改造谷氨酸棒杆菌CmpLs转运系统促进l-谷氨酸合成

Engineering of CmpLs enhances l-glutamate production ofCorynebacterium glutamicum

摘要:

l-谷氨酸的高效生产依赖于产物的快速外排，其转运系统和细胞膜壁结构的人工改造已成为近年来研究的热点。针对谷氨酸棒杆菌特殊的细胞壁结构和组分，本研究在一株能够组成型分泌l-谷氨酸的菌株SCgGC7中验证了CmpLs转运系统对l-谷氨酸合成及转运的影响。首先，构建了不同CmpLs转运蛋白的敲除菌株，明确了CmpL1和CmpL4敲除可以显著提高菌株的l-谷氨酸生产性能；其次，利用温敏发酵工艺在5 L发酵罐中对上述菌株进行了发酵测试，发现CmpL1和CmpL4敲除菌株可以叠加l-谷氨酸生产的温敏特性，进一步强化高温条件下l-谷氨酸的生产，其中CmpL1敲除菌株表现出更好的l-谷氨酸生产性能，产量和糖酸转化率分别比对照菌株提高了69.2%和55.3%，最后，对发酵终点样品进行了胞内和胞外代谢物组分析，明确了CmpLs转运系统的改造可以明显促进l-谷氨酸外排，强化l-谷氨酸合成和转运的代谢通量，并且发现CmpL1敲除菌株胞内三羧酸循环中间代谢物和l-谷氨酸下游代谢物的积累明显更少，与其更强的l-谷氨酸生产性能一致。不同CmpLs蛋白功能上的冗余和互补，不仅为谷氨酸工业菌株的稳定性改造和性能提升提供了理想的靶点，也为改造谷氨酸棒杆菌的细胞膜壁结构强化其他代谢产物的外排提供了新的靶点和策略。

Abstract:

The efficient production of l-glutamate is dependent on the product’s rapid efflux, hence researchers have recently concentrated on artificially modifying its transport system and cell membrane wall structure. Considering the unique composition and structure of the cell wall of Corynebacterium glutamicum, we investigated the effects of CmpLs on l-glutamate synthesis and transport in SCgGC7, a constitutive l-glutamate efflux strain. First, the knockout strains of CmpLs were constructed, and it was confirmed that the deletion of CmpL1 and CmpL4 significantly improved the performance of l-glutamate producers. Next, temperature-sensitive l-glutamate fermentation with the CmpL1 and CmpL4 knockout strains were carried out in 5 L bioreactors, where the knockout strains showcased temperature-sensitive characteristics and enhanced capacities for l-glutamate production under high temperatures. Notably, the CmpL1 knockout strain outperformed the control strain in terms of l-glutamate production, showing production and yield increases of 69.2% and 55.3%, respectively. Finally, the intracellular and extracellular metabolites collected at the end of the fermentation process were analyzed. The modification of CmpLs greatly improved the l-glutamate excretion and metabolic flux for both l-glutamate production and transport. Additionally, the CmpL1 knockout strain showed decreased accumulation of downstream metabolites of l-glutamate and intermediate metabolites of tricarboxylic acid (TCA) cycle, which were consistent with its high l-glutamate biosynthesis capacity. In addition to offering an ideal target for improving the stability and performance of the industrial strains for l-glutamate production, the functional complementarity and redundancy of CmpLs provide a novel target and method for improving the transport of other metabolites by modification of the cell membrane and cell wall structures in C. glutamicum.