食品与发酵工业 >

2024 , Vol. 50 >Issue 20: 9 - 17

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.038171

研究报告

代谢工程改造酿酒酵母高效合成熊果酸

  • 赵星莹 ,
  • 曾伟主 ,
  • 周景文
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  • 1(江南大学 生物工程学院,工业生物技术教育部重点实验室,江苏 无锡,214122)
    2(江南大学,未来食品科学中心,江苏 无锡,214122)
第一作者:硕士研究生(周景文教授为通信作者,E-mail:zhoujw1982@Jiangnan.edu.cn)

收稿日期: 2023-12-05

  修回日期: 2024-01-10

  网络出版日期: 2024-11-01

基金资助

国家自然科学基金创新研究群体项目(32021005)

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Metabolic engineering of Saccharomyces cerevisiae for efficient synthesis of ursolic acid

  • ZHAO Xingying ,
  • ZENG Weizhu ,
  • ZHOU Jingwen
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  • 1(Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China)
    2(Science Center for Future Foods, Jiangnan University, Wuxi 214122, China)

Received date: 2023-12-05

  Revised date: 2024-01-10

  Online published: 2024-11-01

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摘要

熊果酸是一种五环三萜类化合物,具有抗炎、抗氧化等作用,广泛应用于医药、化妆品等领域。为提高酿酒酵母中熊果酸的产量,该研究采用了代谢工程的“推-拉”策略。首先,通过异源表达和优化CADDS、CaCYP716A83和HtCPR,实现了熊果酸的从头合成,其产量为5.4 mg/L。随后,通过扩大内质网面积,为膜蛋白酶提供更多的附着面积,熊果酸产量达到40.3 mg/L。由于熊果酸合成路径中涉及多个氧化还原反应,NADPH是必需的辅因子,强化NADPH供给后,熊果酸产量提升至48.2 mg/L。此外,通过优化电子传递链,利用细胞色素b5参与第二个电子向P450酶转移,增强反应中的电子供给,熊果酸产量达到74.6 mg/L。最后,将扩大内质网、增强NADPH及电子传递、调节CaCYP716A83和HtCPR拷贝数等策略进行迭代整合,熊果酸产量达到84.3 mg/L。该研究为构建高效合成萜类化合物的微生物细胞工厂提供了参考。

关键词： 熊果酸; 酿酒酵母; 内质网; NADPH; 细胞色素b5

本文引用格式

赵星莹 , 曾伟主 , 周景文 . 代谢工程改造酿酒酵母高效合成熊果酸[J]. 食品与发酵工业, 2024 , 50(20) : 9 -17 . DOI: 10.13995/j.cnki.11-1802/ts.038171

Abstract

Ursolic acid, a pentacyclic triterpene compound with anti-inflammatory and antioxidant effects, has been widely applied in medicine, cosmetics, and various other fields.In this study, a “push-pull” metabolic engineering strategy was employed to increase the production of ursolic acid in Saccharomyces cerevisiae.Firstly, de novo biosynthesis of ursolic acid was achieved by heterologous expression and optimization of CADDS, CaCYP716A83, and HtCPR genes, resulting in 5.4 mg/L of ursolic acid.Subsequently, an expansion of the endoplasmic reticulum area was conducted to provide more adherent surface for membrane protease activity, obtaining 40.3 mg/L of ursolic acid.Due to multiple REDOX reactions was involved in the ursolic acid synthesis pathway, in which the NADPH was an essential cofactor.By enhancing the supply of NADPH, the ursolic acid titer was increased to 48.2 mg/L.Additionally, optimization of the electron transport chain and utilization of cytochrome b5 for efficient electron transfer to P450 enzyme further enhanced the electron supply, resulting in 74.6 mg/L of ursolic acid.Finally, by iteratively integrating these strategies including expanding endoplasmic reticulum area, enhancing NADPH availability, and electron transport efficiency and regulating the copy number of CaCYP716A83 and HtCPR genes, the ursoilc acid production was further enhanced to 84.3 mg/L.This study offers valuable insights for the development of efficient microbial cell factories for terpenoid synthesis, serving as a reference in the field.

Key words： ursolic acid; Saccharomyces cerevisiae; endoplasmic reticulum; NADPH; cytochrome b5

参考文献

[1] KASHYAP D, TULI H S, SHARMA A K.Ursolic acid (UA):A metabolite with promising therapeutic potential[J].Life Sciences, 2016, 146:201-213.
[2] GHASEMZADEH F, NAJAFPOUR DARZI G, MOHAMMADI M.Extraction and purification of ursolic acid from the apple peel and in vitro assessment of the biochemical antibacterial, antioxidant and wound healing characteristics[J].Applied Food Biotechnology, 2022, 9(1):17-30.
[3] ZHAO M, WU F Y, TANG Z H, et al.Anti-inflammatory and antioxidant activity of ursolic acid:A systematic review and meta-analysis[J].Frontiers in Pharmacology, 2023, 14:1256946.
[4] WANG J, ZHAO J, YAN Y, et al.Inhibition of glycosidase by ursolic acid:In vitro, in vivo and in silico study[J].Journal of the Science of Food and Agriculture, 2020, 100(3):986-994.
[5] CHEN Z X, LIU Q L, ZHU Z W, et al.Ursolic acid protects against proliferation and inflammatory response in LPS-treated gastric tumour model and cells by Inhibiting NLRP3 inflammasome activation[J].Cancer Management and Research, 2020, 12:8413-8424.
[6] NISTOR G, TRANDAFIRESCU C, PRODEA A, et al.Semisynthetic derivatives of pentacyclic triterpenes bearing heterocyclic moieties with therapeutic potential[J].Molecules, 2022, 27(19):6552.
[7] FUKUSHIMA E O, SEKI H, OHYAMA K, et al.CYP716A subfamily members are multifunctional oxidases in triterpenoid biosynthesis[J].Plant and Cell Physiology, 2011, 52(12):2050-2061.
[8] SPORN M B, LIBY K T, YORE M M, et al.New synthetic triterpenoids:potent agents for prevention and treatment of tissue injury caused by inflammatory and oxidative stress[J].Journal of Natural Products, 2011, 74(3):537-545.
[9] XU Y M, WANG X L, ZHANG C Y, et al.De novo biosynthesis of rubusoside and rebaudiosides in engineered yeasts[J].Nature Communications, 2022, 13(1):3040.
[10] FLETCHER E, KRIVORUCHKO A, NIELSEN J.Industrial systems biology and its impact on synthetic biology of yeast cell factories[J].Biotechnology & Bioengineering, 2016, 113(6):1164-1170.
[11] LIU Q L, LIU Y, LI G, et al.De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories[J].Nature Communications, 2021, 12(1):6085.
[12] LU H Z, KERKHOVEN E J, NIELSEN J.Multiscale models quantifying yeast physiology:Towards a whole-cell model[J].Trends in Biotechnology, 2022, 40(3):291-305.
[13] DAI Z B, WANG B B, LIU Y, et al.Producing aglycons of ginsenosides in bakers′ yeast[J].Scientific Reports, 2014, 4:3698.
[14] HUANG L L, LI J, YE H C, et al.Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus[J].Planta, 2012, 236(5):1571-1581.
[15] LU C Z, ZHANG C B, ZHAO F L, et al.Biosynthesis of ursolic acid and oleanolic acid in Saccharomyces cerevisiae[J].AIChE Journal, 2018, 64(11):3794-3802.
[16] JIN K, SHI X, LIU J H, et al.Combinatorial metabolic engineering enables the efficient production of ursolic acid and oleanolic acid in Saccharomyces cerevisiae[J].Bioresource Technology, 2023, 374:128819.
[17] GAO S, XU X Y, ZENG W Z, et al.Efficient biosynthesis of (2S)-eriodictyol from (2S)-naringenin in Saccharomyces cerevisiae through a combination of promoter adjustment and directed evolution[J].ACS Synthetic Biology, 2020, 9(12):3288-3297.
[18] ZHANG Y L, WANG W G, WEI W Q, et al.Regulation of ethanol assimilation for efficient accumulation of squalene in Saccharomyces cerevisiae[J].Journal of Agricultural and Food Chemistry, 2023, 71(16):6389-6397.
[19] YU Y, CHANG P C, YU H, et al.Productive amyrin synthases for efficient α-amyrin synthesis in engineered Saccharomyces cerevisiae[J].ACS Synthetic Biology, 2018, 7(10):2391-2402.
[20] XIAO F, LIAN J Z, TU S, et al.Metabolic engineering of Saccharomyces cerevisiae for high-level production of chlorogenic acid from glucose[J].ACS Synthetic Biology, 2022, 11(2):800-811.
[21] CHAO L F I, LIU D, SIEWERS V.A highly selective cell-based fluorescent biosensor for genistein detection[J].Engineering Microbiology, 2023, 3(2):100078.
[22] BENVENISTE I, LESOT A, HASENFRATZ M P, et al.Multiple forms of NADPH-cytochrome P450 reductase in higher plants[J].Biochemical and Biophysical Research Communications, 1991, 177(1):105-112.
[23] LV Y Q, GAO S, XU S, et al.Spatial organization of silybin biosynthesis in milk thistle[Silybum marianum (L.) Gaertn][J].The Plant Journal, 2017, 92(6):995-1004.
[24] LIU X N, CHENG J, ZHANG G H, et al.Engineering yeast for the production of breviscapine by genomic analysis and synthetic biology approaches[J].Nature Communications, 2018, 9:448.
[25] XIU X, SUN Y, WU Y K, et al.Modular remodeling of sterol metabolism for overproduction of 7-dehydrocholesterol in engineered yeast[J].Bioresource Technology, 2022, 360:127572.
[26] TAN J X, ZHANG C B, PAI H H, et al.Heterologous biosynthesis of taraxerol by engineered Saccharomyces cerevisiae[J].FEMS Microbiology Letters, 2022, 369(1):fnac070.
[27] STUKEY G J, HAN G S, CARMAN G M.Phosphatidate phosphatase Pah1 contains a novel RP domain that regulates its phosphorylation and function in yeast lipid synthesis[J].Journal of Biological Chemistry, 2023, 299(8):105025.
[28] PAPAGIANNIDIS D, BIRCHAM P W, LÜCHTENBORG C, et al.Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex[J].The EMBO Journal, 2021, 40(22):e107958.
[29] KHONDKER S, KWIATEK J M, HAN G S, et al.Glycogen synthase kinase homolog Rim11 regulates lipid synthesis through the phosphorylation of Pah1 phosphatidate phosphatase in yeast[J].The Journal of Biological Chemistry, 2022, 298(8):102221.
[30] 朱满志, 陈献忠, 沈微, 等.代谢工程改造酿酒酵母高效合成游离脂肪酸[J].食品与发酵工业, 2024, 50(2):15-22.
ZHU M Z, CHEN X Z, SHEN W, et al.Metabolic engineering of Saccharomyces cerevisiae for efficient synthesis of free fatty acids[J].Food and Fermentation Industries, 2024, 50(2):15-22.
[31] 周颂, 李由然, 张梁, 等.代谢工程改造酿酒酵母合成β-胡萝卜素[J].食品与发酵工业, 2024, 50(3): 1-8.
ZHOU S, LI Y R, ZHANG L, et al.Synthesis of β-carotene from Saccharomyces cerevisiae by metabolic engineering[J].Food and Fermentation Industries, 2024, 50(3): 1-8.
[32] HOU J, LAGES N F, OLDIGES M, et al.Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae[J].Metabolic Engineering, 2009, 11(4-5):253-261.
[33] VEMURI G N, EITEMAN M A, MCEWEN J E, et al.Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae[J].Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(7):2402-2407.
[34] GRABOWSKA D, CHELSTOWSKA A.The ALD6 gene product is indispensable for providing NADPH in yeast cells lacking glucose-6-phosphate dehydrogenase activity[J].The Journal of Biological Chemistry, 2003, 278(16):13984-13988.
[35] PARAMASIVAN K, MUTTURI S.Regeneration of NADPH coupled with HMG-CoA reductase activity increases squalene synthesis in Saccharomyces cerevisiae[J].Journal of Agricultural and Food Chemistry, 2017, 65(37):8162-8170.
[36] ZHAO X, SHI F, ZHAN W.Overexpression of ZWF1 and POS5 improves carotenoid biosynthesis in recombinant Saccharomyces cerevisiae[J].Letters in Applied Microbiology, 2015, 61(4):354-360.
[37] CHENG H L, WANG S Q, BILAL M, et al.Identification, characterization of two NADPH-dependent erythrose reductases in the yeast Yarrowia lipolytica and improvement of erythritol productivity using metabolic engineering[J].Microbial Cell Factories, 2018, 17(1):133.
[38] BERGMAN A, VITAY D, HELLGREN J, et al.Effects of overexpression of STB5 in Saccharomyces cerevisiae on fatty acid biosynthesis, physiology and transcriptome[J].FEMS Yeast Research, 2019, 19(3):foz027.
[39] WANG C X, SU X Y, SUN M C, et al.Efficient production of glycyrrhetinic acid in metabolically engineered Saccharomyces cerevisiae via an integrated strategy[J].Microbial Cell Factories, 2019, 18(1):95.
[40] YAZAWA H, IWAHASHI H, KAMISAKA Y, et al.Improvement of polyunsaturated fatty acids synthesis by the coexpression of CYB5 with desaturase genes in Saccharomyces cerevisiae[J].Applied Microbiology and Biotechnology, 2010, 87(6):2185-2193.
[41] PADDON C J, WESTFALL P J, PITERA D J, et al.High-level semi-synthetic production of the potent antimalarial artemisinin[J].Nature, 2013, 496(7446):528-532.
[42] ZHOU Y J, GAO W, RONG Q X, et al.Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production[J].Journal of the American Chemical Society, 2012, 134(6):3234-3241.
[43] NIELSEN J, KEASLING J D.Engineering cellular metabolism[J].Cell, 2016, 164(6):1185-1197.
[44] SHI B, MA T, YE Z L, et al.Systematic metabolic engineering of Saccharomyces cerevisiae for lycopene overproduction[J].Journal of Agricultural and Food Chemistry, 2019, 67(40):11148-11157.
[45] LI T, LIU G S, ZHOU W, et al.Metabolic engineering of Saccharomyces cerevisiae to overproduce squalene[J].Journal of Agricultural and Food Chemistry, 2020, 68(7):2132-2138.
[46] LIU H, FAN J J, WANG C, et al.Enhanced β-amyrin synthesis in Saccharomyces cerevisiae by coupling an optimal acetyl-CoA supply pathway[J].Journal of Agricultural and Food Chemistry, 2019, 67(13):3723-3732.
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