Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

Lian, Jiazhang; Bao, Zehua; Hu, Sumeng; Zhao, Huimin

doi:10.1002/bit.26569

Title: Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

Journal Article · 19 February 2018 · Biotechnology and Bioengineering

DOI: https://doi.org/10.1002/bit.26569 · OSTI ID:1436578

^[1]; Bao, Zehua ^[2]; Hu, Sumeng ^[2];

^[2]

Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States); Zhejiang Univ., Hangzhou (China); University of Illinois
Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States)

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. Furthermore, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.

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Research Organization:: Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States); Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: SC0018420

OSTI ID:: 1436578

Alternate ID(s):: OSTI ID: 1424826

Journal Information:: Biotechnology and Bioengineering, Journal Name: Biotechnology and Bioengineering Journal Issue: 6 Vol. 115; ISSN 0006-3592

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

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A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis Zhang, Lihua; Zhang, Haibing; Liu, Yufei Biotechnology and Bioengineering, Vol. 117, Issue 2 https://doi.org/10.1002/bit.27207	journal	November 2019
CRISPR-Cas9 Approach Constructing Cellulase sestc-Engineered Saccharomyces cerevisiae for the Production of Orange Peel Ethanol Yang, Peizhou; Wu, Yun; Zheng, Zhi Frontiers in Microbiology, Vol. 9 https://doi.org/10.3389/fmicb.2018.02436	journal	October 2018
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CRISPR/Cas9‐RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae Kildegaard, Kanchana Rueksomtawin; Tramontin, Larissa Ribeiro Ramos; Chekina, Ksenia Yeast, Vol. 36, Issue 5 https://doi.org/10.1002/yea.3390	journal	May 2019
Efficient production of S ‐adenosyl‐ l ‐methionine from dl ‐methionine in metabolic engineered Saccharomyces cerevisiae Liu, Wei; Tang, Dandan; Shi, Rui Biotechnology and Bioengineering, Vol. 116, Issue 12 https://doi.org/10.1002/bit.27157	journal	September 2019
CRISPR-Cas9 Approach Constructing Cellulase sestc-Engineered Saccharomyces cerevisiae for the Production of Orange Peel Ethanol Yang, Peizhou; Wu, Yun; Zheng, Zhi Frontiers in Microbiology, Vol. 9 https://doi.org/10.3389/fmicb.2018.02436	journal	October 2018
The renaissance of yeasts as microbial factories in the modern age of biomanufacturing Payen, Celia; Thompson, Dawn Yeast, Vol. 36, Issue 12 https://doi.org/10.1002/yea.3439	journal	September 2019
CRISPR/Cas9‐RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae Kildegaard, Kanchana Rueksomtawin; Tramontin, Larissa Ribeiro Ramos; Chekina, Ksenia Yeast, Vol. 36, Issue 5 https://doi.org/10.1002/yea.3390	journal	May 2019
Synthetic microbial consortia for biosynthesis and biodegradation: promises and challenges Che, Shun; Men, Yujie Journal of Industrial Microbiology & Biotechnology, Vol. 46, Issue 9-10 https://doi.org/10.1007/s10295-019-02211-4	journal	July 2019

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59 BASIC BIOLOGICAL SCIENCES
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