RibM from Streptomyces davawensis is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains

Background: The bacterium Bacillus subtilis, which is not a natural riboflavin overproducer, has been converted into an excellent production strain by classical mutagenesis and metabolic engineering. To our knowledge, the enhancement of riboflavin excretion from the cytoplasm of overproducing cells has not yet been considered as a target for (further) strain improvement. Here we evaluate the flavin transporter RibM from Streptomyces davawensis with respect to improvement of a riboflavin production strain. Results: The gene ribM from S. davawensis, coding for a putative facilitator of riboflavin uptake, was codon optimized (ribM(opt)) for expression in B. subtilis. The gene ribM(opt) was functionally introduced into B. subtilis using the isopropyl-beta-thiogalactopyranoside (IPTG)-inducible expression plasmid pHT01: Northern-blot analysis of total RNA from IPTG treated recombinant B. subtilis cells revealed a ribM(opt) specific transcript. Western blot analysis showed that the his(6)-tagged heterologous gene product RibM was present in the cytoplasmic membrane. Expression of ribM in Escherichia coli increased [C-14] riboflavin uptake, which was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of ribM(opt) supported growth of a B. subtilis Delta ribB:: Erm(r) Delta ribU::Kan(r) double mutant deficient in riboflavin synthesis (Delta ribB) and also deficient with respect to riboflavin uptake (Delta ribU). Expression of ribM(opt) increased roseoflavin (a toxic riboflavin analog produced by S. davawensis) sensitivity of a B. subtilis Delta ribU:: Kan(r) strain. Riboflavin synthesis by a model riboflavin B. subtilis production strain overproducing RibM was increased significantly depending on the amount of the inducer IPTG. Conclusions: The energy independent flavin facilitator RibM could in principle catalyze riboflavin export and thus may be useful to increase the riboflavin yield in a riboflavin production process using a recombinant RibM overproducing B. subtilis strain (or any other microorganism).