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 were eight operons, seven in cluster I and one in 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 the whole length of riboswitches in cluster I were completely removed and promoters regulated by the riboswitches were replaced with strong constitutive ones, B12 biosynthesis was improved by up to 2-fold. Interestingly, modification of the promoter region for cluster II, where many (>10) late genes of B12 biosynthesis belong, always resulted in a significant, greater than 6-fold reduction in B12 biosynthesis.