Analysis, characterization of coenzyme B12 biosynthetic gene clusters and improvement of B12 biosynthesis in Pseudomonas denitrificans ATCC 13867

Abstract

Coenzyme B₁₂ is an essential cofactor for many enzymes such as glycerol dehydratase, methionine synthase and methylmalonyl-CoA mutase. Herein, we revisited the B₁₂ biosynthetic gene clusters (I and II) in Pseudomonas denitrificans, a well-known industrial producer of the coenzyme B₁₂, to understand the regulation of gene expression and improve the production of coenzyme B₁₂. There existed eight operons, seven in the cluster I and one in the cluster II, and four operons were regulated by B₁₂–responsive riboswitches with a switch-off concentration at ∼5 nM coenzyme B₁₂. DNA sequences of the four riboswitches were partially removed, individually or in combination, to destroy the structures of riboswitches, but no improvement was observed. However, when whole length of riboswitches in the cluster I were completely removed and promoters regulated by the riboswitches were replaced with strong constitutive ones, B₁₂ biosynthesis was improved up to two-fold. Interestingly, modification of the promoter region for cluster II, where many (>10) late genes of B₁₂ biosynthesis belong, always resulted in a significant, more than six-fold reduction of B₁₂ biosynthesis.