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

Abstract

Background Sustainable 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. Results The 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. Conclusion This 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.

Model-driven engineering ofCutaneotrichosporon oleaginosusATCC 20509 for improved microbial oil production

“…Implementation of mitochondrial pyruvate dehydrogenase (PDH) bypass into C. oleaginosus by overexpression of pyruvate decarboxylase, acetaldehyde dehydrogenase, and acetyl-CoA synthase from Saccharomyces cerevisiae besides diacylglycerol acyltransferase from Rhizopus oryzae has resulted in an increased triacylglycerol (TAG) yield by using xylose as substrates at an N-limiting minimal medium (Koivuranta et al, 2018). CRISPR-mediated genetic engineering tool was developed and reported by Shaigani et al, 2023 and the ∆9-desaturase gene was overexpressed, and the ∆12-desaturase gene was overexpressed and knocked out to tailor the fatty acid composition. Despite these developments, there are still limited strain engineering studies on C. oleaginosus.…”

Expanding the genetic toolbox for Cutaneotrichosporon oleaginosus employing newly identified promoters and a novel antibiotic resistance marker

“…As the integration site cannot be controlled, genetic insertions can have detrimental effects on the growth of the transformants and on the expression of the gene(s) of interest. While ATMT was successfully used to generate non-native fatty acids in C. oleaginosus , we recently reported a CRISPR-Cas based approach for targeted genomic integration and deletion to yield mutants with modified fatty acid profiles [ 13 , 18 ].…”

“…For Yarrowia lipolytica as well as Rhodosporidum toruloides native and engineered endogenous promoters as well as synthetic combinations have been characterized, which can be used for targeted metabolic engineering approaches [ 19 , 20 ]. For metabolic engineering in C. oleaginosus , only a total of three endogenous promoters have been used for gene expression in previous studies [ 13 , 18 ]. Of these, the glyceraldehyde 3-phosphate dehydrogenase promoter (GAPDHp) was used for the heterologous expression of a dominant marker and other genes of interest.…”

“…Of these, the glyceraldehyde 3-phosphate dehydrogenase promoter (GAPDHp) was used for the heterologous expression of a dominant marker and other genes of interest. The other two promoters used for homologous expression originated from the aldo-keto reductase (AKR) and the transcription elongation factor (TEF), which were combined with different endogenous genes of C. oleaginosus in our previous work [ 18 ]. In the same study, also three native genes (URA5, D9FAD and D12FAD) were used for genetic engineering [ 18 ].…”

“…The other two promoters used for homologous expression originated from the aldo-keto reductase (AKR) and the transcription elongation factor (TEF), which were combined with different endogenous genes of C. oleaginosus in our previous work [ 18 ]. In the same study, also three native genes (URA5, D9FAD and D12FAD) were used for genetic engineering [ 18 ]. As C. oleaginosus is commonly used for lipid accumulation, promoters should be especially active under these conditions to be used for metabolic engineering.…”