Metabolic Flux Analysis and CRISPRi-Mediated Repression of Byproduct Pathways for Optimized L-Lysine Yield in Corynebacterium glutamicum

Industrial production of L-lysine using Corynebacterium glutamicum is pivotal for the global feed and pharmaceutical sectors, yet molar yields remain constrained by carbon diversion into organic acid and competing amino acid byproducts. In this study, we integrated ¹³C-metabolic flux analysis (¹³C-MFA) with CRISPR interference (CRISPRi)-mediated gene repression to systematically identify and eliminate major metabolic sinks in an L-lysine-producing strain. Quantitative flux profiling under hyper-producing conditions revealed significant carbon loss through lactate dehydrogenase (ldhA) and alanine aminotransferase (alaT), which accounted for up to 18% of total consumed glucose. To circumvent the growth defects often associated with complete gene knockouts, a catalytically dead Cas9 (dCas9) platform driven by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter was implemented. Targeted transcriptional silencing achieved a 78% reduction in ldhA expression and a 64% reduction in alaT expression without impairing biomass accumulation. ¹³C-MFA of the optimized strain revealed a 32% increase in net carbon flux through oxaloacetate into the aspartate pathway, coupled with enhanced NADPH regeneration via the pentose phosphate pathway. In fed-batch bioreactor fermentations, the engineered strain C. glutamicum CR-Lys04 achieved an L-lysine yield of 0.54 g/g glucose, representing a 41% improvement over the parental strain with minimal byproduct secretion. This work highlights the synergy between quantitative fluxomics and fine-tuned genetic repression for rational metabolic engineering of high-yielding microbial chassis.