Metabolic engineering of <i>Bacillus subtilis</i> for the production of active hemoglobins and myoglobins by improving heme supply.

Abstract

Hemoglobins (Hb) and myoglobins (Mb) are important hemoproteins with broad applications in food and medicine. Microbial cell factory is a promising approach for the green and sustainable production of hemoproteins. However, current microbial hosts face the challenges of safety and insufficient heme supply. Here, we report a global regulation strategy, "push-restrain-pull-block," to enhance heme supply for producing various active Hb and Mb in food-grade <i>Bacillus subtilis</i>. Initially, the insufficient supply of the precursor 5-aminolevulinate was overcome by relieving feedback inhibition and mitigating the negative effects of HemX on HemA. Next, HemD was identified as the primary uroporphyrinogen III synthase and self-assembled with HemC to minimize the formation of the uroporphyrinogen I by-product. Additionally, the coproporphyrin-dependent pathway was selected as the superior downstream route for heme synthesis, and crucial rate-limiting steps were subsequently enhanced. Moreover, heme consumption was blocked by eliminating protoheme IX farnesyltransferase. Finally, through the combination and fine-tuned expression of key genes, a 221-fold improvement of heme supply was achieved in the engineered strain. Using this stable prokaryotic chassis, we achieved production of 0.81, 0.82, 1.11, and 1.01 g L^-1 of soybean Hb, clover Hb (C-Hb), bovine Mb (B-Mb), and porcine Mb, respectively, through fermentation, marking the highest reported titers in prokaryotic systems. These hemoproteins exhibit properties similar to natural standards. Furthermore, the synthesized C-Hb and B-Mb demonstrated superior effects for preparing plant-based meat analogs as colorants and flavoring agents. This work provides a universal platform for producing other high-value hemoproteins, promising future advancements in food processing and biocatalysis.