In this study, we have expanded the substrates utilized by Y. lipolytica to include xylose. We described a cryptic xylose metabolism pathway in Y. lipolytica, and elucidated the full set of enzymes responsible for the conflicting reports in the literature with regard to its xylose utilization capabilities. To provide a more complete understanding of the innate capabilities of Y. lipolytica for xylose metabolism, we have studied the biochemical and metabolic roles of predicted xylose metabolism enzymes and concluded that XYR1, XYR2, XDH, and XKS are functional, and that XDH and XKS are required for xylose metabolism. Using E. coli complementation assays and measurements in Y. lipolytica with xylitol as a substrate, we identified the XDH and XKS steps as key bottlenecks in xylose metabolism. We also identified the essentiality of these enzymes in xylitol metabolism. Furthermore, we found that overexpression of XDH and XKS resulted in robust growth on xylose, reaching an OD600 of 25 after 7 days. This is approximately three times the cell titer shown by Ryu et al. in a recent study on xylose metabolism that was published, while we prepared this manuscript [35]. Finally, we showed that the lipids produced by Y. lipolytica with overexpression of XDH and XK were nearly identical in composition to those from glucose and yielded similar lipid mass.

By applying a xylose growth challenge strategy using E. coli mutants lacking a native xylose gene (xylA or xylB) and forcing E. coli to utilize the candidate Y. lipolytica enzymes, we were able to quickly confirm the XDH and XKS enzyme in the xylose pathway. The expression of XDH and XKS (Y. lipolytica) allowed the E. coli mutants to grow on xylitol and xylose, respectively. To find the missing XYR gene, 14 candidate XYR genes were tested in the E. coli ?xylA growth challenge. The XYR2 gene was the only candidate gene that enabled E. coli ?xylA to grow on xylose. XYR2 enabled faster growth to higher titers than XYR from S. stipitis in the E. coli growth challenge. Enzymatic characterization of XYR2 and XDH in E. coli lysates showed robust activity from both enzymes. No activity was detected from XYR1, but this enzyme was found to be insoluble in E. coli, so we could not draw a definitive conclusion about the relative activities of these two enzymes. However, overexpression of the XYR1 or XYR2 gene in Y. lipolytica results in similar levels of xylose reductase activity.

We found that XYR1 and XYR2 are constitutively expressed in Y. lipolytica and do not undergo carbon catabolite repression in glucose; however, mRNA expression is still low compared to ?-actin. Activity assays of XYR1 and XYR2 overexpressed in Y. lipolytica show statistically equal activity. We have definitively shown that XYR2 has activity approximately equal to that measured for XYR1 by Ryu et al. and that mRNA expression of these two genes is approximately equal [35]. We suggest that XYR1 and XYR2 may serve redundant roles.

qPCR analysis of the wild type grown on xylitol as compared to glucose showed inducibility of the XDH gene and no inducibility of the XKS gene. In strains where we overexpress XDH and XKS, we observe significant mRNA copy number driven by the strong hybrid promoters used. We do not observe any xylose inducible expression from either XYR1 or XYR2. Ryu et al. measured relative mRNA using their xylose adapted wild-type Y. lipolytica and saw mild inducibility of all three genes [35]. In this work, we measured mRNA levels using gene specific calibration curves instead of simply using the ??Ct method to determine the extent to which genes were being expressed. Overall, the expression level is low relative to the house keeping gene, ?-actin. Strictly going by expression level, XKS is expressed the weakest implying it is a possible bottleneck in xylose metabolism. By overexpressing XDH and XKS genes in Y. lipolytica, we showed that the double overexpression strain grew to ~10-fold higher cell density (OD600 10) on xylitol compared to the wild type. Expressing XKS alone yielded better growth than expressing XDH alone, which is in agreement with the qPCR results. These results indicated that native expression of both XDH and XKS is insufficient to achieve robust grow on xylitol.

Overexpressing XDH and XKS genes in Y. lipolytica also enabled robust growth on xylose as the sole carbon source without the need for adaptation. This surprising result proved that Y. lipolytica contains a complete xylose pathway including functional XYRs, which was not originally identified when we only examined XYR1. Disruption of the XDH and XKS genes using a recently developed CRISPR–Cas9 system [19] showed that both genes are essential for growth on xylitol.

Our approach of activating the cryptic xylose metabolism by overexpression of rate limiting enzymes yielded a xylose metabolizing strain that remarkably did not require any adaptation. Ryu et al. showed that the wild-type strain requires adaptation to get relatively subtle growth on xylose [35]. Likewise, Tai showed that when the oxido-reductase pathway was engineered into Y. lipolytica adaptation was also required [32]. The explanation for what occurs during this adaptation remains an active area of inquiry.

Interestingly, in co-utilization experiments, we observe a much more rapid depletion of xylose, suggesting that the Y. lipolytica is capable of rapid xylose metabolism if additional bottlenecks are addressed. Rate of growth on xylose as a sole carbon source was still was slower than growth on glucose (168 vs ~48 h), indicating that further bottlenecks exist. Potential bottlenecks for xylose metabolism are well appreciated and include limitations in transporter activity/expression, redox balance, enzyme activity, downstream pentose phosphate enzymes, and due to the nature of the hybrid promoters used in this study where expression is several folds lower in early exponential phase as compared to late exponential phase.