谷氨酰胺在生命活动中具有重要的作用，已被广泛应用于食品、医药等诸多领域。谷氨酸是谷氨酰胺生物合成的前体物质，也是谷氨酰胺生产过程中最常见的副产物。研究发现，NCgl1221基因的编码蛋白是谷氨酸分泌的重要载体，丙酮酸羧化酶是谷氨酸棒杆菌回补途径中的关键酶。为减少谷氨酰胺发酵过程中谷氨酸的积累量并提高谷氨酰胺产量，本研究利用同源重组技术敲除谷氨酰胺生产菌GM34的谷氨酸分泌载体编码基因NCgl1221，获得GM34ΔNCgl1221菌株；构建了丙酮酸羧化酶基因pyc过表达菌株GM34-pXMJ19pyc。5 L罐发酵实验表明，NCgl1221基因缺失可以使得谷氨酸积累量降低19.05%，过表达pyc基因使得谷氨酰胺产量和转化率分别提高5.54%和2.37%。可见，敲除NCgl1221、过表达pyc能够有效降低谷氨酸分泌并提高谷氨酰胺产量。

Glutamine has important effects in biological systems and has been widely used in many areas, including in the food and medicine industries. Glutamic acid is the precursor for glutamine biosynthesis, and is the most common by-product in glutamine fermentation. Previous studies have shown that the protein encoded by NCgl1221 is an important glutamic acid exporter and that pyruvate carboxylase is a key enzyme in the feedback pathway of Corynebacterium glutamicum. In order to reduce glutamic acid accumulation during glutamine fermentation and to improve the yield of glutamine, in this study, NCgl1221 of the glutamine-producing strain GM34 was knocked out using homologous recombination technology, resulting in the construction of the mutant strain GM34ΔNCgl1221. Pyc encoding pyruvate carboxylase was also overexpressed in the constructed strain GM34-pXMJ19pyc. Fermentation carried out in 5-L bottles showed that NCgl1221 knockout reduced glutamic acid accumulation by 19.05%. Overexpression of pyc resulted in a promotion of glutamine production and conversion rate by 5.54% and 2.37%, respectively. Taken together, these results demonstrate that NCgl1221 knockout and pyc overexpression can effectively reduce glutamic acid accumulation and improve glutamine production.

国家高技术研究发展计划“863计划”(2013AA102106)