解淀粉芽孢杆菌SYBC H47铁载体合成酶基因dhbA、dhbB、dhbC的克隆表达与序列分析

Cloning, Expression and Sequence Analysis of Siderophore Synthesis Enzyme Genes dhbA, dhbB and dhbC of Strain Bacillus amyloliquefaciens SYBC H47

铁载体是微生物在缺铁环境中产生的小分子螯合物,利用CAS双平板法鉴定出已有菌株产铁载体,经鉴定所产铁载体为儿茶酚型。以解淀粉芽孢杆菌SYBC H47基因组DNA序列为模板,通过PCR克隆得到了铁载体产生途径中前体物质2,3-二羟基苯甲酸(DHB)合成所需酶的3个基因dhb A、dhb B、dhb C,结果表明三基因全长分别为786 bp、927 bp、1 197 bp,诱导表达,经SDS-PAGE电泳表明三蛋白大小分别为27.1 k D、34.3 k D、42.9 k D。用KMB培养基诱导表达三株重组菌株,表明三株重组菌株铁载体产量均有明显提高。经同源性比对和生物信息学分析发现,dhb A、dhb B、dhb C三基因分别编码2,3-二氢-2,3-二羟基苯甲酸脱氢酶、异分支酸酶、异分支酸合酶,dhb A、dhb C蛋白结构稳定,而dhb B蛋白结构则不是很稳定,其二级结构主要由α螺旋、β折叠、延伸链及无规卷曲构成但比例各不相同。以上结果为铁载体合成的研究提供了理论依据。

Siderophores are small molecules produced by microorganisms in the environment which lacks iron.CAS double-layer plates were employed to identify siderophores and the result showed that siderophore was a kind of catechol type. The three genes dhb A, B and C that encoded enzymes included in the synthesis of precursor of the siderophore, 2, 3-dihydroxybenzoate from Bacillus amyloliquefaciens SYBC H47 were obtained by PCR, respectively, with about 786 bp, 927 bp and 1 197 bp in length and inducible expressed. The results of SDS-PAGE indicated that the three recombinant proteins were 27.1 k D, 34.3 k D and 42.9 k D. The results of inducible expression of the three recombinant plasmids in iron absent medium KMB revealed that the sideropjore productions of all the three recombinant strains had improved. The homology analysis revealed that the three genes respectively encoded 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, isochorismatase and isochorismate synthase. Bioinformatic approach was used to analyze the physical and chemical properties and the secondary structure. The results suggested that dhb A and dhb C were stable proteins, while dhb B was unstable protein. The secondary structure of them was mainly composed of α helix, β turn, extended strand and random crimped with different ratios. These studies built a theoretical foundation for the further research in siderophore synthesis.