运动发酵单胞菌糖转运体基因ZMO0293的功能分析

Sugar transporters play an important role in the uptake of carbon source for various organisms. Zymomonas mobilis is considered as a model strain for ethanol fermentation with efficient glucose transportation system and higher ethanol productivity. However, the type and function of glucose transporters associated with glucose uptake are unclear. The previous microarray result illustrated that a sugar transport gene ZMO0293, was significantly up-regulated by 3.7-fold, and maintained high level of transcription for a long time under high glucose conditions. In contrast，the transcription level of the other gene glf encoding glucose-facilitated protein was almost unchanged. To investigate the function and regulation characteristics of the gene ZMO0293, firstly, the uptake pattern and substrate range of sugar transporter gene ZMO0293 will be analyzed using heterologous expression; secondly, knockouts and complementary mutants will be constructed through CRISPR/Cas9 genome editing method to distinguish functional differences of these two genes; at last, the promoter pattern of the gene ZMO0293 will be defined using the reporter gene eGFP. This study can help to elucidate the role of the gene ZMO0293 in glucose uptake process，and provide scientific basis for further modifications of metabolic engineering and sugar transporter engineering.

糖转运体在各种生物的碳源吸收中发挥着重要作用。运动发酵单胞菌是研究乙醇发酵的模式菌株，具有高效的葡萄糖转运系统和较高的乙醇产率，但是与葡萄糖吸收相关的糖转运体种类和功能并不清楚。前期基因芯片结果显示在高浓度葡萄糖条件下，一个糖转运体基因ZMO0293显著上调3.7倍，并且保持长时间、高水平的转录，然而另外一个葡萄糖易化蛋白基因glf的转录水平却几乎不变。为了研究ZMO0293的功能和调控特点，本项目将首先通过异源表达分析糖转运体ZMO0293的葡萄糖吸收方式和底物范围；然后通过CRISPR/Cas9基因编辑技术分别构建ZMO0293和glf的缺失突变体和回补突变体，分析它们的功能差异；最后通过报告基因eGFP确定ZMO0293基因的启动子类型。本项目将有助于理解ZMO0293在葡萄糖吸收过程中的作用，并且为进一步代谢工程或者糖转运体工程改造奠定基础。

运动发酵单胞菌是研究乙醇发酵的模式菌株，具有许多独特的特性，这使它成为理想的用于生物制品生产的微生物生物催化剂。糖转运体在吸收糖类和乙醇发酵的过程中发挥着重要的作用。Z. mobilis利用易化蛋白Glf介导的易化扩散吸收葡萄糖。然而，另一种糖转运蛋白编码基因ZMO0293的功能却不清楚。我们采用CRISPR/Cas方法介导的基因缺失和异源表达来研究ZMO0293的作用。结果表明，在高浓度葡萄糖存在的情况下，ZMO0293基因的缺失减缓生长和降低乙醇的产量，导致参与葡萄糖代谢的关键酶的活性降低。此外，ZMO0293缺失在ZM4-ΔZMO0293菌株中引起Entner-Doudoroff (ED)途径部分基因的不同转录变化，但在ZM4细胞中没有。ZMO0293的整合表达恢复了葡萄糖吸收缺陷菌株Escherichia coli BL21(DE3)-ΔptsG的生长。本研究揭示了Z. mobilis中ZMO0293基因对高浓度葡萄糖的响应功能，为其他微生物提供了一个新的合成生物学元件。