O testLi et al. eLife 2015;four:e05896. DOI: ten.7554eLife.three ofResearch articleComputational and
O testLi et al. eLife 2015;4:e05896. DOI: 10.7554eLife.3 ofResearch articleComputational and systems biology | Ecologywhether S. cerevisiae could make use of xylodextrins, a S. cerevisiae strain was TL1A/TNFSF15 Protein web engineered with the XRXDH pathway derived from Scheffersomyces stipitis–similar to that in N. crassa (Sun et al., 2012)–and a xylodextrin transport (CDT-2) and consumption (GH43-2) pathway from N. crassa. The xylose utilizing yeast expressing CDT-2 in addition to the intracellular -xylosidase GH43-2 was in a position to directly utilize xylodextrins with DPs of two or three (Figure 1B and Figure 1–figure Chemerin/RARRES2 Protein Biological Activity supplement 7). Notably, even though high cell density cultures with the engineered yeast were capable of consuming xylodextrins with DPs as much as 5, xylose levels remained higher (Figure 1C), suggesting the existence of severe bottlenecks within the engineered yeast. These results mirror those of a previous attempt to engineer S. cerevisiae for xylodextrin consumption, in which xylose was reported to accumulate in the culture medium (Fujii et al., 2011). Analyses on the supernatants from cultures from the yeast strains expressing CDT-2, GH43-2 as well as the S. stipitis XRXDH pathway surprisingly revealed that the xylodextrins had been converted into xylosyl-xylitol oligomers, a set of previously unknown compounds as opposed to hydrolyzed to xylose and consumed (Figure 2A and Figure 2–figure supplement 1). The resulting xylosyl-xylitol oligomers have been effectively dead-end goods that couldn’t be metabolized additional. Because the production of xylosyl-xylitol oligomers as intermediate metabolites has not been reported, the molecular components involved in their generation were examined. To test no matter whether the xylosyl-xylitol oligomers resulted from side reactions of xylodextrins with endogenous S. cerevisiae enzymes, we applied two separate yeast strains in a combined culture, 1 containing the xylodextrin hydrolysis pathway composed of CDT-2 and GH43-2, along with the second together with the XRXDH xylose consumption pathway. The strain expressing CDT-2 and GH43-2 would cleave xylodextrins to xylose, which could then be secreted through endogenous transporters (Hamacher et al., 2002) and serve as a carbon source for the strain expressing the xylose consumption pathway (XR and XDH). The engineered yeast expressing XR and XDH is only capable of consuming xylose (Figure 1B). When co-cultured, these strains consumed xylodextrins with out creating the xylosyl-xylitol byproduct (Figure 2–figure supplement 2). These outcomes indicate that endogenous yeast enzymes and GH43-2 transglycolysis activity are not responsible for generating the xylosyl-xylitol byproducts, that is certainly, that they must be generated by the XR from S. stipitis (SsXR). Fungal xylose reductases such as SsXR happen to be widely utilized in industry for xylose fermentation. Even so, the structural details of substrate binding for the XR active web-site have not been established. To explore the molecular basis for XR reduction of oligomeric xylodextrins, the structure of Candida tenuis xylose reductase (CtXR) (Kavanagh et al., 2002), a close homologue of SsXR, was analyzed. CtXR consists of an open active web site cavity where xylose could bind, situated close to the binding website for the NADH co-factor (Kavanagh et al., 2002; Kratzer et al., 2006). Notably, the open shape from the active site can readily accommodate the binding of longer xylodextrin substrates (Figure 2B). Using computational docking algorithms (Trott and Olson, 2010), xylobiose was identified to match well within the pocket. Fu.
