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.
Materials and Methods
Strains, Chemicals and MediumC. aaseri SH14 (ura3) [2], and their mutant strains were cultured in YPD (1% yeast extract, 2% peptone, and 2% glucose). Selection of URA+ transformants was performed on a synthetic complete medium lacking uracil (SCura; 0.67% yeast nitrogen base without amino acids, 0.077% ura dropout supplement, 2% glucose, and 2% agar). For screening of CaAOX mutants, YPD supplemented with 20 μg/ml nourseothricin (NTC, Sigma Chemicals Co.,
The lipolytic yeastCandida aaseri SH14 contains three Acyl-CoA oxidases (ACOXs) which are encoded by the CaAOX2, CaAOX4, and CaAOX5 genes and catalyze the first reaction in the β-oxidation of fatty acids. Here, the respective functions of the three CaAOX isozymes were studied by growth analysis of mutant strains constructed by a combination of three CaAOX mutations in minimal medium containing fatty acid as the sole carbon source. Substrate specificity of the CaAOX isozymes was analyzed using recombinant C. aaseri SH14 strains overexpressing the respective genes. CaAOX2 isozyme showed substrate specificity toward short-and medium-chain fatty acids (C6-C12), while CaAOX5 isozyme preferred long-chain fatty acid longer than C12. CaAOX4 isozyme revealed a preference for a broad substrate spectrum from C6-C16. Although the substrate specificity of CaAOX2 and CaAOX5 covers medium-and long-chain fatty acids, these two isozymes were insufficient for complete β-oxidation of long-chain fatty acids, and therefore CaAOX4 was indispensable.
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