Abstract
BackgroundThe 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.ResultsThe 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.ConclusionsThe 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).
Highlights
Introduction of ribM fromS. davawensis in B. subtilis The ribM gene from S. davawensis has a relatively high G+C-content (72%) and was optimized with respect to the codon usage of B. subtilis in order to allow efficient heterologous expression
Expression of ribM from S. davawensis in E. coli increased riboflavin uptake The wild-type gene ribM from S. davawensis was expressed in E. coli using the plasmid pNCO113ribM, employing an IPTG controlled T5 promotor [15]
Riboflavin uptake mediated by S. davawensis RibM was only slightly affected by flavin adenine dinucleotide (FAD) and the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Figure 1B)
Summary
Introduction of ribM fromS. davawensis in B. subtilis The ribM gene from S. davawensis has a relatively high G+C-content (72%) and was optimized with respect to the codon usage of B. subtilis in order to allow efficient heterologous expression. We evaluate the flavin transporter RibM from Streptomyces davawensis with respect to improvement of a riboflavin production strain. RibM from C. glutamicum is similar (40% at the amino acid level) to RibM (23.7 kDa) from S. davawensis The gene for the latter protein is present in the S. davawensis riboflavin biosynthetic gene cluster ribBMAH which is controlled by an FMN riboswitch [12] directly upstream of ribB [13]. The gene for the flavin facilitator ribM from S. davawensis was codon optimized for expression in B. subtilis. The gene ribM was functionally characterized and was found to encode a transporter for riboflavin and roseoflavin. RibM was evaluated as a possible tool to enhance the productivity of a B. subtilis riboflavin production strain
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