Abstract
INTRODUCTION: Vitamin B(12) (B(12)) is an essential cofactor for key metabolic processes in most living organisms, yet only certain bacteria can synthesize it de novo. Common forms of B(12) include adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl) and cyanocobalamin (CNCbl). This study presents the B(12) production capability of an extremophile-Ectopseudomonas alcaliphila MSJ19, and a multilevel evaluation of bioactivity of various B(12) forms. METHODS: B(12) extracted from Ectopseudomonas alcaliphila MSJ19 was initially analyzed by bioassay and LC-MS to confirm the presence of natural B(12) forms, followed by in vitro enzyme activity assays with glycerol dehydratase (GD) and diol dehydratase (DD). The functionality of various B(12) forms on these enzymes was further evaluated using in-silico molecular docking studies. The bioactivity at the in vivo level was assessed by introducing a coenzyme B(12)-dependent 3-hydroxypropionic acid (3-HP) biosynthetic pathway in E. coli W and Ectopseudomonas alcaliphila MSJ19 for their ability to transform glycerol into 3-HP. RESULTS: Bioassay and LC-MS analysis confirmed the presence of ~7 μg/g cdw B(12) in the processed extract and specific precursor-product ion transitions, indicated the production of natural B(12) forms. To functionally validate the bioactivity of the crude B(12) extract, the coenzyme B(12)-dependent 3-HP biosynthesis pathway was employed in recombinant E. coli W. Supplementation with different B(12) forms revealed a hierarchical GD and DD activity (AdoCbl > MeCbl > CNCbl) and a dose-dependent increase in 3-HP production, with an optimal threshold around 500 nM. The conformational specificity of AdoCbl and competitive inhibition of CNCbl and MeCbl were supported by molecular docking of all 3 B(12) forms with GD and DD. Notably, crude B(12) extract at 0.35 nM yielded 5.9 mM 3-HP titer, closely matching the 7.8 mM obtained with AdoCbl, confirming its bioactive equivalence. Furthermore, recombinant Ectopseudomonas alcaliphila MSJ19 (EaM(r)) harboring the 3-HP pathway produced up to 3.3 mM 3-HP without external B(12) supplementation, highlighting innate capability of the host to produce and utilize bioactive B(12) in vivo. DISCUSSION: Collectively-in vitro, in silico and in vivo approaches establish a functional framework for certifying B(12) bioactivity and demonstrating EaM as a potent chassis for production of value-added chemicals.