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首页> 外文期刊>BMC Biotechnology >RibM from Streptomyces davawensis is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains
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RibM from Streptomyces davawensis is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains

机译：达沃链霉菌的RibM是核黄素/玫瑰黄素转运蛋白，可能对优化核黄素生产菌株有用

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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 ( 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 Δ ribB ::Ermr Δ ribU ::Kanr double mutant deficient in riboflavin synthesis (Δ ribB ) and also deficient with respect to riboflavin uptake (Δ ribU ). Expression of ribM opt increased roseoflavin (a toxic riboflavin analog produced by S. davawensis ) sensitivity of a B. subtilis Δ ribU ::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).
机译：背景技术枯草芽孢杆菌（Bacillus subtilis）细菌不是天然的核黄素过量生产者，已通过经典诱变和代谢工程技术转化为优良的生产菌株。据我们所知，尚未将过量生产细胞的细胞质中核黄素排泄的增强视为（进一步）菌株改良的目标。在这里，我们评估了黄褐链霉菌的黄素转运蛋白RibM，以改善核黄素生产菌株。结果编码S. davawensis的ribM基因（编码假定的核黄素吸收促进剂）经过密码子优化（ribM opt ）以在枯草芽孢杆菌中表达。使用异丙基-β-硫代半乳糖吡喃糖苷（IPTG）诱导型表达质粒pHT01将基因ribM opt 功能性引入枯草芽孢杆菌：总RNA的Northern印迹分析从IPTG处理过的重组枯草芽孢杆菌细胞中发现了ribM opt 特异性转录物。 Western印迹分析表明，胞质膜中存在his 6 标签的异源基因产物RibM。大肠杆菌中ribM的表达增加了[ 14 C]核黄素的摄取，不受质子体羰基氰化物间氯苯hydr（CCCP）的影响。 ribM opt 的表达支持枯草芽孢杆菌的生长ΔribB :: Erm r Δ ribU :: Kan r 双重突变体，缺乏核黄素合成（ΔribB），并且也缺乏核黄素摄取（ΔribU）。 ribM opt 的表达提高了枯草芽孢杆菌ΔribU :: Kan r 菌株。过量产生RibM的模型核黄素枯草芽孢杆菌生产菌株的核黄素合成显着增加，取决于诱导剂IPTG的量。结论能量独立的黄素促进剂RibM原则上可以催化核黄素的输出，因此在使用过量生产的枯草芽孢杆菌重组RibM菌株（或任何其他微生物）的核黄素生产过程中，可用于提高核黄素的产量。

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