食品与发酵工业 >

2019 , Vol. 45 >Issue 20: 7 - 15

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.021558

研究报告

重组酿酒酵母发酵生产瓦伦西亚烯及其衍生物

  • 欧阳小丹 ,
  • 李文 ,
  • 察亚平 ,
  • 朱晁谊 ,
  • 李爽
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  • 华南理工大学 生物科学与工程学院,广东 广州,510006
硕士研究生(李爽教授为通讯作者,E-mail: shuangli@scut.edu.cn)

收稿日期: 2019-07-04

  网络出版日期: 2019-12-20

基金资助

国家自然科学基金项目(21878104);广东省科技计划项目(2017A010105019)

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Production of (+)-valencene and related sesquiterpenoids by engineered Saccharomyces cerevisiae

  • OUYANG Xiaodan ,
  • LI Wen ,
  • CHA Yaping ,
  • ZHU Chaoyi ,
  • LI Shuang
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  • School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China

Received date: 2019-07-04

  Online published: 2019-12-20

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摘要

以酿酒酵母为宿主菌株,重构其体内代谢途径,生物合成瓦伦西亚烯及其衍生物。该研究在酿酒酵母菌株中引入来源于纯天仙子的细胞色素P450 单加氧酶(cytochrome P450 monooxygenase,HPO)、来源于黄扁柏的瓦伦西亚烯氧化酶(valencene oxidase,CnVO),并构建了它们对应的4个突变体HPOM、CnVO-3、CnVO-4、CnVO-34,与来源于拟南芥的细胞色素还原酶(cytochrome P450 reductase,AtCPR) 组合表达,通过静息细胞试验,筛选得到表达HPO和HPOM的菌株催化瓦伦西亚烯的效果最优,选取HPOM作为后续试验的基础。进一步引入来自黄扁柏的瓦伦西亚烯合成酶(valencene synthase,CnVS),过表达醇脱氢酶(alcohol dehydrogenase,ADH1),截短的3-羟基-3-甲基戊二酰辅酶A还原酶(truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase,tHMG1)。最终获得1株原位生产瓦伦西亚烯及其衍生物的重组酵母菌株PK2-24,进行3 L发酵罐的发酵试验,158 h后总萜产量达310.94 mg/L,较原始菌株提高了111倍。该研究为利用酵母规模化生产瓦伦西亚烯及其衍生物奠定了重要基础。

关键词： 酿酒酵母; 瓦伦西亚烯; P450; 倍半萜; 代谢工程

本文引用格式

欧阳小丹 , 李文 , 察亚平 , 朱晁谊 , 李爽 . 重组酿酒酵母发酵生产瓦伦西亚烯及其衍生物[J]. 食品与发酵工业, 2019 , 45(20) : 7 -15 . DOI: 10.13995/j.cnki.11-1802/ts.021558

Abstract

The metabolic pathway of Saccharomyces cerevisiae was modified to produce (+)-valencene and related sesquiterpenoids. The genes of cytochrome P450 monooxygenase HPO derived from Hyoscyamus muticus, valencene oxidase CnVO derived from Callitropsis nootkatensis and four of their mutants (HPOM, CnVO-3, CnVO-4 and CnVO-34) were introduced into S. cerevisiae.Their effect on (+)-valencene hydroxylation were investigated by co-expressed with cytochrome reductase AtCPR derived from Arabidopsis thaliana. Through resting cell assays, the catalytic efficiencies of strains expressing HPO and HPOM showed the best results, and HPOM was selected for future study. Strain S. cerevisiae PK2-24 was constructed by expressing (+)-valencene synthase CnVS from Callitropsis nootkatensis and overexpressing ADH1 (alcohol dehydrogenase) and tHMG1 (truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase). After 158 h fermentation in a 3 L bioreactor, total terpenoid yielded was 310.94 mg/L, which was 111 times higher than that of the original strain. In conclusion, this study presents a new candidate for industrial scale production of (+)-valencene and its related sesquiterpenoids.

Key words： Saccharomyces cerevisiae; (+)-valencene; cytochrome P450; sesquiterpenoids; metabolic engineering

参考文献

[1] GIRHARD M, MACHIDA K, ITOH M, et al. Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system[J]. Microb Cell Fact, 2009, 8(1): 36.
[2] BEEKWILDER J, VAN HOUWELINGEN A, CANKAR K, et al. Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene[J]. Plant Biotechnol J, 2014, 12(2): 174-182.
[3] LEONHARDT R H, BERGER R G. Nootkatone[J]. Adv Biochem Eng Biotechnol, 2015, 148: 391-404.
[4] FURUSAWA M, HASHIMOTO T, NOMA Y, et al. Highly efficient production of nootkatone, the grapefruit aroma from valencene by biotransformation[J]. Chemical and Pharmaceutical Bulletin, 2006, 53(11): 1 513-1 514.
[5] HARING H G, RIJKENS F, BOELENS H, et al. Olfactory studies on enantiomeric eremophilane sesquiterpenoids[J]. Journal of Agricultural and Food Chemistry, 1972, 20(5): 1 018-1 021.
[6] ZHU B C R, HENDERSON G, SAUER A M, et al. Structure-activity of valencenoid derivatives and their repellence to the Formosan subterranean termite[J]. Journal of Chemical Ecology, 2003, 29(12): 2 695-2 701.
[7] WRIESSNEGGER T, AUGUSTIN P, ENGLEDER M, et al. Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris[J]. Metabolic Engineering, 2014, 24: 18-29.
[8] NEMMAR A, AL-SALAM S, BEEGAM S, et al.In vivo protective effects of nootkatone against particles-induced lung injury caused by diesel exhaust is mediated via the NF-κB pathway[J]. Nutrients, 2018, 10(3): 263.
[9] GUO X Y, SUN J, LI D, et al. Heterologous biosynthesis of (+)-nootkatone in unconventional yeast Yarrowia lipolytica[J]. Biochemical Engineering Journal, 2018, 137: 125-131.
[10] HONG B, LEBEUF R, DELBAERE S, et al. One-pot synthesis of (+)-nootkatone via dark singlet oxygenation of valencene: The triple role of the amphiphilic molybdate catalyst[J]. Catalysts, 2016, 6(12): 184-195.
[11] PALMER CARENO D M, RUTIAGA-QUINONES O M, CALVO J R R V, et al. Screening of microorganisms for bioconversion of (+)-valencene to (+)-nootkatone[J]. LWT-Food Science and Technology, 2015, 64(2): 788-793.
[12] CANKAR K, VAN HOUWELINGEN A, BOSCH D, et al. A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene[J].FEBS Letter, 2011, 585(1): 178-182.
[13] EMMERSTORFER A, WIMMER-TEUBENBACHER M, WRIESSNEGGER T, et al. Over-expression of ICE2 stabilizes cytochrome P450 reductase in Saccharomyces cerevisiae and Pichia pastoris[J]. Biotechnol J, 2015, 10(4): 623-635.
[14] TAKAHASHI S, YEO Y S, ZHAO Y, et al. Functional characterization of premnaspirodiene oxygenase, a cytochrome P450 catalyzing regio- and stereo-specific hydroxylations of diverse sesquiterpene substrates[J]. Journal of Biological Chemistry, 2007, 282(43): 31 744-31 754.
[15] VAN EUNEN K, BAKKER B M. The importance and challenges of in vivo-like enzyme kinetics[J]. Perspectives in Science, 2014, 1(1): 126-130.
[16] HENRY K W, NICKELS J T, JOSEPH T, et al. ROX1 and ERG regulation in Saccharomyces cerevisiae: Implications for antifungal susceptibility[J]. Eukaryotic Cell, 2002, 1(6): 1 041-1 044.
[17] MONTANES F M, PASCUAL-AHUIR A, PROFT M. Repression of ergosterol biosynthesis is essential for stress resistance and is mediated by the Hog1 MAP kinase and the Mot3 and Rox1 transcription factors[J]. Mol Microbiol, 2011, 79(4): 1 008-1 023.
[18] SOUZA C M, SCHWABE T M E, PICHLER H, et al. A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic acid resistance[J]. Metab Eng, 2011, 13(5): 555-569.
[19] SCHOLTMEIJER K, CANKAR K, BEEKWILDER J, et al. Production of (+)-valencene in the mushroom-forming fungus S. commune[J]. Appl Microbiol Biotechnol, 2014, 98(11): 5 059-5 068.
[20] URBAN P, MIGNOTTE C, KAZMAIER M, et al. Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5[J]. The Journal of Biological Chemistry, 1997, 272(31): 19 176-19 186.
[21] DING Mingzhu, YAN Huifang, LI Linfeng, et al. Biosynthesis of taxadiene in Saccharomyces cerevisiae: Selection of geranylgeranyl diphosphate synthase directed by a computer-aided docking strategy[J].. PLoS One, 2014, 9(10): e109 348.
[22] LI Qian, SUN Zhiqiang, LI Jing, et al. Enhancing beta-carotene production in Saccharomyces cerevisiae by metabolic engineering[J].. FEMS Microbiol Lett, 2013, 345(2): 94-101.
[23] FARHI M, MARHEVKA E, MASCI T, et al. Harnessing yeast subcellular compartments for the production of plant terpenoids[J]. Metab Eng, 2011, 13(5): 474-481.
[24] MA S M, GARCIA D E, REDDING-JOHANSON A M, et al. Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases[J]. Metab Eng, 2011, 13(5): 588-597.
[25] XIE Wenping, LV Xiaomei, YE Lidan, et al. Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering[J]. Metab Eng, 2015, 30: 69-78.
[26] SCALCINATI G, KNUF C, PARTOW S, et al. Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene alpha-santalene in a fed-batch mode[J].. Metab Eng, 2012, 14(2): 91-103.
[27] ASADOLLAHI M A, MAURY J, SCHALK M, et al. Enhancement of farnesyl diphosphate pool as direct precursor of sesquiterpenes through metabolic engineering of the mevalonate pathway in Saccharomyces cerevisiae[J]. Biotechnol Bioeng, 2010, 106(1): 86-96.
[28] ENGELS B, DAHM P, JENNEWEIN S. Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production[J]. Metab Eng, 2008, 3-4(10): 201-206.
[29] PARTOW S, SIEWERS V, JORN S, et al. Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae[J]. Yeast, 2010, 27(11): 955-964.
[30] XIONG L, ZENG Y, TANG R-Q, et al. Condition-specific promoter activities in Saccharomyces cerevisiae[J]. Microbial Cell Factories, 2018, 17(1): 58.
[31] LIAN J, MISHRA S, ZHAO H. Recent advances in metabolic engineering of Saccharomyces cerevisiae: New tools and their applications[J]. Metabolic Engineering, 2018, 50: 85-108.
[32] YAMADA R, TANAKA T, OGINO C, et al. Gene copy number and polyploidy on products formation in yeast[J]. Appl Microbiol Biotechnol, 2010, 88(4): 849-857.
[33] LIAN J, LI Y, HAMEDIRAD M,et al. Directed evolution of a cellodextrin transporter for improved biofuel production under anaerobic conditions in saccharomyces cerevisiae[J]. Biochemistry and Bioengineering, 2014,111(8):1 521-1 531.
[34] CANKAR K, VAN HOUWELINGEN A, GOEDBLOED M, et al. Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis)[J]. FEBS Letters, 2014, 588(6): 1 001-1 007.
[35] GAVIRA C, HOFER R, LESOT A, et al. Challenges and pitfalls of P450-dependent (+)-valencene bioconversion by Saccharomyces cerevisiae[J]. Metabolic Engineering, 2013, 18: 25-35.
[36] KRIVORUCHKO A, NIELSEN J. Production of natural products through metabolic engineering of Saccharomyces cerevisiae[J]. Curr Opin Biotechnol, 2015, 35: 7-15.
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