Fujian Provincial Key Laboratory of Eco-Industrial Green Technology, College of Ecological and Resource Engineering, Wuyi University, Wuyishan, 354300, People's Republic of China.
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei, People's Republic of China.
Appl Biochem Biotechnol. 2021 Nov;193(11):3540-3552. doi: 10.1007/s12010-021-03619-4. Epub 2021 Jul 26.
Poly-γ-glutamic acid (γ-PGA) is an anionic polymer with wide-ranging applications in the areas of medicine, light chemical industry, wastewater treatment, and agriculture. However, the production cost of γ-PGA is high for the requirement of adding the expensive precursor L-glutamic acid during fermentation, which hinders its widespread application. In this study, in order to improve γ-PGA yield, central carbon metabolism was engineered to enhance the carbon flux of tricarboxylic acid (TCA) cycle and glutamic acid synthesis in a γ-PGA production strain Bacillus licheniformis WX-02. Firstly, pyruvate dehydrogenase (PdhABCD) and citrate synthase (CitA) were overexpressed to strengthen the flux of pyruvate into TCA cycle, resulting in 34.93% and 11.14% increase of γ-PGA yield in B. licheniformis WX-02, respectively. Secondly, the carbon flux to glyoxylate shunt was rewired via varying the expression of isocitrate lyase (AceA), and a 23.24% increase of γ-PGA yield was obtained in AceA down-regulated strain WXPaceBA. Thirdly, deletion of pyruvate formate-lyase gene pflB led to a 30.70% increase of γ-PGA yield. Finally, combinatorial metabolic engineering was applied, and γ-PGA titer was enhanced to 12.02 g/L via overexpressing pdhABCD and citA, repressing aceA, and deleting pflB, with a 69.30% improvement compared to WX-02. Collectively, metabolic engineering of central carbon metabolism is an effective strategy for enhanced γ-PGA production in B. licheniformis, and this research provided a promising strain for industrial production of γ-PGA.
聚γ-谷氨酸（γ-PGA）是一种阴离子聚合物，在医学、轻工、污水处理和农业等领域有广泛的应用。然而，由于发酵过程中需要添加昂贵的前体 L-谷氨酸，γ-PGA 的生产成本很高，这阻碍了其广泛应用。在这项研究中，为了提高 γ-PGA 的产量，对中心碳代谢进行了工程改造，以增强三羧酸（TCA）循环和谷氨酸合成的碳通量，在 γ-PGA 生产菌株地衣芽孢杆菌 WX-02 中。首先，过表达丙酮酸脱氢酶（PdhABCD）和柠檬酸合酶（CitA）以加强丙酮酸进入 TCA 循环的通量，使地衣芽孢杆菌 WX-02 的 γ-PGA 产量分别增加了 34.93%和 11.14%。其次，通过改变异柠檬酸裂解酶（AceA）的表达来重新布线碳通量到乙醛酸支路，在 AceA 下调菌株 WXPaceBA 中获得了 23.24%的 γ-PGA 产量增加。第三，敲除丙酮酸甲酸裂解酶基因 pflB 导致 γ-PGA 产量增加 30.70%。最后，通过过表达 pdhABCD 和 citA、抑制 aceA 和敲除 pflB 进行组合代谢工程，γ-PGA 浓度提高到 12.02 g/L，与 WX-02 相比提高了 69.30%。总之，中心碳代谢的代谢工程是增强地衣芽孢杆菌 γ-PGA 生产的有效策略，该研究为 γ-PGA 的工业生产提供了有前途的菌株。
