Identification, functional characterization of a novel flavohemoglobin from Ensifer adhaerens and its application to enhancing vitamin B12 production.

Abstract

Background: Bacterial flavohemoglobins are well-characterized as versatile proteins that significantly augment the biosynthesis yields of diverse value-added compounds in engineered microbial platforms. Our previous studies revealed marked upregulation of a flavohemoglobin-encoding gene in Ensifer adhaerens under oxygen-limiting conditions, with potential associations to vitamin B₁₂ production.

Results: In this study, a novel flavohemoglobin (FHP, encoded by b12fla gene) from Ensifer adhaerens were characterized. FHP exhibited high sequence and structural similarity to HMP (the flavohaemoglobin from Halalkalibacterium halodurans), yet demonstrating 3-fold higher NADH oxidase activity. Heterologous expression of FHP in Escherichia coli positively influences cell growth, biomass yield on carbon and nitrogen sources, increased OD₆₀₀ and Oxygen Uptake Rate (OUR) in 50-L fermenter of FHP-expressing strain EcFHP (12% and 65% higher compared with control strain respectively). In addition, SEM analysis revealed that cells of overexpression strains exhibited obvious morphological alterations, manifesting as longitudinal extension or radial expansion compared to cells of wild-type strain. Overexpression of b12fla gene in E. adhaerens Casida A generated the recombinant strain Ea-b12fla, which produced 323 mg L^- 1 of vitamin B₁₂ in a 50-L fermentation while the wild-type strain yielded only 247 mg L^- 1. Furthermore, cell growth, OUR, Carbon-dioxide Escape Rate (CER) and sugar utilization were obviously enhanced by overexpression of the flavohemoglobin.

Conclusions: A flavohemoglobin from a new source was identified in this study. Heterologous expression in E. coli validated its ability to promote cell growth under specific oxygen supply conditions. Overexpression in E. adhaerens significantly enhanced cell growth and cell metabolism, while markedly increasing vitamin B₁₂ production. Collectively, these findings demonstrate that flavohemoglobin (FHP) enhances cellular oxygen utilization while simultaneously improving the assimilation efficiency of carbon and nitrogen sources, and reveal the potential of flavohemoglobin for applications in high-density fermentation processes.