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Open Access Research article

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

Sabrina Hemberger1, Danielle B Pedrolli1, Jürgen Stolz2, Christian Vogl2, Martin Lehmann3 and Matthias Mack1*

Author Affiliations

1 Institut für Technische Mikrobiologie, Hochschule Mannheim, 68163 Mannheim, Germany

2 Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL), Lehrstuhl für Ernährungsphysiologie, Technische Universität München, Gregor-Mendel-Str. 2, 85350 Freising-Weihenstephan, Germany

3 Biotechnology R&D, DSM Nutritional Products, P.O. Box 3255, CH-4002 Basel, Switzerland

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BMC Biotechnology 2011, 11:119  doi:10.1186/1472-6750-11-119

Published: 2 December 2011

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).