Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium

Abstract

Cobalamin (vitamin B(12)) production in Bacillus megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome-based overexpression of genes involved in vitamin B(12) biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysG(A)cbiYbtuR, and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding coproporphyrinogen III oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B(12) riboswitch upstream of the cbiXJCDETLFGAcysG(A)cbiYbtuR operon and the recombinant production of three different vitamin B(12) binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B(12)-dependent feedback inhibition. All these strategies increased cobalamin production in B. megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B(12) concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.