Building Microbial Hosts for Heterologous Production of N

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Building Microbial Hosts for Heterologous Production of N-methylpyrrolinium Yu Ping, Xiaodong Li, Baofu Xu, Wei Wei, Wenping Wei, Guoyin Kai, Zhi-Hua Zhou, and Youli Xiao ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00483 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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ACS Synthetic Biology

Building Microbial Hosts for Heterologous Production of N-methylpyrrolinium

Yu Ping1,2,†, Xiaodong Li1,2,†, Baofu Xu1,2, Wei Wei1, Wenping Wei1,2, Guoyin Kai3, Zhihua Zhou1,2,*, Youli Xiao1,2,* 1CAS

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 2University

3Laboratory

of Chinese Academy of Sciences, Beijing 100039, China of Medicinal Plant Biotechnology, College of Pharmacy, Zhejiang Chinese

Medical University, Hangzhou, Zhejiang, 310053, China †These

authors contributed equally to this work.

* Corresponding authors Prof. Youli Xiao, [email protected]; Prof. Zhihua Zhou, [email protected]

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 Phone: +86-21-54924226

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ABSTRACT N-methylpyrrolinium-derived alkaloids like tropane alkaloids, nicotine, and calystegines are valuable plant source specialized metabolites bearing pharmaceutical or biological activity. Microbial synthesis of the critical common intermediate N-methylpyrrolinium (NMP) would allow for sustainable production of N-methylpyrrolinium-derived alkaloids. Here, we achieve the production of N-methylpyrrolinium both in Escherichia coli and Saccharomyces cerevisiae by employment of the biosynthetic genes derived from three different plants. Specifically, the diamine oxidases (DAOs) from Anisodus acutangulus were first characterized. Then, we presented the production of N-methylpyrrolinium in vitro from L-ornithine (ORN) via combination of the three cascade enzymes, ornithine decarboxylase (ODC) from Erythroxylum coca, putrescine N-methyltransferase (PMT) from Anisodus tanguticus, and diamine oxidases (DAOs) from A. acutangulus. Construction of the plant biosynthetic pathway into E. coli and S. cerevisiae resulted in de novo bio-production of N-methylpyrrolinium with titers of 3.02 mg/L and 2.07 mg/L, respectively. Metabolically engineering of the yeast strain to produce N-methylpyrrolinium via decreasing the flux to the product catabolism pathway and improving cofactor supply resulted in a final titer of 17.82 mg/L. This study not only presents the first microbial synthesis of N-methylpyrrolinium but also lays the foundation of heterologous biosynthesis of N-methylpyrrolinium-derived alkaloids. More importantly, the strains constructed can serve as important alternative tools to identify undiscovered pathway enzymes with a synthetic biology strategy. KEYWORDS: N-methylpyrrolinium, microbial synthesis, Escherichia coli, Saccharomyces cerevisiae, diamine oxidase, de novo production

Erythroxylum coca HO NH2 NH2 O L-ornithine

Anisodus tanguticus

EcODC

AtPMT

Anisodus acutangulus

AaDAO

N+ N-methylpyrrolinium

NH2 NH2

NH NH2


NH

O

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A century ago, Robert Robinson published an elegant one-pot chemical synthesis of tropinone at physiological condition which led to the historical Robinson hypothesis about the biological origin of tropine.1 Ever since, the bio-inspired organic synthesis and biogenesis of alkaloids were stimulated and broadly conducted.2 With multiple groups’ feeding experiments using isotopic labeling strategies in plants, the tropane alkaloids’ biosynthetic pathway is paradoxical, but N-methylpyrrolinium (NMP) is still believed as the key common precursor.3 It starts with L-ornithine (ORN) and completes within three consecutive steps. The first step is the decarboxylation of L-ornithine to form putrescine (PU) converted by ornithine decarboxylase (ODC). Subsequently, putrescine was mono-methylated to yield N-methylputrescine (MPU) catalyzed

by

putrescine

N-methyltransferase

(PMT).

N-methylputresicne

was

further

stereoselectively deaminated oxidatively at the pro-S hydrogen position by diamine oxidase (DAO, also known as MPO (N-methylputrescine oxidase)) resulting in the formation of N-methylamino butanal (MBU),4 which spontaneously undergoes a Schiff-base condensation reaction to produce N-methylpyrrolinium (Figure 1).5,

6

As a central common biosynthetic intermediate,

N-methylpyrrolinium is further metabolized to produce nicotine, tropane, and nortropane alkaloids in plants (Figure 1). Nicotine is a widely known ganglion blocking agent mainly produced in tobacco.7, 8 Hyoscyamine and scopolamine, members of tropane alkaloids, are extensively used to treat parasympathetic nervous system related diseases.9 Cocaine, predominantly produced in cultivated Erythroxylum species, is used as an anesthetic.10 Calystegines are nortropane alkaloids acting as inhibitor of glycosidases.11 These pharmaceutical N-methylpyrrolinium-derived important alkaloids with more than 200 specialized metabolites are still mainly extracted from the native Solanaceae, Euphorbiaceae, Proteaceae, Convolvulaceae, Brassicaceae, Rhizophoraceae, and Erythroxylaceae plants.12 Owing to the low accumulation and slow plant growth, alkaloid isolation from plants is often limited. As a potential solution, heterologous biosynthesis of high-value phytochemicals through synthetic biological strategy is an attractive alternative.13, 14 To make it feasible, the biosynthetic pathway need to be elucidated and proper microbial hosts need to be reconstructed. To date, in the N-methylpyrrolinium biosynthetic pathway starting with L-ornithine, only the putrescine biosynthesis has been engineered in microbial hosts.15,16 Nevertheless, bio-based production of neither N-methylpyrrolinium nor its derived alkaloids in



microbes was achieved yet. So there is an urgent demand for building microbial hosts to produce N-methylpyrrolinium for synthetic biology study of its derived alkaloids to mine biosynthetic enzymes and reconstruct synthetic route. In this study, with the N-methylpyrrolinium as the target compound, we firstly identified and characterized key enzymes and established in vitro enzyme cascade production of N-methylpyrrolinium. Then, the constructed plant biosynthetic pathway was introduced into Escherichia coli and Saccharomyces cerevisiae for production of the target compound followed by rationally metabolic engineering in S. cerevisiae for more efficient biosynthesis. To the best of our knowledge, this is the first time that N-methylpyrrolinium has been synthesized in microbes with such great yields. The results presented herein open the door to sustainable production of N-methylpyrrolinium-derived alkaloids. RESULTS AND DISCUSSION Selection of appropriate plant enzymes for N-methylpyrrolinium biosynthesis ODC, PMT, and DAO are cascade enzymes in the N-methylpyrrolinium biosynthetic pathway derived from L-ornithine. To achieve microbial synthesis of N-methylpyrrolinium, it was vital to choose efficient enzyme for each step. EcODC from Erythroxylum coca (E. coca) was selected for decarboxylation step as its catalytic efficiency was almost 20 times greater than Nicotiana glutinosa (N. glutinosa) NgODC.17 For the second committed step about the methylation of putrescine, the reported AtPMT from Anisodus tanguticus (A. tanguticus) was recruited, of which the in vitro activity was well characterized.18 The next-to last step in N-methylpyrrolinium biosynthesis is N-methylputrescine oxidation. Since the solubility of NtMPO1 is poor in E.coli,5, 6 we tried to explore more suitable building block. Anisodus acutangulus (A. acutangulus) is a traditional Chinese Solanaceous medicinal plant and has been used as a common folk herbal anaesthetic medicine for centuries.19 With the availability of the transcriptome data20 and the cDNA of A. acutangulus in hand, we selected DAOs from A. acutangulus (AaDAOs) as candidates and expected combination of the above plant genes would enable efficient production of N-methylpyrrolinium (Figure 2A). Based on the protein sequence alignment of AaDAOs and NtMPO1, AaDAOs possessed a conserved Asn-Tyr-Glu/X motif. Interestingly, the carboxyl terminal - AKL tripeptide of AaDAO2 and AaDAO3 led us to predict their localization in peroxisome like NtMPO1 (Figure S1A).5 However, AaDAO1 lacking the AKL tripeptide may not


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localize to peroxisome. After cloning and expression of the three AaDAOs in E. coli, only AaDAO2 and AaDAO3 are soluble, which were purified to homogeneity (Figure S1B) for enzymatic activity assay with chemically synthesized N-methylputrescine as substrate (Figure S15). Both AaDAO2 and AaDAO3 can catalyze the formation of N-methylpyrrolinium with N-methylputrescine as substrate, which was confirmed by liquid chromatography high-resolution mass spectrometry (LC-HRMS) extracted ion chromatograms (EIC) compared to the chemically synthesized standard analyzed by NaBD4 reduction derivation method (Figure 2B, Figure S5, and S16). We further charaterized the kinetic parametes for AaDAOs using a coupled assay in the presence of L-glutamate dehydrogenase (GLDH) by monitoring the consumption of NADH at 340 nm. The kinetic analysis results are as follows: kcat = 4.44 ± 0.30 s-1 and Km = 0.189 ± 0.040 mM for AaDAO2, and kcat = 19.8 ± 0.72 s-1 and Km = 0.0683 ± 0.011 mM for AaDAO2, respectively (Figure 2C, Table S1). By comparing the specificity constant (kcat/Km), the value of AaDAO3 is 12-fold greater than AaDAO2 implied that AaDAO3 is much more efficient than AaDAO2 and better candidate gene for further engineering. Meanwhile, the activities of EcODC and AtPMT were assayed and evaluated using previously reported derivation method (Figure S2, S3, and S4).17, 18 To determine if the combination of the selected enzymes (EcODC, AtPMT, AaDAO2, and AaDAO3) was appropriate for production of N-methylpyrrolinium, one-pot assay with proper cofactors, purified proteins, and L-ornithine as substrate was conducted and N-methylpyrrolinium was detected (Figure S6). In the presence of EcODC-AtPMT cascade enzymes, the final titers of N-methylpyrrolinium reached to 26.98 mg/L and 37.44 mg/L in 1 hour using AaDAO2, and AaDAO3 according to the standard curve, respectively (Figure 2D, Figure S17). Therefore, combination

of

EcODC,

AtPMT,

and

AaDAO3

was

chosen

for

further

in

vivo

N-methylpyrrolinium biosynthesis.

De novo production of N-methylpyrrolinium in E. coli Aiming to establish a platform for microbial synthesis of N-methylpyrrolinium, we first explored whether E. coli can be a host with its short doubling time. Whole-cell fermentation experiment was performed to test the in vivo catalytic efficiency of selected enzymes (Figure 3A). The vector pACYCDuet-EcODC-AtPMT was constructed for co-expression of the first two pathway enzymes. The plasmid was transformed into BL21(DE3), resulting in strain BL21-OP.



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The strain was cultured in shake flask for 30 hours, after which the fermentation supernatant was analyzed by LC-HRMS. Putrescine and N-methylputrescine were accumulated in the culture supernatant of strain BL21-OP, confirming the function of EcODC and AtPMT in vivo (Figure S7). To initiate the conversion of N-methylputrescine to N-methylpyrrolinium, pET30a-AaDAO3 was introduced into strain BL21-OP, generating strain BL21-OP-A3. Analysis of fermentation supernatant indicated successful production of N-methylpyrrolinium by NaBD4 reduction method (Figure S8). In contrast, N-methylpyrrolinium was not produced in control strains BL21(DE3) and BL21-OP even similar molecular masses were detected with different retention times comparing to the target standard (Figure S8). The extracellular titer of N-methylpyrrolinium increased gradually as the fermentation proceeded (Figure 3B). The N-methylpyrrolinium yield reached to 3.02 mg/L at 48 h after induced by IPTG. At the same time, the putrescine and N-methylputrescine accumulated up to 12.2 mg/mL and 5.45 mg/L, respectively (Figure S9). The result experimentally demonstrated E. coli as a potential host for N-methylpyrrolinium production. De novo production of N-methylpyrrolinium in S. cerevisiae Compared with E. coli, S. cerevisiae is more widely used for functional expression of plant enzymes, like P450s.21 Moreover, alkaloids biosynthesis is affected by intracellular compartmentalization which can only be investigated in eukaryotic host.22,

23

Since

N-methylpyrrolinium-derived alkaloids biosynthesis involve some P450s in later stage steps, we set out to establish a platform for N-methylpyrrolinium biosynthesis in S. cerevisiae (Figure 4A). To begin with, the high-copy plasmid p2M-EcODC-AtPMT harboring EcODC and AtPMT under control of strong constitutive promoter TEF1 and PGK1 respectively was transferred into BY4742 to create strain BY4742-OP. Strain BY4742-OP was able to efficiently produce putrescine and N-methylputrescine (Figure S10). To evaluate the functionality of the newly identified DAO in yeast, the gene AaDAO3 encoding the enzyme was integrated into the X-4 site of the S. cerevisiae genome for stable expression using strong constitutive promoter TEF1, creating strain BY4742-OP-A3. As expected, shake flask culture with strain BY4742-OP-A3 allowed for the conversion of N-methylputrescine into N-methylpyrrolinium (Figure S11). As shown in Figure 4B, N-methylpyrrolinium was produced in a stable increase during the fermentation process. The N-methylpyrrolinium titer excreted into the culture reached 2.07 mg/L at the end of the


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fermentation, indicating successful de novo production of N-methylpyrrolinium in S. cerevisiae. It is also noteworthy that putrescine accumulates a lot (15.15 mg/L) in yeast compared with N-methylputrescine (3.68 mg/L), which indicated that the AtPMT activity is limited (Figure S14A).

Optimization of N-methylpyrrolinium production in S. cerevisiae Given the low yield in S. cerevisiae, further strain optimization was carried out to improve the N-methylpyrrolinium production. To increase the accumulation of a target molecule, it is necessary to block the product catabolism pathway. Since N-methylpyrrolinium formation from N-methylamino butanal is a reversible process and aldehyde was known to be converted into acid by inner aldehyde dehydrogenase (ALDH) in S. cerevisiae,24-26 we speculated that knocking out ALDHs was able to increase accumulation of N-methylpyrrolinium. To test whether these ALDHs act as a catabolism pathway of N-methylpyrrolinium, we purified ALD4, ALD5, and hexadecenal dehydrogenase (HFD1) (Figure S1) and check their ability to convert N-methylamino butanal into N-methylamino butyric acid. Incubation of ALD4, ALD5, and HFD1 individually with AaDAO3 resulted in the formation of N-methylamino butyric acid (MBUA) using N-methylputrescine as substrate (Figure S12). Meanwhile, N-methylamino butyric acid was detected in strain BY4742-OP-A3 (Figure S13). These results demonstrated that inner ALDHs indeed participated in the N-methylpyrrolinium degradation pathway. Accordingly, we constructed strain ΔBY4742-OP-A3 carrying deletions of the three ALDHs and tested their contribution to N-methylpyrrolinium production. Analysis of the culture supernatant of the strain showed that titer of N-methylpyrrolinium was enhanced to 7.68 mg/L, which was 3.7-fold improvement over strain BY4742-OP-A3 (Figure 4C), suggesting the beneficial effect of ALDHs deletions on N-methylpyrrolinium production. In addition, the deletion of the three ALDHs had no negative effect on cell growth from the growth profile. Another beneficial aspect is the decrease of putrescine accumulation with a titer of 6.74 mg/L and increase of N-methylputrescine production with a titer of 5.67 mg/mL (Figure S14B). In a previous report, SAM2 was inserted into the yeast genome for yielding S-adenosyl-L-methionine (SAM) to provide sufficient supply of the cofactor SAM for de novo production of strictosidine.27 As SAM is a necessary cofactor of the second enzyme AtPMT in the



N-methylpyrrolinium biosynthetic pathway, we hypothesized that increasing the SAM availability might enhance the titer of the final product N-methylpyrrolinium. To verify the hypothesis, SAM2 from S. cerevisiae was integrated into the X-4 site of ΔBY4742-OP-A3 genome. The resultant strain ΔBY4742-OP-A3-SAM2 was cultivated under the same shake flask condition. As shown in Figure 4D, the concentration of N-methylpyrrolinium increased from 7.68 mg/L for strain ΔBY4742-OP-A3 to 17.82 mg/L for strain ΔBY4742-OP-A3-SAM2, respectively, indicating overexpression of SAM synthase could significantly improve the N-methylpyrrolinium production. Meanwhile, putrescine was further utilized with a final titer of 3.54 mg/L (Figure S14C). Overall, these results suggested that S. cerevisiae can be used as chassis cells for N-methylpyrrolinium biosynthesis. Moreover, knocking out aldehyde dehydrogenase and overexpressing SAM2 were efficient to enhance N-methylpyrrolinium production. CONCLUSION In the present study, de novo high production of N-methylpyrrolinium is achieved through catalytic module selection and metabolic flux control. As the first step to achieve N-methylpyrrolinium biosynthesis, we characterized AaDAOs from A. acutangulus for the conversion of N-methylputrescine into N-methylpyrrolinium. Subsequent in vitro biosynthesis of N-methylpyrrolinium from L-ornithine was demonstrated with purified EcODC, AtPMT, and AaDAOs. Next, we introduced the encoding genes of EcODC, AtPMT, and AaDAO3 of the plant pathway into E. coli and observed 3.02 mg/L production of N-methylpyrrolinium with simple carbon sources. Moreover, introduction of the plant biosynthetic pathway into S. cerevisiae led to a titer of 2.07 mg/L. In addition, by deletion of several inner ALDHs and overexpression of SAM2, the N-methylpyrrolinium production in S. cerevisiae was remarkably increased 8.6-fold with the titer of 17.82 mg/L. Taken together, our work reported here clearly demonstrated the potential for a viable N-methylpyrrolinium biosynthesis process in microbial hosts. Furthermore, with these engineered microbes in hand, we can identify undiscovered pathway enzymes for the biosynthesis of N-methylpyrrolinium-derived alkaloids. METHODS Protein purification


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E. coli BL21(DE3) was transformed respectively with pET24a-EcODC, pET24a-AtPMT, pET30a-AaDAO1, pET30a-AaDAO2, and pET30a-AaDAO3. Single colonies were inoculated in 4 mL LB with appropriate antibiotics and the cultures were shaken at 37 °C. Overnight cultures were transferred into 1 L LB medium and induced with 0.15 mM β-D-1-thiogalactopyranoside (IPTG) when the OD600 reached 0.6 - 0.8 followed by incubation at 16 °C for another 18 h at 200 rpm before harvested. Cells were suspended in 50 mM potassium phosphate buffer, pH 8.0 containing 100 mM NaCl and lysed. After centrifugation at 35000 g for 50 min, the crude protein was purified with Ni2+ resin (Qiagen) and further analyzed by 10 % SDS-PAGE. The protein concentration was determined with braford assay using BSA as standard. in vitro enzymatic assays The decarboxylation activity of EcODC was detected at 30 °C as described before.17 The assay mixture contained 1 mM L-ornithine, 1 mM PLP, and 10 M EcODC in 50 mM potassium phosphate buffer, pH 8.0, with a total volume of 100 µL. After 30 min, the mixture was added with 50 μL saturated Na2CO3 aqueous solution. Then 100 µL dansyl chloride (5 mg/L in acetonitrile) was added into the mixture and incubated at 58 °C for another 30 min. The organic phase was filtered through 0.22 µm filter for LC-HRMS analysis. The AtPMT assay was conducted in a 100 L mixture containing 1 mM putrescine, 1 mM SAM, and 10 µM AtPMT in 100 mM potassium phosphate buffer, pH 8.0.18 The reaction mixture was incubated at 30 oC for 30 min. The formation of N-methylputrescine was measured in the same way as EcODC assay. The AaDAO2 and AaDAO3 assay were separately performed in a 100 µL reaction system containing 2 mM N-methylputrescine, 20 M CuSO4, and 10 M protein in 50 mM potassium phosphate buffer, pH 8.0. The mixture was incubated at 37 °C in water bath for 30 min. Then the reaction was added with 25 µL 0.5 M borate buffer (pH 10.0) followed by addition of 10 mM NaBD4 (final concentration). The mixture was further incubated at 37 °C for 1 h. 10 L of 4 N HCl was added to quench the chemical reduction. The resultant mixture was filtered through 0.22 µm filter and subjected to LC-HRMS analysis. The one-pot assay was carried out in a 100 L reaction buffer (50 mM potassium phosphate buffer, pH 8.0) containing 5 mM L-ornithine, 5 mM PLP, 5 mM SAM, 20 M CuSO4, 10 µM EcODC, 10 µM AtPMT and 10 M AaDAO2 or AaDAO3. Assay was incubated at 30 oC at varying time and then analyzed with the same method as AaDAO assay. The experiment was repeated for 3 times. For the ALDHs activity assay, a mixture containing 3 mM N-methylputrescine, 1 mM KCl, 1 mM NADP+ or NAD+, 20 µM CuSO4, 10 M AaDAO3 and 10 M ALDHs in 50 mM potassium phosphate buffer, pH 8.0 was incubated at 37 °C in water bath for 1 h. For ALD4 and ALD5 enzyme assay, NADP+ was used. While for HFD1 assay, NAD+ was used instead. The detection method of the product was the same as EcODC assay. To determine the kinetic parameters of AaDAO2 and AaDAO3, the assay was performed in 50 mM potassium phosphate buffer, pH 8.0 with a volume of 200 µL. The mixture contained 20 µM CuSO4, 5 mM α-ketoglutaric acid, 0.15 mM NADH, 3U GLDH (L-glutamate dehydrogenase), 3.0 µM protein, and N-methylputrescine with different concentrations (0.02 mM, 0.05 mM, 0.15 mM,



0.3 mM, 0.625 mM, and 1.25 mM). The change of NADH absorption at 340 nm was used to calculate the velocity. Each experiment was carried out for twice at 37 °C.

De novo production of N-methylpyrrolinium in E. coli Single colonies were inoculated into 4 mL LB media with needed antibiotics and grown overnight. The cultures were diluted at 1:100 in 50 mL LB media containing corresponding antibiotics. When the OD600 reached 0.6 - 0.8, 0.15 mM IPTG and 50 M CuSO4 were added to induce protein expression. The cultures were cultivated in shaker with a speed of 120 rpm at 25 oC for two days. Samples were taken at certain points after induction. OD600 was measured and the production of NMP was analyzed with the same method as AaDAO assay by LC-HRMS. PU and MPU were measured in the same way as EcODC assay using dansyl chloride for derivation followed by quantification with HPLC. Three biological replicates were done for the fermentation experiment. De novo production of N-methylpyrrolinium in S. cerevisiae The genetically engineered yeast strains were inoculated into 5 mL YPD media with 200 mg/L hygromycin B in tubes and cultivated at 30 °C, 250 rpm for 24 hours. Then the strains were inoculated into 50 mL liquid YPD media in 250 ml flasks with 200 mg/L hygromycin B and 50 µM CuSO4 and grown at 30 °C, 200 rpm for fermentation experiment. Samples were taken every 24 h. The quantification of NMP, PU and MPU were same as the E.coli fermentation analysis. Three biological replicates were performed for yeast fermentation. LC-HRMS analysis Putrescine and N-methylamino butyric acid were purchased from Aladdin Chemical Industry and chemically synthesized N-methylputrescine and N-methylpyrrolinium were used as standards. For the LC-HRMS analysis, Agilent 1290 UHPLC coupled to Agilent 6545 Q-TOF ESI high-resolution mass spectrometer (HRMS) was used. For detection of L-ornithine, putrescine, N-methylputrescine, and N-methylbutyric acid, samples were reacted with dansyl chloride for derivation, column Agilent RRHD SB-C18 (2.1 × 100mm, 1.8 m) was adopted for separation with column temperature set at 40 oC. The mobile phase used were Solvent A (0.1% formic acid aqueous solution) and solvent B (0.1% formic acid in acetonitrile). The samples were analyzed under the following gradient program with a flow rate of 0.3 mL/min: 0-1 min, 5 % B; 1-3 min, 5-40 % B; 3-10 min, 40 % B; 10-12 min, 40-75 % B; 12-18 min, 75-80 % B; 18-23 min, 80-95 % B; 23-25 min, 95 % B; 25-26 min, 95-5 % B. For detection of N-methylpyrrolinium, samples were reacted with NaBD4 for reduction, column Agilent 300 Extend-C18 (4.6 × 150mm, 3.5m) was used. The column was set to 40 °C. The following gradient was applied at a flow rate of 0.35 mL/min with solvent A and solvent B: 0-1 min, 2 % B; 1-2 min, 2-3 % B; 2-10 min, 3-5 % B; 10-12 min, 5-95 % B; 12-15 min, 95 % B; 15-16 min, 95-2 % B. The MS was operated in positive ion mode with a mass range of 70–1000 m/z. The acquisition time implemented was 833.3 ms/spectrum. The gas temperature was 300 °C and drying gas was 6 L/min. Nebulizer was 35 psig and the capillary was 4000 V. The fragmentor, skimmer and octupole 1 RF Vpp were 135 V, 65 V and 750 V, respectively. To analyze MS/MS, the collision energy were 20 V and 40 V. The [M+H]+ m/z values of ORN-dan, PU-dan, MPU-dan, and MBUA-dan are 599.1993, 555.2094,


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569.2251, and 351.1373, respectively. With NaBD4 reduction, N-methylpyrrolinium was converted into deuterated N-methylpyrrolidine (d-NMD) with [M+H]+ calculated as 87.1027. Quantification of N-methylpyrrolinium was based on the standard curve made from 3 replicates. For the standard curve preparation, 40 µL NMP with different concentrations (0.025 mM, 0.1 mM, 0.25 mM, 0.4 mM, and 0.5 mM) were added with 10 µL 0.5 M borate buffer (pH 10.0) followed by addition of 10 mM NaBD4 (final concentration). The mixture was incubated at 37 °C for 1 h. Then 4 µL of 4 N HCl was added to quench the chemical reduction. The resultant mixture was filtered through 0.22 µm filter and subjected to LC-HRMS analysis. The peak area of obtained deuterated N-methylpyrrolidine (d-NMD) against the NMP concentration was used to make the curve. As for quantification of putrescine and N-methylputrescine, 100 µL PU and MPU with varying concentrations (0.00375 mM, 0.00750 mM, 0.0150 mM, 0.0300 mM, 0.0600 mM, 0.120 mM, and 0.240 mM) were separately added with 50 μL saturated Na2CO3 aqueous solution. Then 100 µL dansyl chloride (5 mg/L in acetonitrile) was added into the mixture and incubated at 58 °C for 30 min. The organic layer was filtered through 0.22 µm filter for HPLC analysis on Agilent 1260 HPLC. The column phenomenex Luna 5u C18 (250 × 4.6 mm) was used for separation with mobile phase A and B. The flow rate was 1 mL/min and the detection wavelength was 254 nm. The gradient was set as: 0-32 min, 5-58 % B; 32-53 min, 58 % B; 53-58 min, 58-95 %; 58-64 min, 95% B; 64-68 min, 5% B. The peak area was used for standard curve preparation. The data was generated from two independent experiments.

ASSOCIATED CONTENT Supporting information Methods of plasmid, strain construction, and chemical synthesis; supplementary figures, tables, and references. AUTHOR INFORMATION

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

Youli Xiao: 0000-0002-4803-3333 Author Contributions †These

authors contributed equally to this work.

Notes The authors declare no competing financial interests.



ACKNOWLEDGMENTS This work was supported by Chinese Academy of Sciences (CAS) (Grant XDB27020203, ZSYS-016, 153D31KYSB20170121, and 153D31KYSB20160074). We thank Dr. Y. Liu, and Mr. S. Bu in the Core Facility Centre of the Institute of Plant Physiology and Ecology for mass spectrometry and NMR assistance.

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FIGURES

NH2 H 2N

O

ODC

OH L-ornithine (ORN)

CO2

H 2N

PMT

NH2

SAM Putrescine (PU)

NH2

N H

SAH N-methylputrescine (MPU)

DAO

NH3

N

O N H N-methylamino butanal (MBU)

Spontaneous

O N+

O Hyoscyamine

N-methylpyrrolinium (NMP)

OH

O O

N

O

N

O

O

HN HO

O Scopolamine

OH OH

OH

O Cocaine

N N Nicotine

Calystegine

Figure 1. Biosynthetic pathway of N-methylpyrrolinium and its derived representative medicinal natural products. ODC: ornithine decarboxylase; PMT: putrescine N-methyltransferase; DAO: diamine oxidase; SAM: S-Adenosyl-L-methionine.

B

NH2 H 2N

5 x10

OH

ORN

EcODC

CO2 H 2N

NH2

PU SAM

AtPMT SAH NH2

N H

AaDAO O

N H MBU

Spontaneous

N

+

NMP pH=10 NaBD4

N

60

6 4 2 0

i

+ESI EIC(87.1027) x105

6 4 2 0

D

Deuterated N-Methylpyrrolidine (d-NMD)

6 4 2 0

30 AaDAO2

0 0.00

0.25

0.50

0.75

1.00

1.25

1.50

MPU (mM)

iii

5 +ESI EIC(87.1027) x10

5 x10

45

15

ii

6 4 2 0 6 4 2 0

AaDAO3

+ESI EIC(87.1027)

5 +ESI EIC(87.1027) x10

MPU

NH3

C

d-NMD

O

v (M/s)

A

iv +ESI EIC(87.1027)

v

D EcODC

45

NMP titer (mg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 15

AtPMT

AaDAO3

30

15

EcODC

AtPMT

AaDAO2

0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Retention time (min)

0

10

20

30

40

50

60

Time (min)

Figure 2. Characterization enzymatic acitivities of AaDAO2 and AaDAO3 and in vitro N-methylpyrrolinium biosynthesis. (A) Biosynthetic pathway of N-methylpyrrolinium with selected enzymes, the black arrow indicated each step in the pathway, the orange arrow showed the detection method of N-methylpyrrolinium by quantitive NaBD4 reduction derivation. (B) EIC profiles of AaDAO2 and AaDAO3 activities towards N-methylputrescine. (i) deuterated N-methylpyrrolidine (d-NMD) standard with an extracted [M+H]+ m/z value of 87.1027; The whole assay with (ii) boiled AaDAO2; (iii) AaDAO2; (iv) boiled AaDAO3; (v) AaDAO3. More Detailed HRMS of the products can be found in Figure S5. (C) Michaelis-Menten curve for AaDAOs (3.0 M at 37 oC) with MPU as substrate. (D) Profiles of N-methylpyrrolinium production in one-pot assay with EcODC, AtPMT, and AaDAO2 or AaDAO3 using L-ornithine as substrate. The data were generated from three independent experiments.


Page 15 of 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Production of N-methylpyrrolinium in E. coli. (A) Scheme overview of pathway assembly in BL21(DE3). (B) Profiles of N-methylpyrrolinium production in strain BL21-OP-A3. The data were generated from three independent experiments.

Figure 4. Production of N-methylpyrrolinium in S. cerevisiae. (A) Scheme overview of pathway assembly in S. cerevisiae. The pathway was optimized by knocking out ALD4, ALD5, and HFD1 and integrating SAM2 into the genome.

(B)

Production

of

N-methylpyrrolinium

in

strain

BY4742-OP-A3.

(C)

Production

of

N-methylpyrrolinium in strain ΔBY4742-OP-A3. (D) Production of N-methylpyrrolinium in strain

ΔBY4742-OP-A3-SAM2. The data were generated from three independent experiments.


Building Microbial Hosts for Heterologous Production of N

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