Directed evolution of heme biosynthetic enzymes enabled by a growth-coupled heme biosensor in Corynebacterium glutamicum

Heme is an essential cofactor involved in diverse cellular processes and is an important target for microbial biosynthesis. However, engineering microbial heme production remains challenging due to the requirement for coordinated activity of multiple pathway enzymes and the lack of scalable strategies for enzyme-level screening. In this study, we developed a growth-coupled screening system in Corynebacterium glutamicum by establishing a ChrSA-based heme biosensor derived from the native heme-responsive two-component system, which directly converts intracellular heme levels into a selectable growth phenotype. Using this biosensor-enabled system, random mutagenesis libraries were constructed for four key enzymes of the coproporphyrin-dependent (CPD) heme biosynthesis pathway, and improved variants were identified through biosensor-guided selection. The selected variants were subsequently validated by chromosomal allelic replacement, resulting in an overall increase in heme production of approximately 38.9% and a final titer of 308.7 mg/L under native, single-copy regulation. Sequence and structural analyses indicated that the beneficial substitutions were predominantly located outside catalytic residues, suggesting that changes in enzyme stability and conformational properties contributed to the enhanced heme biosynthesis. This work established a heme biosensor-guided strategy for enzyme variant screening in tightly coupled metabolic pathways and provided a practical systems-level approach for microbial heme pathway engineering.