Key Laboratory of Molecular Microbiology and Technology of Ministry of Education, Nankai University, Tianjin, 300071, China.
Appl Microbiol Biotechnol. 2014;98(14):6397-407. doi: 10.1007/s00253-014-5729-0. Epub 2014 Apr 26.
Poly-γ-glutamic acid (γ-PGA) is an important biopolymer with greatly potential in industrial and medical applications. In the present study, we constructed a metabolically engineered glutamate-independent Bacillus amyloliquefaciens LL3 strain with considerable γ-PGA production, which was carried out by single, double, and triple markerless deletions of three degradation genes pgdS, ggt, and cwlO. The highest γ-PGA production (7.12 g/L) was obtained from the pgdS and cwlO double-deletion strain NK-pc, which was 93 % higher than that of wild-type LL3 strain (3.69 g/L). The triple-gene-deletion strain NK-pgc showed a 28 % decrease in γ-PGA production, leading to a yield of 2.69 g/L. Furthermore, the cell morphologies of the mutant strains were also characterized. The cell length of cwlO deletion strains NK-c and NK-pc was shorter than that of the wild-type strain, while the ggt deletion strains NK-g, NK-pg, NK-gc, and NK-pgc showed longer cell lengths. This is the first report concerning the markerless deletion of γ-PGA degradation genes to improve γ-PGA production in a glutamate-independent strain and the first observation that γ-glutamyltranspeptidase (encoded by ggt) could be involved in the inhibition of cell elongation.
聚-γ-谷氨酸（γ-PGA）是一种具有巨大工业和医学应用潜力的重要生物聚合物。在本研究中，我们构建了一株代谢工程谷氨酸非依赖型解淀粉芽孢杆菌 LL3 菌株，具有相当高的γ-PGA 生产能力，这是通过单个、双个和三个无标记缺失 pgdS、ggt 和 cwlO 三个降解基因来实现的。pgdS 和 cwlO 双缺失菌株 NK-pc 的最高 γ-PGA 产量（7.12 g/L）比野生型 LL3 菌株（3.69 g/L）高 93%。三基因缺失菌株 NK-pgc 的 γ-PGA 产量下降了 28%，产量为 2.69 g/L。此外，还对突变株的细胞形态进行了表征。cwlO 缺失菌株 NK-c 和 NK-pc 的细胞长度比野生型菌株短，而 ggt 缺失菌株 NK-g、NK-pg、NK-gc 和 NK-pgc 的细胞长度则较长。这是首次报道在谷氨酸非依赖型菌株中通过无标记缺失 γ-PGA 降解基因来提高 γ-PGA 产量，也是首次观察到 γ-谷氨酰转肽酶（由 ggt 编码）可能参与细胞伸长的抑制。
