d,l-肽链内切酶基因cwlO缺失促进地衣芽孢杆菌利用l-谷氨酰胺合成聚γ-谷氨酸

Deletion of cwlO facilitates the l-glutamine utilization of Bacillus licheniformis for poly-γ-glutamic acid production

摘要:

【背景】聚γ-谷氨酸(poly-γ-glutamic acid, γ-PGA)是一种由芽孢杆菌代谢产生的同质氨基酸聚合物，在众多领域具有广泛的应用潜力。芽孢杆菌cwlO表达一种d,l-肽链内切酶，其对γ-PGA合成的影响机理尚不清晰。【目的】探究缺失cwlO对以不同前体发酵γ-PGA的影响及其机制。【方法】以地衣芽孢杆菌WX-02为出发菌株，构建缺失cwlO的重组菌。在3 L发酵罐条件下对比重组菌和野生菌利用不同前体发酵产γ-PGA的发酵性能，并通过转录水平差异分析、荧光倒置显微镜观察、细胞壁肽聚糖含量和组分分析造成重组菌和野生菌发酵性能差异的原因。【结果】缺失cwlO的重组菌对l-谷氨酰胺的代谢利用效率显著提高，以l-谷氨酰胺和l-谷氨酸为混合前体时，重组菌的γ-PGA产量达到36.3 g/L，比野生菌高48.8%。RT-qPCR结果表明，相较于野生菌，重组菌在利用l-谷氨酰胺时γ-PGA合成途径和呼吸链上关键基因转录水平均上调。荧光倒置显微镜观察发现重组菌细胞形态相比野生菌变短变圆，细胞壁肽聚糖含量和组分测定发现，重组菌细胞壁肽聚糖含量降低，且肽聚糖中蛋白质占比减少。【结论】地衣芽孢杆菌cwlO的缺失引起细胞壁肽聚糖含量降低，促进了菌株对l-谷氨酰胺的利用，强化了γ-PGA的合成，这为探究cwlO对γ-PGA合成的影响提供了新的思路和研究基础。

Abstract:

[Background] Poly-γ-glutamic acid (γ-PGA), a homogeneous amino acid polymer produced by Bacillus, has application potential in a variety of fields. The cwlO in Bacillus can express a d,l-endopeptidase to influence γ-PGA production, the underlying mechanism of which remains to be clarified. [Objective] To explore the effect of cwlO deletion on γ-PGA production by using different precursors and decipher the underlying mechanism. [Methods] The recombinant strain with cwlO deleted was constructed on the basis of Bacillus licheniformis WX-02. The fermentation for γ-PGA production was conducted in 3-L fermenters with different precursors, and the performance was compared between the recombinant and the wild type. Moreover, the mechanism for the performance difference was explored by the analysis of transcriptional levels, cell morphology observation via inverted fluorescence microscopy, and the content measurement of peptidoglycan and its components. [Results] The cwlO-deleted recombinant showed an improved efficiency on l-glutamine assimilation. With l-glutamine and l-glutamic acid as the mixed precursor, the recombinant showed the γ-PGA production of 36.3 g/L, which increased by 48.8% compared with that of the wild type. The results of RT-qPCR indicated that the transcriptional levels of the key genes involved in γ-PGA synthesis and respiratory chain were up-regulated in the recombinant with l-glutamine as the precursor, compared with those of the wild type. The recombinant cells became shorter and rounder. The peptidoglycan content in the cell wall and the protein content in peptidoglycan of the recombinant were greatly lower than those of the wild type. [Conclusion] Deletion of cwlO in B. licheniformis decreased the peptidoglycan content in the cell wall and promoted l-glutamine utilization, which enhanced γ-PGA production. This study provides a new insight and research basis for further research on the role of cwlO in γ-PGA production.