Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

此前，我们课题组构建了一种新型谷氨酸棒杆菌菌株，用于从头生物合成定制的聚-γ-谷氨酸（γ-PGA）。该菌株基于 γ-PGA 合成酶复合物 PgsBCA，它是芽孢杆菌属中唯一负责 γ-PGA 合成的多蛋白复合物。在本研究中，PgsBCA 在谷氨酸棒杆菌中重建并过表达，以进一步增强 γ-PGA 合成。首先，我们确认地衣芽孢杆菌来源的γ-PGA合成酶的所有成分（PgsB、PgsC和PgsA）都是γ-PGA合成所必需的，并且仅当PgsB、PgsC和PgsA表达时才检测到γ-PGA与谷氨酸棒杆菌组合。接下来，调整每个pgsB、pgsC和pgsA的表达水平，以探索每个 γ-PGA 合成酶亚基的表达对 γ-PGA 产生的影响。结果表明，增加pgsB或pgsC的转录水平以及维持中等水平的pgsA转录水平可以使γ-PGA产量（γ-PGA产量-生物量）分别增加35.44%和76.53%。值得注意的是， pgsC的表达水平对γ-PGA合成的影响最大（占68.24%），其次是pgsB。接下来，测试了来自四种不同来源（枯草芽孢杆菌、炭疽芽孢杆菌、甲基营养芽孢杆菌和解淀粉芽孢杆菌）的编码 PgsC 的基因，以确定编码 PgsC 的直系同源物对 γ-PGA 生产的影响，但结果表明，在所有γ-PGA的合成明显受到抑制。同样，我们还探讨了编码 PgsB 的基因直系同源物对 γ-PGA 产量的影响，发现当来自甲基营养芽孢杆菌的 PgsB 取代单独的 PgsB 时，滴度从 8.24 ± 0.10 g/L 增加到 17.14 ± 0.62 g/L。来自地衣芽孢杆菌的 PgsBCA 。选择所得菌株进行进一步优化，通过优化溶解氧水平，我们在 5 L 发酵罐中实现了 38.26 g/L 的 γ-PGA 效价。随后，通过补充葡萄糖，γ-PGA滴度在48小时时增加至50.2 g/L。据我们所知，这项研究在不添加 L-谷氨酸的情况下，从葡萄糖中从头生产 γ-PGA 达到了最高滴度，从而形成了一种提高 γ-PGA 生产的新策略。