrophic markers instead of blank or absent plasmids, and BFP was used as a negative control. The relative accumulation of agmatine, NCP and putrescine in the extracellular medium after the transformed cells were grown in selective medium for 48 hours was analyzed by LC-MS / MS, indicating that all enzymes, except SlCPA and AtARGAH2, retained activity in yeast ( Figure 6 , 7 ). Reconstruction of a plant-specific pathway comprising AsADC, AtAIH, and AtCPA was able to produce putrescine at a titer of 23 mg / L, a 22-fold increase over wild-type titer. When combined with AsADC and AtAIH, an orthologous CPA from tomato (SlCPA) was able to produce putrescine at a titer of 4.5 mg / L, which is similar to putrescine levels in cells expressing AsADC and AtAIH. Reconstruction of a bacterial shortcut pathway by AsADC and E. coli ureahydrolase (speB) enabled the production of putrescine at a titer of 34 mg / L, 32-fold higher than wild-type.

[0320] 1.3) Biosynthetic pathways in engineered strains incorporate overexpression of native yeast genes involved in arginine and polyamine biosynthesis and expression of heterologous biosynthetic enzymes from polyamine production pathways found in organisms other than yeast to further increase putrescine production.

[0321] 1.3.1) The top-performing triad of overexpressed native genes for putrescine biosynthesis (SPE1, ARG2, CAR1; 1.1.2) was combined with the best heterologous putrescine pathway (AsADC, speB; 1.2.3) by co-transforming a wild-type yeast strain with a low-copy plasmid encoding SPE1, AsADC and speB and a low-copy plasmid encoding ARG2 and CAR1. Putrescine titers in the culture medium of transformed cells were measured after 48 hours by LC-MS / MS analysis. The resulting strain produced putrescine at a titer of 47 mg / L ( Fig.10 ).

[0322] 1.4) Regulation of polyamine biosynthesis in yeast through several mechanisms ( Figure 8). The biosynthetic pathways in the engineered strains incorporate disruption of one or more of these regulatory mechanisms to reduce feedback inhibition of putrescine production.

[0323] 1.4.1) Natural yeast genes that are involved in the regulation of polyamine biosynthesis and that may therefore be disrupted to improve intracellular putrescine accumulation include, but are not limited to, the following examples: Figure 8 Methylthioadenosine phosphorylase (Meu1p) catalyzes the driving step in the recycling pathway of decarboxylated S-adenosylmethionine (dcSAM), which constitutes the alkyl donor for the conversion of putrescine to spermidine and spermine catalyzed by spermidine synthase (Spe3p) and spermine synthase (Spe4p) (see Chattopadhyay, MK, Tabor, CW, and Tabor, H. Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1Δmutant. Biochem. Biophys. Res. Commun. 343, 203–207 (2006)). Methylthioadenosine is known to inhibit the activity of spermidine synthase (see Chattopadhyay, MK, Tabor, CW and Tabor, H. Studies on the regulation of ornithine decarboxylase in yeast: Effect of deletion in the MEU1 gene. Proc. Natl. Acad. Sci. 102, 16158-16163 (2005)). Polyamine biosynthesis is regulated by an antizyme-mediated negative feedback loop that is conserved across fungi and metazoans (see Pegg, AE Regulation of ornithine decarboxylase. Journal of Biological Chemistry 281, 14529-14532 (2006)). In yeast, the OAZ1 gene contains two exons separated by a single nucleotide that together encode antizyme-1, a competitive inhibitor of ornithine decarboxylase (Spe1p). The polyamine-induced ribosomal frameshift mechanism enables translation of the full-length antizyme only at high polyamine levels, exerting feedback inhibition on its biosynthesis. Finally, the uptake of polyamines from the extracellular milieu is mediated by a signaling pathway involving Agp2p, a plasma membrane permease with affinity for carnitine, spermidine, and spermine, and Sky1p, a protein kinase thought to interact with Agp2p.

[0324] 1.4.2) Yeast single gene disruption strains for each of MEU1, OAZ1, SPE4, SKY1 and AGP2 were constructed by inserting a series of tandem nonsense mutations within the first third of each open reading frame in wild-type yeast. To characterize the effects of each regulatory disruption in the context of native and heterologous putrescine production pathways, either yeast ODC (SPE1) was overexpressed, or AsADC and speB were co-expressed from a low copy plasmid in each single gene disruption strain. After 72 hours of growth, putrescine titers in the extracellular medium were measured by LC-MS / MS ( Fig. 9 ). When overexpressed through the native putrescine production pathway of SPE1, MEU1 disruption increased putrescine titer by 68%. Similarly, when combined with overexpression of SPE1, OAZ1 disruption significantly increased putrescine production by 174%. Disruption of OAZ1 resulted in a 21-fold increase in putrescine titer in untransformed cells that did not have an overexpressed native or heterologous putrescine pathway. Disruption of SKY1 and AGP2 resulted in a 29% and 14% increase in putrescine titer, respectively, when overexpressed with SPE1. When combined with heterologous expression of AsADC and speB, SKY1 disruption resulted in a 41% decrease in putrescine titer.

[0325] 1.5) Biosynthetic pathways in engineered strains MEU1 and OAZ1 regulatory gene knockouts were combined with overexpression of native and heterologous putrescine biosynthetic genes to further increase putrescine production in engineered strains. Additional copies of native arginine and polyamine biosynthetic genes ARG2, CAR1, and FMS1 were integrated into the genome of the meu1 / oaz1 double disrupted strain. This strain was transformed with a low copy plasmid expressing SPE1, AsADC, and speB. LC-MS / MS analysis of the extracellular medium of the transformed strain showed that after 48 hours of growth in selective medium, putrescine titers reached 86 mg / L ( Fig.10 ).

[0326] Example 2. Engineering yeast strains for producing NMPy

[0327] Saccharomyces cerevisiae strains were developed by modifying the strains that overproduced putrescine developed in Example 1 for the production of the TA precursor NMPy. These strains combined genetic modifications with the goal of increasing carbon and nitrogen fluxes from putrescine to NMPy biosynthesis and included the introduction of key heterologous enzymes to produce the TA precursors NMP, 4MAB, and NMPy. Genetic modifications were used, including modifications of the N- and / or C-terminal domains of the enzymes of interest to improve activity in heterologous hosts, and deletion or disruption of genes encoding enzymes that drive precursor molecules away from the expected pathway.

[0328] 2.1) The biosynthetic pathway in the engineered strain is able to produce NMPy from endogenous putrescine. Putrescine is first converted to N-methylputrescine (NMP) by SAM-dependent N-methyltransferase (PMT), which is subsequently oxidized to 4-methylaminobutyraldehyde (4MAB) by copper-dependent diamine oxidase (MPO). Like many aldehyde compounds, 4MAB is unstable in aqueous solution and spontaneously cyclizes to form NMPy ( Fig.11 ).

[0329] 2.1.1) The putrescine overproducing strain of Example 1.5 containing low copy plasmids expressing SPE1, AsADC and speB to overproduce putrescine was co-transformed with additional low copy plasmids expressing the PMT from Atropa belladonna (AbPMT1) and the subsequent MPO enzyme from Nicotiana tabacum (NtMPO1). After 48 hours of growth, the accumulation of intermediates in the extracellular medium of transformed cells expressing each of the sequential enzymes between putrescine and NMPy was compared by LC-MS / MS analysis. The direct product of NtMPO1 (4MAB) and its spontaneous cyclization product (NMPy) were synthesized by AbPMT1 and NtMPO1 ( Fig.11 ) and their precursors NMP and putrescine ( Fig.12 ) generated.

[0330] 2.1.2) NMP accumulation in the growth medium of putrescine overproducing yeast strains (described in Example 1.4.2) with and without disruption of the MEU1 gene was measured by LC-MS / MS analysis. This analysis showed that the previous disruption of MEU1 in the putrescine overproducing strain and its concomitant effect on SAM recycling did not inhibit putrescine N-methylation by AbPMT1 ( Fig.13 ).

[0331] 2.2) Enzymes can be localized to subcellular compartments different from those in their original host organisms when heterologously expressed, resulting in reduced function. The biosynthetic pathways in engineered strains can incorporate modifications to the polypeptide sequences of native and heterologous enzymes to induce these modified enzymes to be localized to subcellular compartments other than those compartments where they are naturally located. For example, previous studies have shown that although NtPMT is expressed in the cytosol of tobacco cells, NtMPO1 is localized to the peroxisomal cavity (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)).

[0332] 2.2.1) The subcellular localization of NtMPO1 was examined by computer simulation prediction of enzyme subcellular localization using the SherLoc2 utility for signal peptide detection (see Briesemeister, S. et al. SherLoc2: A high-accuracy hybrid method for predicting subcellular localization of proteins. J. Proteome Res. 8, 5363-5366 (2009)). This analysis showed that NtMPO1 contains a strong yeast consensus peroxisome targeting sequence (PTS) (Ala-Lys-Leu, denoted as PTS1) at its C-terminus, indicating that NtMPO1 can be localized to peroxisomes when heterologously expressed in yeast ( Fig.14 ).

[0333] 2.2.2) Fluorescence microscopy of wild-type yeast cells expressing AbPMT1 and NtMPO1 with either N-terminal or C-terminal GFP tags from low-copy plasmids showed that, while AbPMT1 was mainly present in the cytosol, NtMPO1 localized in peroxisomes depending on the exposed C-terminal PTS ( Fig.15 a, 16).

[0334] 2.2.3) Cytosolic expression of NtMPO1 by masking the C-terminal PTS with a GFP fusion did not significantly affect extracellular 4mAb or NMPy levels ( Fig.15 b).

[0335] 2.3) Biosynthetic pathways in engineered strains may incorporate orthologs of biosynthetic enzymes other than those listed in Table 1. Different orthologs of enzymes may exhibit significant differences in activity when expressed in heterologous hosts. Therefore, orthologs of biosynthetic enzymes provided as examples herein and listed in Table 1 may also be used in engineered non-plant cells to perform the same biochemical transformations.

[0336] 2.3.1) Transcriptomes of Atropaeolum belladonna and Datura datura were searched by tBLASTn in the 1000Plants Project database (see Matasci, N. et al. Data access for the 1,000Plants (1KP) project. Gigascience 3, 17 (2014)) using the amino acid sequence of NtMPO1 as the query sequence and 10 -150 Two full-length orthologous sequences, denoted AbMPO1 and DmMPO1, were identified, each sharing 91% sequence identity with NtMPO1 ( Fig.17 a).

[0337] 2.3.2) Yeast codon optimized sequences of AbMPO1 and DmMPO1 were obtained and cloned into low copy expression plasmids. To evaluate their activity, each of the three MPO variants was co-expressed with AbPMT1 from a low copy plasmid in the putrescine overproducing strain of Example 1.5, and after 48 hours of growth in selective medium, the accumulation of 4MAB and NMPy in the extracellular medium was measured by LC-MS / MS. DmMPO1 showed comparable levels of 4MAB and NMPy production to the original NtMPO1 variant ( Fig.17 b).

[0338] 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 NtMPO1, AbMPO1 and DmMPO1 were constructed using the RaptorX web server based on the crystal structure of pea copper-containing amino oxidase (PDB: 1KSI) (see M. et al. Template-based protein structure modeling using the RaptorX web server. Nat. Protoc. 7, 1511–22 (2012)). The homology model showed that the orthologs had long, unstructured N-terminal and C-terminal tail regions ( Fig.17 c).

[0339] 2.3.4) The activity of truncations of two active orthologs, NtMPO1 and DmMPO1, was tested in engineered yeast. The N-terminal truncations removed the first 84 and 81 residues of the two orthologs, respectively. The C-terminal truncations removed the last 21 residues. C-terminal truncations were also constructed in which the unstructured tail was removed but the PTS was retained (denoted as Each MPO truncation was co-expressed with AbPMT1 from a low copy plasmid in the putrescine overproducing strain of Example 1.5, and 4MAB and NMPy accumulation in the culture medium was quantified by LC-MS / MS after 48 h of growth. No significant differences in activity were observed between the NtMPO1 truncations ( Fig.18 Removal of the C-terminal unstructured region from DmMPO1 while retaining the C-terminal PTS tripeptide resulted in a 31% increase in extracellular 4mAb levels relative to the wild-type DmMPO1 enzyme.

[0340] 2.4) Biosynthetic pathways in engineered strains incorporate one or more genetic modifications to reduce or eliminate metabolic flux to undesirable side reactions. Biosynthetic enzymes expressed in heterologous hosts may participate in undesirable side reactions that divert metabolite flux away from the biosynthesis of the desired compound. For example, yeast aldehyde dehydrogenase can oxidize heterologous aldehyde molecules (such as 4MAB) to their cognate carboxylic acids. Based on LC-MS / MS analysis, when AbPMT1 and When co-expressed from a low copy plasmid, but not in the absence of MPO enzyme, accumulation of 4MAB acid was observed in the growth medium of the putrescine overproducing strain of Example 1.5 ( Fig.11 ).

[0341] 2.4.1) Six yeast genes (ALD2-ALD6 and HFD1) have been shown in the literature to encode enzymes with aldehyde dehydrogenase activity (see Datta, S., Annapure, US and Timson, DJ Different specificities of twoaldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii. Biosci. Rep. 37, BSR20160529 (2017); and Nakahara, K. et al. The 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 pantothenate (see White, WH, Skatrud, PL, Xue, Z. and Toyn, JH Specialization of Function Among Aldehyde Dehydrogenases: Genetics 163, 69–77 (2003)). The ALD4, ALD5 and ALD6 genes encode two mitochondrial and one cytosolic acetaldehyde dehydrogenases, respectively, which oxidize acetaldehyde to acetate during fermentative growth on glucose and ethanol (see Saint-Prix, F., L. and Dequin, S. Functional analysis of the ALD gene family of Saccharomyces cerevisiae eduring anaerobic growth on glucose: The NADP + -dependent Ald6p and Ald5p isoforms play a major role in acetate formation. Microbiology 150, 2209–2220 (2004)), the enzyme has been shown to oxidize a series of different aliphatic and aromatic aldehydes to carboxylic acids (see Datta, S., Annapure, US and Timson, DJ Different specificities of two aldehydedehydrogenases from Saccharomyces cerevisiae var. boulardii. Biosci. Rep. 37, BSR 20160529 (2017)). The individual knockout strains of these four target genes were constructed by inserting a series of tandem nonsense mutations in the first third of its open reading frame in the strain that overproduces putrescine in Example 1.5. By co-expressing AbPMT1 and The contribution of each of the four dehydrogenases to 4MAB oxidation was assessed by measuring 4MAB acid accumulation in the culture medium by LC-MS / MS after 48 h of growth. A marginal decrease in 4MAB acid levels was observed in the presence of disruption of HFD1 and ALD4-6 alone ( Fig.19 ).

[0342] 2.4.2) Although ALD4-6 are considered essential genes due to their roles in acetate and acetyl-CoA production, previous studies have shown that these three genes are at least partially redundant and that the lethal phenotype of double and triple knockouts can be rescued by supplementing the medium with acetate (see Saint-Prix, F., et al., 2001). L. and Dequin, S. Functional analysis of the ALD gene family of Saccharomyces cerevisiae eduring anaerobic growth on glucose: The NADP+-dependent Ald6p and Ald5p isoforms play a major role in acetate formation. Microbiology 150, 2209–2220 (2004); and Luo, Z., Walkey, CJ, Madilao, LL, Measday, V. and Van Vuuren, HJJ Functional improvement of Saccharomyces cerevisiae to reduce volatile acidity in wine. FEMS Yeast Res. 13, 485–494 (2013)). A quadruple knockout yeast strain was constructed in which the open reading frames of HFD1 and ALD4-6 were disrupted and AbPMT1 and AbPMT2 were expressed from a low copy plasmid. This strain showed a 45% reduction in 4MAB acid levels compared to the non-disrupted strain ( Fig. 20 a) and a concomitant 46% increase in NMPy production ( Fig. 20 b).

[0343] 2.4.3) Deleting the tandem ALD2-ALD3 genes from the genome of the quadruple knockout strain of Example 2.4.2 and co-expressing AbPMT1 and After 48 h of growth, LC-MS / MS analysis showed that deletion of ALD2 and ALD3 completely eliminated the 4MAB acid byproduct and increased 4MAB and NMPy production by 83% and 75%, respectively, compared with a strain with all six ALD genes intact ( Fig. 20 a, b).

[0344] 2.4.4) by integrating the gene cassette for overproduction of putrescine (SPE1, AsADC, speB) carried by the previous plasmid into the genome of the ALD null strain of Example 2.4.3, and additionally integrating AbPMT1 and LC-MS / MS analysis confirmed that the NMPy production of this strain after 48 h of growth in non-selective medium was comparable to that of the essential putrescine production genes AbPMT1 and The ALD ineffective strain of Example 2.4.3 cultured in a selective medium is equivalent to ( Fig.21 ).

[0345] Example 3. Engineered yeast strains for producing tropine from monosaccharides and nutrients

[0346] Type III polyketide synthases (PKS) and cytochrome P450 can convert NMPy to tropinone via the TA precursor MPOB. Tropinone can be reduced by a stereospecific reductase (denoted as tropinone reductase 1 (TR1)) to produce tropine (see Kim, N., Estrada, O., Chavez, B., Stewart, C., and D'Auria, JC Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis. Molecules 21, (2016)) ( Fig. 22 ).

[0347] 3.1) The biosynthetic pathway in the engineered strain incorporates pyrrolidone synthase, tropinone synthase CYP82M3, one or more cytochrome P450 reductases, and tropinone reductase 1 to convert NMPy to tropine.

[0348] 3.1.1) Yeast codon-optimized DNA sequences encoding belladonna pyrrolidone synthase (AbPYKS), tropinone synthase (AbCYP82M3), and Datura grandiflora tropinone reductase 1 (DsTR1) were obtained. Yeast codon-optimized sequences of a set of four different CPRs were also obtained, including three plant CPRs from Arabidopsis thaliana, Eschscholzia californica (California poppy), and Papaver somniferum (opium poppy), as well as a native yeast CPR (NCP1) for expression in yeast, since P450 enzymes require NADP. + -cytochrome P450 reductase (CPR) partner for continuous electron exchange. Yeast strains were 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 verify enzyme activity and identify potential bottlenecks, the accumulation of NMPy, MPOB, tropinone and tropine in the culture medium of the transformed strains was monitored by LC-MS / MS after 48 hours of growth ( Fig.23 Under the assay conditions, comparable levels of de novo tropine production (175-210 μg / L) were observed with all four CPR partners.

[0349] 3.2) The presence of metabolic bottlenecks defined as biosynthetic enzymes or spontaneous steps whose low activity limits the flux through a portion of the biosynthetic pathway can lead to suboptimal production of desired TAs and precursors.

[0350] 3.2.1) For example, analysis of the accumulation of TA intermediates in the culture medium of the engineered strains of Example 3.1.1 showed that, despite the accumulation of little tropinone, the product of AbCYP82M3, most of the MPOB produced by AbPYKS remained unconsumed by AbCYP82M3 ( Fig.24 ).

[0351] 3.2.2) Integration of tropine biosynthetic genes into the yeast genome can improve tropine production by achieving more stable expression of AbCYP82M3. A tropine-producing platform strain was constructed by integrating AtATR1 together with AbPYKS and AbCYP82M3 into the genome of the NMPy-producing strain of Example 3.1.1. After 48 hours, tropine and scopolamine accumulation of the integrated strain was compared with plasmid-based expression of the same genes by LC-MS / MS analysis ( Fig.28 Genomic expression of AbPYKS, AbCYP82M3, and AtATR1 increased tropine titer by nearly three-fold (565 μg / L) relative to plasmid-based expression (189 μg / L). The engineered strains also showed a 2.6-fold increase in tropine accumulation.

[0352] 3.3) Accumulation of by-products in the biosynthetic pathways of engineered strains can lead to suboptimal production of desired TAs and precursors.

[0353] 3.3.1) For example, analysis of the accumulation of TA intermediates in the culture medium of the engineered strain of Example 3.1.1 showed that NMPy derivative tropine accumulated in large quantities, with a titer almost four times greater than tropine (775-900 μg / L). In the relevant literature, tropine has been observed to accumulate spontaneously by decarboxylation of MPOB (see Bedewitz, MA, Jones, AD, D'Auria, JC and Barry, CS Tropinone synthesis via an atypical polyketide synthase and P450-mediated cyclization. Nat. Commun. 9, 5281 (2018)) ( Fig. 22 As another example, LC-MS / MS analysis of the growth medium of the engineered strain of Example 3.1.1 showed that scopolamine also accumulated in the negative control strain lacking AbPYKS and AbCYP82M3 due to decarboxylative condensation with NMPy ( Fig. 22 ).

[0354] 3.3.2) Regulation of growth temperature can be used to reduce the accumulation of byproducts in the biosynthetic pathway of the engineered strain to increase the flux toward the desired TA and precursors. In one example, the effect of temperature on spontaneous styracine production was evaluated by means of the following kinetic principle: that is, the rates of enzymatic and spontaneous reactions are reduced at lower temperatures. Since Solanum belladonna and other TA-producing Solanaceae plants are adapted to optimal growth under colder climates, growth of yeast strains expressing Solanaceae genes at 25°C can improve enzyme folding and / or activity, thereby enabling enzymatically generated tropine yields comparable to those grown at 30°C, while reducing the rate of spontaneous styracine production. Cultures of the tropine-producing strain of Example 3.2.2 were grown at 30°C and 25°C in a non-selective defined medium and the accumulation of tropine and styracine was compared by LC-MS / MS analysis of the growth medium after 48 hours. Tropine titers are minimally affected by reduced temperature. The accumulation of scutellarine was reduced by 42% at 25°C compared to 30°C, resulting in a 60% increase in the ratio of tropine to scutellarine produced ( Fig.25 ).

[0355] 3.3.3) Reducing or eliminating adverse side reactions can be used to increase the metabolite flux toward the desired TA and TA precursors in the biosynthetic pathway of the engineered strain. In one example, the flux toward the TA precursor tropine can be increased by reducing the production of tropine caused by spontaneous decarboxylation condensation with acetate. The effect of removing the supplemented acetate from the culture medium of the NMPy-producing strain of Example 2.4.4 on the yield of tropine and tropine was evaluated. The effect of eliminating 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 tropine and 4MAB acid by LC-MS / MS analysis after 48 hours of growth. Although the reconstruction of ALD4 or ALD6 enables growth on selective medium in the absence of supplemented acetate ( Fig.26 a), but the addition of ALD4 resulted in a 5-fold increase in the accumulation of 4MAB acid, whereas ALD6 did not produce a significant increase ( Fig.26 b). Furthermore, elimination of acetate feeding in the case of ALD4 or ALD6 resulted in a 38% and 59% reduction in scopolamine accumulation, respectively ( Fig.26 b).

[0356] 3.3.4) A functional copy of the ALD6 gene was reintegrated into the tropine-producing strain of Example 3.2.2 at the previously disrupted ald6 locus. After 48 hours of growth in non-selective medium, the effect of this integration on the accumulation of all metabolites between NMPy and tropine was measured by LC-MS / MS analysis. Restoration of acetate metabolism by Ald6p resulted in a 2.7-fold increase in tropine titer and a 1.6-fold increase in scopolamine accumulation ( Fig.28 In addition, ALD6 integration resulted in significantly increased production of NMPy and tropinone and increased consumption of MPOB ( Fig. 27 ).

[0357] 3.3.5) The biosynthetic enzyme genes between putrescine and tropine (i.e., AbPMT1, After 48 hours of growth in selective medium, the production of TA intermediates was compared with that of the same strain expressing BFP by LC-MS / MS. The expression of an additional copy of AbPYKS resulted in a 4.3-fold increase in NMP accumulation and a 1.3-fold increase in tropine production ( Fig.29 Expression of an additional copy of AbPMT1 resulted in a significant increase in the production of all TA precursors between NMP and tropinone, with a 2.4-fold increase in tropine production ( Fig.29 ). Therefore, additional copies of PMT (AbPMT1 and DsPMT1) and PYKS (AbPYKS) were integrated into the genome of the tropine-producing strain (CSY1249) of Example 3.3.4 at the PAD1 locus. The resulting engineered strain (CSY1251) was grown in non-selective medium at 25°C for 48 hours, resulting in the production of tropine at a titer of 3.4 mg / L, which is 2.2 times greater than the tropine-producing strain (CSY1249) in Example 3.3.4 ( Fig.30 ).

[0358] Example 4: Yeast engineered to produce pseudotropine alkaloids from L-arginine.

[0359] Yeast strains can be engineered to produce non-pharmaceutical TAs from early amino acid precursors such as L-arginine. For example, the platform yeast strain described in Example 3 can be further engineered to produce pseudotropine alkaloids ( Figure 1 ).

[0360] The platform yeast strains that produce tropinone from L-arginine (see description in Example 3) can be further engineered to incorporate a stereospecific reductase, such as tropinone reductase 2 (TR2; EC 1.1.1.236), to convert the biosynthesized tropinone into pseudotropine. An expression cassette containing a strong constitutive promoter (such as TDH3) and the coding sequence of a TR2 variant (e.g., TR2 from Datura grandiflora (DsTR2)) can be integrated into the genome of the platform yeast strain that produces tropinone. The resulting strains can be further engineered to produce hydroxylated derivatives of pseudotropines, such as calanthene, by integrating one or more expression cassettes containing a strong constitutive promoter (such as PGK1) and a hydroxylase (such as a cytochrome P450 that acts on the pseudotropine scaffold). By incorporating multiple P450 enzymes that each act on different positions of the pseudotropine backbone, a variety of calanthene and its derivatives can be biosynthesized. The engineered strains can then be cultured in non-selective synthetic complete medium at 30°C or 25°C for 48 to 96 hours, and the culture medium can then be analyzed for accumulation of pseudotropine alkaloids by LC-MS / MS.

[0361] Example 5: Yeast engineered to overproduce phenylpyruvate and related TA precursors.

[0362] Yeast strains can be engineered to overproduce phenylpyruvate, which represents a precursor for the production of acyl donor molecules required for pharmaceutical TA ( Figure 2 ), with the goal of increasing carbon and nitrogen flux from central metabolism toward desired TAs and TA precursors. Yeast strains can be engineered to overproduce phenylpyruvate by incorporating genetic modifications, including but not limited to fine-tuning of transcriptional regulation of native biosynthetic enzymes, deletion or disruption of genes encoding enzymes that divert precursor molecules from the desired pathway, and introduction of heterologous enzymes for converting endogenous molecules to TA precursor molecules.

[0363] In one example, yeast strains can be engineered to increase the production of phenylpyruvate by incorporating additional copies of natural genes that encode biosynthetic enzymes that produce phenylpyruvate from amino acids or other central metabolites. These additional copies can be controlled by strong constitutive promoters, such as GPD, TEF1 or PGK1. Examples of natural gene targets include, but are not limited to, aromatic acid aminotransferases ARO8 and ARO9, and dehydratase PHA2. 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 a unique strong constitutive promoter.

[0364] Example 6: Yeast engineered to produce acyl donors for biosynthesis of TA scaffolds from L-phenylalanine or L-tyrosine.

[0365] Yeast strains can be engineered to produce various phenylpropanoid donor compounds from L-phenylalanine and L-tyrosine, including PLA, cinnamic acid, coumaric acid, ferulic acid, benzoic acid, and coenzyme A thioesters and glycoside derivatives of these compounds, which can be esterified with tropine, pseudotropine, or their derivatives to biosynthesize pharmaceutical TA, non-pharmaceutical TA, and non-natural TA ( Figure 1-3 ).

[0366] 6.1) Since wild-type yeast only produces trace amounts of PLA, it is necessary to increase the production of this TA precursor to allow sufficient accumulation of downstream TA. In order to increase PLA production, heterologous phenylpyruvate reductase (PPR) can be expressed in engineered host cells. PPR orthologs from Escherichia coli, Lactobacillus, Solanum belladonna and Wick's fluorescens were screened for activity in yeast by expressing each enzyme from a low copy plasmid in CSY1251 and measuring PLA production by LC-MS / MS after 72 h of growth in selective medium, as well as lactate dehydrogenase (LDH) (Table 1) with reported activity on 3-phenylpyruvate from Bacillus and Lactobacillus. Relative to the control, all LDH candidates and PPR from Lactobacillus plantarum, Escherichia coli and Solanum belladonna produced moderate (1.3 to 3.5 times) improvements in PLA production, while the expression of PPR from Wick's fluorescens resulted in a nearly 80-fold increase in PLA production to ~250 mg / L ( Fig.31). Therefore, the integration of WfPPR into CSY1251 was selected to generate strain CSY1287.

[0367] 6.2) As another example, yeast strains can be engineered to produce cinnamic acid and coumaric acid, which are phenylpropanoids that can be used as acyl donor compounds for esterification with tropine or pseudotropine to form non-natural TAs from L-phenylalanine and L-tyrosine, respectively. Yeast can be engineered to produce cinnamic acid from L-phenylalanine by incorporating an ammonia lyase, such as phenylalanine ammonia lyase (PAL; EC 4.3.1.24). Similarly, yeast can be engineered to produce coumaric acid from L-tyrosine by incorporating an ammonia lyase, such as tyrosine ammonia lyase (TAL; EC 4.3.1.23). Yeast strains are engineered to produce cinnamic acid from L-phenylalanine by transformation with a low copy CEN / ARS plasmid with a TRP1 selective marker, a TEF1 promoter, and a coding sequence for a PAL variant from Arabidopsis thaliana (AtPAL1). The resulting strains containing the low copy plasmid were grown in synthetic complete medium with the appropriate amino acid deletion solution (-Ura) at 30°C. After 48 hours of growth, the culture medium was analyzed for cinnamic acid content by LC-MS / MS analysis ( Fig.32 ).

[0368] 6.3) In Atropa belladonna, PLA is activated by glucosylation using UDP-glucosyltransferase 84A27 (AbUGT) to transfer acyl groups to tropine (see Qiu, F. et al., Functional genomics analysis reveals two novelgenes required for littorine biosynthesis. New Phytol., nph. 16317 (2019)). Since plant UGTs are involved in the biosynthesis of various phenylpropanoids and generally exhibit a wide range of substrates (see Ross, J., Li, Y., Lim, E.-K., DJ Bowles, Higher plant glycosyltransferases. Genome Biol. 2, 3004.1–3004.6 (2001)), it is necessary to select UGTs with sufficiently high activity for the desired acyl donor.

[0369] 6.3.1) For example, the activity of AbUGT towards different phenylpropanoyl donors (including the classical substrate PLA) was evaluated by expressing AbUGT from a low copy plasmid in CSY1251 and measuring the conversion of three phenylpropanoyl donors (PLA, cinnamic acid, ferulic acid) to their respective glycosides. While AbUGT glucosylated 60% and 90% of cinnamic acid and ferulic acid, respectively, glucosylation of PLA was the lowest among the substrates tested, with a conversion rate of <3% ( Fig.33 ).

[0370] 6.3.2) Orthologs of AbUGT from other TA-producing Solanaceae plants can be evaluated for activity against PLA and other phenylpropanoids. In this example, transcripts encoding UGT84A27 were obtained from the transcriptomes of Datura stramonium (BsUGT) and Datura datura (DmUGT) in the 1000Plants database using tBLASTn searches. Yeast codon-optimized sequences encoding these orthologous UGTs were screened for activity by expressing AbUGT, BsUGT, DmUGT or BFP negative controls from low-copy plasmids in CSY1251. After 72 h of growth in selective medium supplemented with 500 μM PLA, cinnamic acid (CA) or ferulic acid (FA) as glucose acceptors, glucoside production in cultures of transformed strains was measured by LC-MS / MS. All three UGT orthologs showed significant glycosylation of CA (34-65% conversion) and FA (85-90% conversion) and only trace activity on PLA (<3% conversion), with AbUGT showing the greatest PLA conversion (2.7%) ( Fig.33 , 34 ).

[0371] 6.3.3) Given the disproportionate differences in the activities of AbUGT on structurally similar substrates cinnamate, ferulic acid ester and PLA, a structure-guided rational mutagenesis approach can be implemented to engineer the active site of AbUGT to improve its activity on PLA. In this example, a homology model of AbUGT bound to UDP-glucose was first constructed using the RaptorX web server based on the crystal structure of Arabidopsis thaliana salicylic acid UDP-glucosyltransferase UGT74F2 (PDB: 5V2K) Fig.35 ). Next, the docking of D-PLA in the active site was simulated using the Maestro / GlideXP software suite. Based on the energy-minimized binding model, the aromatic ring of D-PLA is likely stabilized by π-stacking interactions with F130, while its α-hydroxyl and carboxylate groups are stabilized by hydrogen bonds with Q151 and H24, respectively, such that the nucleophilic carboxylate oxygen is located at the electrophilic C1 carbon of UDP-glucose. Within ( Fig.35). D-PLA is also adjacent to residues L205 and I292, neither of which appears to interact with either substrate. This suggests that (i) mutation of F130 to tyrosine preserves π stacking with the aromatic ring of D-PLA while providing an additional hydrogen bond to stabilize the α-hydroxy oxygen of D-PLA, which is absent in cinnamate and ferulate; (ii) mutation of L205 to phenylalanine increases π stacking stabilization of D-PLA with F130Y; and (iii) mutation of I292 to glutamine generates two additional stabilizing hydrogen bonds with D-PLA and UDP-glucose ( Fig.35 The F130Y, L205F, and I292Q point mutants of AbUGT were screened for activity by expressing each mutant, wild-type AbUGT, or a BFP control from a low-copy plasmid in CSY1251, and glucoside production was measured by LC-MS / MS after 72 h of growth in selective medium supplemented with 500 μM PLA, CA, or FA. Although the F130Y and I292Q mutations significantly reduced UGT activity on CA, all three mutants exhibited considerably lower (and statistically indistinguishable) activity on PLA relative to wild-type AbUGT (<3% conversion) ( Fig.36 ).

[0372] 6.3.4) Based on the results described in sections 6.1 and 6.3, strain CSY1288 was constructed by integrating yeast codon-optimized WfPPR and AbUGT into the genome of CSY1251 and verified by verifying PLA production (66 mg / L) and minimal PLA glucoside accumulation ( Fig.37 ).

[0373] 6.4) Since the poor activity of AbUGT towards PLA may limit the flux of TA precursors towards downstream TAs, the flux of phenylalanine to PLA glucosides can be increased by incorporating genetic modifications that promote UDP-glucose accumulation and reduce glycoside degradation.

[0374] 6.4.1) UDP-glucose is essential for the formation of storage polysaccharides, cell wall glucans and glycoproteins, and its biosynthesis is therefore 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 directly produces glucose-6-phosphate along two major metabolic pathways, glycolysis and starch biosynthesis. Since citrate is an allosteric inhibitor of phosphofructokinase, the rate-limiting enzyme of glycolysis (see Li, Y. et al., Production of Rebaudioside A from Stevioside Catalyzed by the Engineered Saccharomyces cerevisiae. Appl. Biochem. Biotechnol. 178, 1586–1598 (2016)), partial inhibition of glycolysis by supplementation with citrate may increase the availability of UDP-glucose and the production of glucosides ( Fig.38 Strain CSY1288, encoding the genomic WfPPR and AbUGT for endogenous PLA glucoside production, was cultured in medium supplemented with 2% citrate and 500 μM CA or FA, and glucoside production was compared by LC-MS / MS after 72 h of growth. Citrate supplementation reduced glucosylation of PLA, CA, and FA by 83%, 56%, and 78%, respectively ( Fig.39 ).

[0375] 6.4.2) Overexpression of PGM2 and UGP1, whose gene products catalyze the isomerization of glucose-6-phosphate to glucose-1-phosphate and the conversion of glucose-1-phosphate to UDP-glucose, respectively, can be used to increase UDP-glucose supply.

[0376] 6.4.2.1) Additional copies of PGM2 and UGP1 were expressed from low copy plasmids in CSY1288 and the production of PLA glucosides was measured after 72 h of growth in selective medium. While PGM2 overexpression did not produce an improvement over the control, overexpression of UGP1 resulted in a ∼1.8-fold increase in PLA glucoside production ( Fig.40 ), which supports that increasing the UDP-glucose pool improves the utilization of PLA by AbUGT.

[0377] 6.4.2.2) Natural glucosidases can act on PLA and other TA precursor glucosides to reduce accumulation, as other heterologous glucosides 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. et al., Engineering Saccharomyces cerevisiae with the deletion of endogenous glucosidases for the production of flavonoid glucosides. Microb. Cell Fact. 15, 1–12 (2016)). In this example, three native glucosidase genes—EXG1, SPR1, and EGH1—were disrupted in CSY1288, and PLA glucosidase production was measured after the disruption mutants were grown in non-selective medium for 72 h. Disruption of EGH1 more than doubled PLA glucosidase production ( Fig.41 ), indicating that hydrolysis by Egh1p constitutes a major loss in TA precursor flux.

[0378] Example 7: Yeast engineered to convert ritolin to scopolaldehyde

[0379] Yeast strains can be engineered to convert ritolin to scopolamine ( Figure 2). For example, the tropane- and PLA-glucoside-producing yeast strains described in Example 6 can be further engineered to express cytochrome P450 CYP80F1 (EC 1.14.19.-) to catalyze the rearrangement of ritolin to scopolaldehyde, and to express cytochrome P450 reductase (CPR; EC 1.6.2.4) to support the activity of the P450 enzyme. The yeast strains were engineered to convert fed ritolin to scopolaldehyde by transforming the yeast strains with a low-copy CEN / ARS plasmid having a LEU2 selection marker, a TDH3 promoter, and a coding sequence for a CYP80F1 variant from Atropa belladonna (AbCYP80F1); and with a low-copy CEN / ARS plasmid having a TRP1 selection marker, a TEF1 promoter, and a coding sequence for a cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae (NCP1) or Arabidopsis thaliana (AtATR1). The resulting strain containing the low copy plasmid was grown in synthetic complete medium with the appropriate amino acid deletion solution ((-Leu-Trp)) supplemented with 1 mM ritolin at 30°C. After 48 hours of growth, the culture medium was analyzed for scopolamine content by LC-MS / MS analysis ( Fig.42 ).

[0380] Example 8: Yeast engineered to convert hyoscyamine to scopolamine.

[0381] Yeast strains can be engineered to convert hyoscyamine to scopolamine ( Figure 2 ). For example, the yeast strain described in Example 7 can be further engineered to incorporate an enzyme having hydroxylase activity at the 6β position of hyoscyamine to form anisodamine and an enzyme having dioxygenase activity at the 6β-hydroxyl position of anisodamine to form scopolamine, or an enzyme having both activities (EC 1.14.11.11). The yeast strain is engineered to convert fed hyoscyamine to scopolamine by transforming it with a low copy CEN / ARS plasmid having a LEU2 selection marker, a TDH3 promoter, and a coding sequence for hyoscyamine 6β-hydroxylase / dioxygenase (H6H) from Datura grandiflora (DsH6H), Datura stramonium (AaH6H), Datura stramonium (BaH6H), or Datura datura (DmH6H). The resulting strains containing the low copy plasmids were grown in synthetic complete medium with the appropriate amino acid deletion solution (-Leu) and supplemented with 1 mM scopolamine at 30°C. After 72 hours of growth, the culture medium was analyzed for scopolamine content by LC-MS / MS analysis ( Fig.43The strain expressing the H6H variant from Datura grandiflora showed the greatest conversion of fed hyoscyamine to scopolamine, although all tested variants showed in vivo H6H activity. The cofactor requirements were further optimized by supplementing the culture medium of this engineered yeast strain with different cofactors and analyzing the culture medium by LC-MS / MS after 72 hours of growth. This analysis determined that supplementation with ferrous iron increased the conversion of hyoscyamine to scopolamine ( Fig.44 ).

[0382] Example 9. Identification of scopolamine dehydrogenase candidates and reduction of scopolamine to scopolamine in engineered non-plant cells

[0383] To identify TA alcohol-aldehyde interconversions suitable for carrying out the methods disclosed herein, in particular dehydrogenases that reduce scopolamine to hyoscyamine, a scopolamine dehydrogenase (HDH) open reading frame was identified from publicly available plant RNA sequencing data.

[0384] 9.1) Tissue-specific abundance (fragments per kilobase of contig per million mapped reads, FPKM) and putative protein structure and function annotations for each of the 43,861 unique transcripts identified from the Atropaeolum solanum transcriptome were obtained from the Michigan State University Medicinal Plant Genomics Resource. Transcripts encoding scopolamine dehydrogenase candidates were identified based on clustering of tissue-specific expression profiles with the bait genes CYP80F1 (ritolinamycin mutase) and H6H (scopolamine 6β-hydroxylase / dioxygenase), which are located before and after the dehydrogenase step in the TA biosynthetic pathway, respectively, using the following computational filtering algorithm.

[0385] First, the complete list of 43,861 transcripts was filtered to find those with annotations with any of the following protein family (PFAM) IDs: PF00106, PF13561, PF08659, PF08240, PF00107, PF00248, PF00465, PF13685, PF13823, PF13602, PF16884, PF00248; or any of the following functional annotation keywords: alcohol dehydrogenase, aldehyde reductase, short chain, aldehyde / ketone. In addition, any transcript with a functional annotation containing the keywords putrescine, tropinone, and tropine was included in the filter as a positive control TA-related gene to verify clustering with the bait genes. Next, average tissue-specific expression profiles were generated for the CYP80F1 and H6H bait genes. For each of the two bait genes, a linear regression model was constructed to represent the bait gene expression profile (in FPKM) as a linear function of each candidate gene profile, and the correlation p-value of each candidate was calculated. Candidates identified using each of the two bait genes were merged and duplicates were removed. The combined p-value of each candidate was calculated as the sum of the log10 p-values ​​of the correlation with each of the two bait genes. Transcripts that matched known dehydrogenases in the TA biosynthetic pathway (i.e., tropinone reductase I and II) were removed, and the remaining candidates were ranked by combined p-values ​​and by hierarchical clustering of tissue-specific expression profiles by distance from the bait gene ( Fig.45 ).

[0386] 9.2) Almost all candidates identified in Example 9.1 showed the same secondary root-specific expression pattern observed for known TA biosynthetic genes. BLASTp search was performed on the obtained ~30 candidates against the UniPROT / SwissPROT database, and it was found that many transcripts lacked terminal or internal sequence regions. To solve this problem, the Trinity software package was used to repeat the de novo transcriptome assembly from the deposited original RNAseq reads (see Haas, BJ et al., De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 8, 1494–512 (2013)), and by BLAST alignment of incomplete sequence regions for the newly assembled transcriptome, all missing sequence fragments of the twelve HDH candidates were reconstructed (Table 2).

[0387] 9.3) Identification of lost HDH activity by screening the candidates generated in Examples 9.1 and 9.2 in yeast.

[0388] 9.3.1) The lack of authentic commercial standards of scopolamine and the insufficient yield of chemical synthesis require that HDH candidates be co-expressed with the upstream biosynthetic enzyme, cytochrome P450 ritolin mutase (CYP80F1), for in vivo activity screening by fed ritolin (see Example 7). Since ritolin exhibits similar chromatographic and mass spectrometric properties to the HDH product scopolamine, an HDH screening strain (CSY1292) was constructed by integrating yeast codon-optimized AbCYP80F1 and DsH6H (see Example 8) into the genome of CSY1251, enabling the detection of the biosynthetic pathway through the three-step ( Figure 2 ) by feeding ritolin (m / z + Scopolamine (m / z 290) + 304) to screen HDH candidates.

[0389] 9.3.2) Yeast codon-optimized sequences encoding each of the 12 HDH candidates were expressed from low-copy plasmids in strain CSY1292, and scopolamine production was measured after 72 h of growth in medium supplemented with 1 mM ritolin. One of the twelve candidates, HDH2 (referred to as AbHDH), exhibited a 35% reduction in scopolamine levels and measurable scopolamine accumulation (7.2 μg / L), indicating that it encodes a lost HDH activity ( Fig.46 ).

[0390] 9.4) Structural and phylogenetic analyses provide further insights into the catalytic mechanism and evolutionary history of HDH.

[0391] 9.4.1) The homology model of AbHDH was constructed based on the crystal structure of Populus tremuloides sinapinic acid dehydrogenase (PtSAD; PDB: 1YQD) ( Fig.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 bilobed structure with a highly 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 the phosphate-stabilized residues S214, T215, S216, and K219 in PtSAD suggests that AbHDH is an NADPH-dependent oxidoreductase. Also typical of ZADH, AbHDH appears to use a tetrad of cysteine ​​residues (C105, C108, C111, and C119) close to the surface of the protein to bind to the structural Zn 2+, and binds to the catalytic Zn in the active site 2+ .

[0392] 9.4.2) The catalytic mechanism of AbHDH was elucidated by docking the substrate scopolamine molecule into the active site using the Maestro / Glide software package ( Fig.47 The most favorable binding mode positions the aldehyde group of the substrate on the catalytic Zn 2+ and NADPH hydride donor The docking results and general mechanism of ZADH (see Bomati, EK, Noel, JP, Structural and kinetic basis for substrate selectivity in Populus tremuloidessinapyl alcohol dehydrogenase. Plant Cell. 17, 1598–1611 (2005)) suggest the following catalytic mechanism of AbHDH. In the absence of substrate, the catalytic Zn in the active site 2+ Stabilized by C52, H74, C168 and a water molecule, which is positioned by polar interactions with S54 and displaced upon binding to scopolaldehyde. The nucleophilic attack of dihydronicotinamide hydride on the aldehyde carbonyl is caused by the catalytic Zn 2+ The oxygen anion intermediate is stabilized by the interaction with NADP + The proton shuttling between the ribose group and S54 causes protonation.

[0393] 9.5) To confirm whether orthologous oxidoreductases catalyze scopolamine biosynthesis in other TA-producing Solanaceae species, variants of the AbHDH coding sequence were identified from the transcriptomes of Datura stramonium and Datura grandiflora using tBLASTx searches ( Fig.48 The HDH activity of the two identified orthologs (DiHDH, DsHDH) was validated by co-expressing the yeast codon-optimized sequence with an additional copy of the flux-limiting DsH6H from a low-copy plasmid in CSY1292 and measuring scopolamine production in medium supplemented with 1 mM ritolin. DsHDH showed the highest substrate consumption and product accumulation of the tested variants ( Fig.49 ).

[0394] 9.6) The overexpressed medicinal TA biosynthetic branch containing the best enzyme variants and flux-limiting enzymes was integrated into a platform yeast strain. Strain CSY1294 was constructed by integrating yeast codon-optimized WfPPR and AbUGT, DsHDH, and a second copy of DsH6H into CSY1292. Scopolamine production from fed ritolin was verified in CSY1294 ( Fig.50 ).

[0395] Example 10: Yeast engineered to perform esterification of acyl donors and acceptors for production of TA scaffolds.

[0396] Yeast strains can be engineered to express enzymes that catalyze the esterification of activated acyl donor compounds and acyl acceptor compounds to produce a variety of TA scaffolds ( Figure 2 , 3 ). Activation of the acyl donor group can be achieved by engineering acyl donor-producing yeast strains to incorporate enzymes that attach chemical moieties with high group transfer potential, such as coenzyme A (CoA) or glucose (glucosides), 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 UDP-glycosyltransferases. Examples of esterifying enzymes that can be used to catalyze the esterification of activated acyl donor compounds and acyl acceptors such as tropine and pseudotropine are acyltransferases, including serine carboxypeptidase-like acyltransferases (SCPL-AT) and BAHD-type acyltransferases. When expressed in a heterologous host such as yeast, the coding sequences of such acyltransferases can be modified to increase their activity.

[0397] 10.1) In plants where SCPL-AT is commonly found naturally occurring, the coding sequence of SCPL-AT includes an N-terminal signal peptide that directs the nascent polypeptide to the endoplasmic reticulum (ER). Once localized in the ER, SCPL-AT polypeptides are transported via the secretory transport pathway through the Golgi apparatus to the vacuole lumen, where they are found to exhibit activity. During this ER-to-vacuole transport process, they undergo several post-translational modifications ( Fig.51 ), including but not limited to signal peptide cleavage, N-glycosylation, removal of internal propeptide sequences and disulfide bond formation (see Stehle, F., Stubbs, MT, Strack, D. and Milkowski, C. Heterologous expression of a serine carboxypeptidase-like acyltransferase and characterization of the kinetic mechanism, FEBS Journal, 275, (2008)). However, since the intracellular transport pathways and post-translational modification patterns vary between organisms, the expression of SCPL-AT in heterologous hosts may result in incorrect subcellular localization and / or incorrect post-translational modifications for activity. For example, when expressed in yeast, the coding sequence of SCPL-AT, such as ritolin synthase (LS) (Table 1), can be modified to increase activity.

[0398] 10.1.1) The signal peptide sequence can affect the processing and localization of SCPL-AT in yeast.

[0399] 10.1.1.1) The presence of a putative N-terminal signal peptide in AbLS suggests that it follows the expected SCPL ER to vacuole trafficking pathway in plants. AbLS localization in yeast was examined by expressing N-terminal and C-terminal GFP fusions of AbLS from low copy plasmids in CSY1294. Fluorescence microscopy revealed that the N-terminal fusion (GFP-AbLS) colocalized with the vacuolar membrane stain FM4-64 ( Fig.52 No fluorescence was detected for the C-terminal fusion (AbLS-GFP), which is consistent with reports that the native C-terminus is critical for the stability of SCPL acyltransferase (see Stehle, F., Stubbs, MT, Strack, D., and Milkowski, C. Heterologous expression of a serine carboxypeptidase-like acyltransferase and characterization of the kinetic mechanism, FEBS Journal, 275, (2008)).

[0400] 10.1.1.2) Vacuolar sequestration of SCPL-AT in yeast may prevent access to the cytosolic substrate pool, as yeast may lack the necessary tonoplastic transporters present in plants for exchanging secondary metabolites with the cytosol. To determine whether forced localization of AbLS to other yeast compartments, perhaps by improving access to cytosolic metabolites, could achieve activity, the wild-type N-terminal SP sequence was replaced with a set of N-terminal signal sequences taken from yeast proteins that target the vacuolar lumen (Prc1p and Pep4p), the lumen-facing tonoplast (Dap2p), the trans-Golgi network (Och1p), the lumen-facing ER membrane (Mns1p), and the mitochondrial matrix (Cit1p) ( Fig.53 ). The wild-type SP was also completely removed, and for another variant, the canonical peroxisome targeting sequence (PTS1) was attached to the C-terminus. These chimeric AbLS variants were expressed from a high copy plasmid in CSY1294, and transformants were screened for activity by LC-MS / MS after 96 h of growth in selective medium. No production of ritolin or downstream intermediates was observed for any of the variants ( Fig.53 ).

[0401] 10.1.2) Incorrect post-translational processing of SCPL-AT in yeast may prevent expression of active enzyme.

[0402] 10.1.2.1) Protein N-glycosylation patterns vary between yeast and plants, and previous reports have shown that correct N-glycosylation of various plant enzymes is essential 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 aβ-glucosidase from Putranjiva roxburghii. Int. J. Biol. Macromol. 112, 490–498 (2018); see also Podzimek, T. et al., N-glycosylation of tomato nuclease TBN1 produced in N. benthamiana and its effect on the enzyme activity. Plant Sci. 276, 152–161 (2018); see also Strasser, R., Plant protein glycosylation. Glycobiology. 26, 926–939 (2016)). Computer simulation analysis of the AbLS peptide predicted four N-glycosylation sites (N152, N320, N376, N416), and no O-glycosylation of the protein was detected in Nicotiana benthamiana ( Fig.54 ). Wild-type AbLS, each of the four N→Q mutants (in which the mutation from N to Q abolishes N-glycosylation (23)), or the quadruple N→Q mutant with a C-terminal HA tag were expressed in CSY1294 and N. benthamiana, and the glycosylation profiles were compared by Western blotting. Whereas wild-type AbLS, the N→Q single mutant, and the quadruple mutant all appeared as a single band in N. benthamiana, indicating a monoglycosylation state, only the quadruple N→Q mutant produced a single band in yeast; all other variants showed double or triple bands, indicating a combination of multiple glycosylation states ( Fig.55 However, since the dense portions of the two wild-type AbLS bands in yeast showed partial overlap with the dense portions of wild-type AbLS in tobacco, at least some fraction of the yeast-expressed AbLS must be in the correct glycosylation state, and misglycosylation is unlikely to explain the complete lack of AbLS activity in yeast. Figure 54-55 ).

[0403] 10.1.2.2) A subset of SCPL acyltransferases, including sinapoylglucose:choline sinapoylglucose acyltransferase (AtSCT) from Arabidopsis thaliana and avenin synthase (AsSCPL1) from Avena strigosa, have been shown to contain an internal propeptide linker that is proteolytically removed to generate an active heterodimer linked by a disulfide bond (see Shirley, AM, Chapple, C., Biochemical characterization of sinapoylglucose:choline sinapoyltransferase, a serine carboxypeptidase-like protein that functions as an acyltransferase in plant secondarymetabolism. J. Biol. Chem. 278, 19870–19877 (2003); see also Mugford, ST et al., Aserine carboxypeptidase-like acyltransferase is required for synthesis ofantimicrobial compounds and disease resistance in oats. Plant Cell. 21, 2473–2484 (2009). Comparison of the AbLS amino acid sequence with those of previously characterized plant serine carboxypeptidases and SCPL acyltransferases revealed the presence of an internal sequence of 25 to 30 residues that aligns with the highly variable propeptides of AtSCT, AsSCPL1, and wheat carboxypeptidase 2 (TaCBP2), indicating that AbLS also undergoes endoproteolytic cleavage to form heterodimers ( Fig.56 ). Furthermore, homology modeling of AbLS indicated that the predicted internal propeptide blocked the active site and was therefore required for its removal ( Fig.57 However, wild-type AbLS expressed in N. benthamiana does not appear to undergo proteolytic cleavage, as the expected ∼20-25 kDa C-terminal fragment was not detected by Western blotting under disulfide-reducing conditions ( Fig.54 , 55 ). Since the putative propeptide does not appear to be cleaved or removed in planta, it is possible that AbLS adopts a native conformation in planta that displaces the propeptide away from the active site, but differences in the biochemical environment of the yeast secretory pathway and / or vacuole prevent this displacement, thereby blocking activity.

[0404] To address this failure mode, a split AbLS control was constructed in which the N- and C-terminal domains flanking the putative propeptide linker were ex...

Claims

1. An engineered non-plant cell that produces a tropane alkaloid product, wherein the tropane alkaloid product is selected from the group consisting of hyoscyamine, atropine and scopolamine, wherein the engineered non-plant cell comprises a heterologous coding sequence encoding hyoscyamine dehydrogenase (HDH), wherein the HDH consists of an amino acid sequence as shown in SEQ ID No.: 13, SEQ ID No.: 14 or SEQ ID No.:

15.

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

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

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

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

6. The cell of any one of claims 1-3, wherein the engineered cell further 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 ureahydrolase, agmatinease, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidone synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylalanine ammonia lyase, tyrosine ammonia lyase, phenylpyruvate reductase, 4-coumarate-CoA ligase, 3-phenyllactate UDP-glucosyltransferase 84A27, ritolin synthase, ritolin mutase, scopolamine 6β-hydroxylase / dioxygenase, and cocaine synthase.

7. The cell of claim 2, wherein endogenous arginine metabolism in the cell is altered by modifying 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-γ-glutamyl-phosphate reductase, acetylornithine aminotransferase, ornithine acetyltransferase, ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lyase, and arginase.

8. The cell of claim 2, wherein endogenous phenylalanine and phenylpropanoid metabolism in the cell is altered by modifying 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 a pentafunctional AROM polypeptide, a chorismate synthase, a chorismate mutase, a prephenate dehydratase, an aromatic aminotransferase, and a phenylacrylic acid decarboxylase.

9. The cell of claim 2, wherein the endogenous polyamine regulatory mechanism in the cell is altered by modifying 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 antienzyme, polyamine oxidase, spermidine synthase, spermine synthase, polyamine transporter and polyamine permease.

10. The cell of claim 2, wherein endogenous acetate metabolism in the cell is altered by modifying 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 of claim 2, wherein endogenous glycoside metabolism in the cell is altered by modifying 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-β-glucosidase and steryl-β-glucosidase.

12. The cell according to any one of claims 7-11, wherein the modification of one or more coding sequences is selected from the group consisting of: feedback inhibition relief mutations in biosynthetic enzymes or regulatory protein genes inherent to the cell, transcriptional regulation modifications of biosynthetic enzyme genes inherent to the cell, and inactivating mutations in enzymes or proteins inherent to the cell.

13. The cell of any one of claims 1-3, 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-like acyltransferase domain such that the acyltransferase domain can be functionally expressed in a subcellular compartment of the engineered cell.

14. The cell of any one of claims 1-3, wherein the cell produces a precursor for a tropane alkaloid product selected from the group consisting of agmatine, N-carbamoylputrescine, N-methylputrescine, 4-methylaminobutyraldehyde, N-methylpyrrolinium, 4-(1-methyl-2-pyrrolidinyl)-3-oxobutyric acid, tropinone, tropine, pseudotropine, ecgonine, methylecgonine, coenzyme A covalently bonded to phenyllactic acid through a thioester bond, or a sugar covalently bonded to cinnamic acid, ferulic acid, coumaric acid, or phenyllactic acid through a glycosidic bond.

15. The cell of any one of claims 1-3, wherein the cell produces a derivative of a scopolamine alkaloid product selected from the group consisting of p-hydroxyatropine, p-hydroxyscopolamine, p-fluscopolamine, p-chloroscopolamine, p-bromoscopolamine, p-fluoroscopolamine, p-chloroscopolamine, p-bromoscopolamine, N-methylscopolamine, N-butylscopolamine, N-methylscopolamine, N-butylscopolamine, N-acetylscopolamine, and N-acetylscopolamine.

16. The cell according to any one of claims 1-3, wherein transport of one or more TAs, one or more TA precursors and / or one or more TA derivatives across an intracellular membrane or across the plasma membrane is modified in the cell.

17. The cell of claim 16, wherein the modified transport is achieved 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: multidrug and toxin efflux transporters, nitrate / peptide family transporters, ATP binding cassette transporters, and pleiotropic resistance transporters.

18. A method for producing a tropane alkaloid product or a derivative of a tropane alkaloid product, comprising (a) culturing the cell according to any one of claims 1 to 17 under conditions suitable for protein production; (b) adding a starting compound to the cell culture; and (c) recovering the tropane alkaloid product or the derivative of the tropane alkaloid product from the culture.

19. The method of claim 18, wherein the cells are cultured in a fed-batch or batch fermentation.

20. The method of claim 18 or 19, wherein the starting compound added to the cell culture is a sugar or a substrate which contains one or more sugars or is converted to one or more sugars during microbial fermentation.

21. The method according to claim 18 or 19, 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 converted into one or more amino acids during microbial fermentation.

22. The method of claim 18 or 19, wherein the starting compound added to the cell culture is a precursor of a tropane alkaloid product.

23. The method of claim 18 or 19, wherein the tropane alkaloid product or the derivative of the tropane alkaloid product is recovered by a process comprising liquid-liquid extraction, chromatography, distillation or recrystallization.

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