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

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.

CRISPR-Based Genetic Manipulation of Candida Species: Historical Perspectives and Current Approaches

“…While the most characterized CRISPR systems exist in many medically relevant Candida species, CRISPR has also been applied to other members of the genus. In the field of biotechnology, CRISPR systems similar to the ones described above have been applied to species such as Candida aaseri and Candida glycerinogenes (Zhu et al, 2019 ; Ibrahim et al, 2020 ). As CRISPR-Cas9 continues to gain prominence for its streamlined metabolic engineering potential, the number of industrially-relevant Candida species with well-defined CRISPR systems will likely continue to increase.…”

“…The innate potential of several environmental species of Candida could also be exploited using CRISPR technology, in addition to using them as vessels for heterologous synthetic pathways. Candida aaseri SH14 was first isolated from the compost of oil palm; its lipolytic nature and ability to survive solely off of atypical carbon sources, such as fatty acids and alkanes, could make C. asseri SH14 a platform for chemical synthesis from plant oils (Picataggio et al, 1992 ; Ibrahim et al, 2020 ). CRISPR-Cas9 was employed to impair the ß-oxidation pathway in C. asseri SH14, to divert from fatty acid catabolism into acetyl-CoA to long-chain dicarboxylic acid production instead, as was done in the past with C. tropicalis using older genetic tools.…”

“…CRISPR-Cas9 was employed to impair the ß-oxidation pathway in C. asseri SH14, to divert from fatty acid catabolism into acetyl-CoA to long-chain dicarboxylic acid production instead, as was done in the past with C. tropicalis using older genetic tools. The CRISPR system promoted HDR in this species and enabled multiple related genes to be disrupted with just one sgRNA that targeted conserved sequences at an efficiency of 70% (Picataggio et al, 1992 ; Ibrahim et al, 2020 ). While conversion of fatty acids to dicarboxylic acids was still slow, yield could be improved by the introduction of overexpression cassettes for rate-limiting enzymes in the process, a strategy that is currently underway using the efficient CRISPR-Cas9 tool (Picataggio et al, 1992 ; Ibrahim et al, 2020 ).…”

“…The CRISPR system promoted HDR in this species and enabled multiple related genes to be disrupted with just one sgRNA that targeted conserved sequences at an efficiency of 70% (Picataggio et al, 1992 ; Ibrahim et al, 2020 ). While conversion of fatty acids to dicarboxylic acids was still slow, yield could be improved by the introduction of overexpression cassettes for rate-limiting enzymes in the process, a strategy that is currently underway using the efficient CRISPR-Cas9 tool (Picataggio et al, 1992 ; Ibrahim et al, 2020 ).…”

Characterization of Acyl-CoA Oxidases from the Lipolytic Yeast Candida aaseri SH14

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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).

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