Enhancing Biosynthesis of a Ginsenoside Precursor by Self-Assembly

Publication Date (Web): April 13, 2016. Copyright © 2016 American Chemical Society. *(F.W.) E-mail: [email protected]. Phone: +86 21 6425 3156...
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Enhancing Biosynthesis of a Ginsenoside Precursor by Self-Assembly of Two Key Enzymes in Pichia pastoris Chengcheng Zhao,†,‡ Xin Gao,†,‡ Xinbin Liu,† Yong Wang,§ Shengli Yang,† Fengqing Wang,*,† and Yuhong Ren*,† †

State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China § Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China S Supporting Information *

ABSTRACT: Ginsenosides from the edible and medicinal plant ginseng have demonstrated various pharmacological activities. However, producing ginsenoside efficiently remains a challenge. Engineering metabolic pathways through protein assembly in yeast is a promising way for ginsenoside production. In the biosynthetic pathway of ginsenosides, dammarenediol-II synthase and squalene epoxidase are two key enzymes that determine the production rate of the dammarane-type ginsenoside precursor dammarenediol-II. In this work, a strategy to enhance the biosynthesis of dammarenediol-II in Pichia pastoris was developed by the self-assembly of the two key enzymes via protein−protein interaction. After being modified by interacting proteins, the two enzymes were successfully co-localized, resulting in a 2.1-fold enhancement in dammarenediol-II yields. KEYWORDS: ginsenosides, self-assembly, Pichia pastoris, dammarenediol-II

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INTRODUCTION Ginsenosides are bioactive triterpenoids found in the popular oriental medicinal plant, ginseng, and exhibit diverse pharmacological effects such as anticarcinogenic, immunomodulatory, anti-inflammatory, and antiallergic effects.1 Dammarane-type tetracyclic triterpenoids, as the major components of ginsenosides, are divided into panaxadiol and panaxatriol classes.2,3 Currently, ginsenosides are mainly obtained by extraction from ginseng roots. However, field cultivation of ginseng is a time-consuming and labor-intensive process that is susceptible to many environmental factors, which render cultivation inefficient.4 Recently, biotechnological strategies, including tissue culture, transgenic plants, and engineered yeast cells, have been used for the production of ginsenosides.5−8 Yeast is one of the best candidates for terpene production through synthetic biology methods due to its sufficient pool of precursors and tolerance to plant-derived heterologous proteins.9−12 With the development of metabolic engineering and synthetic biology, ginsenosides (such as protopanaxadiol and protopanaxatriol) and their precursors (dammarenediol-II) have been successfully and efficiently produced in Saccharomyces cerevisiae.8,13,14 Tansakul et al. were able to produce dammarane-type ginsenosides in S. cerevisiae by heterologous expression of dammarenediol-II synthase, which catalyzes the synthesis of dammarane triterpene.15 Ginsenosides are derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are synthesized by the mevalonic acid (MVA) pathway in eukaryotic cells (see Figure 1). Squalene, transformed from IPP and DMAPP, is catalyzed to 2,3-oxidosqualene by squalene epoxidase (ERG1). In Panax ginseng, cyclization of 2,3© 2016 American Chemical Society

oxidosqualene to dammarenediol-II by dammarenediol synthase (PgDDS) is the first reaction that leads toward the biosynthesis of dammarane-type ginsenosides, followed by cytochrome P450 enzyme-mediated hydroxylation and glycosylation by glycosyltransferase.3,16 ERG1 and PgDDS are the key enzymes required for the synthesis of ginsenosides. However, heterologous expression of PgDDS alone is insufficient to achieve a satisfactory yield of dammarenediolII. Moreover, in yeast, most of the 2,3-oxidosqualene precursors enter the ergosterol synthetic pathway by ERG7,7 which results in competition between PgDDS and ERG7 for the substrate 2,3- oxidosqualene. Therefore, to enhance the competitiveness of PgDDS and to improve the catalytic efficiency of ERG1 and PgDDS in yeast, strategies involving the assembly of multienzyme cascades should be considered. Designing protein assemblies has been an attractive goal in synthetic biology because strategies involving multienzyme assembly facilitate the substrate transfer between enzymes, limit the cross-talk between pathways, and increase the product yields in a cascade reaction. Although strategies such as enzyme fusion and modular scaffolds have been shown to enhance the cascade efficiency in many instances, some disadvantages also exist. A major disadvantage of enzyme−enzyme fusion is that it often shows decreased enzymatic activity. The drawback of modular scaffold methods is that expression of the target enzymes and linked scaffolds might be a heavy burden on host cells.17 Therefore, a scaffold-free self-assembly method for Received: Revised: Accepted: Published: 3380

February April 12, April 13, April 13,

6, 2016 2016 2016 2016 DOI: 10.1021/acs.jafc.6b00650 J. Agric. Food Chem. 2016, 64, 3380−3385

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Journal of Agricultural and Food Chemistry

Figure 1. Biosynthetic pathway of dammarane-type ginsenosides in engineered yeasts. 2,3-Oxidosqualene stands at the branch-point of the exogenous dammarenediol-II synthetic pathway and the native ergosterol synthetic pathway in yeast. Single solid arrows represent one-step conversions and triple solid arrows represent multiple steps. Dashed arrows represent exogenous steps, and thick arrows (including dashed arrows) represent rate-limiting steps. IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; FPP, farnesyl diphosphate; FPS, farnesyl diphosphate synthase; SS, squalene synthase; SE (ERG1), squalene epoxidase; PgDDS, dammarenediol-II synthase from Panax ginseng. pPIC3.5k-ERG1-PgDDS (including p-[PgDDS-ER/k-ERG1], p[PgDDS-L3-ERG1], p-[ERG1-ER/k-PgDDS] and p-[ERG1-L3PgDDS]) was constructed using a normal restriction enzyme digestion and ligation method (see Figure S1A). ER/k, a rigid α-helical motif, and L3, a flexible motif, acted as linkers of the two genes. The DNA fragments (PgDDS with BamHI and SpeI restriction sites and ERG1 with Acc65I and NotI restriction sites, or PgDDS with Acc65I and NotI restriction sites and ERG1 with BamHI and SpeI restriction sites) were cloned into the template with linker. The self-assembly plasmid p[PgDDS-PDZlig]/[ERG1-PDZ] was constructed using a seamless cloning method (see Figure S1C). The oligonucleotide sequences of the PDZ domain and PDZ ligand were previously constructed in our laboratory. The PDZ domain and its corresponding ligand (PDZlig) were fused to the C-termini of ERG1 and PgDDS, with linking motifs, the construction of which was the same as the construction of enzymefusion plasmids described above, thus generating ERG1-PDZ and PgDDS-PDZlig, respectively. The PgDDS-PDZlig, ERG1-PDZ, and TT-AOX1 fragments were respectively acquired by PCR for seamless cloning. Likewise, the PgDDS, ERG1, and TT-AOX1 fragments were respectively amplified for seamless cloning, producing p-[PgDDS]/ [ERG1]) as a control plasmid (see Figure S1B). The mCherry coding sequence was divided into mN159 and mC160 fragments19 that were separately amplified from the pRSET BmCherry vector. Fluorescence complementation experiments were carried out by fusing mN159 and mC160 to the site between ERG1 and PDZ or to the site between PgDDS and PDZlig. The chimeric genes PgDDS-mC160-PDZlig and ERG1-mN159-PDZ domain were constructed by respectively cloning mC160 and mN159 into pET28a vector containing the PgDDS-PDZlig gene and the pET28a vector containing the ERG1-PDZ domain gene by overlap extension PCR. The two gene fragments from the pET-28a vector and the TT-AOX1 fragment from the p-[PgDDS]/[ERG1] plasmid were inserted into the linearized pPIC3.5k vector through seamless cloning after PCR amplification to produce p-[PgDDS-mC160-PDZlig]/[ERG1-mN159PDZ] (see Figure S1D). Similarly, p-[PgDDS-mC160]/[ERG1mN159] was constructed as a control (see Figure S1F). The genes and plasmids used in this study are summarized in Table S1. Strain Construction. The four strains generated for the expression of fused ERG1 and PgDDS were named DEE for PgDDS-ER/k-ERG1, DLE for PgDDS-L3-ERG1, EED for ERG1-ER/k-PgDDS, and ELD for ERG1-L3-PgDDS, respectively. The self-assembly system of

multiple enzymes was needed for the biosynthesis of valuable products such as ginsenosides.18 In this study, a strategy for the self-assembly of key enzymes of the pathway via the interactional protein domain and its ligand was developed in yeast to enhance the biosynthesis of the ginsenoside precursor, dammarenediol-II. Because P. pastoris requires similar genetic manipulations as S. cerevisiae and shows higher expression of exogenous proteins, it was used as the host to produce dammarenediol-II. The strategy for self-assembly of ERG1 and PgDDS is outlined in Figure 2. Self-assembly was realized by fusing PgDDS and EGR1 with the PDZ domain and its corresponding ligand (PDZlig) (see Figure S1C), respectively. To overcome the interaction between the enzymes and PDZ/PDZlig, a rigid α-helical ER/K motif was used as a linker between ERG1 and the PDZ domain, whereas a flexible L3 motif was used as a linker between PgDDS and PDZlig. After the coexpression of these two fusion enzymes in P. pastoris, PgDDS and EGR1 would get close in space driven by the interactive force between PDZ/PDZlig, resulting in a two-enzyme complex. Additionally, the fusion of ERG1 and PgDDS enzymes in different orders using various linkers was also performed (see Figure S1A) to compare the effect of protein assembly and fusion on the production of dammarenediol-II.

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MATERIALS AND METHODS

Materials and Reagents. The authentic dammarenediol-II was purchased from BioBioPha Co. (Kunming, Yunnan, China). P. pastoris GS115 and the vector pPIC3.5k from Multi-Copy Pichia Expression Kit of Invitrogen (catalog no. K1750-01, Thermo Fisher Scientific, Rockford, IL, USA) were used for gene expression. A ClonExpress MultiS Kit from Vazyme Biotech Co., Ltd. (Nanjing, Jiangsu, China), was used for seamless cloning. Plasmid Construction. The ERG1 gene was PCR amplified from the genomic DNA of P. pastoris GS115. The complete gene PgDDS was synthesized with codon-optimization (Generay Biotech Co., Ltd., Shanghai, China) and then PCR amplified for fusion with ERG1 or PDZ ligand. The enzyme-fusion plasmid pPIC3.5k-PgDDS-ERG1 or 3381

DOI: 10.1021/acs.jafc.6b00650 J. Agric. Food Chem. 2016, 64, 3380−3385

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Figure 2. Schematic view of transformation of the engineered plasmids and expression of target genes. PgDDS and EGR1 would be brought together in space by the interactional force between PDZ and its ligand, PDZlig. was performed after screening of a number of His+ transformants. Geneticin hyper-resistant (4 mg/mL) colonies were cultivated, and their genomes were isolated to identify the integrants. Amplification of the gene of interest was performed with the 5′-AOX1 primer paired with the 3′-AOX1 primer. Yeast Cultivation and Protein Expression. YPD medium was used for activating recombinant P. pastoris strains and cultivating GS115 strains for further preparation of competent cells. Preculture of recombinant P. pastoris strains was performed in BMGY medium with an initial pH of 6.0 at 28 °C in a rotary shaking incubator (220 rpm) until the culture reached an OD600 = 2−6 (approximately 18−22 h). The cells were harvested by centrifuging at 6000g for 10 min at room temperature and were resuspended to an OD600 of 1.0 in BMMY medium (initial pH 6.0) to induce expression. Methanol (100%) was added to the BMMY medium to a final concentration of 0.5% methanol every 24 h to maintain induction.

PgDDS and ERG1 in vivo was constructed by coexpressing PgDDSPDZlig and ERG1-PDZ in P. pastoris GS115 (the strain was named KDPEP). The expression of the PgDDS-PDZlig and ERG1-PDZ fragments was controlled by their upstream AOX1 promoters. Coexpression of PgDDS and ERG1 genes, controlled by the AOX1 promoters, acted as a control (the strain was named KDE). After transformation with p-[PgDDS-mC160-PDZlig]/[ERG1-mN159PDZ], the strain was named KMPMP. Similarly, the control strain with p-[PgDDS-mC160]/[ERG1-mN159] was named KMM. The strains used in this study are summarized in Table S2. Preparation of electrocompetent cells of P. pastoris GS115 and transformation through electroporation with plasmids linearized by the restriction enzymes SalI or SacI were performed according to the protocol provided by Invitrogen. The parent vector pPIC3.5k was also digested and transformed into GS115 as a background control for expression. Screening of multiple inserts by Geneticin hyper-resistance 3382

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Journal of Agricultural and Food Chemistry Product Determination. The yeast cells were collected at 8000g for 10 min, and dry cell weight was measured after vacuum freezedrying. Freeze-dried cells were refluxed with 20% KOH/50% ethanol aqueous in centrifuge tubes at 90 °C for 2 h. After extraction with the same volume of n-hexane, the extract was concentrated and chromatographed on silica gel to determine the presence of dammarenediol-II. The TLC plate was developed twice with benzene/acetone 19:1 and visualized with 20% sulfuric acid. For further confirmation, the extracts were applied to LC-MS equipped with HPLC (Agilent, 1100 series) and an Eclipse Plus C18 column (5 μm, 4.6 × 250 mm) maintained at 40 °C, eluted with 85% CH3CN at 1.0 mL/min, and monitored by absorbance at UV 203 nm. Quantitative analysis of dammarenediol-II was examined by a standard curve method. In APCI-MS analysis, authentic dammarenediol-II gave the ion peaks at m/z 427 and 409, which come from [M + H+] by elimination of one and two water molecule(s), respectively. Fluorescence Complementation Assay. Bimolecular fluorescence complementation is a technology typically used to validate protein interactions. Protein interactions could bring the fluorescent fragments within proximity, allowing the fluorescent protein to re-form its native three-dimensional structure and emit fluorescent signal. The fluorescence complementation assay was performed in this study on the basis of the mCherry fluorophore. Following transformation, screening, and induction, cells were collected and visualized by epifluorescence (excitation, 562−640 nm; emission, 590−650 nm) and differential interference contrast on Ti-E and A1R confocal laserscanning microscopes (both from Nikon, Tokyo, Japan). Cells coexpressing mC160 and mN159 without the PDZlig and PDZ domain were visualized in a similar manner. The fluorescence intensity of 5.0 OD600 cells collected at different times after methanol induction was measured using a SpectraMax M5 microplate reader (Synergy Mx, Bio-Tek Instruments, Inc., Winooski, VT, USA) at sensitivity = 90.

Figure 3. Dammarenediol-II yield (A) and fluorescence intensity (B) of engineered yeast strains over induction time. All strains were induced in BMMY medium (pH 6.0) with 0.5% methanol for 4 days. Data represent the mean ± standard deviation of three measurements.

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RESULTS AND DISCUSSION Co-localization of Cascade Enzymes in P. pastoris. To construct the dammarenediol-II biosynthetic pathway in P. pastoris, the PgDDS gene from P. ginseng and the ERG1 gene from P. pastoris were integrated into the genome of P. pastoris GS115. The colonies verified by PCR amplification were cultivated and induced for 4 days. LC-MS analysis of cell extracts gave the same ion peaks as authentic dammarenediol-II at m/z 427 and 409, which confirmed the production of dammarenediol-II (see Figure S2). To compare the efficiency of the self-assembly and enzyme fusion strategies, dammarenediol-II yields from four fusion expression strains and one self-assembly strain were determined. Cell extracts obtained after 96 h of induction were examined for the quantitative analysis of dammarenediol-II yield. The yields of four fusion expression strains ranged from 0.022 to 0.038 mg/g dry cell weight (DCW), whereas the KDPEP strain achieved a yield of 0.078 mg/g DCW (see Table S3), which was more than twice the yield of the fusion expression strains. The reasons for the difference in yield between the fusion expression and the self-assembly strains might be that fusion of the two enzymes resulted in reduced activity and steric hindrance to substrate transfer. The significant difference observed between the yields from the two strategies suggested that the self-assembly strategy was more efficient than the enzyme fusion strategy for the biosynthesis of dammarenediol-II. To further determine the level of intracellular dammarenediol-II production in the self-assembly strain, the cell extracts were measured at a regular time daily after the addition of methanol. As shown in Figure 3A, dammarenediol-II yields of both strains KDE and KDPEP were gradually accumulated after methanol induction (see Table S4). Strain KDE produced

0.011 mg/g DCW, whereas strain KDPEP produced 0.045 mg/ g DCW dammarenediol-II, which was about 4-fold that of KDE. Dammarenediol-II yields from 72 to 96 h showed a modest increase for both KDE and KDPEP strains. Dammarenediol-II titers reached 0.048 mg/g DCW for KDE and 0.10 mg/g DCW for KDPEP at 96 h. The self-assembly strategy achieved a positive result compared to the unassembled system, showing a maximum enhancement of 4.1-fold and an ultimate enhancement of 2.1-fold in dammarenediol-II yield and the dammarenediol-II production in the self-assembled strain at 24 h could reach the level of that in the unassembled strain at 72 h. Increased production may be a result of substrate channeling by the assembly of PgDDS and ERG1 via the interaction between the PDZ domain and its ligand, which brought them closer in space. Assembly Identification by Fluorescence Complementation. The multiples of increased production dropped with the induction time. On the first day after methanol induction, KDPEP had a significant advantage over the KDE strain in dammarenediol-II production. In the later phase of induction, the gap between the yields was relatively reduced. To investigate this phenomenon, fluorescence complementation experiments were performed to examine the assembly dynamics of ERG1 and PgDDS in vivo. The fluorescence of yeast cells was observed by inverted fluorescence microscope and confocal laser scanning microscope (CLSM). As shown in Figure 4, a weak fluorescence signal was detected at 12 h, when PgDDS-mC160 was coexpressed with ERG1-mN159 (KMM), but an obvious fluorescence signal was observed when PgDDS-mC160-PDZlig 3383

DOI: 10.1021/acs.jafc.6b00650 J. Agric. Food Chem. 2016, 64, 3380−3385

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These results also explained why the multiples of increased dammarenediol-II production in strain KMPMP dropped off gradually with induction time. Notably, even though the advantage of self-assembly was weakened compared to the unassembled, the engineered yeast still achieved 2.1-fold enhancement in dammarenediol-II production. The distribution of fluorescence-labeled enzymes indicated that the selfassembly method improved the efficiency of substrate transfer and decreased the loss of intermediates to the ergosterol synthetic pathway, thus increasing dammarenediol-II production. The self-assembly method in this study could also be applied to the biosynthesis of other high value-added natural products, which presents a potential method in synthetic biology.

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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/acs.jafc.6b00650. Figures S1−S3, Tables S1−S4, protein sequences, and references (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(F.W.) E-mail: [email protected]. Phone: +86 21 6425 3156. Fax: +86 21 6425 0068. *(Y.R.) E-mail: [email protected]. Phone: +86 21 6425 2163. Fax: +86 21 6425 0068. Author Contributions

Figure 4. Visualization of self-assembled and unassembled yeast cells by inverted fluorescence microscope (A, C, E, F) and by confocal laser scanning microscope (B, D): (A, B) KMM-12h; (C, D) KMPMP-12h; (E) KMM-24h; (F) KMPMP-24h.

‡

C.Z. and X.G. contributed equally to this paper. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. C.Z. and X.G. designed and preformed most of experiments, participated in all data analysis, and drafted much of the manuscript. X.L. and F.W. provided advice on metabolic pathway analysis and molecular biology experiments. Y.W. provided advice on cultivation of yeasts. S.Y. contributed to the conception. Y.R. conceived the experiments, provided regular advice as the study progressed, and revised the manuscript.

and ERG1-mN159-PDZ were coexpressed (KMPMP). After 12 h, the fluorescence of KMM and KMPMP kept increasing until 24 h, at which point KMPMP showed a higher fluorescence signal than KMM. To further clarify the changes in fluorescence between KMPMP and KMM, fluorescence intensity was measured with a microplate reader at regular intervals after methanol induction. As shown in Figure 3B, the fluorescence of both KMPMP and KMM was steadily increased with time. At 24 h, KMPMP showed a nearly 2-fold greater fluorescence intensity than KMM. However, the multiples of increased fluorescence intensity dropped with the induction time, in a manner similar to the phenomenon of increased dammarenediol-II production mentioned previously. According to previous studies on the subcellular localization of ERG1 and PgDDS in yeast, PgDDS is almost exclusively located in lipid particles, whereas ERG1 is distributed among lipid particles and the endoplasmic reticulum.20 Localization of the two enzymes in lipid particles was confirmed by the fluorescence complementation experiments, including the observations of fluorescence with CLSM (Figure 4) and its quantitative detection (Figure 3B) using a microplate reader. As indicated by fluorescence complementation experiments with KMM, localization of PgDDS was originally observed to be close to that of ERG1 in lipid particles. The specific interaction between PDZ and its ligand brought PgDDS and ERG1 closer in space, resulting in higher fluorescence intensity. The drop in the multiples of increased fluorescence intensity indicated that PgDDS and ERG1 could also get closer in the KMM strain, which weakened the advantage of self-assembly in KMPMP.

Funding

This work was funded by the National Special Fund for the State Key Laboratory of Bioreactor Engineering (2060204) and the National Basic Research Program of China (973, Program No. 2012CB721003, No. 2012CB721103). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; MVA, mevalonic acid; SE (ERG1), squalene epoxidase; PgDDS, Panax ginseng dammarenediol-II synthase; PDZ, PDZ domain; PDZlig, ligand for PDZ domain; TLC, thin layer chromatography; HPLC, high-performance liquid chromatography; LC-MS, liquid chromatography−mass spectrometry; DCW, dry cell weight

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REFERENCES

(1) Christensen, L. P. Chapter 1: Ginsenosides: chemistry, biosynthesis, analysis, and potential health effects. In Advances in Food and Nutrition Research; Steve, L. T., Ed.; Academic Press: Burlington, MA, USA, 2008; Vol. 55, pp 1−9910.1016/S10434526(08)00401-4. 3384

DOI: 10.1021/acs.jafc.6b00650 J. Agric. Food Chem. 2016, 64, 3380−3385

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Journal of Agricultural and Food Chemistry (2) Helms, S. Cancer prevention and therapeutics: Panax Ginseng. Altern. Med. Rev. 2004, 9, 259−274. (3) Han, J. Y.; Kim, H. J.; Kwon, Y. S.; Choi, Y. E. The Cyt P450 enzyme CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediol-II during ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol. 2011, 52, 2062−2073. (4) Paek, K. Y.; Murthy, H. N.; Hahn, E. J.; Zhong, J. J. Large scale culture of ginseng adventitious roots for production of ginsenosides. Adv. Biochem. Eng./Biotechnol. 2009, 113, 151−176. (5) Zhang, Y. H.; Zhong, J. J.; Yu, J. T. Enhancement of ginseng saponin production in suspension cultures of Panax notoginseng: manipulation of medium sucrose. J. Biotechnol. 1996, 51, 49−56. (6) Kim, Y. S.; Yeung, E. C.; Hahn, E. J.; Paek, K. Y. Combined effects of phytohormone, indole-3-butyric acid, and methyl jasmonate on root growth and ginsenoside production in adventitious root cultures of Panax ginseng C. A. Meyer. Biotechnol. Lett. 2007, 29, 1789−1792. (7) Kim, O. T.; Bang, K. H.; Kim, Y. C.; Hyun, D. Y.; Kim, M. Y.; Cha, S. W. Upregulation of ginsenoside and gene expression related to triterpene biosynthesis in ginseng hairy root cultures elicited by methyl jasmonate. Plant Cell, Tissue Organ Cult. 2009, 98, 25−33. (8) Liang, Y. L.; Zhao, S. J.; Xu, L. X.; Zhang, X. Y. Heterologous expression of dammarenediol synthase gene in an engineered Saccharomyces cerevisiae. Lett. Appl. Microbiol. 2012, 55, 323−329. (9) Ro, D. K.; Paradise, E. M.; Ouellet, M.; Fisher, K. J.; Newman, K. L.; Ndungu, J. M.; Ho, K. A.; Eachus, R. A.; Ham, T. S.; Kirby, J.; Chang, M. C.; Withers, S. T.; Shiba, Y.; Sarpong, R.; Keasling, J. D. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 2006, 440, 940−943. (10) Westfall, P. J.; Pitera, D. J.; Lenihan, J. R.; Eng, D.; Woolard, F. X.; Regentin, R.; Horning, T.; Tsuruta, H.; Melis, D. J.; Owens, A.; Fickes, S.; Diola, D.; Benjamin, K. R.; Keasling, J. D.; Leavell, M. D.; McPhee, D. J.; Renninger, N. S.; Newman, J. D.; Paddon, C. J. Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, E111−E118. (11) Scalcinati, G.; Knuf, C.; Partow, S.; Chen, Y.; Maury, J.; Schalk, M.; Daviet, L.; Nielsen, J.; Siewers, V. Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fed-batch mode. Metab. Eng. 2012, 14, 91−103. (12) Chen, Y.; Daviet, L.; Schalk, M.; Siewers, V.; Nielsen, J. Establishing a platform cell factory through engineering of yeast acetylCoA metabolism. Metab. Eng. 2013, 15, 48−54. (13) Dai, Z. B.; Liu, Y.; Zhang, X. A.; Shi, M. Y.; Wang, B. B.; Wang, D.; Huang, L. Q.; Zhang, X. L. Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. Metab. Eng. 2013, 20, 146− 156. (14) Dai, Z. B.; Wang, B. B.; Liu, Y.; Shi, M. Y.; Wang, D.; Zhang, X. A.; Liu, T.; Huang, L. Q.; Zhang, X. L. Producing aglycons of ginsenosides in bakers’ yeast. Sci. Rep. 2014, 4, 3698−3701. (15) Tansakul, P.; Shibuya, M.; Kushiro, T.; Ebizuka, Y. Dammarenediol-II synthase, the first dedicated enzyme for ginsenoside biosynthesis. FEBS Lett. 2006, 580, 5143−5149. (16) Jung, S. C.; Kim, W.; Park, S. C.; Jeong, J.; Park, M. K.; Lim, S.; Lee, Y.; Im, W. T.; Lee, J. H.; Choi, G.; Kim, S. C. Two ginseng UDPglycosyltransferases synthesize ginsenoside Rg3 and Rd. Plant Cell Physiol. 2014, 55 (12), 2177−2188. (17) Lee, H.; DeLoache, W. C.; Dueber, J. E. Spatial organization of enzymes for metabolic engineering. Metab. Eng. 2012, 14, 242−251. (18) Gao, X.; Yang, S.; Zhao, C. C.; Ren, Y. H.; Wei, D. Z. Artificial multienzyme supramolecular device: highly ordered self-assembly of oligomeric enzymes in vitro and in vivo. Angew. Chem. 2014, 126, 14251−14254. (19) Fan, J. Y.; Cui, Z. Q.; Wei, H. P.; Zhang, Z. P.; Zhou, Y. F.; Wang, Y. P.; Zhang, X. E. Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein interactions in living cells. Biochem. Biophys. Res. Commun. 2008, 367, 47−53.

(20) Milla, P.; Athenstaedt, K.; Viola, F.; Oliaro-Bosso, S.; Kohlwein, S. D.; Daum, G.; Balliano, G. Yeast oxidosqualene cyclase (Erg7p) is a major component of lipid particles. J. Biol. Chem. 2002, 277 (4), 2406−2412.

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DOI: 10.1021/acs.jafc.6b00650 J. Agric. Food Chem. 2016, 64, 3380−3385