De Novo Production of the Plant-Derived Tropine and Pseudotropine

Letter Cite This: ACS Synth. Biol. 2019, 8, 1257−1262

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De Novo Production of the Plant-Derived Tropine and Pseudotropine in Yeast Yu Ping,†,‡,# Xiaodong Li,†,‡,# Wenjing You,†,‡,# Guoqiang Li,†,‡,# Mengquan Yang,†,‡ Wenping Wei,†,‡ Zhihua Zhou,*,†,‡ and Youli Xiao*,†,‡ †

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CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China ‡ University of Chinese Academy of Sciences, Beijing, 100039, China S Supporting Information *

ABSTRACT: Tropine and pseudotropine with opposite stereospecific configurations as platform compounds are central building blocks in both biosynthesis and chemical synthesis of pharmacologically important tropane and nortropane alkaloids. The supply of plant-derived tropine and pseudotropine still heavily depends on either plant extraction or chemical synthesis. Advances in synthetic biology prompt the microbial synthesis of various valuable chemicals. With the biosynthetic pathway elucidation of tropine and pseudotropine in several Solanaceae plants, the key genes were sequentially identified. Here, the enzymes responsible for converting N-methylpyrrolinium into tropine and pseudotropine from Anisodus acutangulus were characterized. Reconstruction of the six-step biosynthetic pathways into Saccharomyces cerevisiae provides cell chassis producing tropine and pseudotropine with 0.13 and 0.08 mg/L titers from simple feedstocks in a shake flask, respectively. The strains described not only offer alternative sources of these central intermediates and their derived alkaloids but also provide platforms for pathway enzyme discovery. KEYWORDS: tropine, pseudotropine, tropane alkaloid, Anisodus acutangulus, Saccharomyces cerevisiae

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not been elucidated to date.8 The traditional extraction scale from plant material could not meet the higher demand for TAs supply, and new approaches for the production of key metabolites urgently need to be developed. With a synthetic biology strategy, reconstitution of the plant biosynthetic pathway in microbial hosts is a promising path and some complex plant-derived alkaloids have been successfully produced in engineered yeast,9 such as dihydrosanguinarine,10 strictosidine,11 opioids,12 and noscapine.13 Previously, we have successfully built a microbial cell factory for heterologous production of N-methylpyrrolinium with a final titer of 17.82 mg/L in Saccharomyces cerevisiae.14 We conducted the biosynthesis pathway of TAs using the transcriptomic approach in Anisodus acutangulus which is a well-known traditional Chinese herb (San-Fen-San in Chinese) rich in TAs. Type III polyketide synthases involved in the tropinone ring formation (condensation) was deduced.15 Only one transcript (comp83664_c1_seq1) with full length was identified by searching against the A. acutangulus transcriptome database. This transcript is probably involved in the tropane alkaloids biosynthesis and annotated as type III polyketide synthase. The enzymatic assay with malonyl-Coenzyme A and N-methylpyrrolinium as substrates was performed to test the ability of condensation. Meanwhile, Bedewitz et al. reported

INTRODUCTION As documented in Ebers Papyrus, one of the oldest and most important herbal papyri of ancient Egypt in 1500 BC, tropane alkaloids (TAs) from the plant Hyoscyamus were recorded for the medicinal use of releasing abdominal pain.1 Since then, they have been used for centuries in folk medicine and modern pharmacology due to their anticholinergic activities.2 “Tropane” is named by its unique bicyclic saturated structure (Nmethyl-8-azabicyclo[3.2.1]octane) (Figure 1, the red colored skeleton). With this common tropane ring, TAs are identified as a group of more than 200 specialized metabolites naturally produced in most Solanaceous plants.3 These structurally diverse compounds, such as hyoscyamine, scopolamine, and calystegines are believed to be biosynthesized in two branches from the two biosynthetic intermediates, tropine and pseudotropine, with opposite stereospecific configurations, respectively (Figure 1).2 In the later TAs biosynthetic pathway, tropine is supposed to condense with activated (R)-phenyllactate (phenyllactyl-CoA) to produce littorine. The enzymes catalyzing the conversion of phenylalanine into phenyllactate, namely ArAT4 and AbPPAR were characterized in Atropa belladonna.4,5 Littorine is further rearranged into hyoscyamine aldehyde by CYP80F1 followed by reduction to produce hyoscyamine.6 Then hyoscyamine is hydroxylated into anisodamine followed by epoxidation to form scopolamine, which is catalyzed by the bifunctional enzyme H6H.7 However, the biosynthetic pathway from pseudotropine to calystegines has © 2019 American Chemical Society

Received: April 4, 2019 Published: June 10, 2019 1257

DOI: 10.1021/acssynbio.9b00152 ACS Synth. Biol. 2019, 8, 1257−1262

Letter

ACS Synthetic Biology

Figure 1. Biosynthetic pathway of tropine and pseudotropine and their derived representative medicinal natural products. Abbreviations: ODC, ornithine decarboxylase; PMT, putrescine N-methyltransferase; MPO (also named AaDAO in A. acutangulus), N-methylputrescine oxidase; SAM, S-adenosyl-L-methionine; PKS, polyketide synthase; TRI, tropinone reductase I; TRII, tropinone reductase II; ArAT4, aromatic amino acid aminotransferase; PPAR, phenylpyruvic acid reductase; CYP80F1, littorine mutase; H6H, hyoscyamine 6β-hydroxylase. The reconstituted biosynthetic pathways in yeast are in green background and the intermediates detected are in the red squares.

97.4% and 96.3% to those of A. belladonna16 were selected as candidates to synthesize tropinone from N-methylpyrrolinium (Figures S2 and S3). To assess the biochemically functional role of AaPYKS, the enzyme was heterologously expressed and purified in E. coli (Figure S4). With malonyl-Coenzyme A generated by AAE13 in situ from malonic acid and Coenzyme A as cosubstrate,19 AaPYKS indeed utilizes N-methylpyrrolinium to produce 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid ([M + H]+ m/z = 186.1125) in vitro evidenced by the same MS fragmentation identical to the report (Figure 2A and Figure S5A).16 Meanwhile, the side products hygrine and cuscohygrine were also detected according to the MS fragmentation (Figure S5B−E). To confirm the biochemical function of AaCYP82M3, we also cloned the cytochrome P450 reductase (CPR) genes from A. acutangulus (AaCPR1−3), which takes charge of transporting electrons for P450 catalysis by searching transcriptome data using ATR1 and ATR2 from Arabidopsis thaliana as templates. Sequence alignments show 97.5% identity between AaCPR1 and AaCPR2, while 65.7% identity for AaCPR1 and AaCPR3 (Figure S6). Microsomes were isolated from yeast expressing AaCYP82M3 and AaCPRs. Incubation of these microsomes, 4-(1-methyl-2pyrrolidinyl)-3-oxobutanoic acid provided by AaPYKS assay, along with DTT and the cofactor NADPH, a product with m/z 140.107 ([M + H]+) at the same retention time as the tropinone standard, was detected by liquid chromatography− high resolution mass spectroscopy (LC−HRMS) (Figure 2B and Figure S7). As for the branching steps to produce tropane and nortropane alkaloids, AaTRI and AaTRII from A. acutangulus were also cloned. A previous study demonstrated

that two genes, AbPYKS and AbCYP82M3 are involved in tropinone biosynthesis in Atropa belladonna.16 As A. acutangulus and A. belladonna are in the same plant family of Solanaceae. In this study, by comparing the homologous genes from the transcriptomic data published,15 we were able to further characterize the biosynthetic genes from A. acutangulus and reconstitute the plant-pathway into S. cerevisiae for de novo production of tropine and pesudotropine for the first time from simple carbon and nitrogen sources.

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RESULTS AND DISCUSSION Biochemical Characterization of AaPYKS-AaCYP82M3-AaTRI/AaTRII. Starting from the common biosynthetic precursor, namely, N-methylpyrrolinium, tropine and pseudotropine are generated by three structural genes, pks, P450, and TRI (tropinone reductase I) or TRII (tropinone reductase II), respectively (Figure 1).16−18 Previously, with the microbial hosts we had built for biosynthesis of Nmethylpyrrolinium in hands,14 the only one type III PKS (comp83664_c1_seq1) was identified from the transcriptome database of A. acutangulus hairy roots. Phylogenetic analysis was performed to compare the public plant polyketide synthases with AaPYKS sequence (Figure S1). To expand the heterologous biosynthetic pathway further to produce tropine and pseudotropine on the basis of a previous study,14 we put effort to identify the related enzymes in A. acutangulus considering that it is reported to possess the high abundance of TAs in vivo and also a convenient plant source for our research.14,15 By transcriptome analysis, the corresponding PKS (AaPYKS) and P450 (AaCYP82M3) with identities of 1258

DOI: 10.1021/acssynbio.9b00152 ACS Synth. Biol. 2019, 8, 1257−1262

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Figure 2. Characterization of AaPYKS, AaCYP82M3, AaTRI, and AaTRII enzymatic activities. (A) EIC profiles of AaPYKS assay; (i) AaPYKS assay; (ii) with boiled AaPYKS. The [M + H]+ m/z value of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid calculated is 186.1125. (B) EIC profiles of AaCYP82M3 assay using AaCPRs for electron transfer: (i) tropinone standard; (ii) AaCYP82M3 assay using AaCPR1 for electron transfer; (iii) AaCYP82M3 assay using AaCPR2 for electron transfer; (iv) AaCYP82M3 assay using AaCPR3 for electron transfer; (v) BY4742. The [M + H]+ m/z value of tropinone calculated is 140.107. (C) EIC profiles of AaTRI and AaTRII assay: (i) tropine standard; (ii) AaTRI assay; (iii) with boiled AaTRI; (iv) pseudotropine standard; (v) AaTRII assay; (vi) with boiled AaTRII.

the activity of AaTRI in vivo by overexpression in a hairy root culture.20 However, the in vitro biochemical properties are really lacking. As the first step to validate in vitro activities, AaTRI and AaTRII were expressed in E. coli followed by purification to homogeneity (Figure S4). Enzymes were added into the mixture of tropinone and NADPH, and the spectra of the obtained products were consistent with the MS fragmentation of tropine and pseudotropine standards (Figure 2C, Figure S8). To assess the catalytic efficiency, the kinetic parameters of AaTRI and AaTRII were determined for tropinone (Table S1, Figure S9). AaTRI exhibited a lower Km value and higher specificity constant (Km = 0.170 ± 0.020 mM, kcat/Km = 61.5 s−1 mM−1) than that of DsTRI (Km = 4.18 ± 0.51 mM, kcat/Km = 0.574 s−1 mM−1), BaTRI (Km = 2.65 ± 0.19 mM, kcat/Km = 1.10 s−1 mM−1),21 and WsTRI (Km = 1.27 mM, kcat/Km = 7.49 s−1 mM−1)22 representing higher catalytic efficiency. Whereas AaTRII shows lower substrate-binding affinity (Km = 0.076 ± 0.008 mM) compared with StTRII (Km = 0.033 mM),23 DsTRII (Km = 0.048 mM)24 and HnTRII (Km = 0.034 mM),25 the turnover rate of AaTRII (kcat = 13.62 s−1) was higher than that of DsTRII (kcat = 2.73 ± 0.16 s−1).24 Together, the above results clearly illustrated the activities of AaPYKS, AaCYP82M3, AaTRI, and AaTRII, providing guidance for the tropine and pseudotropine biosynthetic pathway reconstitution in vivo. Reconstitution of Tropinone Biosynthetic Pathway in S. cerevisiae. To achieve de novo biosynthesis of tropine and pseudotropine in yeast (Figure 3), we set out to build a yeast cell factory producing the last common central intermediate, namely tropinone. For stable protein expression, we integrated

Figure 3. Scheme overview of pathway construction in yeast: (A) pathway construction for tropine production; (B) pathway construction for pseudotropine production.

EcODC and AtPMT into the genome of ΔBY4742-AaDAO3 (namely AaMPO3)-SAM2 with deletions of three aldehyde dehydrogenases (ALD4, ALD5, and HFD1) and overexpression of SAM2, which was built in our previous study.14 The resultant base N-methylpyrrolinium-producing strain PL1 1259

DOI: 10.1021/acssynbio.9b00152 ACS Synth. Biol. 2019, 8, 1257−1262

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Figure 4. Biosynthesis of tropinone, tropine, and pseudotropine in yeast: (A) profiles of tropinone production in strain PL2-ACC1; (B) profiles of tropine production in strain PL3-TRI; (C) profiles of pseudotropine production in strain PL3-TRII.

definitely produces N-methylpyrrolinium as analyzed by LC− HRMS with a final concentration of 5.06 mg/L in a shake flask (Figure S10). On the basis of this strain, AaPYKS and AaCYP82M3 coupled with a cytochrome P450 reductase (CPR) partner from Arabidopsis thaliana (ATR1) were introduced into the YPRC△15 integration site of PL1 using a combination of TEF1 promoter/ENO2 terminator, TCCTDH promoter/PDC1 terminator, and TEF2 promoter/ PYK1 terminator as expression cassettes (Figure 3). Since two molecules of malonyl-CoA are required in the step catalyzed by AaPYKS, an extra copy of endogenous ACC1 from yeast encoding acetyl-CoA carboxylase was simultaneously introduced into PL1 to allow for ample cosubstrate production under the control of the PGK1 promoter. The resultant strain PL2-ACC1 was cultivated at 30 °C for 120 h in a shake flask, and the fermentation supernatant was analyzed using LC− HRMS. As indicated in Figure S11, strain PL2-ACC1 synthesized a compound ([M + H]+ m/z = 140.107) with the same fragmentation pattern as the tropinone standard. In agreement with in vitro activities of AaPYKS and AaCYP82M3, we also detected the formation of side-product hygrine (Figure S11). The extracellular titer of tropinone in the medium reached 0.10 mg/L after 120 h based on the standard curve (Figure 4A and Figure S12A). The concentration of Nmethylpyrrolinium in the fermentation supernatant decreased 1.2 fold compared to that of PL1 to a final concentration of 2.33 mg/L (Figure S13A). Biosynthesis of Tropine and Pseudotropine in S. cerevisiae. To complete the synthesis of tropine and pseudotropine, AaTRI and AaTRII were introduced into strain PL2-ACC1, respectively, generating strain PL3-TRI and PL3-TRII. Metabolites analysis showed production of tropine in PL3-TRI and pseudotropine in PL3-TRII instead of PL2ACC1 (Figure S14). After 120 h of growth in a shake flask, 0.13 mg/L tropine and 0.08 mg/L pseudotropine accumulated in the fermentation supernatant of PL3-TRI and PL3-TRII,

respectively (Figure 4B,C). The tropinone concentration in the supernatant indicated extremely low yields compared to that of PL2-ACC1, namely 0.014 mg/L and 0.007 mg/L suggesting excellent performance of AaTRI and AaTRII in vivo. However, the N-methylpyrrolinium remained in the same concentration level as PL2-ACC1 (Figure S13B,C). On the basis of these results, we further attempted to improve the titer of the two target compounds, namely tropine and pseudotropine. Since the reaction catalyzed by AaDAO3 (MPO) releases H2O2 as coproduct which may impair the pathway enzymes, we explored whether scavenging H2O2 would benefit. However, the addition of 1 mM sodium-L-ascorbate to the medium culturing PL3-TRI and PL3-TRII led to no increase in tropine and pseudotropine titers (PL3-TRI, 0.13 mg/L; PL3-TRII, 0.08 mg/L) (Figure S15). In this study, we demonstrated the capability of yeast to produce the critical intermediates (tropine and pseudotropine) of tropane alkaloids, however, the present yields are still quite low now. This is perhaps not unexpected given the high reactivity and instability of some pathway intermediates, for example, N-methylpyrrolinium and 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. Further enhancing the titers of tropine and pseudotropine is of great significance as an initial step to meet the commercial need. We suppose strain metabolic engineering combined with protein engineering will realize improved yields in the future. In the biosynthetic pathway of tropine and pseudotropine, the related enzymes AaCYP82M3, AaTRI, and AaTRII all utilize the cofactor NADPH to transfer the electron. Previous studies demonstrated the beneficial effect of increasing the supply of NADPH on the production of strictosidine and noscapine, via overexpressing ZWF1 and Tyr1.11,13 Therefore, we surmise introducing the NADPH regeneration system would result in higher titers of tropine and pseudotropine. Meanwhile, introducing an extra copy of AaCYP82M3 into PL3-TRI and PL3-TRII may also benefit the production of tropine and pseudotropine considering that 1260

DOI: 10.1021/acssynbio.9b00152 ACS Synth. Biol. 2019, 8, 1257−1262

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The AaTRI and AaTRII activities were assayed separately at 37 °C in 100 μL of reaction system containing 1 mM tropinone, 1 mM NADPH, 100 mM potassium phosphate buffer (pH 8.0), and 20 μM protein (AaTRI or AaTRII) for 30 min. The reaction was stopped by adding 100 μL of methanol, and then the mixture was centrifuged to remove the precipitant followed by LC−HRMS analysis. The standard reaction mixture for kinetics parameters determination of AaTRI and AaTRII contained 0.1 mM NADPH, 100 mM potassium phosphate buffer (pH 6.4), 0.16 μM AaTRI or 0.10 μM AaTRII, and different concentrations of tropinone (0.01−4 mM for AaTRI, 0.01−0.25 mM for AaTRII). The decrease in absorption at 340 nm was used to calculate the reaction velocity. The results were from triplicate experiments. Pathway Construction in Yeast. ΔBY4742-AaDAO3SAM2 was used as the starting strain in our study.14 The promoters and terminators were amplified from S288C genomic DNA with exception of PTCCTDH from plasmid using the primers listed in Table S4. The marker genes (HIS3, URA3, LEU2) used for strain selection were amplified from corresponding pESC vectors. The desired genes, promoters, terminators, markers, and homologous arms of integration sites were flanked by ∼80 bp homologous to either the adjacent DNA fragment or the yeast genomic integration site. The assembled fragments by overlap PCR were cotransformed into yeast for integration.28 Transformants were selected on either amino acid-lacking or antibiotic-containing plates and further verified by PCR analysis. The detailed information about strains and genes for pathway construction were listed in Table S2 and S3. Shake Flask Cultivation. The yeast strains were restreaked and single colonies were inoculated into 4 mL of YPD medium. After cultivation at 30 °C shaking at 200 rpm for 24 h, the cultures were diluted into 50 mL shake flasks containing 10 mL of YPD medium with a starting optical density (OD600) of 0.1 (1 mM sodium-L-ascorbate was added when needed). Then the cultures were grown at 30 °C with constant shaking at 250 rpm. Samples were taken at certain time points for OD600 measurement and metabolite analysis by LC−HRMS.

the AaCYP82M3-catalyzed reaction maybe a limiting step in the biosynthetic pathway. Moreover, further increasing the malonyl-CoA availability through improving the precursor acetyl-CoA pool may lead to improved titers, which has been demonstrated in producing some valuable plant-derived compounds.26,27 Protein engineering has been widely applied to achieve higher activity and broader substrate scope. We observed large amount of N-methylpyrrolinium accumulation in strain PL3-TRI and PL3-TRII (Figure S13) compared to other metabolites, which promote us to hypothesize that more sufficient consumption of intermediates will increase the final products titer. Thus, we expect efforts on engineering the biosynthetic pathway enzymes of tropine and pseudotropine will realize higher titers in future work.

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CONCLUSIONS Herein, we successfully characterized the function of AaPYKS, AaCYP82M3, AaTRI, and AaTRII from A. acutangulus in vitro. With synthetic biology strategy, we reconstructed the six-step biosynthetic pathways in Saccharomyces cerevisiae. The resultant cell factory produced tropine and pseudotropine with 0.13 and 0.08 mg/L titers, respectively, which is the first report to our knowledge. Development of these strains serves as an important step toward heterologous biosynthesis of bulk tropine and pseudotropine-derived alkaloids. More importantly, these yeast platforms will allow us to further explore the miraculous biosynthetic pathway of TAs in plants.

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METHODS Plasmids Construction. Genes encoding AaPYKS, AaCYP82M3, AaTRI, AaTRII, and AaCPRs (AaCPR1, AaCPR2, and AaCPR3) were amplified with cDNA from hairy root of Anisodus acutangulus as template as reported in the method.14 AaPYKS was ligated into pET24a using BamHI and XhoI restriction sites generating pET24a-AaPYKS. Recombinant vector pESC-URA-AaCYP82M3 was constructed by inserting AaCYP82M3 into the pESC-URA between BamHI and KpnI. The genes encoding AaTRI and AaTRII were separately constructed into pET28a using BamHI and NotI. AaCPRs (AaCPR1, AaCPR2, and AaCPR3) were introduced into pESC-HIS via BamHI and XhoI for expression, respectively. The plasmids and primers used were listed in Tables S2 and S4. Enzymatic Assay. To test the activity of AaPYKS, the assay mixture (50 μL) contained 10 mM malonic acid, 10 mM CoA, 10 mM ATP, 2 mM N-methylpyrrolinium (NMP), 10 μM AAE13 (AMP-dependent synthetase catalyzing the formation of malonyl-CoA from malonic acid and CoA) and 20 μM AaPYKS in 10 mM PBS at pH 6.8. After incubation at 30 °C for 30 min, an equal volume of methanol was added to stop the reaction followed by centrifugation. The supernatant was subjected to LC−HRMS analysis. The AaCYP82M3 activity was measured in a coupled reaction with AaPYKS given the unavailability of the direct substrate 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. The AaPYKS assay was first performed in a volume of 50 μL at 30 °C for 30 min followed by addition of 500 μg of microsomal protein, 1 mM NADPH, and 1 mM DTT. The reactions were incubated for 12 h at 30 °C. Reactions were quenched by adding an equal volume of methanol. The mixture was centrifuged, and the supernatant was further analyzed by LC− HRMS.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.9b00152. Methods of strains and medium, protein preparation and analytic methods of metabolites; supplementary figures, tables, and references (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-21-54924226. E-mail: [email protected]. *Tel.: +86-21-54924050. E-mail: [email protected]. ORCID

Mengquan Yang: 0000-0001-6473-1409 Youli Xiao: 0000-0002-4803-3333 Author Contributions #

These authors contributed equally to this work. Y.X. conceived the project. Y.X. Y.P. X.L. and Z.Z. designed the experiments. Y.P., W.Y., G.L., and W.W. carried out the 1261

DOI: 10.1021/acssynbio.9b00152 ACS Synth. Biol. 2019, 8, 1257−1262

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ACS Synthetic Biology

(15) Cui, L., Huang, F., Zhang, D., Lin, Y., Liao, P., Zong, J., and Kai, G. (2015) Transcriptome exploration for further understanding of the tropane alkaloids biosynthesis in Anisodus acutangulus. Mol. Genet. Genomics 290, 1367−1377. (16) Bedewitz, M. A., Jones, A. D., D’Auria, J. C., and Barry, C. S. (2018) Tropinone synthesis via an atypical polyketide synthase and P450-mediated cyclization. Nat. Commun. 9, 5281. (17) Humphrey, A. J., and O’Hagan, D. (2001) Tropane alkaloid biosynthesis. A century old problem unresolved. Nat. Prod. Rep. 18, 494−502. (18) Nakajima, K., and Hashimoto, T. (1999) Two tropinone reductases, that catalyze opposite stereospecific reductions in tropane alkaloid biosynthesis, are localized in plant root with different cellspecific patterns. Plant Cell Physiol. 40, 1099−1107. (19) Chen, H., Kim, H. U., Weng, H., and Browse, J. (2011) Malonyl-CoA synthetase, encoded by ACYL ACTIVATING ENZYME13, is essential for growth and development of Arabidopsis. Plant Cell 23, 2247−2262. (20) Kai, G., Li, L., Jiang, Y., Yan, X., Zhang, Y., Lu, X., Liao, P., and Chen, J. (2009) Molecular cloning and characterization of two tropinone reductases in Anisodus acutangulus and enhancement of tropane alkaloid production in AaTRI-transformed hairy roots. Biotechnol. Appl. Biochem. 54, 177−186. (21) Qiang, W., Xia, K., Zhang, Q., Zeng, J., Huang, Y., Yang, C., Chen, M., Liu, X., Lan, X., and Liao, Z. (2016) Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids. Phytochemistry 127, 12−22. (22) Kushwaha, A. K., Sangwan, N. S., Trivedi, P. K., Negi, A. S., Misra, L., and Sangwan, R. S. (2013) Tropine forming tropinone reductase gene from Withania somnifera (Ashwagandha): biochemical characteristics of the recombinant enzyme and novel physiological overtones of tissue-wide gene expression patterns. PLoS One 8, e74777. (23) Keiner, R., Kaiser, H., Nakajima, K., Hashimoto, T., and Dräger, B. (2002) Molecular cloning, expression and characterization of tropinone reductase II, an enzyme of the SDR family in Solanum tuberosum (L.). Plant Mol. Biol. 48, 299−308. (24) Nakajima, K., Kato, H., Oda, J. i., Yamada, Y., and Hashimoto, T. (1999) Site-directed mutagenesis of putative substrate-binding residues reveals a mechanism controlling the different stereospecificities of two tropinone reductases. J. Biol. Chem. 274, 16563− 16568. (25) Hashimoto, T., Nakajima, K., Ongena, G., and Yamada, Y. (1992) Two tropinone reductases with distinct stereospecificities from cultured roots of Hyoscyamus niger. Plant Physiol. 100, 836−845. (26) Liu, X., Cheng, J., Zhang, G., Ding, W., Duan, L., Yang, J., Kui, L., Cheng, X., Ruan, J., Fan, W., Chen, J., Long, G., Zhao, Y., Cai, J., Wang, W., Ma, Y., Dong, Y., Yang, S., and Jiang, H. (2018) Engineering yeast for the production of breviscapine by genomic analysis and synthetic biology approaches. Nat. Commun. 9, 448. (27) Chen, Y., Bao, J., Kim, I.-K., Siewers, V., and Nielsen, J. (2014) Coupled incremental precursor and co-factor supply improves 3hydroxypropionic acid production in Saccharomyces cerevisiae. Metab. Eng. 22, 104−109. (28) Gietz, R. D., and Schiestl, R. H. (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 31−34.

molecular cloning and biochemical experiments. X.L. constructed the strains. Y.P. did metabolites measurement. M.Y. performed bioinformatic analysis. Y.X. and Y.P. wrote and revised the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Chinese Academy of Sciences (CAS) (Grant Nos. XDB27020203, ZSYS-016, KFJ-BRP-009, 153D31KYSB20170121, and 153D31KYSB20160074) and National Natural Science Foundation of China (NSFC) (Grant 91856112). We thank Dr. Yining Liu in the Core Facility Centre of the Institute of Plant Physiology and Ecology for mass spectrometry assistance. The authors also appreciate anonymous reviewers for their helpful suggestions.

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DOI: 10.1021/acssynbio.9b00152 ACS Synth. Biol. 2019, 8, 1257−1262