Shi Feng, Zhang Ming, Li Yongfu
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
World J Microbiol Biotechnol. 2017 Jun;33(6):122. doi: 10.1007/s11274-017-2289-3. Epub 2017 May 22.
L-Glutamate decarboxylase (GAD) transforms L-glutamate into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene(s) can synthesize GABA from its own produced L-glutamate. To enhance GABA production in recombinant C. glutamicum strain SH, metabolic engineering strategies were used to improve the supply of the GABA precursor, L-glutamate. Five new strains were constructed here. First, the ppc gene was coexpressed with two GAD genes (gadB1 and gadB2). Then, the mdh gene was deleted in C. glutamicum SH. Next, gadB1-gadB2 and gadB1-gadB2-ppc co-expression plasmids were transformed into C. glutamicum strains SH and Δmdh, resulting in four recombinant GAD strains SE1, SE2, SDE1, and SDE2, respectively. Finally, the mdh gene was overexpressed in mdh-deleted SDE1, generating the mdh-complemented GAD strain SDE3. After fermenting for 72 h, GABA production increased to 26.3 ± 3.4, 24.8 ± 0.7, and 25.5 ± 3.3 g/L in ppc-overexpressed SE2, mdh-deleted SDE1, and mdh-deleted ppc-overexpressed SDE2, respectively, which was higher than that in the control GAD strain SE1 (22.7 ± 0.5 g/L). While in the mdh-complemented SDE3, GABA production decreased to 20.0 ± 0.6 g/L. This study demonstrates that the recombinant strains SE2, SDE1, and SDE2 can be used as candidates for GABA production.
L-谷氨酸脱羧酶（GAD）可将L-谷氨酸转化为γ-氨基丁酸（GABA）。表达外源GAD基因的谷氨酸棒杆菌能够利用自身产生的L-谷氨酸合成GABA。为提高重组谷氨酸棒杆菌菌株SH中GABA的产量，采用代谢工程策略改善GABA前体L-谷氨酸的供应。在此构建了五个新菌株。首先，ppc基因与两个GAD基因（gadB1和gadB2）共表达。然后，在谷氨酸棒杆菌SH中删除mdh基因。接下来，将gadB1-gadB2和gadB1-gadB2-ppc共表达质粒分别转化到谷氨酸棒杆菌菌株SH和Δmdh中，分别得到四个重组GAD菌株SE1、SE2、SDE1和SDE2。最后，在缺失mdh的SDE1中过表达mdh基因，得到mdh互补的GAD菌株SDE3。发酵72小时后，过表达ppc的SE2、缺失mdh的SDE1和过表达ppc且缺失mdh的SDE2中GABA产量分别提高到26.3±3.4、24.8±0.7和25.5±3.3 g/L，高于对照GAD菌株SE1（22.7±0.5 g/L）。而在mdh互补的SDE3中，GABA产量降至20.0±0.6 g/L。本研究表明，重组菌株SE2、SDE1和SDE2可作为GABA生产的候选菌株。
