苦瓜中重组降糖多肽的制备-毕业设计论文(学术).docVIP

苦瓜中重组降糖多肽的自备 摘要 Bagremycin A和B是由Streptomyces sp. Tü 4128生产的两种抗生素，它们对一些革兰氏阳性细菌和真菌具有抗菌活性。为了阐明Bagremycins的生物合成机制，在bagremycins生产菌Streptomyces sp. Tü 4128中克隆和分析了bagE基因，序列分析BagE属于CoA连接酶家族，与苯乙酰-CoA连接酶（PCL）有较高的相似性，ORF全长1329bp，推测编码442个氨基酸，蛋白分子量为48.03 kDa。在敲除bagE基因的发酵提取物中未检测到Bagremycins的生产，证实bagE基因与Bagremycins的生物合成相关，在敲除突变株中回补bagE基因能恢复Bagremycins的生产，在Streptomyces sp. Tü 4128中过表达bagE基因导致菌丝体的生长比野生型延迟，而Bagmycins的产量并没有显著增长。BagE在大肠杆菌中异源表达证实它具有苯乙酰-CoA连接酶活性，其动力学参数分别为：Km=5.14mM，Kcat=6.09s-1，Kcat/Km=1.18mM-1s-1。这项研究使我们在分子水平上进一认识Streptomyces sp. Tü 4128中bagE基因在Bagremycins的生物合成中的作用。 关键词：Bagremycin；苯乙酰-CoA连接酶；基因敲除；链霉菌. Abstract Bagremycin A and B, produced by Streptomyces sp. Tü 4128, are two antibiotics against gram-positive bacteria and fungi. In an effort to elucidate the biosynthetic mechanism of bagremycins, a novel phenylacetate-CoA ligase gene, designated as bagE, was cloned and sequenced from bagremycins producing strain Streptomyces sp. Tü 4128. The ORF of bagE was 1329 bp that putatively encoded a polypeptide of 442 amino acids, with a predicted molecular mass of 48.03 kDa. The deduced amino acid sequence of BagE shared high similarity with other known PCL. The disruption of the bagE gene abolished bagremycins production in the fermented cultures, strongly indicating that bagE gene was involved in bagremycins biosynthesis. Complemention of bagE gene could recover bagremycins accumulation. Overexpression of bagE gene in strain Streptomyces sp. Tü 4128 did not promote an increase of bagremycins accumulation but retarded mycelial growth in comparison with the wild-type strain. Heterologous expression experiments in recombinant Escherichia coli demonstrated phenylacetate-CoA ligase enzyme activity of BagE, with Km=5.14mM, Kcat=6.09s-1, Kcat/Km=1.18mM-1s-1. This study will enable us to further understand the role of bagE in the synthesis of bagremycins in Streptomyces sp. Tü 4128 at the molecular level. Keywords: Bagremycin; phenylacetate-CoA ligase; gene disru