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

Abstract

Pentoses, including xylose and arabinose, are the second most prevalent sugars in lignocellulosic biomass that can be harnessed for biological conversion. Although 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 (ATCC MYA-2613) has endogenous enzymes for d-xylose assimilation, but inefficient xylitol dehydrogenase causes to assimilate xylose poorly. In this study, we investigated the functional roles of native sugar-specific transporters for activating the dormant pentose metabolism in 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 parental engineered strain overexpressing solely the native xylitol dehydrogenase gene. Further, we activated and elucidated a widely unknown native l-arabinose assimilation pathway in through transcriptomic and metabolic analyses. We discovered that can coconsume xylose and arabinose, where arabinose utilization shares transporters and metabolic enzymes of some intermediate steps of the xylose assimilation pathway. Arabinose assimilation is synergistically enhanced in the presence of xylose, while xylose assimilation is competitively inhibited by arabinose. l-Arabitol dehydrogenase is the rate-limiting step responsible for poor arabinose utilization in Overall, this study sheds light on the cryptic pentose metabolism of and, further, helps guide strain engineering of for enhanced assimilation of pentose sugars. The oleaginous yeast is a promising industrial-platform microorganism for production of high-value chemicals and fuels. For decades since its isolation, has 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 Remarkably, unlike most yeast strains, which share the same transporters for importing hexose and pentose sugars, we discovered that possesses 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 Overall, this study provides a fundamental understanding of the dormant pentose metabolism of and guides future metabolic engineering of for enhanced conversion of pentose sugars to high-value chemicals and fuels.

Rye and Oat Agricultural Wastes as Substrate Candidates for Biomass Production of the Non-Conventional Yeast Yarrowia lipolytica

“…Rye bran hydrolysate, in addition to glucose, also contains significant amounts of xylose (less than 10%) and less than 5% of arabinose. What is important to note, is that the yeast Y. lipolytica can utilize both of these sugars, although pentose metabolism is poorly understood [32] and the xylose utilization pathway has to be induced [33]. Similar to wood, straw mainly consists of hemicelluloses-the main sugar found in this fraction is xylose [34].…”

Alternative Substrate Metabolism in Yarrowia lipolytica

“…Another pentose which is present in hemicellulose and could serve as an alternative carbon source is arabinose. Arabinose catabolism has not been well-characterized in Y. lipolytica ; however, limited work has been done to elucidate this pathway ( Ryu and Trinh, 2017 ). L -Arabinose metabolism has been engineered in the conventional yeast, S. cerevisiae , by pairing its endogenous galactose permease ( GAL2 ) with bacterial arabinose metabolizing genes: L -Arabinose isomerase ( araA ) from Bacillus subtilis , L -ribulokinase ( araB ) from E. coli , and L -ribulose-5-phosphate 4-epimerase ( araD ) also from E. coli ( Becker and Boles, 2003 ).…”

“…Thirteen other transporters were observed to be upregulated in xylose, suggesting that there could be multiple transporters which help to transport D -xylose across the cell membrane ( Nicaud et al, 2017 ). More recently, Ryu and Trinh (2017) has reiterated that YALI0C04730 and YALI0B00396 function as pentose-specific transporters in Y. lipolytica . These data, along with the fact that xylose metabolism can be enabled with the overexpression of xylose pathway genes, suggest that xylose transport is not a bottleneck for cells utilizing D -xylose as a sole carbon source.…”

Understanding and Eliminating the Detrimental Effect of Thiamine Deficiency on the Oleaginous Yeast Yarrowia lipolytica

“…Primers used for rt-PCR are listed in Table 2. The gene expression levels were investigated after normalization to levels of the actin housekeeping gene, as described elsewhere (25). (ii) Promoter characterization with hrGFP.…”

“…While mammals require nutritional supplementation of thiamine from dietary sources, most bacteria, fungi, and plants can synthesize thiamine endogenously (20). One of the exceptions is the thiamine-auxotrophic oleaginous yeast Yarrowia lipolytica, which has recently emerged as an important industrial microbe with broad biotechnological applications due to its generally regarded as safe (GRAS) status (21), metabolic capability (22)(23)(24)(25)(26), and robustness (27)(28)(29). Hence, Y. lipolytica is an ideal eukaryotic host to study the fundamental effects of thiamine deficiency on cellular health.…”