Adaptive laboratory evolution boosts Yarrowia lipolytica tolerance to vanillic acid.

Microbial tolerance to lignocellulose-derived inhibitors, such as aromatic acids, is critical for the economical production of biofuels and biochemicals. Here, adaptive laboratory evolution was applied to improve the tolerance of Yarrowia lipolytica to a representative aromatic acid inhibitor vanillic acid. The transcriptome profiling of evolved strain suggested that the tolerance could be related to the up-regulation of RNA processing and multidrug transporting pathways. Further analysis by reverse engineering confirmed that the amplification of YALI0_F13475g coding for transcriptional coactivator and YALI0_E25201g coding for multidrug transporter conferred tolerance not only to vanillic acid but also towards ferulic acid, p-coumaric acid, p-hydroxybenzoic acid and syringic acid. These findings suggested that regulation of RNA processing and multidrug transporting pathways may be important for enhanced aromatic acid tolerance in Y. lipolytica. This study provides valuable genetic information for robust strain construction for lignocellulosic biorefinery.

Combined adaptive evolution and transcriptomic profiles reveal aromatic aldehydes tolerance mechanisms in Yarrowia 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.

Adaptive laboratory evolution of Yarrowia lipolytica improves ferulic acid tolerance.

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.