Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2017, Vol. 37 Issue (9): 89-97    DOI: 10.13523/j.cb.20170912
技术与方法     
产对香豆酸酿酒酵母菌株的构建及优化
张伟1,2, 刘夺1,2, 李炳志1,2, 元英进1,2
1. 天津大学化工学院制药工程系系统生物工程教育部重点实验室 天津 300072;
2. 天津化学化工协同创新中心合成生物学平台 天津 300072
Construction and Optimization of p-coumaric Acid Producing Saccharomyces cerevisiae
ZHANG Wei1,2, LIU Duo1,2, LI Bing-zhi1,2, YUAN Ying-jin1,2
1. Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University;Key Laboratory of System Bioengineering(Ministry of Education), Tianjin 300072, China;
2. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering(Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
 全文: PDF(1306 KB)   HTML
摘要: 对香豆酸(p-coumaric acid)作为苯丙素类物质、芪类物质及黄酮类物质的重要前体化合物,在生物医药、化妆品及食品工业中均有广泛的应用价值。以酿酒酵母作为底盘菌株,利用合成生物学原理构建一株高产对香豆酸的人工酵母细胞。通过对比不同拷贝数的酪氨酸解氨酶(tyrosine ammonia lyase)合成的对香豆酸产量,发现随着基因拷贝数的增加对香豆酸的产量也相应提高;同时对酪氨酸的负反馈调控相关的蛋白质进行氨基酸定点突变得到Aro4pK229L和Aro7pG141S,利用delta位点将突变后的基因整合至酵母基因组,并挑取24株构建成功的酵母细胞进行发酵验证,发现菌株最高产量与最低产量相差28.87mg/L;为了进一步增加对香豆酸的代谢通量,对生成芳香醇类物质的旁路基因 ARO10PDC5进行敲除,发现同时敲除两个基因的菌株对香豆酸的产量最高,是敲除前产量的2.05倍(从42.71mg/L到87.56mg/L)。此外,通过设计前体酪氨酸的梯度添加实验,发现当添加1mmol/L的酪氨酸时,对香豆酸产量达到峰值(174.57±0.30)mg/L,相较于未添加时提高了将近1倍。通过运用合成生物学原理在酿酒酵母中实现了对香豆酸的高产,为后续的芪类化合物和黄酮类化合物生物合成奠定了基础。
关键词: 对香豆酸合成生物学酿酒酵母内源改造    
Abstract: p-coumaric acid is widely used in bio-pharmaceutical, cosmetics and food industry as a very important precursor compound of phenylpropanoids, stilbenes and flavonoids. Saccharomyces cerevisiae was used as the host cell to synthesize p-coumaric acid with the methods of synthetic biology. It was demonstrated that the multi-copies plasmid harbored TAL showed a larger accumulation of p-coumaric acid; meanwhile, to eliminate the feedback inhibition of L-tyrosine, specific amino acid mutation Aro4pK229L and Aro7pG141S was obtained. The relevant mutated gene was integrated into yeast genome using delta site integration. 24 strains were picked out to verify the production of p-coumaric acid, the different yield between the highest strain and the lowest strain was 28.87mg/L. To strength the metabolic flux to p-coumaric, gene ARO10 and PDC5 which involved in the biosynthesis of byproduct aromatic alcohols were knocked out. Production of p-coumaric acid in strain with two gene knock out was improved to 87.56mg/L, 2.05-fold to the control one. Furthermore, when 1mmol/L L-tyrosine was added, the production of p-coumaric acid arrived the peak, about (174.57±0.30)mg/L. The p-coumaric acid over-producing S. cerevisiae using the synthetic biology method as well as lay a foundation of the biosynthesis of subsequent stilbenes and flavonoids has been successfuly constructed.
Key words: p-coumaric acid    Synthetic biology    Saccharomyces cerevisiae    Endogenous modifications
收稿日期: 2017-03-08 出版日期: 2017-09-25
ZTFLH:  Q815  
基金资助: 国家自然科学基金(21576198,21622605),天津市科技计划项目(13RCGFSY19800)资助项目
通讯作者: 李炳志     E-mail: bzli@tju.edu.cn
服务  
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章  
元英进
张伟
刘夺
李炳志

引用本文:

张伟, 刘夺, 李炳志, 元英进. 产对香豆酸酿酒酵母菌株的构建及优化[J]. 中国生物工程杂志, 2017, 37(9): 89-97.

ZHANG Wei, LIU Duo, LI Bing-zhi, YUAN Ying-jin. Construction and Optimization of p-coumaric Acid Producing Saccharomyces cerevisiae. China Biotechnology, 2017, 37(9): 89-97.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170912        https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I9/89

[1] 冉桂梅, 何彬, 杨凌,等. 对香豆酸在大鼠体内的药动学研究. 中国药学杂志, 2005, 40(24):1889-1891. Ran G M, He B, Yang L, et al. Study on pharmacokinetics of p-coumaric acid in rat. Chinese Pharmaceutical Journal, 2005, 40(24):1889-1891.
[2] Cheng J C, Dai F, Zhou B, et al. Antioxidant activity of hydroxycinnamic acid derivatives in human low density lipoprotein:Mechanism and structure-activity relationship. Food Chemistry, 2007, 104(1):132-139.
[3] Abdelwahab M H, Elmahdy M A, Abdellah M F, et al. Influence of p-coumaric acid on doxorubicin-induced oxidative stress in rat's heart. Pharmacological Research, 2003, 48(5):461-465.
[4] Bodini S F, Manfredini S, Epp M, et al. Quorum sensing inhibition activity of garlic extract and p-coumaric acid. Letters in Applied Microbiology, 2009, 49(5):551-555.
[5] Alam M A, Subhan N, Hossain H, et al. Hydroxycinnamic acid derivatives:a potential class of natural compounds for the management of lipid metabolism and obesity. Nutrition & Metabolism, 2016, 13(1):1-13.
[6] Fowler Z L, Koffas M A. Biosynthesis and biotechnological production of flavanones:current state and perspectives. Applied Microbiology and Biotechnology, 2009, 83(5):799-808.
[7] Shin S Y, Jung S M, Kim M D, et al. Production of resveratrol from tyrosine in metabolically engineered Saccharomyces cerevisiae. Enzyme & Microbial Technology, 2012, 51(4):211-216.
[8] Yan Y, Chemler J, Huang L, et al. Metabolic engineering of anthocyanin biosynthesis in Escherichia coli. Applied & Environmental Microbiology, 2005, 71(7):3617-3623.
[9] Zhao S, Jones J A, Lachance D M, et al. Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering. Metabolic Engineering, 2015, 28:43-53.
[10] Trantas E, Panopoulos N, Ververidis F. Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae. Metabolic Engineering, 2009, 11(6):355-366.
[11] Yan Y, Kohli A, Koffas M A. Biosynthesis of natural flavanones in Saccharomyces cerevisiae. Applied & Environmental Microbiology, 2005, 71(9):5610-5613.
[12] Hwang E I, Kaneko M, Ohnishi Y, et al. Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster. Applied & Environmental Microbiology, 2003, 69(5):2699-2706.
[13] Vannelli T, Wei Q W, Sweigard J, et al. Production of p-hydroxycinnamic acid from glucose in Saccharomyces cerevisiae and Escherichia coli by expression of heterologous genes from plants and fungi. Metabolic Engineering, 2007, 9(2):142-151.
[14] Santos C N, Koffas M, Stephanopoulos G. Optimization of a heterologous pathway for the production of flavonoids from glucose. Metabolic Engineering, 2011, 13(4):392-400.
[15] Xue Z, Mccluskey M, Cantera K, et al. Improved production of p -hydroxycinnamic acid from tyrosine using a novel thermostable phenylalanine/tyrosine ammonia lyase enzyme. Enzyme & Microbial Technology, 2007, 42(1):58-64.
[16] Li M, Kildegaard K R, Chen Y, et al. De novo production of resveratrol from glucose or ethanol by engineered Saccharomyces cerevisiae. Metabolic Engineering, 2015, 32:1-11.
[17] Siddiqui M S, Thodey K, Trenchard I, et al. Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools. Fems Yeast Research, 2012, 12(2):144.
[18] Liu D, Li B, Liu H, et al. Profiling influences of gene overexpression on heterologous resveratrol production in Saccharomyces cerevisiae. Frontiers of Chemical Science & Engineering, 2017,11(1):117-125.
[19] Liu D, Jin D U, Zhao G, et al. Applications of synthetic biology in medicine and energy. Ciesc Journal, 2011, 62(9):2391-2397.
[20] 肖文海, 周嗣杰, 王颖,等. 如何工程化生物学. 化工进展, 2016, 35(6):1827-1836. Xiao W H, Zhou S J, Wang Y, et al. How to make biology more "engineering". Chemical industry and Engineering Progress, 2016, 35(6):1827-1836.
[21] Fink G, Farabaugh P, Roeder G, et al. Transposable elements (Ty) in yeast. Cold Spring Harbor Symposia on Quantitative Biology, 1981, 45(pt 2):575-580.
[22] Dujon B. The yeast genome project:what did we learn. Trends in Genetics, 1996, 12(7):263-270.
[23] Luttik M A, Vuralhan Z, Suir E, et al. Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis:quantification of metabolic impact. Metabolic Engineering, 2008, 10(4):141-153.
[24] Brachmann C B, Davies A, Cost G J, et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C:A useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast, 1998, 14(2):115-132.
[25] Jendresen C B, Stahlhut S G, Li M, et al. Novel highly active and specific tyrosine ammonia-lyases from diverse origins enable enhanced production of aromatic compounds in bacteria and yeast. Applied & Environmental Microbiology, 2015, 81(13):4458-4476.
[26] Gietz R D, Schiestl R H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols, 2007, 2(1):38-41.
[27] Wang R Z, Gu X L, Yao M D, et al. Engineering of β-carotene hydroxylase and ketolase for astaxanthin overproduction in Saccharomyces cerevisiae. Frontiers of Chemical Science & Engineering, 2017,11(1):89-99.
[28] Hartmann M, Schneider T R, Pfeil A, et al. Evolution of feedback-inhibited beta/alpha barrel isoenzymes by gene duplication and a single mutation. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(3):862-867.
[29] Krappmann S, Lipscomb W N, Braus G H. Coevolution of transcriptional and allosteric regulation at the chorismate metabolic branch point of Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, 2000, 97(25):13585-13590.
[30] F W F L, Silva N A D. Sequential δ-Integration for the regulated insertion of cloned genes in Saccharomyces cerevisiae. Biotechnology Progress, 1997, 13(4):368-373.
[31] Sakai A, Shimizu Y, Hishinuma F. Integration of heterologous genes into the chromosome of Saccharomyces cerevisiae using a delta sequence of yeast retrotransposon Ty. Applied Microbiology and Biotechnology, 1990, 33(3):302-306.
[32] 王瑞钊, 潘才惠, 王颖,等. 高产β-胡萝卜素酿酒酵母菌株的设计与构建. 中国生物工程杂志, 2016, 36(7):83-91. Wang R Z, Pan C H, Wang Y, et al. Design and construction of highβ-carotene producing Saccharomyces cerevisiae. China Biotechnology, 2016, 36(7):83-91.
[33] Koopman F, Beekwilder J, Crimi B, et al. De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae. Microbial Cell Factories, 2012, 11(1):155.
[34] Hegemann J H, Güldener U, Köhler G J. Gene disruption in the budding yeast Saccharomyces cerevisiae. Methods in Molecular Biology, 2006, 313:129-144.
[35] Rodriguez A, Kildegaard K R, Li M, et al. Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis. Metabolic Engineering, 2015, 31:181-188.
[36] Zhang H, Stephanopoulos G. Co-culture engineering for microbial biosynthesis of 3-amino-benzoic acid in Escherichia coli. Biotechnology Journal, 2016, 11(7):149-150.
[37] Shin S Y, Han N S, Park Y C, et al. Production of resveratrol from p-coumaric acid in recombinant Saccharomyces cerevisiae expressing 4-coumarate:coenzyme A ligase and stilbene synthase genes. Enzyme & Microbial Technology, 2011, 48(1):48-53.
[38] Sun P, Liang J L, Kang L Z, et al. Increased resveratrol production in wines using engineered wine strains Saccharomyces cerevisiae EC1118 and relaxed antibiotic or auxotrophic selection. Biotechnol Prog, 2014, 31(3):650-655.
[39] Wang S, Zhang S, Xiao A, et al. Metabolic engineering of Escherichia coli for the biosynthesis of various phenylpropanoid derivatives. Metabolic Engineering, 2015, 29(9):153-159.
[40] Santos C N, Koffas M, Stephanopoulos G. Optimization of a heterologous pathway for the production of flavonoids from glucose. Metabolic Engineering, 2011, 13(4):392-400.
[41] Shin S Y, Jung S M, Kim M D, et al. Production of resveratrol from tyrosine in metabolically engineered Saccharomyces cerevisiae. Enzyme & Microbial Technology, 2012, 51(4):211-216.
[1] 马宁,王汉杰. 光遗传学在细菌生产调控中的应用进展[J]. 中国生物工程杂志, 2021, 41(9): 101-109.
[2] 黄焕邦,吴洋,杨友辉,王兆官,齐浩. 基于古菌酪氨酰tRNA合成酶非天然氨基酸插入的研究进展[J]. 中国生物工程杂志, 2021, 41(9): 110-125.
[3] 郭曼曼,田开仁,乔建军,李艳妮. 噬菌体重组酶系统在合成生物学中的应用*[J]. 中国生物工程杂志, 2021, 41(8): 90-102.
[4] 董曙馨,秦磊,李春,李珺. 利用转录因子工程重塑代谢网络实现细胞工厂高效生产[J]. 中国生物工程杂志, 2021, 41(4): 55-63.
[5] 薛志勇,代红生,张显元,孙艳颖,黄志伟. 表达透明颤菌血红蛋白基因对酿酒酵母生长及细胞内氧化状态的影响*[J]. 中国生物工程杂志, 2021, 41(11): 32-39.
[6] 郑义,郭世英,隋凤翔,杨骐羽,卫雅萱,李晓岩. 群体感应系统在合成生物学中的应用*[J]. 中国生物工程杂志, 2021, 41(11): 100-109.
[7] 陈鑫洁,钱芷兰,刘启,赵清,张元兴,蔡孟浩. 毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸*[J]. 中国生物工程杂志, 2021, 41(10): 52-61.
[8] 察亚平, 朱牧孜, 李爽. 体内连续定向进化研究进展 *[J]. 中国生物工程杂志, 2021, 41(1): 42-51.
[9] 石鹏程, 纪晓俊. 酵母系统表达人表皮生长因子研究进展 *[J]. 中国生物工程杂志, 2021, 41(1): 72-79.
[10] 郭二鹏, 张建志, 司同. 羊毛硫肽的高通量工程改造方法新进展 *[J]. 中国生物工程杂志, 2021, 41(1): 30-41.
[11] 常璐, 黄娇芳, 董浩, 周斌辉, 朱小娟, 庄英萍. 合成生物学改造微生物及生物被膜用于重金属污染检测与修复 *[J]. 中国生物工程杂志, 2021, 41(1): 62-71.
[12] 饶海密,梁冬梅,李伟国,乔建军,财音青格乐. 真菌芳香聚酮化合物的合成生物学研究进展*[J]. 中国生物工程杂志, 2020, 40(9): 52-61.
[13] 张玉婷,李伟国,梁冬梅,乔建军,财音青格乐. P450s在萜类合成方面的合成生物学研究进展 *[J]. 中国生物工程杂志, 2020, 40(8): 84-96.
[14] 王震,李霞,元英进. 微生物异源合成咖啡酸及其酯类衍生物研究进展 *[J]. 中国生物工程杂志, 2020, 40(7): 91-99.
[15] 岑黔鸿,高彤,任怡,雷涵. 重组酿酒酵母表达幽门螺杆菌VacA蛋白及其免疫原性分析*[J]. 中国生物工程杂志, 2020, 40(5): 15-21.