Engineering Corynebacterium glutamicum with Effective Heme Supply for the Synthesis of High-Activity Hemoglobins and Myoglobins

Hemoglobin (Hb) and myoglobin (Mb) have been extensively employed as oxygen carriers, iron supplements, and food additives, but the microbial synthesis of functional Hb and Mb remains a challenge. Herein, we engineered a Corynebacterium glutamicum strain with efficient heme supply for producing active Hb and Mb. Through the iterative multimodule strategy, including improvement of precursor supply (5-aminolevulinate), redirection of unstable hydroxymethylbilane, selection of the coproporphyrin-dependent pathway as the superior downstream route, enhancement of rate-limiting steps, and prevention of heme secretion, the heme titer was increased by 90.75-fold. Combined with the optimized expression of globins, the engineered HEME3-ΔhrtBA strain achieved the highest titers at the shake-flask level for soybean Hb (1.10 g/L), clover Hb (1.12 g/L), bovine Mb (0.56 g/L), and porcine Mb (1.14 g/L), respectively. Notably, the biosynthesized Hb and Mb presented biochemical properties similar to those of their natural counterparts. This engineered strain provides a scalable platform for synthesizing diverse functional hemoproteins.