RibM from <it>Streptomyces davawensis </it>is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains
<p>Abstract</p> <p>Background</p> <p>The bacterium <it>Bacillus subtilis</it>, 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 en...
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| Format: | Article |
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BMC
2011-12-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/11/119 |
| _version_ | 1818664058150191104 |
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| author | Hemberger Sabrina Pedrolli Danielle B Stolz Jürgen Vogl Christian Lehmann Martin Mack Matthias |
| author_facet | Hemberger Sabrina Pedrolli Danielle B Stolz Jürgen Vogl Christian Lehmann Martin Mack Matthias |
| author_sort | Hemberger Sabrina |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>The bacterium <it>Bacillus subtilis</it>, 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 <it>Streptomyces davawensis </it>with respect to improvement of a riboflavin production strain.</p> <p>Results</p> <p>The gene <it>ribM </it>from <it>S. davawensis</it>, coding for a putative facilitator of riboflavin uptake, was codon optimized (<it>ribM<sub>opt</sub></it>) for expression in <it>B. subtilis</it>. The gene <it>ribM<sub>opt </sub></it>was functionally introduced into <it>B. subtilis </it>using the isopropyl-β-thiogalactopyranoside (IPTG)-inducible expression plasmid pHT01: Northern-blot analysis of total RNA from IPTG treated recombinant <it>B. subtilis </it>cells revealed a <it>ribM<sub>opt </sub></it>specific transcript. Western blot analysis showed that the his<sub>6</sub>-tagged heterologous gene product RibM was present in the cytoplasmic membrane. Expression of <it>ribM </it>in <it>Escherichia coli </it>increased [<sup>14</sup>C]riboflavin uptake, which was not affected by the protonophore carbonyl cyanide <it>m</it>-chlorophenylhydrazone (CCCP). Expression of <it>ribM<sub>opt </sub></it>supported growth of a <it>B. subtilis </it>Δ<it>ribB</it>::Erm<sup>r </sup>Δ<it>ribU</it>::Kan<sup>r </sup>double mutant deficient in riboflavin synthesis (Δ<it>ribB</it>) and also deficient with respect to riboflavin uptake (Δ<it>ribU</it>). Expression of <it>ribM<sub>opt </sub></it>increased roseoflavin (a toxic riboflavin analog produced by <it>S. davawensis</it>) sensitivity of a <it>B. subtilis </it>Δ<it>ribU</it>::Kan<sup>r </sup>strain. Riboflavin synthesis by a model riboflavin <it>B. subtilis </it>production strain overproducing RibM was increased significantly depending on the amount of the inducer IPTG.</p> <p>Conclusions</p> <p>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 <it>B. subtilis </it>strain (or any other microorganism).</p> |
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| publishDate | 2011-12-01 |
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| spelling | doaj.art-cec0d3ed8865438baba2583398aebfd32022-12-21T22:01:50ZengBMCBMC Biotechnology1472-67502011-12-0111111910.1186/1472-6750-11-119RibM from <it>Streptomyces davawensis </it>is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strainsHemberger SabrinaPedrolli Danielle BStolz JürgenVogl ChristianLehmann MartinMack Matthias<p>Abstract</p> <p>Background</p> <p>The bacterium <it>Bacillus subtilis</it>, 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 <it>Streptomyces davawensis </it>with respect to improvement of a riboflavin production strain.</p> <p>Results</p> <p>The gene <it>ribM </it>from <it>S. davawensis</it>, coding for a putative facilitator of riboflavin uptake, was codon optimized (<it>ribM<sub>opt</sub></it>) for expression in <it>B. subtilis</it>. The gene <it>ribM<sub>opt </sub></it>was functionally introduced into <it>B. subtilis </it>using the isopropyl-β-thiogalactopyranoside (IPTG)-inducible expression plasmid pHT01: Northern-blot analysis of total RNA from IPTG treated recombinant <it>B. subtilis </it>cells revealed a <it>ribM<sub>opt </sub></it>specific transcript. Western blot analysis showed that the his<sub>6</sub>-tagged heterologous gene product RibM was present in the cytoplasmic membrane. Expression of <it>ribM </it>in <it>Escherichia coli </it>increased [<sup>14</sup>C]riboflavin uptake, which was not affected by the protonophore carbonyl cyanide <it>m</it>-chlorophenylhydrazone (CCCP). Expression of <it>ribM<sub>opt </sub></it>supported growth of a <it>B. subtilis </it>Δ<it>ribB</it>::Erm<sup>r </sup>Δ<it>ribU</it>::Kan<sup>r </sup>double mutant deficient in riboflavin synthesis (Δ<it>ribB</it>) and also deficient with respect to riboflavin uptake (Δ<it>ribU</it>). Expression of <it>ribM<sub>opt </sub></it>increased roseoflavin (a toxic riboflavin analog produced by <it>S. davawensis</it>) sensitivity of a <it>B. subtilis </it>Δ<it>ribU</it>::Kan<sup>r </sup>strain. Riboflavin synthesis by a model riboflavin <it>B. subtilis </it>production strain overproducing RibM was increased significantly depending on the amount of the inducer IPTG.</p> <p>Conclusions</p> <p>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 <it>B. subtilis </it>strain (or any other microorganism).</p>http://www.biomedcentral.com/1472-6750/11/119 |
| spellingShingle | Hemberger Sabrina Pedrolli Danielle B Stolz Jürgen Vogl Christian Lehmann Martin Mack Matthias RibM from <it>Streptomyces davawensis </it>is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains BMC Biotechnology |
| title | RibM from <it>Streptomyces davawensis </it>is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains |
| title_full | RibM from <it>Streptomyces davawensis </it>is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains |
| title_fullStr | RibM from <it>Streptomyces davawensis </it>is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains |
| title_full_unstemmed | RibM from <it>Streptomyces davawensis </it>is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains |
| title_short | RibM from <it>Streptomyces davawensis </it>is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains |
| title_sort | ribm from it streptomyces davawensis it is a riboflavin roseoflavin transporter and may be useful for the optimization of riboflavin production strains |
| url | http://www.biomedcentral.com/1472-6750/11/119 |
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