地衣芽胞杆菌DW2中敲除氨基酸转运蛋白基因yhdG提高杆菌肽产量

Enhanced production of bacitracin by knocking out of amino acid permease gene yhdG in Bacillus licheniformis DW2

摘要:

杆菌肽是微生物产生的由11种氨基酸残基组成的广谱性抗生素，前体物的供应可能是限制杆菌肽高产的重要因素。文中通过支链氨基酸 (异亮氨酸、亮氨酸、缬氨酸) 的添加实验考察了前体物质支链氨基酸对杆菌肽高产的影响，证实了异亮氨酸 (Ile) 和亮氨酸 (Leu) 的添加可以提高杆菌肽的效价，其中Ile的添加对杆 菌肽效价提高的效果较为明显。随后，文中以地衣芽胞杆菌DW2为出发菌株，分别构建了支链氨基酸转运蛋白YhdG的缺失和强化表达菌株。发酵结果表明，转运蛋白YhdG缺失工程菌DW2△yhdG的杆菌肽效价达到917.35 U/mL，与原始菌DW2相比提高了11%，而强化YhdG则会使杆菌肽效价下降25%。最后通过分析胞内胞外支链氨基酸含量，发现缺失转运蛋白YhdG能够在发酵中后期显著提高胞内支链氨基酸含量，表明氨基酸转运蛋白YhdG在地衣芽胞杆菌DW2中可能发挥着氨基酸输出的功能。综上，文中通过缺失转运蛋白YhdG显著提高了地衣芽胞杆菌胞内支链氨基酸的供给水平，从而提高了杆菌肽效价，为杆菌肽高产菌株的构建提供了一种新的策略。

Abstract:

Bacitracin is a broad-spectrum polypeptide antibiotic, which is formed by 11 amino acids residues. Precursor amino acids supply might be the limit factor during bacitracin fermentation. First, our results demonstrated that increasing Ile and Leu supplies were regarded as the efficient strategies for the enhanced titer of bacitracin. Then, the amino acid permease YhdG, which was identified as the BCAA permease, was deleted and overexpressed in DW2, respectively. Our results showed that knocking out of permease YhdG could improve bacitracin production remarkablely. The bacitracin titer of the yhdG deficient strain DW2△yhdG reached 917.35 U/mL by flask fermentation, increased by 11% compared with that of DW2. In addition, the bacitracin titer was decreased by 25% in the YhdG overexpressed strain. Meanwhile, the intracellular concentrations of BCAA were higher than DW2 during the biosynthesis of bacitracin. The above results suggested that the permease YhdG might act as an exporter for branched chain amino acids in B. licheniformis DW2. Taken together, the increasing intracellular concentrations of branched chain amino acids by deleting amino acid permease YhdG could improve bacitracin titer. This study provided a new strategy for high-level production of bacitracin.