Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS–PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology.

In lactic acid bacteria, the global transcriptional regulator CcpA regulates carbon metabolism by repressing and activating the central carbon metabolism pathway, thus decreasing or increasing the yield of certain metabolites to maximize carbon flow. However, there are no reports on the deregulation of the inhibitory effects of CcpA on the metabolism of secondary metabolites. In this study, we identified a single-base mutant strain of Lactococcus lactis N8-2 that is capable of metabolizing 2,3-butanediol. It has been established that CcpA dissociates from the catabolite responsive element (cre) site due to a mutation, leading to the activation of derepression and expression of the 2,3-butanediol dehydrogenase gene cluster (butB and butA). Transcriptome analysis and quantitative polymerase chain reaction (Q-PCR) results showed significant upregulation of transcription of butB and butA compared to the unmutated strain. Furthermore, micro-scale thermophoresis experiments confirmed that CcpA did not bind to the mutated cre. Furthermore, in a bacterial two-plasmid fluorescent hybridization system, it was similarly confirmed that the dissociation of CcpA from cre eliminated the repressive effect of CcpA on downstream genes. Finally, we investigated the differing catalytic capacities of the 2,3-butanediol dehydrogenase gene cluster in L. lactis N8-1 and L. lactis N8-2 for 2,3-butanediol. This led to increased expression of butB and butA, which were deregulated by CcpA repression. This is the first report on the elimination of the deterrent effect of CcpA in lactic acid bacteria, which changes the direction of enzymatic catalysis and alters the direction of carbon metabolism. This provides new perspectives and strategies for metabolizing 2,3-butanediol using bacteria in synthetic biology.