☆ 4.7 Article

Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector

MICROBIAL CELL FACTORIES (2023)

Journal

MICROBIAL CELL FACTORIES

Volume 22, Issue 1, Pages -

Publisher

BMC

DOI: 10.1186/s12934-023-02033-1

Keywords

Oleaginous yeast; Yeast oil; Genome engineering; CRISPR; Cas; Tailored plant oil alternatives; Cutaneotrichosporon oleaginosus; High-oleic sunflower oil; Cocoa butter; Fatty acid biosynthesis

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Abstract

This study successfully performed targeted gene editing in Cutaneotrichosporon oleaginosus using CRISPR/Cas system, establishing a strong foundation for sustainable microbial lipid production.

BackgroundSustainable production of triglycerides for various applications is a major focus of microbial factories. Oleaginous yeast species have been targeted for commercial production of microbial oils. Among all the oleaginous yeasts examined in a previous comparative study, Cutaneotrichosporon oleaginosus showed the highest lipid productivity. Moreover, a new lipid production process for C. oleaginosus with minimal waste generation and energy consumption resulted in the highest lipid productivity in the history of oleaginous yeasts. However, productivity and product diversity are restricted because of the genetic intractability of this yeast. To date, successful targeted genetic engineering of C. oleaginosus has not yet been reported.ResultsThe targeted gene editing was successfully carried out in C. oleaginosus using CRISPR/Cas system. A tailored enzyme system isolated to degrade the C. oleaginosus cell wall enabled the isolation of viable spheroplasts that are amenable to in-cell delivery of nucleic acids and proteins. The employment of both Cas9 protein and Cas mRNA was effective in obtaining strains with URA5 knockout that did not exhibit growth in the absence of uracil. Subsequently, we successfully created several strains with enhanced lipid yield (54% increase compared to that in wild type) or modified fatty acid profiles comparable with those of cocoa butter or sunflower oil compositions.ConclusionThis study establishes the first targeted engineering technique for C. oleaginosus using the CRISPR/Cas system. The current study creates the foundation for flexible and targeted strain optimizations towards building a robust platform for sustainable microbial lipid production. Moreover, the genetic transformation of eukaryotic microbial cells using Cas9 mRNA was successfully achieved.

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Macadamia oil-based oleogels as cocoa butter alternatives: Physical properties, oxidative stability, lipolysis, and application

Xixiang Shuai, David Julian McClements, Qin Geng, Taotao Dai, Roger Ruan, Liqing Du, Yuhuan Liu, Jun Chen

Summary: Macadamia oil-based oleogels were prepared using monoglyceride stearate (MG) as the gelator with a low critical gelation concentration. The physical properties of the oleogels were evaluated, and it was found that increasing MG concentration improved the hardness, oil binding capacity, and thermal stability of the oleogels. Rheological analysis showed that the oleogels exhibited a solid-to-liquid transition and viscoelastic behavior at ambient temperatures. The formation of oleogels also increased fatty acid release during lipid digestion and improved the oxidative stability of the macadamia oil. Moreover, the potential application of these oleogels as replacements for saturated fats in foods was demonstrated.

FOOD RESEARCH INTERNATIONAL (2023)