Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies

Abstract

Background: Hydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B 12. The introduction of a heterologous de novo vitamin B 12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. Although E. coli avoids major limitations that currently faced by industrial producers of vitamin B 12 , such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B 12 production. Results: By designing combinatorial ribosomal binding site libraries of the hemABCD genes in vivo, we found that their optimal relative translational initiation rates are 10:1:1:5. The transcriptional coordination of the uroporphyrinogen III biosynthetic module was realized by promoter engineering of the hemABCD operon. Knockdown of competitive heme and siroheme biosynthesis pathways by RBS engineering enhanced the hydrogenobyrinic acid titer to 20.54 and 15.85 mg L −1 , respectively. Combined fine-tuning of the heme and siroheme biosynthetic pathways enhanced the hydrogenobyrinic acid titer to 22.57 mg L −1 , representing a remarkable increase of 1356.13% compared with the original strain FH215-HBA. Conclusions: Through multi-level metabolic engineering strategies, we achieved the metabolic balance of the uroporphyrinogen III biosynthesis pathway, eliminated toxicity due to by-product accumulation, and finally achieved a high HBA titer of 22.57 mg L −1 in E. coli. This lays the foundation for high-yield production of vitamin B 12 in E. coli and will hopefully accelerate its industrial production.

Optimization of Hydrogenobyrinic Acid Synthesis in a Cell-Free Multienzyme Reaction by Novel S-Adenosyl-methionine Regeneration

“…1 Recently, after complicated synthetic module integration and adjustment, the artificially engineered Escherichia coli was developed to produce vitamin B 12 and also HBA de novo. 6 After that, the reconstructed HBA-producing strains were also developed and optimized through multilevel metabolic engineering strategies to improve the titer of HBA in vivo to 22.57 mg/L, 7 making it possess the potential to be applied in industrial production. However, limited by complicated metabolic engineering and complex intracellular metabolic network, optimizing the production of HBA faces a big challenge, and enzyme-based total synthesis of artificial compounds in a cell-free system might be a good alternative strategy.…”

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

“…Pathway assembly strategies for metabolic engineering, illustrated by the production of hydrogenobyrinic acid and curcumin in Escherichia coli. Top panel: in work by Jiang et al (2020), classical combinatorial pathway assembly via multivariate optimisation (left) was augmented with debottlenecking approaches (right) to increase the titre of a vitamin B 12 precursor. For step 1, different RBS libraries were used for each gene, each comprising 8 variants, and 288 clones were tested.…”