title: RibM from Streptomyces davawensis is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains.
abstract: 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 (ribMopt) for expression in B. subtilis. The gene ribMopt was functionally introduced into B. subtilis using the isopropyl-β-thiogalactopyranoside (IPTG)-inducible expression plasmid pHT01: Northern-blot analysis of total RNA from IPTG treated recombinant B. subtilis cells revealed a ribMopt specific transcript. Western blot analysis showed that the his6-tagged heterologous gene product RibM was present in the cytoplasmic membrane. Expression of ribM in Escherichia coli increased [14C]riboflavin uptake, which was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of ribMopt supported growth of a B. subtilis ΔribB::Ermr ΔribU::Kanr double mutant deficient in riboflavin synthesis (ΔribB) and also deficient with respect to riboflavin uptake (ΔribU). Expression of ribMopt increased roseoflavin (a toxic riboflavin analog produced by S. davawensis) sensitivity of a B. subtilis ΔribU::Kanr 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).
authors.affiliation: Institut für Technische Mikrobiologie, Hochschule Mannheim, Mannheim, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Hemberger
authors.forename: Danielle B
authors.initials: DB
authors.lastname: Pedrolli
authors.forename: Jürgen
authors.initials: J
authors.lastname: Stolz
authors.forename: Christian
authors.initials: C
authors.lastname: Vogl
authors.forename: Martin
authors.initials: M
authors.lastname: Lehmann
authors.forename: Matthias
authors.initials: M
authors.lastname: Mack
mesh_terms: D001412:Bacillus subtilis; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D015152:Blotting, Northern; D015153:Blotting, Western; D002250:Carbon Radioisotopes; D004926:Escherichia coli; D018014:Gene Transfer Techniques; D007544:Isopropyl Thiogalactoside; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D012256:Riboflavin / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
chemical_list: D002250:Carbon Radioisotopes; D026901:Membrane Transport Proteins; D007544:Isopropyl Thiogalactoside; C009191:roseoflavin; D012256:Riboflavin