全基因组水平扫描鉴定粗糙脉孢菌糖转运蛋白及其在酿酒酵母己糖发酵中的评价

Genome-wide screening of predicted sugar transporters in Neurospora crassa and the application in hexose fermentation by Saccharomyces cerevisiae

摘要:

木质纤维素降解真菌粗糙脉孢菌天然具有吸收利用多种单糖和寡糖的能力，但是目前基因组中注释的预测糖转运蛋白仍然有过半功能未知。本研究从全基因组水平系统分析了粗糙脉孢菌预测糖转运蛋白的转运底物。研究发现两个转运蛋白 (NCU01868和NCU08152) 具有转运多种己糖底物的功能，因此分别命名为NcHXT-1和NcHXT-2。利用荧光共振能量转移技术 (FRET) 确认了NcHXT-1/-2具有葡萄糖转运功能。在己糖转运蛋白全缺酿酒酵母EBY.VW4000中分别过表达NcHXT-1/-2，能恢复其在葡萄糖、半乳糖或甘露糖的液体培养基中生长并生成乙醇的能力。NcHXT-1/-2在很多纤维素降解真菌中均具有保守的同源蛋白。本研究通过全基因组扫描鉴定，发现了两个保守的丝状真菌己糖转运蛋白，为真菌降解利用木质纤维素及酵母利用单糖发酵提供了新的改造靶点。

关键词:粗糙脉孢菌，NcHXT-1，NcHXT-2，己糖转运

Abstract:

The lignocellulolytic filamentous fungus Neurospora crassa is able to assimilate various mono- and oligo-saccharides. However, more than half of predicted sugar transporters in the genome are still waiting for functional elucidation. In this study, system analysis of substrate spectra of predicted sugar transporters in N. crassa was performed at genome-wide level. NCU01868 and NCU08152 have the capability of uptaking various hexose, which are named as NcHXT-1 and NcHXT-2 respectively. Their transport activities for glucose were further confirmed by fluorescence resonance energy transfer analysis. Over-expression of either NcHXT-1 or NcHXT-2 in the null-hexose-transporter yeast EBY.VW4000 restored the growth and ethanol fermentation under submerged fermentation with glucose, galactose, or mannose as the sole carbon source. NcHXT-1/-2 homologues were found in a variety of cellulolytic fungi. Functional identification of two filamentous fungal-conserved hexose transporters NcHXT-1/-2 via genome scanning would represent novel targets for ongoing efforts in engineering cellulolytic fungi and hexose fermentation in yeast.

Key words:Neurospora crassa, NcHXT-1, NcHXT-2, hexose transport