枯草芽孢杆菌基因修饰生产核黄素

Riboflavin production by a genetically modified Bacillus subtilis

【目的】研究枯草芽孢杆菌核黄素合成途径、木糖代谢相关基因修饰对核黄素合成的影响。【方法】单独过表达或共同过表达核黄素操纵子中的基因、过表达木糖代谢相关基因构建相应的重组菌株。通过测定和比较重组菌株摇瓶发酵的核黄素产量和生物量,表征各个基因修饰的效应。采用摇瓶和5 L罐发酵,考察木糖作为主要碳源以及木糖与蔗糖共代谢对核黄素发酵的影响。【结果】ribA基因单独过表达,使核黄素产量提高99%,但生物量降低30%,出现细胞自溶现象。ribA-ribH基因共表达,使核黄素产量提高280%,并且无细胞自溶和生物量下降现象。1.5%蔗糖与6.5%木糖作为碳源,5 L发酵罐发酵70 h,核黄素产量达到3.6 g/L,与8%蔗糖为碳源的发酵相比,核黄素产量提高80%。木糖代谢相关基因过表达,均明显降低核黄素产量。【结论】与ribA基因单独过表达相比,ribA-ribH基因共表达可有效避免细胞自溶现象,并能进一步提高核黄素产量。蔗糖与木糖共代谢,能够改善前体物供给,有利于提高核黄素产量。

[Objective] We modified genes responsible for xylose metabolism and riboflavin biosynthesis in Bacillus subtilis. [Methods] Genes responsible for riboflavin biosynthesis were overexpressed or co-overexpressed with genes of xylose metabolism in Bacillus subtilis. Recombinant strains were evaluated by measuring the riboflavin yield and biomass. Fermentation was done in shake flask and fermenter to produce riboflavin with sucrose as sole carbon source or a mixture of sucrose/xylose. Meanwhile, riboflavin yield, xylose consumption and biomass were analyzed. [Results] Overexpression of ribA gene increased riboflavin yield by 99% and reduced maximum biomass by 30% because of autolysis. With ribA and ribH genes co-overexpression, riboflavin yield increased by 280% without biomass decrease and autolysis. Through a 70 h batch fermentation with 6.5% xylose and 1.5% sucrose as carbon source in a 5 L fermenter, riboflavin yield of 3.6 g/L was obtained, 80% higher than that with 8% sucrose as carbon source. Riboflavin yield decreased significantly by overexpressing the genes involved in xylose metabolism. [Conclusion] Co-overexpression of ribA and ribH genes avoided the cytotoxicity effectively and further increased riboflavin production.