Functional Research Of The Key Genes Of Xylose Metabolic Pathway From Neurospora Crassa

Biofuels are promising alternative energy to liquid fossil fuels for reducing bothpetroleum dependence and the environmental impact of combustion processes.D-xylose, the second most abundant monosaccharide in nature following glucose, isthe potential bio-energy for producing bioethanol. The conversion of xylose intoethanol has received considerable attention for a long time. Neurospora crassa, amodel filamentous fungus, could metabolize xylose quickly due to its XR-XDHmetabolic pathway. Genome annotations predicted there is a xylose isomerase likeprotein encoding by NCU00838(NcXIL) in N.crassa. When△xr is induced by xylose,RNA level of NcXIL in△xr is much higher than in wild type. We guess if there is XIpathway in N.crassa,△xr will grow in the xylose medium. However, our experimentshowed that NcXIL do not participate xylose utilization directly in N.crassa.When NcXIL was heterologous expressed in yeast W303, we found that theNcXIL-expressing yeast could not utilize xylose for growth or metabolism, althoughNcXIL expression in W303in transcriptional and protein levels could be detected.Even if the recombinant yeast was cultured in mix sugars containing glucose andxylose, it could not still grow after glucose was exhausted. Based on the method of XIfrom Piromyces sp.E2, we got two NcXIL mutants:4NcXIL and20NcXIL, encodingproteins that were four and twenty amino acids longer than NcXIL. However, neither4NcXIL nor20NcXIL-expressing yeast can utilize xylose for growth.On the other hand, NcXR-NcXDH pathway was constructed in W303using DNAassembler，yielding yeast S3. Xylulokinase from S.cerevisiae (ScXK) and N.crassa(NcXK) were also expressed in yeast S1, respectively, named S1and S2. Growthcurve indicated that S1enter the logarithmic phase36h earlier than S2when they arecultured in the xylose medium. What’s more, the grow rate of S3is much lower thanS1and S2, suggesting that XK plays important roles in xylose utilization.In conclusion, NcXIL do not participate xylose utilization directly, although itresponds to xylose inducement. Expressing NcXIL yeast could not utilize xylose forgrowth. The functions of NcXIL are still mysterious. Besides, the xylose-utilizedefficiency of ScXK is much higher than NcXK, which provides clue for subsequentconstruction of engineering yeast.