理性代谢工程改造促进谷氨酸棒杆菌高效合成L-谷氨酸

Rational metabolic engineering of Corynebacterium glutamicum for efficient synthesis of L-glutamate

L-谷氨酸是世界上第一大宗氨基酸产品,广泛应用于食品医药及化工等行业。以谷氨酸高产菌谷氨酸棒杆菌(Corynebacterium glutamicum)G01为出发菌株,首先通过敲除主要副产物丙氨酸合成相关基因-丙氨酸氨基转移酶编码基因(alaT),降低了发酵副产物丙氨酸含量。其次,α-酮戊二酸节点碳流量对谷氨酸合成起重要作用,因此,采用核糖体结合位点(ribosome-binding site,RBS)序列优化降低了α-酮戊二酸脱氢酶的活性,强化了谷氨酸合成代谢流。同时通过筛选不同来源的谷氨酸脱氢酶,加强了α-酮戊二酸内源转化为谷氨酸的能力。接着,对谷氨酸转运蛋白进行理性设计,提高了谷氨酸的外排能力。最后,对基于以上策略构建的整合菌株进行了5L发酵罐发酵优化,通过梯度升温结合分批补料策略,谷氨酸产量为(136.33±4.68)g/L,较原始菌的产量(96.53±2.32)g/L提高了41.2%;糖酸转化率为55.8%,较原始菌的44.2%提高了11.6%;且降低了副产物丙氨酸的含量。以上策略一定程度上提高了谷氨酸的产量与糖酸转化率,可为谷氨酸生产菌株的代谢改造提供参考。

L-glutamic acid is the world’s largest bulk amino acid product that is widely used in the food,pharmaceutical and chemical industries.Using Corynebacterium glutamicum G01 as the starting strain,the fermentation by-product alanine content was firstly reduced by knocking out the gene encoding alanine aminotransferase(alaT),a major by-product related to alanine synthesis.Secondly,since theα-ketoglutarate node carbon flow plays an important role in glutamate synthesis,the ribosome-binding site(RBS)sequence optimization was used to reduce the activity ofα-ketoglutarate dehydrogenase and enhance the glutamate anabolic flow.The endogenous conversion ofα-ketoglutarate to glutamate was also enhanced by screening different glutamate dehydrogenase.Subsequently,the glutamate transporter was rationally desgined to improve the glutamate efflux capacity.Finally,the fermentation conditions of the strain constructed using the above strategy were optimized in 5 L fermenters by a gradient temperature increase combined with a batch replenishment strategy.The glutamic acid production reached(135.33±4.68)g/L,which was 41.2%higher than that of the original strain(96.53±2.32)g/L.The yield was 55.8%,which was 11.6%higher than that of the original strain(44.2%).The combined strategy improved the titer and the yield of glutamic acid,which provides a reference for the metabolic modification of glutamic acid producing strains.