阿维链霉菌ilvBNC基因簇的克隆、测序及表达

Cloning, sequencing and expression of the ilvBNC gene cluster from Streptomyces avermitilis.

作者信息

De Rossi E, Leva R, Gusberti L, Manachini P L, Riccardi G

机构信息

Department of Genetics and Microbiology, University of Pavia, Italy.

出版信息

Gene. 1995 Dec 1;166(1):127-32. doi: 10.1016/0378-1119(95)00563-4.

DOI:10.1016/0378-1119(95)00563-4

PMID:8529876

Abstract

The metabolism of the branched-chain amino acids (BCAA) isoleucine, leucine and valine is correlated to the production of polyketide antibiotics in many streptomycetes. Despite its significance, this biosynthetic pathway is poorly understood in Streptomyces. In order to develop a better understanding of Streptomyces BCCA biosynthesis, two genes, ilvBN and ilvC, encoding acetohydroxy acid synthase (AHS) and acetohydroxy acid isomeroreductase (IR), respectively, were cloned from Streptomyces avermitilis, a strain producing avermectins, potent antiparasitic compounds. The genes were isolated by applying a combination of PCR and genomic library screening. The deduced amino-acid sequences revealed significant homology to the AHS and IR proteins from other bacterial species. The ilvBN gene, expressed in Escherichia coli (Ec) by using the expression vector pGEX-4T-1, complemented the ilv- mutation of Ec PS1283. Ec transformants produced high levels of AHS, whose activity was feedback inhibited by valine.

摘要

在许多链霉菌中，支链氨基酸（BCAA）异亮氨酸、亮氨酸和缬氨酸的代谢与聚酮类抗生素的产生相关。尽管其具有重要意义，但在链霉菌中对该生物合成途径的了解却很少。为了更好地理解链霉菌BCAA的生物合成，从产生阿维菌素（一种强效抗寄生虫化合物）的阿维链霉菌中克隆了分别编码乙酰羟酸合酶（AHS）和乙酰羟酸异构还原酶（IR）的两个基因ilvBN和ilvC。通过PCR和基因组文库筛选相结合的方法分离出这些基因。推导的氨基酸序列显示与其他细菌物种的AHS和IR蛋白具有显著同源性。使用表达载体pGEX-4T-1在大肠杆菌（Ec）中表达的ilvBN基因，互补了Ec PS1283的ilv-突变。Ec转化体产生了高水平的AHS，其活性受到缬氨酸的反馈抑制。