茂原链霉菌luxR基因对TGase生物合成的作用

Effects of luxR gene in the biosynthesis of transglutaminase in Streptomyces mobaraensis

摘要:

前期研究表明，MgCl₂胁迫不但可以提高TGase的产量，还会影响微生物次级代谢产物合成的一个重要调节因子LuxR家族蛋白的表达.为了研究LuxR家族蛋白对茂原链霉菌(Streptomyces mobaraensis) TGase生物合成以及菌体生长的调控作用，利用pKC1139为载体，构建luxR基因阻断质粒，通过PEG介导的质粒转化将其转入S. mobaraensis原生质体中，利用单交换同源重组法将整个重组质粒插入到茂原链霉菌基因组中构建S. mobaraensis luxR家族蛋白基因阻断突变菌.利用菌体干重法对比突变菌和野生菌菌体生长变化，利用比色法研究两株菌TGase生物合成的变化，找到LuxR家族蛋白基因表达与TGase生物合成之间的关系.结果发现，本研究采用单交换同源重组的方法可以成功地构建出S. mobaraensis luxR基因阻断突变菌株.通过对比野生菌与阻断菌株生长和TGase活力的变化发现阻断luxR基因会导致菌体生长受阻，TGase合成延迟，并且表达量显著下降.这些研究结果表明本文研究的luxR基因可能是S. mobaraensis合成TGase必不可少的正向调控因子.

Abstract:

Our previous studies indicated that MgCl₂ stress could not only increase the yield of TGase, but also affect the expression of LuxR family protein, which is an important regulator of microbial metabolism. In order to study the effects of LuxR family protein on the biosynthesis of TGase, the luxR gene disruption plasmid was constructed with the vector of pKC1139. The plasmid was transferred into the protoplasts of Streptomyces mobaraensis by PEG mediated transformation. The recombinant plasmid was inserted into the genome of Streptomyces by a single exchange of homologous recombination. The cell growth and TGase activity of luxR gene disruption mutant of S. mobaraensis were compared with the wild stain. The results showed that the luxR gene disruption mutant was successfully constructed, and that the block of the luxR gene leads to a decrease of the biomass, a synthesis delaying and expression decrease of TGase. These results showed that luxR gene might be an important positive factor on TGase production.