CcpA mutants influence selective carbon source utilization by changing interactions with target genes in Bacillus licheniformis

Abstract

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 that are 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 has yet to be uncovered. 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 DNAbinding 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. This study provides a new strategy for understanding the relationship between the function and structure of regulatory proteins. This study also used a new strategy was used to regulate carbon source utilization beyond CCR engineering.

A New Mechanism of Carbon Metabolism and Acetic Acid Balance Regulated by CcpA

“…Glucose is a preferred carbon source that can be quickly used to produce energy and metabolites in bacilli, including for the production of acetoin, 2,3-butanediol, acetic acid and so on [ 35 ]. A previous study has demonstrated that CcpA mutants can affect the use of other carbon sources and the synthesis of related metabolites in the presence of glucose [ 36 , 37 , 38 ]. In order to further research the regulation mechanism of CcpA on glucose utilization.…”

“…B. licheniformis CA is a strain constructed by knocking out the ccpA on the basis of B. licheniformis . The method of ccpA gene knockout is carried out according to the method we reported earlier [ 36 ]. pET28a-CcpA was the plasmid used for the expression of CcpA.…”

Transcriptome Analysis of Bacillus licheniformis for Improving Bacitracin Production

“…Subsequently, the ps09-vgb fragment was cloned into pNZTT-D1 using the Sal I restriction site, and plasmid pNZTT-D2 was obtained. The gene knockout was performed according to the homologous recombination method, 43 and then, mutants BL2S and BL2K were constructed (Figure S4).…”

Understanding and application of Bacillus nitrogen regulation: A synthetic biology perspective

“…Improvement can be made by artificial mutation in the recognized nucleic acid binding region [76] , [77] , [78] . For the modification of transcription factors, site-directed mutagenesis strategy is usually adopted to meet the specific needs in application [79] , and the understanding of the mechanism can be built at the molecular-level [80] , [81] , [82] .…”