Production of Triterpene Ginsenoside Compound K in the Non

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Biotechnology and Biological Transformations

Production of triterpene ginsenoside compound K in the nonconventional yeast Yarrowia lipolytica Dashuai Li, Yufen Wu, Chuanbo Zhang, Jie Sun, Zhijiang Zhou, and Wenyu Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00009 • Publication Date (Web): 13 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

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Journal of Agricultural and Food Chemistry

Production of triterpene ginsenoside compound K in the nonconventional yeast Yarrowia lipolytica Dashuai Lia#, Yufen Wua#, Chuanbo Zhanga#, Jie Suna, Zhijiang Zhoua, Wenyu Lua, b, c*

(a) School of Chemical Engineering and Technology, Tianjin University, Tianjin, People’s Republic of China (b) Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, People’s Republic of China (c) SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, People’s Republic of China #these

authors contributed equally to this work.

*Corresponding

author: Wenyu Lu

Tel: +86-022-27892132; Fax: +86-022-27892132. E-mails: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Compound K (CK) is a rare, tetracyclic, triterpenoid compound with important

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medical properties, such as antitumor and anti-inflammatory activity. Herein, an

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efficient biosynthetic pathway of CK was constructed in metabolically engineered

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Yarrowia lipolytica for the first time, and the engineered strain, YL-CK0, produced 5.1

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mg/L of CK. The production of CK was further increased by 5.96-fold to 30.4 mg/L

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with overexpression of key genes in the MVA pathway and fusion of cytochrome P450

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monooxygenase (PPDS) and NADPH-P450 reductase. Finally, 161.8 mg/L CK

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production was achieved by fed-batch fermentation in a 5-L fermenter using the strain

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YL-MVA-CK. To the best of our knowledge, this is the first report on heterologous CK

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synthesis with the highest titer in Y. lipolytica. This study demonstrates the feasibility

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of producing high-value triterpenoid compounds using Y. lipolytica as a platform.

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Keywords:

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Yarrowia lipolytica; compound K; cytochrome P450 monooxygenase; metabolic

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engineering

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1. Introduction

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Triterpenoids, a member of large isoprene families, are C30 natural compounds

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with important medical values, such as anticancer, antitumor, cytotoxic, antiviral, and

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antibiotic properties1,

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belongs to dammarane-type ginsenosides. CK, as one of the active ingredients in the

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valuable Chinese medicine, ginseng, has been reported to exhibit strong antitumor

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activity and to have an effective role in treating heart disease and depression, improving

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immunity, and enhancing learning ability4-7. Phytoextraction, enzymatic conversion,

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and chemical synthesis have been applied to produce CK8, 9, 10. However, these methods

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were limited owing to the shortage of raw materials and the complexity of traditional

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preparation processes. On one hand, CK is rarely accumulated in ginseng plants whose

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growth is affected by climate and seasons11, 12. On the other hand, CK production via

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enzymatic conversion and chemical synthesis led to high production costs and

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complicated by-products13, so it is necessary to explore other methods for CK

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production.

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Compound K (CK), a tetracyclic triterpenoid compound,

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Synthetic biology has made significant progress in producing natural products via

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different microorganism hosts, such as ginsenoside Rh2 production in Saccharomyces

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cerevisiae14, β-carotene production in Escherichia coli15, and dammarenediol-II

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production in Pichia pastoris or E. coli16, 17. In a previous study, the biosynthesis of CK

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has been successfully realized in S. cerevisiae by introducing an exogenous CK

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synthesis pathway, including the dammarenediol-II synthase (DS), cytochrome P450

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enzyme (PPDS), and UDP-glycosyltransferase (UGT71A28) from P. ginseng, together 3



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with an NADPH-cytochrome P450 reductase ATR2-1 from Arabidopsis thaliana. The

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engineered S. cerevisiae produced only 1.4 mg/L of CK18. The CK pathway was also

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introduced into the tobacco genome by Gwak19, and the transgenic plant yielded 1.55–

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2.64 μg/g DCW of CK. However, the production volume cannot meet the consumer

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demand.

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An unconventional yeast, Y. lipolytica, generally regarded as safe (GRAS), has

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been modified to produce valuable compounds such as fatty acids, oils, biofuels, and

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few terpenoids. Due to its convenient genetic manipulation, the robust acetyl-CoA

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synthesis, and energy supply system, Y. lipolytica is considered as a potential host for

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heterologous terpenoids synthesis20-23. Like S. cerevisiae, Y. lipolytica contains a native

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mevalonate (MVA) pathway that provides the prerequisite compounds, IPP and

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DMAPP24, which further demonstrates that Y. lipolytica could serve as a better natural

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host for terpenoids synthesis, as S. cerevisiae lacks acetyl-CoA25, 26. In recent years,

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engineering Y. lipolytica has been applied to synthesis of terpenoids, such as

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monoterpene (limonene), sesquiterpenoid (α-farnesene), and tetraterpene (β-carotene,

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lycopene). Various metabolic strategies have been applied to enhance the yield of these

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terpenoids, such as increasing the cytosolic acetyl-CoA supply, optimizing the

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mevalonate (MVA) pathway, and balancing redox cofactors. The overexpression of

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HMG1 and ERG12 genes, involved in the MVA pathway, can effectively increase the

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production of limonene27. The sesquiterpene α-farnesene yield was increased by 20.8-

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fold by tHMG1, IDI, and ERG20 overexpression28. Besides, HMG1 overexpression has

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also been reported to increase the production of tetraterpene, lycopene, in Y. lipolytica29. 4


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However, metabolic engineering Y. lipolytica for triterpenoid synthesis, especially for

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triterpenoid saponins, has been rarely reported. Moreover, Y. lipolytica can use a variety

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of cheap hydrophobic or hydrophilic carbon sources30, such as fatty acids and waste oil,

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which makes it more suitable for industrial applications31.

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In this study, Y. lipolytica was selected as the chassis to de novo synthesize

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triterpenoid saponin CK. Multi-step metabolic engineering strategies were applied to

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increase the production of CK to 30.4 mg/L. The CK production was further increased

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to 161.8 ± 9.3 mg/L via fed-batch fermentation in the 5-L fermenter. To our knowledge,

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this is the first report on the production of triterpenoid saponin in Y. lipolytica and the

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highest titer of CK in microbial cell factories, which implys the feasibility for

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production of other triterpenoids in Y. lipolytica.

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2. Materials and Methods

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2.1 Strains, Plasmids, and Medium

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Y. lipolytica ATCC 201249 was used as the parental strain for strain engineering

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and plasmid pINA1269 was used as the vector for gene expression32, 33. Y. lipolytica

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ATCC 201249 and plasmid pINA1269 were kindly provided by Professor Yingjin Yuan

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(School of Chemical Engineering and Technology, Tianjin University). E. coli DH5α

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was grown in LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) with

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ampicillin (100 mg/L) at 37 °C for plasmid construction. All Y. lipolytica strains were

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cultivated in YPD medium (2% glucose, 2% peptone, and 1% yeast extract) at 30 °C

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and 200 rpm for 4 days. SC medium (0.67% yeast nitrogen base, 2% glucose, and 2%

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agar) lacking leucine or uracil was used for screening Y. lipolytica transformants. Feed

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solution (containing: glucose, 400 g/L; KH2PO4, 9 g/L; MgSO4· 7H2O, 5.12 g/L; K2SO4,

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3.5 g/L; Na2SO4, 0.28 g/L; adenine, 0.5 g/L; uracil, 0.6 g/L; lysine, 1.2 g/L) at pH 5.5

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was used for fed-batch fermentation.

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2.2 Plasmid construction

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Primers used for plasmids and expression cassettes constructing are listed in Table

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S1. Promoters and terminators were amplified from the genomic DNA of Y. lipolytica

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ATCC 201249. The DS (GenBank: AB265170.1), PPDS (GenBank: JN604537.1),

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UGT1 (GenBank: AIE12479.1), and ATR1 (GenBank: BT008426. 1) genes were

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synthesized and cloned into pUC57 plasmids by GENEWIZ (Suzhou, China) with

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codon optimization for Y. lipolytica (Codon optimized sequences are showed in Table

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S2.). Other genes used for this study were all amplified from the genomic DNA of Y.

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lipolytica ATCC 201249. ClonExpress® II One Step Cloning Kit (Vazyme, Nanjing)

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was used for inserting PCR fragments into proper restriction sites of the parental

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plasmid. Figure S1 illustrates the construction processes of pINA1269-HE9-20 from

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pINA1269. For single gene insertion, primer pairs PmlI-gene-F and PmlI-gene-R were

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used for gene amplification by PCR, and the PCR fragment was then cloned into the

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PmlI site of pINA1269 resulting in pINA1269-gene. Subsequently, ERG1, ERG9,

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ERG12, ERG19, and ERG20 expression cassettes were amplified from pINA1269-

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ERG1, pINA1269-ERG9, pINA1269-ERG12, pINA1269-ERG19, and pINA1269-

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ERG20 with primers P-X-T-SalI-F and P-X-T-SalI-R, and inserted into the SalI site of

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pINA1269-tHMG1 resulting in pINA1269-HE1, pINA1269-HE9, pINA1269-HE12, 6


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pINA1269-HE19, and pINA1269-HE20, respectively. The ERG20 gene expression

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cassette was amplified from pINA1269-ERG20 using the primers P-X-T-ClaI-F and P-

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X-T-ClaI-R; the fragments were inserted into the ClaI site of pINA1269-HE9 to get

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pINA1269-HE9-20. The pINA1269-HE1-9 was constructed by inserting ERG9

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expression cassette (hp4d-ERG9-XPR2t) into the ClaI site of pINA1269-HE1, and

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pINA1269-HE9-UGT1 was constructed by inserting UGT1 expression cassette (hp4d-

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UGT1-XPR2t) into the ClaI site of pINA1269-HE9. Constructed plasmids are shown in

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Table 1.

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2.3 Strain construction

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The pINA1269 derived plasmids were digested with Not I, and the gene expression

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cassettes used for genome integration were constructed by fusion PCR as shown in

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Figure S2. The linearized plasmids and gene expression cassettes were transformed to

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Y. lipolytica by the LiAc/ssDNA/PEG method according to our previously reported

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methods34, 35, 36. Constructed strains are shown in Table 2.

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2.4 Copy number assay

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Gene copy number assays were performed by qPCR with absolute quantification

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method37. ERG5 gene was selected as a reference gene. Open reading frames of ERG5,

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OpDS, PPDS, and ATR1 were amplified and quantified to generate standard curves.

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Genome DNA of different strains were extracted using a TIANamp Yeast DNA kit

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(Tiangen, Beijing, China). TaqMan probe-based determinations were performed as

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described by Zhang38. All experiments were performed in triplicates. Primers used for

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qPCR are listed in Table S3.

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2.5 Flask and 5-L bioreactor fermentation

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Y. lipolytica strains were inoculated into 5 mL of YPD medium as the pre-culture

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for 24 h at 30 °C and 200 rpm and the culture was transferred to 250-mL shake flasks

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loaded with 50 mL YPD medium for 5 days under the same cultivation conditions. All

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shake flask fermentation experiments were carried out in 3 parallel experimental groups.

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For batch fermentation in the 5-L bioreactor (Bailun, Shanghai, China), 5 mL of pre-

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culture was transferred to 150 mL of YPD medium in 500-mL shake flasks, and

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cultivated for 24 h at 30 °C and 220 rpm. The seed medium was inoculated into 5-L

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bioreactor with an initial OD600 of 0.5 in 2 L of YPD medium. The temperature was

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maintained at 30 °C. The pH was maintained at 6.8 by automatically adding 5 M

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ammonia water or 3 M H2SO4. The rotating speed was set at 500 rpm with an air flow

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rate of 2 vvm. When the glucose concentration was lower than 2 g/L, the feed solution

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was added to maintain the glucose concentration at below 5 g/L.

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2.6 Metabolite extraction and analysis

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Cell growth was determined by measuring the optical density at 600 nm (OD600)

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using a UV-VIS spectrophotometer. Glucose concentration was measured via a

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bioanalyzer (SBA-40C, Shandong Academy of Sciences, China). Squalene, 2, 3-

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oxidosqualene, dammarenediol-II, and PPD were extracted with ethyl acetate,

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determined by LC/APCI/MS, and quantified by HPLC following the protocols

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previously reported39.

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CK was extracted with n-butanol. Five hundred microliters of n-butanol and 0.5 g

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SiO2 (0.5 mm diameter) were added into 1 mL of culture broth, and the mixture was

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agitated by vortexing for 20 min. Then, the mixture was centrifuged at 11564 (× g) rpm

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for 10 min, and the n-butanol phase was collected for HPLC analysis. HPLC analysis

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was carried out on an Elite (Dalian, China) HPLC system equipped with an Elite P230II

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pump and an UV230II detector at 203 nm. The mobile phase was 80% acetonitrile at a

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flow rate of 1 mL/min for CK. Standards which were purchased from Meilun

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Biotechnology Co., Ltd (Dalian, China). The LC-MS analysis of DMD, PPD, and CK

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is shown in Figure S3.

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3. Results and Discussion

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3.1 Production of DMD and PPD in Y. lipolytica ATCC 201249

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PPD, precursor of CK, cannot be directly generated in Y. lipolytica. Although Y.

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lipolytica has a native MVA pathway, but it cannot directly synthesize CK due to the

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lack of dammarenediol-II synthase (DS), cytochrome P450 enzyme (PPDS), and UDP-

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glycosyltransferase (UGT1) (Figure 1).

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The plasmid pINA1269 (pINA1269-DS) containing a DS gene derived from

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Panax ginseng was transformed into Y. lipolytica ATCC 201249 resulting in strain YL-

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DS. However, DMD was not detected in strain YL-DS. It has been reported that Y.

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lipolytica has a special codon preference for some heterologous genes27, 28. Here, plant-

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derived DS gene might not be correctly expressed in Y. lipolytica. To solve this problem,

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a codon-optimized DS gene was integrated into single (pINA1269-OpDS) and muti-

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copy sites (rDNA)33 of the Y. lipolytica genome resulting in YL-OpDS0 and YL-OpDS1,

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respectively. As shown in Figure 2, DMD was detected in both strains and the yield of

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DMD in YL-OpDS1 (17.9 ± 2.3 mg/L) was 2.2-fold higher than that in YL-OpDS0 (7.9

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± 1.3 mg/L). The levels of DMD precursors, squalene and 2, 3-oxidosqualene, also

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decreased in YL-OpDS1. It has been reported that the expression level of the first

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exogenous gene that competed with precursors directly affected the yield of the target

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product during heterologous steroid synthesis40. Then, we inserted TEF1p-OpDS-

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TXPR2t, EXP1p-PPDS-MIG1t, and GPD1p-ATR1-LIP2t expression modules into the

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Y. lipolytica ATCC 201249 multi-copy rDNA sites obtaining strain YL-PPDS0. The

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YL-PPDS0 strain produced 12.9 mg/L of PPD which was mostly detected in

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extracellular (10.4 mg/L) medium. However, DMD was only detected in intracellular

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(4.8 mg/L) compartment. These results demonstrated Y. lipolytica has the ability to

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express heterologous P450 systems, which indicated the feasibility of synthesizing

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tetracyclic triterpenoid products in Y. lipolytica.

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However, the production of PPD was still low, presumably due to the following

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reasons. On one hand, the precursor flux is low in the endogenous MVA pathway, such

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as acetyl-CoA, IPP, and FPP. On the other hand, low PPD production may be due to

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the compatibility issues between the heterologous genes and the endogenous

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metabolisms, such as the endogenous P450 enzymes competed with the introduced

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PPDS for the electrons transmitted by ATR1. In fact, DMD was not fully converted

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with a conversion rate of 72%. Therefore, we speculated that increasing the flux of

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precursors in the triterpene synthesis pathway and optimizing the heterologous P450 10


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oxidation system could improve PPD production.

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3.2 Overexpression of key genes of MVA pathway to improve the production of

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PPD in Y. lipolytica

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It has been reported that β-carotene production increased by 134% when truncated

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hydroxymethylglutaryl-CoA reductase (tHMGR) from Y. lipolytica was overexpressed.

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Moreover, additional two tHMGR1 copy numbers effectively increased β-carotene

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production in Y. lipolytica41. Overexpression of hydroxymethylglutaryl-CoA reductase

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(HMGR1) was beneficial to lycopene production in Y. lipolytica29, and to beta-carotene

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production in S. cerevisiae42. Overexpression of enzymes, tHMG1 and ERG9, in the

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MVA pathway increased betulinic acid production in Y. lipolytica36. Therefore, to

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evaluate the influences on the production of PPD in Y. lipolytica, the MVA pathway

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(Figure 1) genes were analyzed in this study. Firstly, MVA pathway genes including

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ERG1, ERG8, ERG9, ERG10, ERG12, ERG13, ERG19, ERG20, IDI, HMG1, and

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tHMG1 (The sequence encoding the first 511 amino acids at the N-terminal of HMG1

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was deleted) were expressed in YL-PPDS0 using the single copy vector pINA1269

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(Figure 3A). Except for YL-PPDS9 that overexpressed IDI, the synthesis of rest of the

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PPDs were improved in strains that overexpressed other genes. Particularly, PPD

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production in YL-PPDS11 (tHMG1 overexpression) increased by 230% compared to

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that in YL-PPDS0. Overexpression of ERG1, ERG9, ERG12, ERG19, ERG20, and

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HMG1 increased PPD production by 84%, 110%, 96%, 38%, 87%, and 180%,

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respectively. The results indicated that HMG1 is also an important rate-limiting enzyme

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for triterpenoid synthesis in Y. lipolytica, followed by ERG9 and ERG20. In addition, 11



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these results also corroborated with previous studies that overexpressed key enzymes

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in the MVA pathway could significantly enhance the yield of terpenoids in the Y.

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lipolytica or S. cerevisiae43, 44, 45. In this study, both overexpressed HMG1 and tHMG1

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increased the titer of PPD, but the effect of tHMG1 was more prominent. The

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hydroxymethylglutaryl-CoA reductase HMG1 contains an N-terminal transmembrane

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region and a C-terminal catalytically active region, which respectively play a role in

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transmembrane localization and catalysis. HMG1 has been recognized as the most

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important rate-limiting enzyme in the MVA pathway, and is an important regulatory

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point in the metabolism of terpenoids in the cytoplasm. The increase in HMG1

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expression level was beneficial to increase the supply of mevalonate in the MVA

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pathway. Overexpression of HMG1 resulted in feedback inhibition of the mevalonate

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metabolic pathway, during which downstream products activate HMG1 on the

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endoplasmic reticulum for degradation. The tHMG1 (deleted N-terminal) avoids the

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self-degradation mediated by its N-terminal domain, which enhances protein stability

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and increases the metabolic flow of mevalonate46, 47. Therefore, the tHMG1 was used

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for later strain construction instead of HMG1.

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In order to further increase the titer of PPD, ERG1, ERG9, ERG12, ERG19, and

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ERG20 were selected to co-express with tHMG1 in YL-PPDS0 respectively resulting

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in strains YL-PPDS12-16 (Figure 3B). The highest PPD production of 65.5 mg/L was

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achieved by overexpressing tHMG1 and ERG9 (YL-PPDS13). However, the PPD

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production in YL-PPDS14 and YL-PPDS15 strains did not significantly increase

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compared to its parental strain, YL-PPDS11. Therefore, ERG20 or ERG1 was co-

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expressed with tHMG1 and ERG9 to obtain strain YL-PPDS17 and YL-PPDS18,

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respectively. The production of PPD in YL-PPDS17 increased by 5-fold compared to

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the original strain (YL-PPDS0), reaching 82.5 mg/L. The above results suggested that

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the synergy between tHMG1 and ERG9 was the most effective for synthesizing high

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levels of PPD, followed by ERG20. Therefore, the strategy of metabolic engineering to

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overexpress key genes in the MVA pathway exhibited a significant effect on increasing

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the metabolic flux of the precursors and promoting the accumulation of products.

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As shown in Table 3, when the upstream MVA pathway was overexpressed,

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although the PPD production was enhanced, DMD and endogenous sterol accumulation

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was also increased. It is possible to explain why the ergosterol content increased as the

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PPD synthesis yield increased. Therefore, it was speculated that DMD could not be

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completely transformed, which was the key to strengthen the potentiality of

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heterologous triterpenes. Finding a more suitable CPR (ATR1) for P450 (PPDS) was

247

an effective way to improve the P450 catalytic efficiency. In previous studies, we found

248

that PPDS catalytic efficiency plays an important role in balancing the heterologous

249

pathway in S. cerevisiae, and PPDS-ATR1 fusion could increase the catalytic efficiency

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in S. cerevisiae39. Therefore, the fusion protein strategy was implemented in Y.

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lipolytica. Strains YL-fuPPDS1 and YL-fuPPDS2 were obtained by PPDS and

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t46AtCPR1 fusion with linker1 (GSTSSGSSG) and linker2 (GSTSSG)34,

253

fuPPDS2 accumulated 109.6 mg/L of PPD and 2.3 mg/L of DMD with a conversion

254

rate of 98%, which was in accordance with the effects in S. cerevisiae (Figure 3C). The

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artificially constructed PPDS-ATR1 fusion protein improved the efficiency of electron

13


39.

YL-


256

transfer and the activity of P450 oxidation system in Y. lipolytica. This indicated that

257

fusion of key enzymes in the pathway may reduce loss of intermediates by diffusion,

258

degradation, or conversion of the intermediate through competitive pathways48.

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3.3 Construction of CK synthesis pathway in Y. lipolytica

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Protopanaxadiol (PPD) is a direct precursor compound of CK. In order to produce

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CK in Y. lipolytica, the plasmid pINA1269-UGT1 carrying the codon-optimized

262

glycosyltransferase gene was integrated into the genome of YL-PPDS0 to obtain the

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strain YL-CK0. Then, UGT1 was co-expressed with tHMG1, ERG20, and ERG9

264

resulting in YL-CK1. As shown in Figure 4, the production of CK in YL-CK1 was 21.7

265

mg/L which was 4.2-fold higher than that in YL-CK0 (5.1 mg/L). The results showed

266

that enhancing the metabolic flux of the precursor was important for increase in CK

267

production via the overexpression of the MVA pathway. Expressing the key genes of

268

MVA pathway, including tHMG1, ERG20, and ERG9, and genes involved in CK

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synthesis pathway, including OpDS, PPDS, ATR1, and UGT1, into the Y. lipolytica

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genome muti-copy sites obtained the strain YL-MVA-CK (Figure S2). YL-MVA-CK

271

accumulated 30.4 mg/L of CK which was 5.96-fold higher than that of YL-CK0. It

272

exhibited a significant increase compared with previous reported 1.4 mg/L yield in S.

273

cerevisiae18. To our knowledge, this is the first report of CK biosynthesis in oleaginous

274

yeast Y. lipolytica. Multi-copy integration strategy in key genes of MVA pathway and

275

fusion strategy between PPDS and ATR1 effectively increased the yield of CK.

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However, PPD was highly accumulated in the final CK producing strain YL-MVA-CK.

277

Thus, enhancement in the UDP-glucose supply and glycosyltransferase activity could 14


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be the feasible methods to solve this problem.

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3.4 Copy number assay

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In order to verify the effect of copy number on the results, we used qPCR to detect

281

the copy numbers of DS, PPDS, and ATR1 in screened strains (YL-OpDS1, YL-PPDS0,

282

YL-fuPPDS1, YL-fuPPDS2, and YL-MVA-CK). Based on the results of qPCR (Figure

283

S4), we found that the copy numbers of the coding genes DS, PPDS, and ATR1 at muti-

284

copy sites were more than one in different engineered strains, which indicated that the

285

multi-copy integration strategy has a positive impact on improving substrate conversion

286

rate. In Saccharomyces cerevisiae, the conversion of DMD and the reactive oxygen

287

species (ROS) level increased with increasing copy numbers of PPDS and ATR1, but

288

three copies of PPDS-ATR1 fusion proteins could convert 96.8% DMD to PPD39. In

289

this study, two copies of PPDS-ATR1 fusion proteins were sufficient to convert 98%

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DMD to PPD in Y. lipolytica. Compared with the starting strain Y. lipolytica 201249,

291

there was no significant difference in the growth of strains YL-fuPPDS1 and YL-

292

fuPPDS2. Therefore, in this study, we believed that the ROS level caused by two copy

293

numbers has no significant effect on cell growth and the synthesis of product. Further

294

research is needed to explore the effect of more copy number or ROS level on the

295

synthesis of PPD and CK.

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3.5 Fed-batch fermentation for CK production in the 5-L bioreactor

297

According to the growth curve and sugar consumption characteristics of the

298

engineered strain, YL-MVA-CK, in flaks, fed-batch fermentation was performed in a

299

5-L bioreactor with a working volume of 2.5 L. The results of biomass and CK 15



300

production during fermentation course are shown in Figure 5. In this study, after 72

301

hours of fermentation, we found a CK titer of 161.8 mg/L from 100 g (total glucose)/L

302

(yield = 1.61 mg/g sugars, productivity = 2.25 mg/L/h) feed. This was the highest yield

303

reported. In the next 48 hours, the production of CK had mostly increased. It can be

304

considered that the short fermentation cycle for the production of CK in Y. lipolytica

305

has important industrial value.

306

Conflicts of interest

307 308

The authors declare no conflicts of interest. Acknowledgments

309

This work was financially supported by the National Natural Science Foundation

310

of China (No. 21878220) and the Major Research Plan of Tianjin [No.

311

16YFXTSF00460].

312

Supporting information

313

Table S1. All the primers used in this study.

314

Table S2. Codon optimized sequences of DS, PPDS, ATR1 and UGT1 for Y.lipolytica.

315

Table S3. Primers for qPCR.

316

Figure S1. The diagram of plasmid p1269-HE9-20 construction process.

317

Figure S2. The gene expression cassettes used in engineered strain YL-MVA-CK.

318

Figure S3. LC/MS analysis of DMD, PPD and CK.

319

Figure S4. Copy number of OpDS, PPDS and ATR1 in different engineered strains.

16


Page 16 of 37

Page 17 of 37


320

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45. Westfall, P. J.; Pitera, D. J.; Lenihan, J. R.; Eng, D.; Woolard, F. X.; Regentin, R.;

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47. Donald, K.; Hampton, R. Y.; Fritz, I. B., Effects of overproduction of the catalytic

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48. Albertsen, L.; Chen, Y.; Bach, L. S.; Rattleff, S.; Maury, J.; Brix, S.; Nielsen, J.;

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24


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Page 25 of 37


480

Figure Captions

481

Figure 1. Biosynthesis pathway of CK in Yarrowia lipolytica.

482

ERG10,

483

synthase; HMG1/HMG2, HMG-CoA reductase, ERG12, mevalonate kinase; ERG8,

484

phosphomevalonate kinase; ERG19, diphosphomevalonate decarboxylase; IDI,

485

Isopentenyl pyrophosphate isomerase; ERG20, FPP synthase; ERG9, squalene

486

synthase; ERG1, squalene monooxygenase; DS, DMD synthase; PPDS, PPD synthase;

487

ATR1, NADPH-P450 reductase; UGT1, UDP-glucose glucosyltransferase. HMG-CoA,

488

hydroxymethylglutaryl-CoA; MVA, Mevalonate acid; DMD, Dammarenediol-II; PPD,

489

Protopanaxadiol; CK, Compound K. Green represents the overexpressed genes in the

490

final strain. Blue represents the overexpressed native genes and red represents the

491

optimized heterologous genes. Three dotted arrows represent multi-step reactions.

492

Figure 2. Metabolites analysis of different engineered strains. The production of

493

DMD, squalene, and 2, 3-oxidosqualene in strains YL-OpDS0 and YL-OpDS1. YL-

494

OpDS0 represents the strain in which DS was overexpressed with single copy in Y.

495

lipolytica ATCC 201249, and YL-OpDS1 represents the strain in which DS was

496

overexpressed with multiple copies in Y. lipolytica ATCC 201249. Each data represents

497

an average ± 1 standard deviation of three parallel fermentations.

498

Figure 3. Production of PPD by metabolic engineering. (A) Comparison of PPD

499

yields between YL-PPDS0 and the strains YL-PPDS1-11. YL-PPDS1-11 represent the

500

Y. lipolytica strains that overexpressed ERG1, ERG8, ERG9, ERG10, ERG12, ERG13,

acetyl-CoA

C-acetyltransferase;

ERG13,

25


hydroxymethylglutaryl-CoA


501

ERG19, ERG20, IDI, HMG1 and tHMG1, respectively. (B) Production of PPD by

502

multiple genes overexpression. YL-PPDS12-18 represents the Y. lipolytica strains YL-

503

PPDS0 were inserted into the overexpression plasmids pINA1269-HE1, pINA1269-

504

HE9, pINA1269-HE12, pINA1269-HE19, pINA1269-HE20, pINA1269-HE9-20 and

505

pINA1269-HE1-9, respectively. (C) The PPD and DMD production of fusion P450

506

oxidation system in Y. lipolytica. Each data represents an average ± 1 standard deviation

507

of three parallel fermentations.

508

Figure 4. The biosynthesis of CK in engineered Y. lipolytica. YL-CK0 represents the

509

Y. lipolytica YL-PPDS0 was inserted into the plasmid pINA1269-UGT1. YL-CK1

510

represents the Y. lipolytica YL-CK0 was inserted in pINA1269-HE9-20. YL-MVA-CK

511

represents Y. lipolytica ATCC 201249 overexpressed tHMG1, ERG9 and ERG20 in zeta

512

sites, and overexpressed OpDS, PPDS-linker2-ATR1 and UGT1 in rDNA sites. Each

513

data represents an average ± 1 standard deviation of three parallel fermentations.

514

Figure 5. Fed-batch fermentation of the engineered strain YL-MVA-CK in 5-L

515

bioreactor. The feed solution was added at 24 h. Each data point represents an average

516

± 1 standard deviation of three individual bioreactor fermentations.

517

26


Page 26 of 37

Page 27 of 37


Table 1. Plasmids used in this study. Plasmids

Description

Source

Y. lipolytica integrative plasmid, hp4d promoter, (Nicaud et pINA1269 XPR2 terminator, leu2 selection marker pINA1269-DS

al. 2002)

pINA1269 vector containing DS from Panax This study ginseng

pINA1269-OpDS

pINA1269 vector containing codon-optimized DS

This study

pINA1269-UGT1

pINA1269 vector containing codon-optimized This study UGT1

pINA1269-ERG1

pINA1269

vector

containing

ERG1

from This study

vector

containing

ERG8

from This study

vector

containing

ERG9

from This study

vector

containing

ERG10

from This study

vector

containing

ERG12

from This study

vector

containing

ERG13

from This study

Y.lipolytica pINA1269-ERG8

pINA1269 Y.lipolytica

pINA1269-ERG9


pINA1269-ERG10


pINA1269-ERG12


pINA1269-ERG13


27



pINA1269-ERG19

pINA1269

Page 28 of 37

vector

containing

ERG19

from This study

vector

containing

ERG20

from This study

Y.lipolytica pINA1269-ERG20


pINA1269-IDI

pINA1269 vector containing IDI from Y.lipolytica

pINA1269-HMG1

pINA1269

This study

vector

containing

HMG1

from This study

vector

containing

tHMG1

from This study

Y.lipolytica pINA1269-tHMG1


pINA1269-HE1

pINA1269 vector containing tHMG1 and ERG1

This study

pINA1269-HE9


This study

pINA1269-HE12


This study

pINA1269-HE19


This study

pINA1269-HE20


This study

pINA1269-HE9-20

pINA1269 vector containing tHMG1, ERG9 and This study ERG20

pINA1269-HE1-9

pINA1269 vector containing tHMG1, ERG1 and This study ERG9

pINA1269-HE9-

pINA1269 vector containing tHMG1, ERG9 and This study

UGT1

UGT1

28


Page 29 of 37


Table 2.

Strains used in this study.

Strains Y.

Description

Source

lipolytica MATA，ura3-302，leu2-270，lys8-11，pex17-ha

ATCC 201249

(Papanikola ou S 2002)

YL-DS

ATCC 201249-pINA1269-DS

This study

YL-OpDS0

ATCC 201249-pINA1269-OpDS

This study

YL-OpDS1

ATCC 201249 rDNA :: TEF1p-OpDS-TXPR2t

This study

YL-PPDS0

ATCC 201249 rDNA :: TEF1p-OpDS-TXPR2t, This study EXP1p-PPDS-MIG1t, GPD1p-ATR1-LIP2t

YL-PPDS1

YL-PPDS0-pINA1269-ERG1

This study

YL-PPDS2


This study

YL-PPDS3


This study

YL-PPDS4


This study

YL-PPDS5


This study

YL-PPDS6


This study

YL-PPDS7


This study

YL-PPDS8


This study

YL-PPDS9

YL-PPDS0-pINA1269-IDI

This study

YL-PPDS10

YL-PPDS0-pINA1269-HMG1

This study

YL-PPDS11

YL-PPDS0-pINA1269-tHMG1

This study

YL-PPDS12

YL-PPDS0-pINA1269-HE1

This study

29



Page 30 of 37

YL-PPDS13


This study

YL-PPDS14


This study

YL-PPDS15


This study

YL-PPDS16


This study

YL-PPDS17

YL-PPDS0-pINA1269-HE9-20

This study

YL-PPDS18

YL-PPDS0-pINA1269-HE1-9

This study

YL-fuPPDS1

ATCC 201249 rDNA :: TEF1p-OpDS-TXPR2t, This study EXP1p-PPDS-linker1-ATR1-MIG1t

YL-fuPPDS2

ATCC 201249 rDNA :: TEF1p-OpDS-TXPR2t, This study EXP1p-PPDS-linker2-ATR1-MIG1t

YL-CK0

YL-PPDS0-pINA1269-UGT1

This study

YL-CK1

YL-CK0-pINA1269-HE9-20

This study

YL-MVA

ATCC 201249 zeta :: FBAINp-tHMG1-XPR2t ， This study EXP1p-ERG9-MIG2t, GPD1p-ERG20-CYC1t

YL-MVA-CK

YL-MVA rDNA :: TEF1p-OpDS-XPR2t, EXP1p- This study PPDS-linker2-ATR1-MIG1t, GPD1p-UGT1-LIP2t

30


Page 31 of 37


Table 3. Metabolite analysis in different strains Strain

DMD

PPD

Squalene

2,3-oxidosqualene

Ergosterol

(mg/L)

(mg/L)

(mg/L)

(mg/L)

(mg/L)

YL-DS1

17.9±2.3

-

6.5±1.8

5.6±1.3

29.4±1.3

YL-PPDS0

4.8±0.6

12.9±1.3

5.9±1.4

4.8±0.7

27.1±2.7

YL-PPDS17 32.1±4.4

82.5±7.5

9.1±0.5

13.6±1.2

38.5±2.2

31



Figure 1. Biosynthesis pathway of CK in Yarrowia lipolytica.

32


Page 32 of 37

Page 33 of 37


Figure 2. Metabolites analysis of different engineered strains.

33



Figure 3. Production of PPD by metabolic engineering.

34


Page 34 of 37

Page 35 of 37


Figure 4. The biosynthesis of CK in engineered Y. lipolytica.

35



Figure 5. Fed-batch fermentation of the engineering strain YL-MVA-CK in 5-L bioreactor.

36


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Page 37 of 37


For Table of Contents Only

37


Production of Triterpene Ginsenoside Compound K in the Non

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