Understanding Functional Roles of Native Pentose-Specific Transporters for Activating Dormant Pentose Metabolism inYarrowia lipolytica

Abstract

Pentoses including xylose and arabinose are the second-most prevalent sugars of lignocellulosic biomass that can be harnessed for biological conversion. Although Yarrowia lipolytica has emerged as a promising industrial microorganism for production of high-value chemicals and biofuels, its native pentose metabolism is poorly understood. Our previous study demonstrated that Y. lipolytica (ATCC MYA-2613) has endogenous enzymes for D-xylose assimilation, but inefficient xylitol dehydrogenase causes Y. lipolytica to assimilate xylose poorly. In this study, we investigated the functional roles of native sugar-specific transporters for activating the dormant pentose metabolism in Y. lipolytica. By screening a comprehensive set of 16 putative pentose-specific transporters, we identified two candidates, YALI0C04730p and YALI0B00396p, that enhanced xylose assimilation. The engineered mutants YlSR207 and YlSR223, overexpressing YALI0C04730p and YALI0B00396p, respectively, improved xylose assimilation approximately 23% and 50% in comparison to YlSR102, a parent engineered strain overexpressing solely the native xylitol dehydrogenase gene. Further, we activated and elucidated a widely unknown, native L-arabinose-assimilating pathway in Y. lipolytica through transcriptomic and metabolic analyses. We discovered that Y. lipolytica can co-consume xylose and arabinose, where arabinose utilization shares transporters and metabolic enzymes of some intermediate steps of the xylose-assimilating pathway. Arabinose assimilation was synergistically enhanced in the presence of xylose while xylose assimilation was competitively inhibited by arabinose. L-arabitol dehydrogenase is the rate-limiting step responsible for poor arabinose utilization in Y. lipolytica. Overall, this study sheds light on the cryptic pentose metabolism of Y. lipolytica and further helps guide strain engineering of Y. lipolytica for enhanced assimilation of pentose sugars.IMPORTANCEThe oleaginous yeast Yarrowia lipolytica is a promising industrial platform microorganism for production of high-value chemicals and fuels. For decades since its isolation, Y. lipolytica has often been known to be incapable of assimilating pentose sugars, xylose and arabinose, that are dominantly present in lignocellulosic biomass. Through bioinformatic, transcriptomic and enzymatic studies, we have uncovered the dormant pentose metabolism of Y. lipolytica. Remarkably, unlike most yeast strains that share the same transporters for importing hexose and pentose sugars, we discovered that Y. lipolytica possess the native pentose-specific transporters. By overexpressing these transporters together with the rate-limiting D-xylitol and L-arabitol dehydrogenases, we activated the dormant pentose metabolism of Y. lipolytica. Overall, this study provides a fundamental understanding of the dormant pentose metabolism of Y. lipolytica and guides future metabolic engineering of Y. lipolytica for enhanced conversion of pentose sugars to high-value chemicals and fuels.

Evolutionary Engineering Improved d-Glucose/Xylose Cofermentation of Yarrowia lipolytica

“…Interestingly, not all genes annotated as pentose transporters are candidate gene targets for pentose engineering. We also noted that overexpression level of some genes such as YALI0_D01111g and YALI0_B00396g that have been previously annotated as pentose specific transporter even decreased by 47.6% and 48.8% in our evolved strains, respectively [11,12,16] (Fig. 7).…”

“…Interestingly, Y. lipolytica has been experimentally confirmed to harbor endogenous xylose-specific transporters YALI0_C04730 and YALI0_B00396 as well glucose. The phenomenon is common in most microorganisms [17], which may be attributed to the low efficiency of xylose transporters, complex regulation of metabolism, and the un-fluent pentose phosphate pathway [10,11,[13][14][15][16] [10,11,[13][14][15][16] [10,11,[13][14][15][16] However, CCR can be relieved or even eliminated by metabolic and evolutionary engineering [18,19].…”

Synthetic microbial communities of heterotrophs and phototrophs facilitate sustainable growth

“…2b, d). Meanwhile, SPCs containing B. subtilis or Y. lipolytica did not grow experimentally as predicted, possibly due to the high sensitivity of amino acids to folding events of metal transporters 40 , deactivation of hexose transporters in Y. lipolytica 41 , as well as to the presence of reactive oxygen species 7,8 potentially affecting growth of B. subtilis. Furthermore, sugar assimilation in Y. lipolytica, including the uptake mechanism, metabolic pathways, and regulatory mechanisms, is currently poorly understood 26,41 .…”

Draft Genome Assemblies of Five Robust Yarrowia lipolytica Strains Exhibiting High Lipid Production, Pentose Sugar Utilization, and Sugar Alcohol Secretion from Undetoxified Lignocellulosic Biomass Hydrolysates

“… Yarrowia lipolytica is a dimorphic, generally regarded as safe (GRAS) oleaginous budding yeast (subphylum Saccharomycotina ). It possesses unique phenotypes, including hydrocarbon assimilation ( 1 – 5 ), specialty lipid and organic acid production ( 6 – 11 ), and resistance to harsh environments, including high salinity ( 12 ), broad-range pH ( 13 ), and ionic liquid ( 14 ). By screening a comprehensive set of 45 Y. lipolytica strains with genetic diversity from the Agricultural Research Service Culture Collection ( https://nrrl.ncaur.usda.gov/ ), we identified five promising candidate strains, YB-392, YB-419, YB-420, YB-566, and YB-567, exhibiting beneficial phenotypes for industrial biocatalysis, including biomass hydrolysate consumption, inhibitor tolerance, and lipid and fatty acid production ( 15 ).…”