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This content will become publicly available on February 20, 2019

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

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