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 WXP_bacAaceBA. 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，在医药、轻化工、污水处理、农业等领域有着广泛的应用。然而，γ-PGA的生产成本较高，需要在发酵过程中添加昂贵的前体L-谷氨酸，阻碍了其广泛应用。在本研究中，为了提高 γ-PGA 产量，对中心碳代谢进行了改造，以增强 γ-PGA 生产菌株Bacillus licheniformis WX-02的三羧酸 (TCA) 循环和谷氨酸合成的碳通量。首先，过表达丙酮酸脱氢酶（PdhABCD）和柠檬酸合酶（CitA）以增强丙酮酸进入TCA循环的通量，导致地衣芽孢杆菌γ-PGA产量增加34.93%和11.14%WX-02，分别。其次，通过改变异柠檬酸裂解酶（AceA）的表达，重新连接乙醛酸分流器的碳通量，在AceA下调的菌株WXP _bacA aceBA中获得了23.24％的γ-PGA产量增加。第三，丙酮酸甲酸裂解酶基因pflB的缺失导致γ-PGA产量增加30.70%。最后，应用组合代谢工程，通过过表达pdhABCD和citA、抑制aceA、删除pflB，γ-PGA滴度提高到12.02 g/L，与WX-02相比提高了69.30%。总的来说，中心碳代谢的代谢工程是提高 γ-PGA 产量的有效策略。B. licheniformis，该研究为γ-PGA的工业化生产提供了有希望的菌株。