产蒎烯人工酵母细胞的构建

陈天华; 张若思; 姜国珍; 姚明东; 刘宏; 王颖; 肖文海; 元英进

doi:10.11949/j.issn.0438-1157.20180784

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产蒎烯人工酵母细胞的构建

生物化学工程与技术 | 更新时间：2020-06-24

- 产蒎烯人工酵母细胞的构建
- Metabolic engineering of Saccharomyces cerevisiae for pinene production
- 化工学报 2019年70卷第1期页码：179-188
- 作者机构：
  
  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
- 作者简介：
  
  陈天华（1993—），女，硕士研究生，13102217875@163.com
  王颖（1983—），女，副研究员，ying.wang@tju.edu.cn
- 基金信息：
  
  国家自然科学基金面上项目(21621004;21676192)
- DOI：10.11949/j.issn.0438-1157.20180784
  中图分类号： Q 819
- 收稿：2018-07-11，
  
  修回：2018-09-09，
  
  纸质出版：2019-01-05
- 稿件说明：
移动端阅览
陈天华, 张若思, 姜国珍, 姚明东, 刘宏, 王颖, 肖文海, 元英进. 产蒎烯人工酵母细胞的构建[J]. 化工学报, 2019, 70(1): 179-188

CHEN Tianhua, ZHANG Ruosi, JIANG Guozhen, YAO Mingdong, LIU Hong, WANG Ying, XIAO Wenhai, YUAN Yingjin. Metabolic engineering of Saccharomyces cerevisiae for pinene production[J]. CIESC Journal, 2019, 70(1): 179-188
陈天华, 张若思, 姜国珍, 姚明东, 刘宏, 王颖, 肖文海, 元英进. 产蒎烯人工酵母细胞的构建[J]. 化工学报, 2019, 70(1): 179-188 DOI： 10.11949/j.issn.0438-1157.20180784.

CHEN Tianhua, ZHANG Ruosi, JIANG Guozhen, YAO Mingdong, LIU Hong, WANG Ying, XIAO Wenhai, YUAN Yingjin. Metabolic engineering of Saccharomyces cerevisiae for pinene production[J]. CIESC Journal, 2019, 70(1): 179-188 DOI： 10.11949/j.issn.0438-1157.20180784.

摘要

蒎烯可衍生为高能量密度燃料，但在酿酒酵母中的全生物合成却未见报道。酿酒酵母由于拥有强大的蛋白表达和翻译后修饰系统以及完整的内膜系统，相比于大肠杆菌等原核生物更适于P450等蛋白的表达，因此将酿酒酵母作为宿主细胞，对于蒎烯或者其他物质实现如“疯狂碳环”的高能量化是至关重要的。本研究在酿酒酵母底盘中表达内源焦磷酸香叶酯合成酶（ERG20）的突变体ERG20

和火炬松来源的蒎烯合酶（PtPS）构建了蒎烯的合成路径。通过截短PtPS N端2～51位氨基酸残基（tPtPS），蒎烯产量较初始产量（0.329 mg·L

-1

）提高了2.23倍。在过表达异戊二烯焦磷酸异构酶（IDI1）和RNA聚合酶Ш负调控因子（MAF1）的基础上，表达ERG20

和tPtPS的融合蛋白，蒎烯产量进一步提高了5.16倍。通过将内源基因

ERG20

启动子原位替换为弱启动子

HXT1

，下调

ERG20

的转录，蒎烯的产量提高了26.0%。最终通过调节发酵过程中的培养基pH使蒎烯产量达11.7 mg·L

-1

，较初始产量提高了34.5倍。本研究在酿酒酵母中实现蒎烯的从头合成，并获得已知蒎烯摇瓶水平的最高产量。

Abstract

The derivatives of pinene can be employed as high energy density fuels. However

the biosynthesis of pinene from single carbon sources has not been realized in yeast. As

Saccharomyces cerevisiae

has stronger ability on protein expression and post-translational modification as well as processes a maturity endomembrane system

it is more suitable for expressing complex proteins (

e.g.

cytochrome P450) by yeast than by prokaryotic hosts such as

Escherichia coli

. Therefore

it is crucial to engineer

S. cerevisiae

as the host cell to produce high energy density fuels (like “Crazy Carbon Ring”) based on the derivatization of pinene or other terpenes compounds. Here

in order to achieve pinene synthesis in yeast

endogenous farnesyl diphosphate synthase (ERG20) mutant ERG20

and

Pinus taeda

pinene synthase (PtPS) were expressed in

Saccharomyces cerevisiae

strain

obtaining an initial pinene titer of 0.329 mg·L

-1

. N-terminus truncation (from 2A to 51P) of PtPS (obtaining tPtPS) improve the pinene production by 2.23-fold. The pinene titer was further enhanced by 5.16-fold by expression the fusion of ERG20

/tPtPS on the basis of overexpression of isoprene pyrophosphate isomerase (IDI1) and the repressor of RNA polymerase III (MAF1). Replacing the promoter of gene

ERG20

by a weaker promoter

HXT1

down-regulated the transcription of

ERG20

. And correspondingly the pinene output was increased by 26.0%. Eventually

adjusting the pH of the fermentation medium further increased the pinene production by 42.2% (to 11.7 mg·L

-1

). This titer was 34.5-fold higher than the initial one. This study is the first to achieve de novo synthesis of terpenes in

Saccharomyces cerevisiae

and to obtain the highest yield of known terpene shake flasks.

关键词

Keywords

references

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