The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi, 214122, China; Jiangsu Provincial Engineering Research Center for Bioactive Product Processing Technology, Jiangnan University, Wuxi, 214122, China; Yixing Institute of Food and Biotechnology, Yixing, 214200, China.
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi, 214122, China; Jiangsu Provincial Engineering Research Center for Bioactive Product Processing Technology, Jiangnan University, Wuxi, 214122, China.
Metab Eng. 2024 Jan;81:238-248. doi: 10.1016/j.ymben.2023.12.008. Epub 2023 Dec 29.
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.
先前，我们小组构建了一株用于从头生物合成定制聚γ-谷氨酸（γ-PGA）的新型谷氨酸棒杆菌菌株。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，我们在谷氨酸棒杆菌中重新构建和过表达 PgsBCA，以进一步提高 γ-PGA 的合成。首先，我们证实来源于地衣芽孢杆菌的 γ-PGA 合成酶的所有成分（PgsB、PgsC 和 PgsA）对于 γ-PGA 合成都是必需的，并且只有当 PgsB、PgsC 和 PgsA 组合表达时，才能检测到 γ-PGA。接下来，我们调整了每个 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 产量的影响。结果表明，增加 pgsB 或 pgsC 的转录水平并保持 pgsA 的中等转录水平可分别使 γ-PGA 产量（γ-PGA 产量与生物量的比值）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，我们测试了来自四个不同来源（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 PgsC 编码基因，以鉴定 PgsC 编码同源物对 γ-PGA 生产的影响，但结果表明，在所有情况下，γ-PGA 的合成均受到显著抑制。同样，我们还探索了基因同源物编码 PgsB 对 γ-PGA 生产的影响，发现当来自甲基营养芽孢杆菌的 PgsB 替代地衣芽孢杆菌来源的 PgsBCA 中的 PgsB 时，γ-PGA 的产量增加到 17.14±0.62g/L，而不是 8.24±0.10g/L。选择该菌株进行进一步优化，我们通过优化溶解氧水平，在 5L 发酵罐中实现了 38.26g/L 的 γ-PGA 产量。随后，通过补充葡萄糖，在 48h 时 γ-PGA 的产量增加到 50.2g/L。据我们所知，这项研究实现了从葡萄糖从头生产 γ-PGA 的最高产量，而无需添加 L-谷氨酸，这为提高 γ-PGA 产量提供了一种新策略。
