高浓度2-KLG对氧化葡萄糖酸杆菌WSH-003中2-KLG合成途径关键基因表达的影响

【目的】研究高浓度的2-KLG对其生产菌株氧化葡萄糖酸杆菌生产过程中关键的脱氢酶合成基因、辅因子合成基因及其转运蛋白编码基因的影响。【方法】测定高浓度梯度2-KLG下氧化葡萄糖酸杆菌的生长情况,确定合适的添加浓度对氧化葡萄糖酸杆菌进行胁迫。使用实时定量PCR技术检测2-KLG合成中关键山梨醇脱氢酶基因sld AB、关键辅因子PQQ合成基因pqq ABCDE及5个潜在转运蛋白合成基因的变化。【结果】根据氧化葡萄糖酸杆菌在2-KLG高浓度梯度下生长测定实验结果,选定40、80和120 g/L 2-KLG作为添加浓度。实时定量PCR结果显示,在高浓度的2-KLG压力下,PQQ合成基因pqq ABCDE未受到显著影响,山梨醇脱氢酶基因sld AB以及部分PQQ潜在转运蛋白编码基因的表达均显著下调。【结论】高浓度2-KLG会抑制氧化葡萄糖酸杆菌中山梨醇脱氢酶基因的表达,有可能会影响辅酶PQQ的转运,但不会显著影响辅酶PQQ的合成。

Effect of high 2-KLG concentration on expression of pivotal genes involved in 2-KLG synthesis in Gluconobacter oxydans WSH-003

Objective] To analyze the effect of high 2-keto-L-gulonic acid (2-KLG) on important dehydrogenase, cofactor and transport proteins involved in 2-KLG synthesis. Methods] First, the growth of Gluconobacter oxydans under high 2-KLG was observed. The real-time PCR was used to detect the expression of key sorbitol dehydrogenase gene sldAB, pyrroloquinoline quinone (PQQ) biosynthesis gene cluster pqqABCDE, and five genes encoding hypothetic PQQ transport proteins. Results] According to results of the growth of G. oxydans under different 2-KLG concentration, 40, 80 and 120 g/L 2-KLG were decided to stimulate strains. Real-time PCR showed that PQQ synthesis genes pqqABCDE were not affected by high 2-KLG, but sorbitol dehydrogenase genes sldAB and part of genes encoding PQQ transport proteins were down-regulated under high 2-KLG stress. Conclusion] The expression of sorbitol dehydrogenase genes was restrained by high 2-KLG, PQQ transport was probably inhibited, but PQQ synthesis was not affected.