Functional Characterization of ccpA in Heyndrickxia coagulans Reveals Coordinated Regulation of Carbon Catabolite Repression and L-Lactic Fermentation

Heyndrickxia coagulans is widely used for industrial L-lactic acid production, but carbon catabolite repression (CCR) and its link to fermentative metabolism remain poorly understood. A ccpA deletion mutant (ΔccpA) and a complementation strain (C-ccpA) were constructed to investigated the physiological, enzymatic, and transcriptomic consequences of CcpA loss. Deletion of ccpA completely abolished glucose-mediated CCR, enabling simultaneous glucose–xylose co-utilization, and triggered a marked shift from L-lactic to mixed-acid fermentation, with an 82.5% reduction in lactate titer accompanied by 24.1-fold and 51.6-fold increases in acetate and formate, respectively. Enzyme activity assays showed that L-lactate dehydrogenase activity was reduced by half, whereas acetate kinase activity increased nearly six-fold. Transcriptomic analysis revealed downregulation of ldhL and upregulation of pflB and ackA. Scale-up fermentation in a 5 L bioreactor confirmed that the wild type directed 90.2% of carbon flux to lactate (yield, 0.95 g/g glucose), compared with only 24.5% in the mutant. All phenotypes were fully restored upon complementation. These results demonstrate that CcpA is as an indispensable dual regulator of both CCR and L-lactic fermentation, providing a foundation for rational metabolic engineering of H. coagulans.