Genetic manipulation of a lipolytic yeast Candida aaseri SH14 using CRISPR-Cas9 system

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Title
Genetic manipulation of a lipolytic yeast Candida aaseri SH14 using CRISPR-Cas9 system
Author(s)
Z H Ibrahim; Bae Jung Hoon; Sun Hee Lee; Bong Hyun Sung; A H Ab Rashid; Jung Hoon Sohn
Bibliographic Citation
Microorganisms, vol. 8, no. 4, pp. 526-526
Publication Year
2020
Abstract
A lipolytic yeast Candida aaseri SH14 that can utilise long-chain fatty acids as the sole carbon source was isolated from oil palm compost. To develop this strain as a platform yeast for the production of bio-based chemicals from renewable plant oils, a genetic manipulation system using CRISPR-Cas9 was developed. Episomal vectors for expression of Cas9 and sgRNA were constructed using an autonomously replicating sequence isolated from C. aaseri SH14. This system guaranteed temporal expression of Cas9 for genetic manipulation and rapid curing of the vector from transformed strains. A β-oxidation mutant was directly constructed by simultaneous disruption of six copies of acyl-CoA oxidases genes (AOX2, AOX4 and AOX5) in diploid cells using a single sgRNA with 70% efficiency and the Cas9 vector was efficiently removed. Blocking of β-oxidation in the triple AOX mutant was confirmed by the accumulation of dodecanedioic acid from dodecane. Targeted integration of the expression cassette for C. aaseri lipase2 was demonstrated with 60% efficiency using this CRISPR-Cas9 system. This genome engineering tool could accelerate industrial application of C. aaseri SH14 for production of bio-based chemicals from renewable oils.
Keyword
CRISPRCandida aaseriacyl-CoA oxidasegenetic manipulation
ISSN
2076-2607
Publisher
MDPI
DOI
http://dx.doi.org/10.3390/microorganisms8040526
Type
Article
Appears in Collections:
Division of Biomaterials Research > Synthetic Biology and Bioengineering Research Center > 1. Journal Articles
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