基于代谢途径改造谷氨酸棒杆菌生产L-缬氨酸

Metabolic engineering modification of Corynebacterium glutamicum for L-valine production

L-缬氨酸是一种重要的支链氨基酸,随着市场对其需求量的不断提升,进一步提高L-缬氨酸的产量和糖酸转化率具有重要意义。在该研究中,使用实验室保藏的谷氨酸棒杆菌VHL-1作为出发菌株,通过对L-缬氨酸合成路径进行代谢改造显著提高了L-缬氨酸的产量和丙酮酸前体物的供应。首先,通过敲除ldh(编码乳酸脱氢酶)、poxB(编码丙酮酸氧化酶)、pyc(编码丙酮酸羧化酶)基因以及弱化alaT(编码丙氨酸转氨酶)基因表达来实现丙酮酸的富集。其次,通过强启动子P_(tuf)替换ilvBNC操纵子原始启动子并增加ilvBN(编码乙酰羟酸合酶)基因拷贝数来增强丙酮酸向L-缬氨酸合成的碳代谢流。最后,通过过表达支链氨基酸转运蛋白编码基因brnFE和调节蛋白编码基因lrp增强L-缬氨酸胞外输出效率。最终构建的重组菌株VHL-9在5 L生物反应器中进行补料分批培养,L-缬氨酸产量可到达(82.5±5.6)g/L,生产强度为1.15 g/(L·h),糖酸转化率为0.302 g/g葡萄糖。

L-valine is an indispensable branched-chain amino acid.With the increasing market demand for L-valine,it is of great significance to further improve the production and the conversion rate of glucose of L-valine.In this study,the laboratory-conserved Corynebacterium glutamicum VHL-1 was used as the starting strain,the production of L-valine and the supply of pyruvate precursors were significantly improved by metabolic modification of the L-valine synthesis pathway.First,the pyruvate enrichment was achieved by knocking out the ldh(encoding lactate dehydrogenase),poxB(encoding pyruvate oxidase),and pyc(encoding pyruvate carboxylase)genes and by weakening alaT(encoding alanine transaminase)gene expression.Second,the original promoter of the ilvBNC manipulator was replaced with the strong promoter P_(tuf) and the copy number of the ilvBN(encoding acetylhydroxylate synthase)gene was increased to enhance the carbon metabolic flow of pyruvate to L-valine synthesis.Finally,the exocytosis efficiency of L-valine was enhanced by overexpressing of the branched-chain amino acid transfer protein-encoding gene brnFE and regulating protein-encoding gene lrp.The final constructed recombinant strain VHL-9 was incubated in a 5 L bioreactor with fed-batch culture.The yield of L-valine reached(82.5±5.6)g/L with a production intensity of 1.15 g/(L·h),and the sugar-acid conversion rate was 0.302 g/g glucose.