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

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.