O testLi et al. eLife 2015;4:e05896. DOI: 10.7554eLife.3 ofResearch articleComputational and
O testLi et al. eLife 2015;4:e05896. DOI: 10.7554eLife.three ofResearch articleComputational and systems biology | Ecologywhether S. cerevisiae could make use of xylodextrins, a S. cerevisiae strain was engineered using 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 able to directly utilize xylodextrins with DPs of two or 3 (Figure 1B and Figure 1–figure supplement 7). Notably, although higher cell density cultures with the engineered yeast were capable of consuming xylodextrins with DPs as much as five, xylose levels remained high (Figure 1C), suggesting the existence of extreme bottlenecks in the engineered yeast. These benefits mirror these of a prior attempt to engineer S. cerevisiae for xylodextrin consumption, in which xylose was reported to accumulate in the culture medium (Fujii et al., 2011). Analyses of the supernatants from cultures in the yeast strains expressing CDT-2, GH43-2 and also the S. stipitis XRXDH pathway surprisingly revealed that the xylodextrins have been converted into xylosyl-xylitol oligomers, a set of previously unknown compounds rather than hydrolyzed to xylose and consumed (Figure 2A and Figure 2–figure supplement 1). The resulting xylosyl-xylitol oligomers have been proficiently dead-end merchandise that couldn’t be metabolized additional. Since the production of xylosyl-xylitol oligomers as intermediate metabolites has not been reported, the molecular elements involved in their generation have been examined. To test irrespective of whether the xylosyl-xylitol oligomers resulted from side reactions of xylodextrins with endogenous S. cerevisiae enzymes, we used two separate yeast strains within a combined culture, a single containing the xylodextrin hydrolysis pathway composed of CDT-2 and GH43-2, plus the second 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 Desmin/DES Protein manufacturer transporters (Hamacher et al., 2002) and serve as a carbon supply 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 without the need of generating the xylosyl-xylitol byproduct (Figure 2–figure supplement 2). These outcomes indicate that endogenous yeast enzymes and GH43-2 transglycolysis activity usually are not responsible for producing the xylosyl-xylitol byproducts, that’s, that they must be generated by the XR from S. stipitis (SsXR). Fungal xylose reductases like SsXR have already been widely used in business for xylose fermentation. Nonetheless, the structural specifics of substrate binding to the XR active web-site have not been established. To discover the molecular basis for XR IL-13 Protein Gene ID 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 website cavity where xylose could bind, positioned near the binding web site for the NADH co-factor (Kavanagh et al., 2002; Kratzer et al., 2006). Notably, the open shape on the active web page can readily accommodate the binding of longer xylodextrin substrates (Figure 2B). Utilizing computational docking algorithms (Trott and Olson, 2010), xylobiose was identified to fit properly within the pocket. Fu.
