Genetic Manipulation of a Lipolytic Yeast Candida aaseri SH14 Using CRISPR-Cas9 System.

Zool Hilmi Ibrahim,Bong Hyun Sung,Ahmad Hazri Ab Rashid,Jung-Hoon Bae,Sun-Hee Lee,Jung-Hoon Sohn

doi:10.3390/microorganisms8040526

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.

Highlights

Yeasts are unicellular eukaryotes with a long history of use in the biopharmaceutics and food industries [1,2]
Since C. aaseri SH14 showed higher sensitivity to NTC (10 μg/mL) than hygromycin (100 μg/mL), NAT1 gene from Streptomyces noursei was employed as a selection marker gene
To construct an episomal vector of C. aaseri SH14, autonomously replicating sequence (ARS) were isolated from a genomic library of C. aaseri SH14 constructed in the pBluescript II KS+ plasmid containing NAT1 gene

Summary

Introduction

Yeasts are unicellular eukaryotes with a long history of use in the biopharmaceutics and food industries [1,2]. Owing to their robust physiology in harsh conditions, such as elevated temperatures and low pH, many different yeasts have been explored for the production of renewable bio-based chemicals to replace environmentally detrimental petrochemicals [3]. Different dicarboxylic acids were produced from alkanes or fatty acids by blocking or engineering of the β-oxidation pathway of a pathogenic yeast Candida tropicalis and the conversion efficiency was much improved by amplification of the ω-oxidation pathway [9]. To produce long-chain dicarboxylic acids from renewable plant oils using microbial cell factories, a safe and robust strain that is able to utilise various plant oils as carbon sources and tolerant to organic acids is desirable [10,11]

Methods

Results

Conclusion