合成8二甲基异戊烯基柚皮素的人工酿酒酵母菌株构建

doi:10.13523/j.cb.20170910

中国生物工程杂志

2017, Vol. 37

Issue (9): 71-81 DOI: 10.13523/j.cb.20170910

技术与方法

合成8二甲基异戊烯基柚皮素的人工酿酒酵母菌株构建

李博^1,2, 梁楠^1,2, 刘夺^1,2, 刘宏^1,2, 王颖^1,2, 肖文海^1,2, 姚明东^1,2, 元英进^1,2

1. 天津大学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

Metabolic Engineering of Saccharomyces cerevisiae for Production of 8-Dimenthylally Naringenin

LI Bo^1,2, LIANG Nan^1,2, LIU Duo^1,2, LIU Hong^1,2, WANG Ying^1,2, XIAO Wen-hai^1,2, YAO Ming-dong^1,2, YUAN Ying-jin^1,2

1. Key Laboratory of Systems Bioengineering(Ministry of Education), Tianjin University, Tianjin 300072, China;
2. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China

全文: PDF(2389 KB) HTML

摘要： 8二甲基异戊烯基柚皮素（8DN）作为生产黄酮类药物淫羊藿苷的重要前体，在医药合成领域具有重大应用潜力。由于其合成路径及相关基因的复杂性，目前主要通过饲喂8DN的直接前体（柚皮素、异黄腐酚等）的方式合成8DN，而在生物体内全合成8DN的研究工作还未见报道。为了实现8DN在酿酒酵母体内的生物全合成，通过组合筛选8DN前体物柚皮素合成所需的多种外源基因（TAL、4CL、CHS、CHI），获得30株柚皮素生产菌，发现不同来源的基因组合使柚皮素产量的具有明显差异（0.37~22.33mg/L）。并且利用Delta位点将较优的基因组合整合至酵母基因组，实现了稳定的柚皮素高产菌株（SyBE_Sc02050031）构建。在此基础上进一步导入带有苦参来源的异戊烯基转移酶基因（N8DT）多拷贝质粒，实现8DN合成的完整反应过程，8DN的摇瓶发酵产量达到36.7μg/L。另外，通过关键限速酶N8DT的序列优化策略，发现截断定位信号肽序列的N8DT明显提高了从柚皮素到8DN这一关键反应的催化效果，8DN的产量提高到52.6μg/L（144.2%）。首次在酿酒酵母中成功构建高产8DN的生物全合成路径，为在微生物体内合成其他黄酮类天然产物提供了参考，具有重要的指导意义。

关键词： 蛋白质截断; 合成生物学; 异源表达; 酿酒酵母; 8二甲基异戊烯基柚皮素

Abstract: 8-dimenthylallynaringenin(8DN) is the important precursor for the flavonoids medicine Icaritin, which has wide applications in pharmaceutical area. Due to the complexity of its biosynthetic pathway and related genes, main attentions were paied to biotransfering by procursor (like naringenin or isoxanthohumol) feeding. To date, no report about de novo biosynthesis of 8DN has been uncovered. In present study, in order to realize 8DN production in Saccharomyces cerevisiae, four key genes involved in naringenin biosynthesis, TAL,4CL,CHS and CHI from different species, were combinatorially screened, obtaining 30 naringenin producing strains, in which significant production variation ranged from 0.37mg/L to 22.34mg/L. Furthermore, the best combination of TAL,4CL,CHS and CHI genes were integrated into chromosome for better genetic stability by delta integration, generating strain SyBE_Sc02050031. Subsequently, prenyltransferase gene (N8DT) from Sophora flavescens incorporated with multicopy plasmid was introduced into SyBE_Sc02050031 (gaining strain SyBE_Sc02050032) and a titer of 8DN at 36.7μg/L in shaking flask was observed accordingly, indicating de novo biosynthesis of 8DN in yeast was successfully achieved. In addition, truncation tailoring strategy was explored to improve the catalysis function of N8DT, leading to 8DN production (up to 52.6μg/L) increasing by 44% compared to that in strain SyBE_Sc02050032. de novo biosynthesis of 8DN in microbes is firstly accomplished, which provides a good reference for microbial production of other natural flavonoids.

Key words: Saccharomyces cerevisiae 8-dimenthylally Synthetic biology naringenin Heterologous expression Protein truncation

收稿日期: 2017-03-15 出版日期: 2017-09-25

ZTFLH:

Q815

基金资助: 国家自然科学基金资助项目（31600052）

通讯作者: 姚明东 E-mail: yao.1982@163.com

	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
	李博
	梁楠
	刘宏
	肖文海
	元英进
	王颖
	姚明东
	刘夺

引用本文:

李博, 梁楠, 刘夺, 刘宏, 王颖, 肖文海, 姚明东, 元英进. 合成8二甲基异戊烯基柚皮素的人工酿酒酵母菌株构建[J]. 中国生物工程杂志, 2017, 37(9): 71-81.

LI Bo, LIANG Nan, LIU Duo, LIU Hong, WANG Ying, XIAO Wen-hai, YAO Ming-dong, YUAN Ying-jin. Metabolic Engineering of Saccharomyces cerevisiae for Production of 8-Dimenthylally Naringenin. China Biotechnology, 2017, 37(9): 71-81.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170910 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I9/71

[1] Yan Y J, Kohli A, Koffas M A. Biosynthesis of natural flavanones in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 2005, 71(9):5610-5613.
[2] Chtourou Y, Aouey B, Aroui S, et al. Anti-apoptotic and anti-inflammatory effects of naringin on cisplatin-induced renal injury in the rat. Chemico-biological Interactions, 2016, 243:1-9.
[3] Harborne J B, Williams C A. Advances in flavonoid research since 1992. Phytochemistry, 2000, 55(6):481-504.
[4] Maitrejean M, Comte G, Barron D, et al. The flavanolignan silybin and its hemisynthetic derivatives, a novel series of potential modulators of glycoprotein. Bioorg Med Chem Lett, 2000, 10(2):157-160.
[5] Sasaki K, Tsurumaru Y, Yazaki K. Prenylation of flavonoids by biotransformation of yeast expressing plant membrane-bound prenyltransferase SfN8DT-1. Biosci Biotechnol Biochem, 2009, 73(3):759-761.
[6] Wu J, Du G, Chen J, et al. Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli. Scientific Reports, 2015, 5:13477.
[7] Zhang G, Liu S, Tan W, et al. Synthesis and biological evaluations of chalcones, flavones and chromenes as farnesoid x receptor (FXR) antagonists. Eur J Med Chem, 2017, 129(3):303-309.
[8] 李毅林. 一种天然产物淫羊藿苷类化合物的全合成方法:中国, 200610165354. 2008-06-25. Li Y L, A Method of Total Synthesis for the Natural Product Lcariin Compound:Chinese, 200610165354. 2008-06-25.
[9] Geissman T A, David K F. Flavonones and related compounds. V. the oxidation of 2'-hydroqchalcones with alkaline hydrogen peroxide. J Am Chem Soc, 1948, 50(5):1686-1689.
[10] Aurangzeb H, Sadipa A, Abbasa A, et al. Isolation and synthesis of flavonols and comparison of their antioxidant activity. Natural Product Research, 2010, 24(11):995-1003.
[11] Fu M L, Wang W, Chen F, et al. Production of 8-phrenylnaringenin from isoxanthohumol through biotransformation by fungi cells. J Agric Food Chem, 2011, 59(13):7419-7426.
[12] Sasaki K, Tsurumaru Y, Yazaki K, et al. Molecular characterization of a membrane-bound prenyltransferase specific for isoflavone from Sophora flavescens. The Journal of Biological Chemistry, 2011, 286(8):24125-24134.
[13] 傅明亮, 刘婧, 陈苗苗, 等. RP-HPLC法同步检测酒花中黄腐酚、异黄腐酚与8异戊烯基柚皮素. 中国食品学报, 2010, 10(6):193-198. Fu M L, Liu J, Chen M M, et al. Synchronous detected xanthohumol, isoxanthohumol and 8-prenylnaringenin in Humulus lupulus by RP-HPLC. Journal of Chinese Institute of Food Science and Technology, 2010, 10(6):193-198.
[14] Vickers C E, Bongers M, Liu Q, et al. Metabolic engineering of volatile isoprenoids in plants and microbes. Plant, Cell & Environment, 2014, 37(8):1753-1775.
[15] Fowler Z L, Koffas M A. Biosynthesis and biotechnological production of flavanones:current state and perspectives. Appl Microbiol Biotechnol, 2009, 83(5):799-808.
[16] Wang Y, Chen S, Yu O. Metabolic engineering of flavonoids in plants and microorganisms. Appl Microbiol Biotechnol, 2011, 91(4):949-956.
[17] Limem I, Guedonc E, Hehn A, et al. Production of phenylpropanoid compounds by recombinant microorganisms expressing plant-specific biosynthesis genes. Process Biochem, 2008, 43:463-479.
[18] Wilhelm H, Wessjohann L A. An efficient synthesis of the phytoestrogen 8-prenylnaringenin from xanthohumol by a novel demethylation process. Tetrahedron, 2006, 62(29):6961-6966.
[19] MacDonald I C, Deans T L. Tools and applications in synthetic biology. Adv Drug Deliv Rev, 2016, 105(A):20-34.
[20] Gibson D G, Venter J C. Construction of a yeast chromosome. Nature, 2014, 509(5):168-169.
[21] Sun J, Shao Z, Zhao H, et al. Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae. Biotechnol Bioeng, 2012, 109(8):2082-2092.
[22] Fink G, Farabaugh P, Roeder G, et al. Transposable elements (Ty) in yeast. Cold Spring Harb Symp Quant Biol, 1981, 45(2):575-580.
[23] Siddiqui M S, Thodey K, Trenchard I, et al. Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools. Fems Yeast Res, 2012, 12(2):144-170.
[24] Jiang H X, Karl V, John A. Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 2005, 71(6):2962-2969.
[25]. Lee F W, Da S N. Sequential delta-integration for the regulated insertion of cloned genes in Saccharomyces cerevisiae. Biotechnol Prog, 1997, 13(4):368-373.
[26] Sasaki K, Tsurumaru Y, Yazaki K, et al. Cloning and characterization of naringenin 8-prenyltransferase, a flavonoid-specific prenyltransferase of Sophora flavescens. Plant Physiology, 2008, 146(3):1075-1084.
[27] Sugiyama A, Linley P J, Sasaki K, et al. Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases. Metabolic Engineering, 2011, 13(6):629-637.
[28] Kim I K, Roldao A, Siewers V, et al. A systems-level approach for metabolic engineering of yeast cell factories. Fems Yeast Res, 2012, 12(2):228-248.
[29] Shao Z, Zhao H, Zhao H. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res, 2008, 37(2):e16.

[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]	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.
[8]	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.
[9]	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.
[10]	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.
[11]	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.
[12]	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.
[13]	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.
[14]	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.
[15]	位薇,常保根,王英,路福平,刘夫锋. Tau蛋白核心片段306~378的异源表达、纯化及聚集特性验证^*[J]. 中国生物工程杂志, 2020, 40(5): 22-29.

Viewed

Full text

Abstract

Cited

Shared

Discussed