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

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.