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 · Tue Feb 20 00:00:00 EST 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)
Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States)

Abstract The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae . However, 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, 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)

Grant/Contract Number:: SC0018420

OSTI ID:: 1436578

Alternate ID(s):: OSTI ID: 1424826

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

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

Citation Metrics:

Cited by: 40 works

Citation information provided by
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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
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‐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
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
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
New Applications of Synthetic Biology Tools for Cyanobacterial Metabolic Engineering Santos-Merino, María; Singh, Amit K.; Ducat, Daniel C. Frontiers in Bioengineering and Biotechnology, Vol. 7 https://doi.org/10.3389/fbioe.2019.00033	journal	February 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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59 BASIC BIOLOGICAL SCIENCES
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