TY - JOUR
T1 - Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains
AU - Lian, Jiazhang
AU - Bao, Zehua
AU - Hu, Sumeng
AU - Zhao, Huimin
N1 - Publisher Copyright:
© 2018 Wiley Periodicals, Inc.
PY - 2018/6
Y1 - 2018/6
N2 - 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.
AB - 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.
KW - CRISPR/Cas9
KW - multiplex genome editing
KW - polyploid industrial yeast
KW - xylose utilization
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U2 - 10.1002/bit.26569
DO - 10.1002/bit.26569
M3 - Article
C2 - 29460422
AN - SCOPUS:85043366666
SN - 0006-3592
VL - 115
SP - 1630
EP - 1635
JO - Biotechnology and bioengineering
JF - Biotechnology and bioengineering
IS - 6
ER -