Abstract

BackgroundHydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B12. The introduction of a heterologous de novo vitamin B12 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 B12, such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B12 production.ResultsBy 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.ConclusionsThrough 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 B12 in E. coli and will hopefully accelerate its industrial production.

Highlights

  • Hydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin ­B12

  • We introduced an exogenous urogen Uroporphyrinogen III (III) biosynthesis pathway into E. coli to realize the biosynthesis of vitamin B­ 12, and an siRNA strategy was used to knock down the heme branch pathway [6]

  • Optimizing the expression ratio of aminolevulinic acid synthase (ALAS), porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD) and uroporphyrinogen III synthase (UROS) using ribosome binding site (RBS) Libraries Urogen III is the common precursor of vitamin ­B12, heme and siroheme

Read more

Summary

Introduction

Hydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin ­B12. The introduction of a heterologous de novo vitamin ­B12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. 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 ­B12 production. Hydrogenobyrinic acid (HBA) is the first stable intermediate in the oxygen-dependent (aerobic) biosynthetic pathway of vitamin ­B12 (cobalamin) [1, 2]. It is a modified tetrapyrrole that belongs to the same class of compounds. Jiang et al Microb Cell Fact (2020) 19:118. Precursor module a Glycine Succinyl-CoA Glutamate HemA. RBS hemA hemB hemC hemD O H2N ALA HemB OH O

Methods
Results
Discussion
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call