AdpA_(sd)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

Effects of AdpA_(sd) on Morphological Differentiation and Toyocamycin Production in Streptomyces diastatochromogenes 1628

为阐明Adp A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adp Asd基因(Gen Bank登录号:JX847412)。结果发现,Adp Asd的氨基酸序列与灰色链霉菌Streptomyces griseus的Adp Ag及天然色链霉菌Streptomyces coelicolor的Adp Ac的同源性分别为80.7%和78.9%。将adp Asd基因置于载体p IB139启动子Perm E*下游,构建了重组质粒p IB139-adp Asd,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adp Asd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adp Asd的分子调节机制奠定了基础。

In several streptomycetes, Adp A is a pleiotropic transcription factor involved in regulation of morphological development and secondary metabolites biosynthesis. The relevance of Adp A to morphological development and toyocamycin（TM） production in Streptomyces diastatochromogenes 1628 was investigated in this paper.Firstly, 1263 bp adp Asd gene（Gen Bank accession No. JX847412） was successfully cloned from S. diastatochromogenes 1628 by using degenerate primer. The deduced amino acid sequence of Adp Asd showed similarity to Adp Ag（80.7%） of S. griseus and Adp Ac（78.9%） of S. coelicolor, respectively. Subsequently, adp Asd gene was placed under control of the Perm E＊ in plasmid p IB139 to generate p IB139-adp Asd. The constructed plasmid p IB139-adp Asd was introduced by using intergeneric conjugation and integrated into chromosome of rifampicin resistance（Rifr） 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-T15 A and 1628-T62 A, respectively. The results indicated that compared with that of control strain 1628-T62, sporulation of recombinant strain 1628-T62 A was recovered with a certain degree. In contrast, there was no significant difference between control strain 1628-T15 and recombinant strain 1628-T15 A in term of morphological differentiation. Moreover, at flask-shake fermentation condition, TM production of strain 1628-T62 A was improved 3-times higher than that of control 1628-T62, while recombinant strain 1628-T15 A showed only a slight increase in TM production, compared with control strain 1628-T15. These data confirm that adp Asd 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 adp Asd at molecular level in S. diastatochromogenes.