Yu Wencheng, Chen Zhen, Ye Hong, Liu Peize, Li Zhipeng, Wang Yuanpeng, Li Qingbiao, Yan Shan, Zhong Chuan-Jian, He Ning
Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People's Republic of China.
The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, People's Republic of China.
Microb Cell Fact. 2017 Feb 8;16(1):22. doi: 10.1186/s12934-017-0642-8.
Poly-gamma-glutamic acid (γ-PGA) is a promising macromolecule with potential as a replacement for chemosynthetic polymers. γ-PGA can be produced by many microorganisms, including Bacillus species. Bacillus licheniformis CGMCC2876 secretes γ-PGA when using glycerol and trisodium citrate as its optimal carbon sources and secretes polysaccharides when using glucose as the sole carbon source. To better understand the metabolic mechanism underlying the secretion of polymeric substances, SWATH was applied to investigate the effect of glucose on the production of polysaccharides and γ-PGA at the proteome level.
The addition of glucose at 5 or 10 g/L of glucose decreased the γ-PGA concentration by 31.54 or 61.62%, respectively, whereas the polysaccharide concentration increased from 5.2 to 43.47%. Several proteins playing related roles in γ-PGA and polysaccharide synthesis were identified using the SWATH acquisition LC-MS/MS method. CcpA and CcpN co-enhanced glycolysis and suppressed carbon flux into the TCA cycle, consequently slowing glutamic acid synthesis. On the other hand, CcpN cut off the carbon flux from glycerol metabolism and further reduced γ-PGA production. CcpA activated a series of operons (glm and epsA-O) to reallocate the carbon flux to polysaccharide synthesis when glucose was present. The production of γ-PGA was influenced by NrgB, which converted the major nitrogen metabolic flux between NH and glutamate.
The mechanism by which B. licheniformis regulates two macromolecules was proposed for the first time in this paper. This genetic information will facilitate the engineering of bacteria for practicable strategies for the fermentation of γ-PGA and polysaccharides for diverse applications.
聚γ-谷氨酸（γ-PGA）是一种有前景的大分子物质，有潜力替代化学合成聚合物。γ-PGA可由包括芽孢杆菌属在内的多种微生物产生。地衣芽孢杆菌CGMCC2876在以甘油和柠檬酸钠作为最佳碳源时分泌γ-PGA，而以葡萄糖作为唯一碳源时分泌多糖。为了更好地理解聚合物分泌的代谢机制，采用了数据非依赖采集（SWATH）技术在蛋白质组水平研究葡萄糖对多糖和γ-PGA产生的影响。
添加5 g/L或10 g/L葡萄糖分别使γ-PGA浓度降低了31.54%或61.62%，而多糖浓度从5.2增加到43.47%。使用SWATH采集液相色谱-串联质谱法鉴定了几种在γ-PGA和多糖合成中起相关作用的蛋白质。碳代谢物阻遏蛋白A（CcpA）和碳代谢物阻遏蛋白N（CcpN）共同增强糖酵解并抑制碳流入三羧酸循环，从而减缓谷氨酸合成。另一方面，CcpN切断了甘油代谢的碳流，进一步降低了γ-PGA的产生。当存在葡萄糖时，CcpA激活一系列操纵子（glm和epsA-O）以将碳流重新分配到多糖合成。γ-PGA的产生受NrgB影响，NrgB在铵离子和谷氨酸之间转换主要的氮代谢通量。
本文首次提出了地衣芽孢杆菌调节两种大分子物质的机制。这些遗传信息将有助于对细菌进行工程改造，以制定切实可行的策略，用于γ-PGA和多糖的发酵，以实现多种应用。
