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Modulating the selective utilization of carbon sources by engineering the 3rd and 4th helices of the DNA-binding domain of catabolite control protein A (CcpA) in Bacillus licheniformis

The gram-positive bacterium Bacillus licheniformis exhibits obvious selective utilization on carbon sources. This process is mainly governed by the global regulator catabolite control protein A (CcpA), which can recognize and bind to multiple target genes widely distributed in metabolic pathways. Although the DNA-binding domain of CcpA has been predicted, the influence of key amino acids on target gene recognition and binding remains elusive. In this study, the impact of Lys31, Ile42 and Leu56 on in vitro protein-DNA interactions and in vivo carbon source selective utilization was investigated. The results showed that alanine substitution of Lys31 and Ile42, located within the 3rd helices of the DNA-binding domain, significantly weakened the binding strength between CcpA and target genes. These mutations also lead to alleviated repression of xylose utilization in the presence of glucose. On the other hand, the Leu56Arg mutant in the 4th helices exhibited enhanced binding affinity compared with that of the wild-type one. When this mutant was used to replace the native one in B. licheniformis cells, the selective utilization of glucose over xylose increased. The above research results are helpful for a deep understanding of how microorganisms can flexibly sense and adapt to changes in the external environment. Additionally, they can provide important theoretical basis for the rational design of biomass utilization and environmental adaptability of B. licheniformis cell factories. Importance Bacillus licheniformis is widely used in producing various valuable products, such as α enzymes, industrial chemicals and biocides. The carbon catabolite regulation process in the utilization of raw materials is crucial to maximizing the efficiency of this microbial cell factory. CcpA plays an important role in this process. This study represents a new paradigm to investigate the structure–function relationship in CcpA by fluorescence polarization experiments in vitro. The results also uncover key amino acids in the DNA-binding domain that affect the selective utilization of carbon sources. These results provide a theoretical basis for the rational design of industrial microorganisms.