RibM from Streptomyces davawensis is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains
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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.
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-β-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 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 ribM opt 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 ribM opt 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.
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).
KeywordsRiboflavin Flavin Adenine Dinucleotide Corynebacterium Glutamicum Subtilis Strain Lysogeny Broth
Riboflavin (vitamin B2) is a direct precursor to the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Riboflavin is synthesized by plants and many microorganisms; it is not synthesized by animals . Many Gram-positive bacteria seem to be capable of acquiring riboflavin from the environment, whereas most Gram-negative bacteria depend on the endogenous synthesis of this vitamin . Riboflavin transporters (uptake systems) have been identified and characterized in B. subtilis , in Lactococcus lactis [4, 5] and in a few other bacteria. Three classes of riboflavin transporters seem to exist: (1) homologs of ribU of B. subtilis, (2) homologs of ribM of Corynebacterium glutamicum and (3) homologs of impX of Fusobacterium nucleatum . The latter class has not been functionally characterized . B. subtilis RibU is part of a modular multi-subunit riboflavin transporter and belongs to the recently identified family of energy-coupling factor (ECF) transporters [7, 8, 9, 10, 11]. L. lactis RibU  has also been included in the latter classification, the driving force behind transport activity was shown to be ATP hydrolysis . Notably, RibU of Staphylococcus aureus has been crystallized and its three-dimensional structure has been determined . B. subtilis RibU is a proton-riboflavin symporter with high affinity for its substrate (K m = 5-20 nM) . RibU is strikingly different from the Corynebacterium glutamicum riboflavin transporter RibM, which was characterized as an energy-independent facilitator for riboflavin with much lower affinity (K m = 11 μM) . 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  directly upstream of ribB . S. davawensis is the only known producer of the riboflavin analog roseoflavin, which has antibiotic activity . Highly similar (> 65% similarity) RibM proteins (all containing five putative trans membrane domains) are present in other species of the genus Streptomyces. 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. Finally, ribM was evaluated as a possible tool to enhance the productivity of a B. subtilis riboflavin production strain.
Expression of ribM from S. davawensis in E. coli increased riboflavin uptake
Introduction of ribM from S. 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. The artificial gene was named ribM opt and was inserted into the expression vector pHT01 to give pHT01ribMopt. Expression using pHT01 is based on the strong σA-dependent promoter (P grac ) preceding the groE operon of B. subtilis which has been converted into an efficiently controllable (IPTG-inducible) promoter by addition of the lac operator from E. coli. A B. subtilis wild-type strain (Marburg 168) and a riboflavin overproducing B. subtilis strain (BSHP) were transformed with pHT01ribMopt. From the resulting strains, B. subtilis 168 < pHT01ribMopt > and BSHP < pHT01ribMopt > plasmids were isolated. DNA sequencing revealed the presence of the ribM opt gene under control of P grac in all strains.
The gene ribM opt from S. davawensis was functionally expressed in B. subtilis
Synthesis of RibM in B. subtilis mutants supported growth and enhanced roseoflavin sensitivity
RibM enhanced riboflavin production in a high performance B. subtilis production strain
Our results suggest that RibM from S. davawensis mediates flavin (riboflavin/roseoflavin) translocation via an energy independent facilitated diffusion mechanism. The probable physiological role of RibM is the acquirement of riboflavin from the environment. The genes ribBMAH from S. davawensis form a transcription unit and are expressed only when riboflavin is limiting in the growth medium . The genes ribB (riboflavin synthase, alpha-chain; EC 126.96.36.199), ribA (bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase; EC 188.8.131.52) and ribH (lumazine synthase; EC 184.108.40.206) are responsible for riboflavin biosynthesis. The fact that ribM is cotranscribed with the genes ribB, ribA and ribH suggests that a transporter is produced in parallel to the riboflavin biosynthetic enzymes. This appears not to be economical. If no riboflavin is present in the cytoplasm, the corresponding biosynthetic enzymes must be synthesized. The coproduction of a riboflavin transporter, however, is reasonable only when external riboflavin indeed is present. In turn, if riboflavin is present in the growth medium, the production of biosynthetic enzymes apparently constitutes a waste of metabolic energy. Notably, in the Streptomycetes S. avermitilis, S. coelicolor, S. scabiei and S. griseus ribBMAH clusters (containing ribM transporter genes) are present as well. It seems that Streptomycetes play it safe with respect to riboflavin supply. If riboflavin is growth limiting, proteins for transport and biosynthesis are synthesized in parallel to ensure availability of the vitamin.
The transport activity and direction of an energy independent facilitator depends on the concentration of the metabolite in the cytoplasm and the surrounding medium, respectively. Thus, RibM in principle may also confer roseoflavin resistance to S. davawensis (by exporting roseoflavin from the cytoplasm), which naturally produces this antibiotic in the stationary growth phase. The export activity of RibM (or other riboflavin transporters) in principle may also be of importance with respect to metabolic engineering of riboflavin production strains. In order to test this idea, ribM was functionally expressed in a production strain of B. subtilis, an organism, which is used for the commercial production of riboflavin . The introduction of ribM into a high performance B. subtilis riboflavin production strain apparently improved riboflavin production. Although the increase was relatively small, for a large scale production process the improvement may be relevant. We suggest that the export activity of RibM leads to reduced levels of riboflavin in the cytoplasm possibly enhancing the carbon flux through the pathway. This may enhance productivity during the exponential growth phase (when riboflavin concentration outside is low) or during stationary growth (when riboflavin levels inside the cells are extremely high). Wild-type B. subtilis cells rapidly and almost quantitatively convert intracellular riboflavin to FMN and FAD , a reaction, which is catalyzed by the bifunctional flavokinase/FAD synthetase RibC. All production strains have a strongly reduced RibC activity (1%) [20, 21]. Thus, production strains contain unusually high amounts of free riboflavin, which, in principle, might reduce the activity of the riboflavin biosynthetic enzymes.
The introduction of a protein catalyzing riboflavin export may also improve current production processes employing other microorganisms such as Ashbya gossypii or Candida famata . For A. gossypii interesting physiological studies suggest riboflavin export across the plasma membrane . To our knowledge the corresponding exporter has not yet been identified.
Successful examples for strain optimization on the basis of metabolite export (L-threonine and L-lysine) have been reported for Corynebacterium glutamicum [24, 25, 26]. Our work is the first to use a similar approach for optimizing one of the most successful biotechnological processes, the commercial synthesis of riboflavin .
The gene ribM from S. davawensis encodes a membrane protein which is able to catalyze the uptake of riboflavin but also of the antibiotic roseoflavin, a structural riboflavin analog. The functional expression of ribM in E. coli and B. subtilis apparently was possible without the cosynthesis of other protein components. Furthermore, our previous  and our present data data suggest that RibM proteins are energy independent flavin facilitators. Consequently, S. davawensis RibM could in principle also catalyze riboflavin export and be useful to increase the riboflavin yield in a riboflavin production process. Most riboflavin currently is produced using genetically engineered microorganisms, whereby B. subtilis is an important host. Classic random mutagenesis and methods of metabolic engineering have been used in order to optimize B. subtilis for the production of riboflavin. Most effort was directed at impairment of regulation of the riboflavin biosynthetic operon and amplification of the copy number of the structural genes ribGBAH(T) . However, import of substrates and export of the product have not yet been considered as strategies for further improvement. In order to partially fill this gap, we introduced ribM into a high-performance B. subtilis riboflavin production strain. We could show that the gene was actively transcribed and that the gene product RibM was directed to the cytoplasmic membrane. Shake flask experiments routinely used to evaluate the performance of riboflavin overproducing strains suggest that upon induction with 100 μM IPTG, the amount of riboflavin at the end of growth indeed is higher as compared to the controls. Possibly, riboflavin transporters from other microorganisms may even be more useful and may show even better results.
Bacterial strains, plasmids and growth conditions
E. coli DH5α was used as a host for gene cloning experiments and was aerobically grown at 37°C on lysogeny broth (LB) [27, 28]. E. coli BL21  was used as a host for the uptake experiments with [14C]riboflavin. A recombinant E. coli BL21 strain overexpressing ribM from S. davawensis was generated by transformation using the plasmid pNCO113ribM which was described earlier . The plasmid pET21 was obtained from Stratagene (Waldbronn, Germany). If not otherwise indicated B. subtilis was aerobically grown at 37°C in 2 × Spizizen's minimal medium  supplemented with 0.02% casamino acids, 2% yeast extract and 10% glucose or in LB. B. subtilis 168 (trpC2)  is a wild-type strain with respect to riboflavin biosynthesis and uptake and was used as a control. The B. subtilis double mutant ΔribB::Ermr ΔribU::Kanr is auxotrophic for riboflavin and does not contain a functional riboflavin uptake system (ribU) . It was grown in media supplemented with 20 mg/L riboflavin, 1 μg/ml erythromycin and 5 μg/ml kanamycin. B. subtilis 168 ΔribU::Kanr  was grown in media supplemented with 5 μg/ml kanamycin. The B. subtilis high-performance riboflavin production strain BSHP was constructed by introducing additional copies of the B. subtilis ribGBAHT genes controlled by strong constitutive phage promoters (P spo ) into the genome of B. subtilis strain 3979 [18, 32].
Expression of S. davawensis ribM in B. subtilis
B. subtilis strains overproducing RibM (B. subtilis < pHT01ribMopt >) were generated using the expression vector pHT01 (Mobitech, Göttingen, Germany) replicating in Bacillus species from the pUB110 origin . The gene ribM from S. davawensis was codon-optimized by GENEART (Regensburg, Germany) using overlapping oligonucleotides and PCR. In addition, codons specifying a his6-tag were introduced at the 3'-end. The gene (ribM opt ) (# FR719838, European Nucleotide Archive) was delivered in pGA4ribMopt. The latter plasmid was used as a template for PCR amplification of ribM opt using the modifying oligonucleotides RibMopt fw BamHI (5'-ACA GGA TCC ATG AAT TGG CTG AAT AGC-3') and RibMopt rv AatII (5'-ATT GAC GTC CTA TTA GTG GTG GTG ATG GTG-3'). The PCR product was purified and digested with BamHI and AatII to allow cloning in pHT01. The resulting plasmid pHT01ribMopt was used to transform B. subtilis using a standard protocol . Expression of ribM opt in B. subtilis was stimulated by adding IPTG in the early exponential growth phase. For selection of pHT01ribMopt 30 μg ml-1 chloramphenicol was added to the growth media. Growth was monitored using a photometer at μ = 600 nm.
Uptake experiments in E. coli
E. coli BL21 cells were grown in M9 minimal medium  supplemented with antibiotics as required. The growth medium was inoculated to an OD600 of 0.15, and the cells were grown at 37°C until they reached an OD600 of 0.5. E. coli cells were treated with 0.5 mM IPTG and grown for 3 h (to an OD600 of 0.8). Cells were harvested, washed once with ice-cold water and once with transport buffer (50 mM KH2PO4/K2HPO4, 50 mM MgCl2 pH 7.0). The cells were finally resuspended in transport buffer (10 OD600 ml-1) and stored on ice. Uptake experiments were performed with 500 μl of cells that were vigorously stirred at 30°C. After warming for 2 min, glucose was added to a final concentration of 1 mM, and the assay was started by adding [14C]riboflavin (specific activity, 5.54 MBq/mg; a generous gift of R. Krämer, Köln, Germany) to a final concentration of 2 μM. Several aliquots were removed at minute intervals, filtered on 0.45-μm GN-6 membrane filters (Pall, Dreieich, Germany), washed with an excess of water, and analysed by liquid scintillation counting. The transport activity was determined without further additions or after adding carbonyl cyanide m-chlorophenylhydrazone (CCCP; 130 μM), unlabeled riboflavin, FMN, FAD or roseoflavin (MP Biomedicals, Montreal, Canada) 3 min before addition of the labeled substrate. The latter assay was done only once.
RNA preparation, Northern (RNA) blotting and hybridisation
The preparation of total RNA from B. subtilis, the transfer of RNA to a solid support and the hybridisation to a ribM opt specific digoxigenin-labelled DNA probe was carried out according to standard procedures . The detection of the digoxigenin labelled probe was performed as suggested by the supplier of the "DIG DNA Labeling and Detection Kit" (Roche Diagnostics, Mannheim, Germany).
Preparation of B. subtilis membranes and Western blot analysis
Stationary phase cultures (50 ml) were harvested by centrifugation and washed twice with cold 50 mM Tris-HCl (pH 8.0). Pellets were dissolved in 1 ml TMS buffer (50 mM Tris-HCl, pH 8.0; 16 mM MgCl2; 33% sucrose (w/v); 300 μg/ml lysozyme; 1 mM phenylmethanesulfonylfluoride, PMSF) and incubated for 60 min at 37°C. Protoplasts were harvested by centrifugation (10 min at 7,500 × g at 4°C). Pellets were suspended in 1 ml lysis buffer (50 mM Tris-HCl, pH 8.0; 5 mM MgSO4; 2 mM PMSF). Cell free extracts were produced by sonication (1 min at 60% of the maximal power and 50% interval) on ice. Membranes were collected by ultracentrifugation for 30 min at 100,000 × g and washed once with 50 mM Tris-HCl (pH 8.0). Pellets were dissolved in 50 mM Tris-HCl (pH 8.0). The protein concentration was estimated by the method of Bradford . The samples were analyzed by SDS-PAGE on 4-20% gradient polyacrylamide gels using 50 μg of protein per lane. After transfer to nitrocellulose membranes his6-tagged RibM was immunologically detected (mouse anti-penta-his primary antibodies/goat anti-mouse IgG alkaline phosphatase(AP)-coupled secondary antibodies; Novagen, Darmstadt, Germany). AP was detected with the "AP Detection Reagent Kit" (Novagen) using 3-bromo-4-chloro-5-indolyl phosphate and nitro blue tetrazolium chloride.
Monitoring of riboflavin production
Riboflavin synthesis by B. subtilis in the time course of fermentation was monitored as follows. An aliquot from the culture containing cells and medium (500 μl) was combined with 465 μl 4 N NaOH and vigorously mixed for 1 min. The mixture was neutralized by adding potassium phosphate (1 M pH 8.0) and centrifuged for 5 min at 13,000 × g at room temperature. Riboflavin in the supernatant was determined by using a standard procedure employing HPLC . Using the above described protocol the total amount of riboflavin (intracellular riboflavin and riboflavin present in the fermentation broth) was measured. For the statistical analysis of the data student's t test was applied comparing two unknown means based on independent samples.
This work was supported by the state of Baden-Württemberg (Germany) ("Ideenwettbewerb"). We thank Dr. E. Huebener for critically reading the manuscript.
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