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Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains
Author(s) -
Lian Jiazhang,
Bao Zehua,
Hu Sumeng,
Zhao Huimin
Publication year - 2018
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26569
Subject(s) - crispr , multiplex , polyploid , yeast , biology , genome , cas9 , computational biology , genetics , gene
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.