基因增变器驱动的酿酒酵母基因组连续进化

潘颖佳; 夏思杨; 董昌; 蔡谨; 连佳长

doi:10.12211/2096-8280.2022-051

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基因增变器驱动的酿酒酵母基因组连续进化

研究论文 | 更新时间：2025-03-20

- 基因增变器驱动的酿酒酵母基因组连续进化
- Mutator-driven continuous genome evolution of Saccharomyces cerevisiae
- 合成生物学 2023年4卷第1期页码：225-240
- 作者机构：
  
  1.浙江大学化学工程与生物工程学院，浙江杭州 310027
  2.浙江大学杭州国际科创中心，浙江杭州 310000
- 作者简介：
  
  [ "潘颖佳（1992—），女，博士研究生。研究方向为合成生物学。E-mail：12228048@zju.edu.cn" ]
  [ "夏思杨（1996—），女，硕士研究生。研究方向为基因组进化。E-mail：21828174@zju.edu.cn" ]
  [ "蔡谨（1960—），男，博士，副教授。研究方向为工业微生物学。E-mail：caij@zju.edu.cn" ]
  [ "连佳长（1984—），男，博士，研究员。研究方向为合成生物学。E-mail：jzlian@zju.edu.cn" ]
- 基金信息：
  
  国家自然科学基金(21808199);国家重点研发计划(2018YFA0901800;2021YFC2103200);浙江省杰出青年基金(LR20B060003);中央高校基本科研业务费专项资金(226-2022-00214)
- DOI：10.12211/2096-8280.2022-051
  中图分类号： Q812
- 收稿：2022-09-21，
  
  修回：2022-12-14，
  
  纸质出版：2023-02-28
- 稿件说明：
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潘颖佳，夏思杨，董昌，蔡谨，连佳长. 基因增变器驱动的酿酒酵母基因组连续进化［J］. 合成生物学， 2023， 4（1）： 225-240

PAN Yingjia， XIA Siyang， DONG Chang， CAI Jin， LIAN Jiazhang. Mutator-driven continuous genome evolution of Saccharomyces cerevisiae［J］. Synthetic Biology Journal， 2023， 4（1）： 225-240
潘颖佳，夏思杨，董昌，蔡谨，连佳长. 基因增变器驱动的酿酒酵母基因组连续进化［J］. 合成生物学， 2023， 4（1）： 225-240 DOI： 10.12211/2096-8280.2022-051.

PAN Yingjia， XIA Siyang， DONG Chang， CAI Jin， LIAN Jiazhang. Mutator-driven continuous genome evolution of Saccharomyces cerevisiae［J］. Synthetic Biology Journal， 2023， 4（1）： 225-240 DOI： 10.12211/2096-8280.2022-051.

摘要

酿酒酵母是工业生物技术最常用的底盘细胞之一，被广泛应用于生物基化学品和高附加值产品的大规模生产。鉴于生物体系代谢和调控网络的复杂性，由多基因协同控制的复杂生理性状的改造通常需要采取全基因组进化来实现。为实现酿酒酵母基因组的快速进化，本研究采用CRISPR干扰技术（CRISPRi）调控与染色体复制和稳定性相关基因（

MSH2、TSA1、RAD27

和

CLB5，

即增变基因）的表达水平，构建了能够调控酿酒酵母基因组突变率和突变类型的基因增变器。利用基因增变器提高酿酒酵母的β-胡萝卜素合成水平、木糖利用效率和异丁醇耐受性。此外，构建了混合基因增变器和多重基因增变器，进一步探究了不同表型基因组进化所需的最佳突变类型以及不同突变类型之间的协同进化机制。本研究不仅可用于创建高性能酿酒酵母细胞工厂，还有可能发展一个具有普遍适用性的基因组连续进化策略。

Abstract

Saccharomyces cerevisiae

one of the most commonly used cell factories for industrial biotechnology

is widely employed for mass production of bio-based chemicals and value-added compounds. Due to complicated cellular metabolism and regulatory network of biological systems

genome evolution is generally required for engineering with complicated phenotypes

which are coordinated and regulated by multiple genes. To achieve rapid evolution of

S. cerevisiae

genome

this study employed Clustered Regularly Interspaced Short Palindromic Repeats Interference (CRISPRi) to regulate the expression of genes closely related to chromosome replication and maintenance

such as

MSH2

TSA1

RAD27

and

CLB5

known as mutator genes. By designing guide RNAs (gRNAs) with differential repression efficiency

four mutators:

MSH2

mutator mainly for point mutations and small InDels (MM)

TAS1

mutator mainly for point mutations

small InDels

and structural variants (TM)

RAD27

mutator mainly for small InDels and structural variants (RM)

and

CLB5

mutator mainly for structural variants and aneuploidy chromosomes (CM) were constructed to control genome mutation rates and types (

e.g

. point mutation

small InDels

structural variant

and aneuploid chromosomes). These mutators were used for continuous evolution of a series of industrially relevant phenotypes

such as isobutanol tolerance

xylose utilization

and β-carotene biosynthesis. Interestingly

TM was more efficient for evolving isobutanol tolerance

but TM and CM were preferred for evolving β-carotene overproduction

and all mutators were verified to have comparable performance for evolving xylose utilization with yeast strains. We also discovered that the effectiveness of mutators was dependent on the phenotype to be evolved. To address challenges in the evolution of phenotypes without pre-determined knowledge on mutation rates and types

a mixed mutator (MTRC) was constructed to rapidly evaluate the mutator-phenotype relationship. Finally

a combinatorial mutator (MTRC*2) were constructed to explore synergistic interactions among various mutators for the continuous genome evolution of

S. cerevisiae

. The established mutators can not only be used for constructing robust yeast cell factories

but also be further developed as a generally applicable genome evolution tool.

关键词

Keywords

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