Towards a cell factory for vitamin B12 production in Bacillus megaterium: bypassing of the cobalamin riboswitch control elements.

Bacillus megaterium is a bacterium that has been used in the past for the industrial production of vitamin B12 (cobalamin), the anti-pernicious anaemia factor. Cobalamin is a modified tetrapyrrole with a cobalt ion coordinated within its macrocycle. More recently, B. megaterium has been developed as a host for the high-yield production of recombinant proteins using a xylose inducible promoter system. Herein, we revisit cobalamin production in B. megaterium DSM319. We have investigated the importance of cobalt for optimum growth and cobalamin production. The cobaltochelatase (CbiX(L)) is encoded within a 14-gene cobalamin biosynthetic (cbi) operon, whose gene-products oversee the transformation of uroporphyrinogen III into adenosylcobyrinic acid a,c-diamide, a key precursor of cobalamin synthesis. The production of CbiX(L) in response to exogenous cobalt was monitored. The metal was found to stimulate cobalamin biosynthesis and decrease the levels of CbiX(L). From this we were able to show that the entire cbi operon is transcriptionally regulated by a B12-riboswitch, with a switch-off point at approximately 5 nM cobalamin. To bypass the effects of the B12-riboswitch the cbi operon was cloned without these regulatory elements. Growth of these strains on minimal media supplemented with glycerol as a carbon source resulted in significant increases in cobalamin production (up to 200 μg L(-1)). In addition, a range of partially amidated intermediates up to adenosylcobyric acid was detected. These findings outline a potential way to develop B. megaterium as a cell factory for cobalamin production using cheap raw materials.