Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production.
Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70Δ. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore.
Ai-Qun
AQ
Yu
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore.
Nina
N
Pratomo
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore.
Tee-Kheang
TK
Ng
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore.
Hua
H
Ling
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore.
Han-Saem
HS
Cho
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore; Food Science and Chemical Engineering, Singapore Institute of Technology.
Susanna Su Jan
SS
Leong
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore; matthew_chang@nuhs.edu.sg.
Matthew Wook
MW
Chang
D056804:Biofuels; D001709:Biotechnology / Q000379:methods; D005818:Genetic Engineering; D059765:Homologous Recombination; D012441:Saccharomyces cerevisiae; D025062:Yarrowia
D056804:Biofuels
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