Mutator-driven continuous genome evolution of <italic style="font-style: italic">Saccharomyces cerevisiae</italic>

PAN Yingjia; XIA Siyang; DONG Chang; CAI Jin; LIAN Jiazhang

doi:10.12211/2096-8280.2022-051

您当前的位置：

首页 >

文章列表页 >

Mutator-driven continuous genome evolution of Saccharomyces cerevisiae

Research Article | 更新时间：2025-03-20

- Mutator-driven continuous genome evolution of Saccharomyces cerevisiae
- Synthetic Biology Journal Vol. 4, Issue 1, Pages: 225-240(2023)
- 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：
- 基金信息：
- DOI：10.12211/2096-8280.2022-051
  CLC： Q812
- Received：21 September 2022，
  
  Revised：2022-12-14，
  
  Published：28 February 2023
- 稿件说明：
移动端阅览
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

references

李祎 , 林振泉 , 刘子鹤 . 酿酒酵母适应性实验室进化工具的最新进展 [J ] . 合成生物学 , 2021 , 2 ( 2 ): 287 - 301 .

LI Y , LIN Z Q , LIU Z H . Advances in yeast based adaptive laboratory evolution [J ] . Synthetic Biology Journal , 2021 , 2 ( 2 ): 287 - 301 .

夏思杨 , 江丽红 , 蔡谨 , 等 . 酿酒酵母基因组进化的研究进展 [J ] . 合成生物学 , 2020 , 1 ( 5 ): 556 - 569 .

XIA S Y , JIANG L H , CAI J , et al . Advances in genome evolution of Saccharomyces cerevisiae [J ] . Synthetic Biology Journal , 2020 , 1 ( 5 ): 556 - 569 .

SI T , XIAO H , ZHAO H M . Rapid prototyping of microbial cell factories via genome-scale engineering [J ] . Biotechnology Advances , 2015 , 33 ( 7 ): 1420 - 1432 .

LUAN G D , CAI Z , LI Y , et al . Genome replication engineering assisted continuous evolution (GREACE) to improve microbial tolerance for biofuels production [J ] . Biotechnology for Biofuels , 2013 , 6 ( 1 ): 137 .

BADRAN A H , LIU D R . In vivo continuous directed evolution [J ] . Current Opinion in Chemical Biology , 2015 , 24 : 1 - 10 .

ARNOLD F H , VOLKOV A A . Directed evolution of biocatalysts [J ] . Current Opinion in Chemical Biology , 1999 , 3 ( 1 ): 54 - 59 .

WANG Y J , XUE P , CAO M F , et al . Directed evolution: methodologies and applications [J ] . Chemical Reviews , 2021 , 121 ( 20 ): 12384 - 12444 .

祁延萍 , 朱晋 , 张凯 , 等 . 定向进化在蛋白质工程中的应用研究进展 [J ] . 合成生物学 , 2022 , 3 ( 6 ): 1 - 29 .

QI Y P , ZHU J , ZHANG K , et al . Recent development of directed evolution in protein engineering [J ] . Synthetic Biology Journal , 2022 , 3 ( 6 ): 1 - 29 .

SCHELLER J , SCHÜRER A , RUDOLPH C , et al . MPH1 , a yeast gene encoding a DEAH protein, plays a role in protection of the genome from spontaneous and chemically induced damage [J ] . Genetics , 2000 , 155 ( 3 ): 1069 - 1081 .

SPEYER J F . Mutagenic DNA polymerase [J ] . Biochemical and Biophysical Research Communications , 1965 , 21 ( 1 ): 6 - 8 .

CHOU H H , KEASLING J D . Programming adaptive control to evolve increased metabolite production [J ] . Nature Communications , 2013 , 4 : 2595 .

DROTSCHMANN K , CLARK A B , TRAN H T , et al . Mutator phenotypes of yeast strains heterozygous for mutations in the MSH2 gene [J ] . Proceedings of the National Academy of Sciences of the United States of America , 1999 , 96 ( 6 ): 2970 - 2975 .

SERERO A , JUBIN C , LOEILLET S , et al . Mutational landscape of yeast mutator strains [J ] . Proceedings of the National Academy of Sciences of the United States of America , 2014 , 111 ( 5 ): 1897 - 1902 .

REENAN R A , KOLODNER R D . Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate mitochondrial and nuclear functions [J ] . Genetics , 1992 , 132 ( 4 ): 975 - 985 .

KATJU V , KONRAD A , DEISS T C , et al . Mutation rate and spectrum in obligately outcrossing Caenorhabditis elegans mutation accumulation lines subjected to RNAi-induced knockdown of the mismatch repair gene msh-2 [J ] . G 3 Genes|Genomes|Genetics , 2021 , 12 ( 1 ): jkab364 .

GOTO M , SASAKI M , KOBAYASHI T . The S-phase cyclin Clb5 promotes rRNA gene (rDNA) stability by maintaining replication initiation efficiency in rDNA [J ] . Molecular and Cellular Biology , 2021 , 41 ( 5 ): e00324 - e00320 .

TISHKOFF D X , FILOSI N , GAIDA G M , et al . A novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair [J ] . Cell , 1997 , 88 ( 2 ): 253 - 263 .

MCCULLEY J L , PETES T D . Chromosome rearrangements and aneuploidy in yeast strains lacking both Tel1p and Mec1p reflect deficiencies in two different mechanisms [J ] . Proceedings of the National Academy of Sciences of the United States of America , 2010 , 107 ( 25 ): 11465 - 11470 .

PENNANEACH V , KOLODNER R D . Recombination and the Tel1 and Mec1 checkpoints differentially effect genome rearrangements driven by telomere dysfunction in yeast [J ] . Nature Genetics , 2004 , 36 ( 6 ): 612 - 617 .

PARK H , KIM S . Gene-specific mutagenesis enables rapid continuous evolution of enzymes in vivo [J ] . Nucleic Acids Research , 2021 , 49 ( 6 ): e32 .

YONA A H , MANOR Y S , HERBST R H , et al . Chromosomal duplication is a transient evolutionary solution to stress [J ] . Proceedings of the National Academy of Sciences of the United States of America , 2012 , 109 ( 51 ): 21010 - 21015 .

GILBERT L A , LARSON M H , MORSUT L , et al . CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes [J ] . Cell , 2013 , 154 ( 2 ): 442 - 451 .

YAO Z , WANG Q H , DAI Z J . Recent advances in directed yeast genome evolution [J ] . Journal of Fungi , 2022 , 8 ( 6 ): 635 .

LIAN J Z , HAMEDIRAD M , HU S M , et al . Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system [J ] . Nature Communications , 2017 , 8 : 1688 .

GIETZ R D , SCHIESTL R H . High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method [J ] . Nature Protocols , 2007 , 2 ( 1 ): 31 - 34 .

PATTERSON M N , MAXWELL P H . Combining magnetic sorting of mother cells and fluctuation tests to analyze genome instability during mitotic cell aging in Saccharomyces cerevisiae [J ] . Journal of Visualized Experiments: JoVE , 2014 ( 92 ): e51850 .

BOEKE J D , LACROUTE F , FINK G R . A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance [J ] . Molecular & General Genetics: MGG , 1984 , 197 ( 2 ): 345 - 346 .

WHELAN W L , GOCKE E , MANNEY T R . The CAN1 locus of Saccharomyces cerevisiae : fine-structure analysis and forward mutation rates [J ] . Genetics , 1979 , 91 ( 1 ): 35 - 51 .

QI M M , ZHANG B , JIANG L H , et al . PCR & Go: a pre-installed expression chassis for facile integration of multi-gene biosynthetic pathways [J ] . Frontiers in Bioengineering and Biotechnology , 2021 , 8 : 613771 .

TSAI C S , KONG I I , LESMANA A , et al . Rapid and marker-free refactoring of xylose-fermenting yeast strains with Cas9/CRISPR [J ] . Biotechnology and Bioengineering , 2015 , 112 ( 11 ): 2406 - 2411 .

丁明珠 , 李炳志 , 王颖 , 等 . 合成生物学重要研究方向进展 [J ] . 合成生物学 , 2020 , 1 ( 1 ): 7 - 28 .

DING M Z , LI B Z , WANG Y , et al . Significant research progress in synthetic biology [J ] . Synthetic Biology Journal , 2020 , 1 ( 1 ): 7 - 28 .

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Enabling technology for the biosynthesis of cosmetic raw materials with Saccharomyces cerevisiae

Production of sesquiterpenoids α-neoclovene and β-caryophyllene by engineered Saccharomyces cerevisiae

Regulation on oxidation selectivity for β-amyrin by Class Ⅱ cytochrome P450 enzymes

Advances in yeast based adaptive laboratory evolution

Related Author

LIAN Jiazhang

ZHANG Jiaojiao

ZUO Yimeng

ZHOU Zhihua

LI Xiaodong

YANG Chengshuai

WANG Pingping

YAN Xing

Related Institution

Key Laboratory of Biomass Chemical Engineering of Ministry of Education & National Key Laboratory of Biobased Transportation Fuel Technology， College of Chemical and Biological Engineering， Zhejiang University

CAS Key Laboratory of Quantitative Engineering Biology， Shenzhen Institute of Synthetic Biology， Shenzhen Institutes of Advanced Technology， Chinese Academy of Sciences

University of Chinese Academy of Sciences

CAS-key Laboratory of Synthetic Biology， Center for Excellence in Molecular Plant Sciences， Chinese Academy of Sciences

School of Chemistry and Chemical Engineering，Beijing Institute of Technology

⁰

Mutator-driven continuous genome evolution of Saccharomyces cerevisiae

DOI：10.12211/2096-8280.2022-051

摘要

Abstract

关键词

Keywords

references