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Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica. [ABSTRACT FROM AUTHOR]

[Display omitted] • Adaptive evolution improved the aromatic aldehydes tolerance of Y. lipolytica. • The transcriptomics of the aromatic aldehydes tolerate strain was analyzed. • Two genes were confirmed as the key targets for detoxification in Y. lipolytica. • The first report on the aromatic aldehyde conversion in Y. lipolytica. Yarrowia lipolytica is an efficient oleaginous yeast, whereas its activity is typically reduced by inhibitors present in lignocellulosic hydrolysate. Understanding the response mechanism of Y. lipolytica to hydrolysate inhibitors and developing inhibitor tolerant strains are vital to lignocellulose valorization by this promising species. In this study, through adaptive laboratory evolution on three representative aromatic aldehyde inhibitors, evolved strains were obtained. Fermentation phenotype suggested that aromatic aldehydes conversion was one main reason for high tolerance of adapted strains. Transcriptome profiling analysis and reverse metabolic engineering confirmed that overexpressing the aldehyde ketone reductase gene YALI0_B07117g and aldehyde dehydrogenase gene YALI0_B01298g effectively converted aromatic aldehyde to corresponding alcohols and acids. The potential degradation pathways for aromatic aldehyde inhibitors in Y. lipolytica XYL+ were then discussed. This study provided insights to the aromatic aldehyde degradation in Y. lipolytica and a reliable basis for the development of aromatic aldehyde tolerant strains. [ABSTRACT FROM AUTHOR]

Yarrowia lipolytica strain is a promising cell factory for the conversion of lignocellulose to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to lignocellulose-derived inhibitors toxicity tolerance of Y. lipolytica are also required to achieve industrial application. Here, adaptive laboratory evolution was employed with increasing concentrations of ferulic acid. The adaptive laboratory evolution experiments led to evolve Y. lipolytica strain yl-XYL + *FA*4 with increased tolerance to ferulic acid as compared to the parental strain. Specifically, the evolved strain could tolerate 1.5 g/L ferulic acid, whereas 0.5 g/L ferulic acid could cause about 90% lethality of the parental strain. Transcriptome analysis of the evolved strain revealed several targets underlying toxicity tolerance enhancements. YALI0_E25201g, YALI0_F05984g, YALI0_B18854g, and YALI0_F16731g were among the highest upregulated genes, and the beneficial contributions of these genes were verified via reverse engineering. Recombinant strains with overexpressing each of these four genes obtained enhanced tolerance to ferulic acid as compared to the control strain. Fortunately, recombinant strains with overexpression of YALI0_E25201g, YALI0_B18854g, and YALI0_F16731g individually also obtained enhanced tolerance to vanillic acid. Overall, this work demonstrated a whole strain improvement cycle by "non-rational" metabolic engineering and presented new targets to modify Y. lipolytica for microbial lignocellulose valorization. Key points: • Adaptive evolution improved the ferulic acid tolerance of Yarrowia lipolytica • Transcriptome sequence was applied to analyze the ferulic acid tolerate strain • Three genes were demonstrated for both ferulic acid and vanillic acid tolerance [ABSTRACT FROM AUTHOR]