AdpAsd对于淀粉酶产色链霉菌1628的形态分化和丰加霉素合成的影响

为阐明AdpA与淀粉酶产色链霉菌Streptomyces diastatochromogenes 1628形态分化和合成丰加霉素（Toyocamycin,TM）的相关性。本研究设计简并引物成功克隆了来源于淀粉酶产色链霉菌1628大小为1263 bp的adpA_sd基因（GenBank登录号：JX847412）。结果发现,AdpA_sd的氨基酸序列与灰色链霉菌Streptomyces griseus的AdpA_g及天然色链霉菌Streptomyces coelicolor的AdpA_c的同源性分别为80.7%和78.9%。将adpA_sd基因置于载体pIB139启动子PermE^*下游,构建了重组质粒pIB139-adpA_sd,将其通过接合转移法分别转入菌株1628-T15（高产TM）和1628-T62（低产TM,孢子合成受阻）中,获得重组菌1628-T15A和1628-T62A。结果表明,与出发菌株1628-T62相比,重组菌1628-T62A产孢能力得到一定程度的恢复;重组菌1628-T15A与出发菌株1628-T15相比,整体形态无明显变化。摇瓶发酵试验表明,重组菌1628-T62A较出发菌株1628-T62的TM合成能力显著提高,最终TM产量提高了近3倍,而重组菌1628-T15A的TM产量比出发菌株1628-T15仅有小幅提升。以上结果证实adpA_sd参与淀粉酶产色链霉菌1628形态分化以及TM合成,为今后深入理解adpA_sd的分子调节机制奠定了基础。

Effects of AdpAsd on Morphological Differentiation and Toyocamycin Production in Streptomyces diastatochromogenes 1628

In several streptomycetes, AdpA is a pleiotropic transcription factor involved in regulation of morphological development and secondary metabolites biosynthesis. The relevance of AdpA to morphological development and toyocamycin (TM) production in Streptomyces diastatochromogenes 1628was investigated in this paper.Firstly, 1263 bp adpA_sd gene (GenBank accession No. JX847412) was successfully cloned from S. diastatochromogenes 1628 by using degenerate primer. The deduced amino acid sequence of AdpA_sd showed similarity to AdpA_g(80.7%) of S. griseus and AdpA_c (78.9%) of S. coelicolor, respectively. Subsequently, adpA_sd gene was placed under control of the PermE^* in plasmid pIB139 to generate pIB139-adpA_sd. The constructed plasmid pIB139-adpA_sd was introduced by using intergeneric conjugation and integrated into chromosome of rifampicin resistance (Rif^r) mutants 1628-T15 (high-level TM producer) and 1628-T62 (low-level TM producer, poor ability of sporulation), which were isolated and selected by using ribosome engineering technology, to create recombinant strains 1628-T15A and 1628-T62A, respectively. The results indicated that compared with that of control strain 1628-T62, sporulation of recombinant strain 1628-T62A was recovered with a certain degree. In contrast, there was no significant difference between control strain 1628-T15 and recombinant strain 1628-T15A in term of morphological differentiation. Moreover, at flask-shake fermentation condition, TM production of strain 1628-T62A was improved 3-times higher than that of control 1628-T62, while recombinant strain 1628-T15A showed only a slight increase in TM production, compared with control strain 1628-T15. These data confirm that adpA_sd gene is involved in morphological differentiation and TM biosynthesis in S. diastatochromogenes 1628. Also, this work will provide the basis for studying regulatory mechanism of adpA_sd at molecular level in S. diastatochromogenes.