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

Abstract

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.

Authors:
ORCiD logo [1];  [2];  [2]; ORCiD logo [2]
  1. Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States); Zhejiang Univ., Hangzhou (China)
  2. Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States)
Publication Date:
Research Org.:
Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States); Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1436578
Alternate Identifier(s):
OSTI ID: 1424826
Grant/Contract Number:  
SC0018420
Resource Type:
Accepted Manuscript
Journal Name:
Biotechnology and Bioengineering
Additional Journal Information:
Journal Volume: 115; Journal Issue: 6; Journal ID: ISSN 0006-3592
Publisher:
Wiley
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Polyploid industrial yeast; CRISPR/Cas9; multiplex genome editing; xylose utilization

Citation Formats

Lian, Jiazhang, Bao, Zehua, Hu, Sumeng, and Zhao, Huimin. Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains. United States: N. p., 2018. Web. doi:10.1002/bit.26569.
Lian, Jiazhang, Bao, Zehua, Hu, Sumeng, & Zhao, Huimin. Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains. United States. doi:10.1002/bit.26569.
Lian, Jiazhang, Bao, Zehua, Hu, Sumeng, and Zhao, Huimin. Tue . "Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains". United States. doi:10.1002/bit.26569. https://www.osti.gov/servlets/purl/1436578.
@article{osti_1436578,
title = {Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains},
author = {Lian, Jiazhang and Bao, Zehua and Hu, Sumeng and Zhao, Huimin},
abstractNote = {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.},
doi = {10.1002/bit.26569},
journal = {Biotechnology and Bioengineering},
number = 6,
volume = 115,
place = {United States},
year = {2018},
month = {2}
}

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Cited by: 5 works
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