Dataset Information

Improving the homologous recombination efficiency of Yarrowia lipolytica by grafting heterologous component from Saccharomyces cerevisiae.

ABSTRACT: The oleaginous non-conventional yeast Yarrowia lipolytica has enormous potential as a microbial platform for the synthesis of various bioproducts. However, while the model yeast Saccharomyces cerevisiae has very high homologous recombination (HR) efficiency, non-homologous end-joining is dominant in Y. lipolytica, and foreign genes are randomly inserted into the genome. Consequently, the low HR efficiency greatly restricts the genetic engineering of this yeast. In this study, RAD52, the key component of the HR machinery in S. cerevisiae, was grafted into Y. lipolytica to improve HR efficiency. The gene ade2, whose deletion can result in a brown colony phenotype, was used as the reporter gene for evaluating the HR efficiency. The HR efficiency of Y. lipolytica strains before and after integrating the ScRad52 gene was compared using insets with homology arms of different length. The results showed that the strategy could achieve gene targeting efficiencies of up to 95% with a homology arm length of 1000 bp, which was 6.5 times of the wildtype strain and 1.6 times of the traditionally used ku70 disruption strategy. This study will facilitate the further genetic engineering of Y. lipolytica to make it a more efficient cell factory for the production of value-added compounds.

SUBMITTER: Ji Q

PROVIDER: S-EPMC7691175 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Improving the homologous recombination efficiency of Yarrowia lipolytica by grafting heterologous component from Saccharomyces cerevisiae.

Ji Qingchun Q Mai Jie J Ding Ying Y Wei Yongjun Y Ledesma-Amaro Rodrigo R Ji Xiao-Jun XJ

Metabolic engineering communications 20201113

The oleaginous non-conventional yeast Yarrowia lipolytica has enormous potential as a microbial platform for the synthesis of various bioproducts. However, while the model yeast Saccharomyces cerevisiae has very high homologous recombination (HR) efficiency, non-homologous end-joining is dominant in Y. lipolytica, and foreign genes are randomly inserted into the genome. Consequently, the low HR efficiency greatly restricts the genetic engineering of this yeast. In this study ...[more]

PMID: 33294367

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Project description:The low secretion levels of cellobiohydrolase I (CBHI) in yeasts are one of the key barriers preventing yeast from directly degrading and utilizing lignocellulose. To overcome this obstacle, we have explored the approach of genetically linking an easily secreted protein to CBHI, with CBHI being the last to be folded. The Trichoderma reesei eg2 (TrEGII) gene was selected as the leading gene due to its previously demonstrated outstanding secretion in yeast. To comprehensively characterize the effects of this fusion protein, we tested this hypothesis in three industrially relevant yeasts: Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi. Our initial assays with the L. starkeyi secretome expressing differing TrEGII domains fused to a chimeric Talaromyces emersonii-T. reesei CBHI (TeTrCBHI) showed that the complete TrEGII enzyme, including the glycoside hydrolase (GH) 5 domain is required for increased expression level of the fusion protein when linked to CBHI. We found that this new construct (TrEGII-TeTrCBHI, Fusion 3) had an increased secretion level of at least threefold in L. starkeyi compared to the expression level of the chimeric TeTrCBHI. However, the same improvements were not observed when Fusion 3 construct was expressed in S. cerevisiae and Y. lipolytica. Digestion of pretreated corn stover with the secretomes of Y. lipolytica and L. starkeyi showed that conversion was much better using Y. lipolytica secretomes (50% versus 29%, respectively). In Y. lipolytica, TeTrCBHI performed better than the fusion construct. Furthermore, S. cerevisiae expression of Fusion 3 construct was poor and only minimal activity was observed when acting on the substrate, pNP-cellobiose. No activity was observed for the pNP-lactose substrate. Clearly, this approach is not universally applicable to all yeasts, but works in specific cases. With purified protein and soluble substrates, the exoglucanase activity of the GH7 domain embedded in the Fusion 3 construct in L. starkeyi was significantly higher than that of the GH7 domain in TeTrCBHI expressed alone. It is probable that a higher fraction of fusion construct CBHI is in an active form in Fusion 3 compared to just TeTrCBHI. We conclude that the strategy of leading TeTrCBHI expression with a linked TrEGII module significantly improved the expression of active CBHI in L. starkeyi.

Project description:Fatty alcohols are widely used in various applications within a diverse set of industries, such as the soap and detergent industry, the personal care, and cosmetics industry, as well as the food industry. The total world production of fatty alcohols is over 2 million tons with approximately equal parts derived from fossil oil and from plant oils or animal fats. Due to the environmental impact of these production methods, there is an interest in alternative methods for fatty alcohol production via microbial fermentation using cheap renewable feedstocks. In this study, we aimed to obtain a better understanding of how fatty alcohol biosynthesis impacts the host organism, baker's yeast Saccharomyces cerevisiae or oleaginous yeast Yarrowia lipolytica. Producing and non-producing strains were compared in growth and nitrogen-depletion cultivation phases. The multi-omics analysis included physiological characterization, transcriptome analysis by RNAseq, 13Cmetabolic flux analysis, and intracellular metabolomics. Both species accumulated fatty alcohols under nitrogen-depletion conditions but not during growth. The fatty alcohol-producing Y. lipolytica strain had a higher fatty alcohol production rate than an analogous S. cerevisiae strain. Nitrogen-depletion phase was associated with lower glucose uptake rates and a decrease in the intracellular concentration of acetyl-CoA in both yeast species, as well as increased organic acid secretion rates in Y. lipolytica. Expression of the fatty alcohol-producing enzyme fatty acyl-CoA reductase alleviated the growth defect caused by deletion of hexadecenal dehydrogenase encoding genes (HFD1 and HFD4) in Y. lipolytica. RNAseq analysis showed that fatty alcohol production triggered a cell wall stress response in S. cerevisiae. RNAseq analysis also showed that both nitrogen-depletion and fatty alcohol production have substantial effects on the expression of transporter encoding genes in Y. lipolytica. In conclusion, through this multi-omics study, we uncovered some effects of fatty alcohol production on the host metabolism. This knowledge can be used as guidance for further strain improvement towards the production of fatty alcohols.

Dataset Information

Improving the homologous recombination efficiency of Yarrowia lipolytica by grafting heterologous component from Saccharomyces cerevisiae.

Publications

Improving the homologous recombination efficiency of <i>Yarrowia lipolytica</i> by grafting heterologous component from <i>Saccharomyces cerevisiae</i>.

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