YCR007c基因抑制木糖异构酶途径酵母木糖发酵的分子机制研究

Xylose isomerase (XI) pathway does not require extensive cofactors as in the oxidoreductase pathway and thus has higher potential in terms of theoretical yield. However, nearly all reported xylose isomerase-based pathways in Saccharomyces cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. In previous research, we successfully constructed glucose and xylose co-fermenting yeast strain C5D-M-P for ethanol production. Then by adaptive strain evolution, we get the strain C5D-M-P-E with enhanced xylose fermentation and without any mutations in XI Gene sequence. By identifying differentially expressed genes between C5D-M-P-E and C5D-M-P, We already found target gene YCR007c inhibiting xylose fermentation of S. cerevisiae with engineered xylose-isomerase pathways. By using research methods such as knockout and overexpression, we will identify YCR007c function in xylose isomerase-based pathways yeast for xylose metabolism and research the interaction between YCR007c and genes in xylose isomerase-based pathways. In addition, the work would illustrate the molecular mechanism of YCR007c gene inhibiting xylose fermentation of S. cerevisiae with engineered XI pathways.

木糖异构酶(XI)途径和氧化还原途径相比，无需辅酶参与，理论产率更高，是建立木糖发酵的优选途径。然而，与氧化还原途径相比几乎现有的XI途径酵母工程菌木糖代谢和乙醇产率较低，特别是需要自适应进化，这暗示酵母存在未知的XI途径木糖发酵内源抑制基因。本项目组构建完成共发酵葡萄糖木糖的XI途径酵母工程菌C5D-M-P，驯化C5D-M-P得到木糖发酵效率提高、XI基因序列没有发生任何突变的进化菌株C5D-M-P-E。通过分析C5D-M-P和C5D-M-P-E基因差异表达，我们最近已经找到酿酒酵母内源抑制XI途径木糖发酵效率的靶标基因YCR007c，在此基础上，本项目拟采用基因敲除和过表达等研究方法，鉴定YCR007c基因在酿酒酵母木糖代谢中的功能，研究YCR007c和XI途径基因的相互作用，进一步阐明YCR007c抑制XI途径木糖发酵效率的分子机理。

木糖异构酶(XI)途径和氧化还原途径相比，无需辅酶参与，理论产率更高，是建立木糖发酵的优选途径。然而，与氧化还原途径相比几乎现有的XI途径酵母工程菌木糖代谢和乙醇产率较低，特别是需要自适应进化，这暗示酵母存在未知的XI途径木糖发酵内源抑制基因。本项目组构建完成共发酵葡萄糖木糖的XI途径酵母工程菌C5D-M-P，驯化C5D-M-P得到木糖发酵效率提高、XI基因序列没有发生任何突变的进化菌株C5D-M-P-E。通过分析C5D-M-P和C5D-M-P-E基因差异表达，找到酿酒酵母内源抑制XI途径木糖发酵效率的靶标基因YCR007c。在此基础上，本项目明确YCR007c基因在酿酒酵母木糖代谢中的功能，YCR007c敲除后木糖利用率提高了18.5%，乙醇产量提高了8.9%。进一步通过基因表达谱差异分析发现YCR007c降低了DUP380家族成员YGL263W的表达活性，YGL263W参与物质的跨膜运输过程。构建YGL263W缺失突变株C5D-P12，其木糖利用率降低了23.2%，推测YCR007c抑制木糖异构酶途径木糖发酵效率的分子机理是降低木糖的运输效率。本研究为进一步利用基因工程和代谢工程的方法改造酿酒酵母提高其木糖发酵效率提供理论及实践基础。