Metabolic engineering of Bacillus subtilis for the production of active hemoglobins and myoglobins by improving heme supply

• 成果类型: Article
• 署名作者: Wang, Song; Zhou, Jingwen; Li, Jianghua; Chen, Jian; Du, Guocheng; Li, Zhengqiang; Lee, Sang Yup; Zhao, Xinrui
• 署名单位: Jiangnan University; Jiangnan University; Jiangnan University; Jiangnan University; Jilin University; Korea Advanced Institute of Science & Technology (KAIST); Korea Advanced Institute of Science & Technology (KAIST)
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8717
• DOI: 10.1073/pnas.2504795122
• 发表日期: 2025-07-08
• 关键词: escherichia-coli; protein; meat; biosynthesis; purification; synthase; encodes; PATHWAY; complex; genes

摘要

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 Bacillus subtilis. 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. Significance The large-scale biosynthesis of active hemoproteins for applications in the manufacture of food and medicine remains a significant challenge. In this study, we comprehensively identified bottlenecks in the heme biosynthetic pathway and addressed them using a push-restrain-pull-block strategy. Based on these metabolic modifications, we developed a stable food-grade Bacillus subtilis chassis with efficient and moderate heme supply. Consequently, various active hemoglobins (Hb) and myoglobins (Mb) were successfully produced, achieving higher titers and productivities. Additionally, the biosynthesized clover Hb and bovine Mb demonstrated excellent properties for enhancing the color and flavor of plant-based meat analogs. This work lays a foundation for scalable biosynthesis of valuable hemoproteins used in food technology, healthcare, and green chemistry.