以前,我们小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,这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,在谷氨酸棒杆菌中重构并过表达PgsBCA以进一步增强γ-PGA合成。首先,我们确认了所有的成分(PgsB,PgsC,和来自地衣芽孢杆菌的γ-PGA合成酶的PgsA)是γ-PGA合成所必需的,只有当PgsB时,γ-PGA才被检测到,PgsC,和PgsA在谷氨酸棒杆菌中组合表达。接下来,每个pgsB的表达水平,PGSC,和调节pgsA以探索每个γ-PGA合成酶亚基的表达对γ-PGA生产的影响。结果表明,增加pgsB或pgsC的转录水平并保持pgsA的中等水平转录水平导致γ-PGA产量(γ-PGA产量对生物量)增加35.44%和76.53%,分别。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,编码来自四种不同来源的PgsC的基因(枯草芽孢杆菌,炭疽杆菌,嗜甲基芽孢杆菌,和解淀粉芽孢杆菌)进行了测试,以鉴定编码PgsC的直系同源物对γ-PGA生产的影响,但结果表明,在所有情况下,γ-PGA的合成都受到显著抑制。同样,我们还探索了编码PgsB的基因直向同源物对γ-PGA产生的影响,并发现当来自甲基嗜酸性芽孢杆菌的PgsB替代来自地衣芽孢杆菌的PgsBCA中的PgsB时,滴度从8.24±0.10g/L增加到17.14±0.62g/L。选择所得应变进行进一步优化,通过优化溶解氧水平,我们在5L发酵罐中获得了38.26g/L的γ-PGA滴度。随后,通过补充葡萄糖,γ-PGA滴度在48h时增加到50.2g/L。据我们所知,这项研究实现了从葡萄糖重新生产γ-PGA的最高滴度,不添加L-谷氨酸,从而产生了一种提高γ-PGA产量的新策略。
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.