教酒链霉菌NRRL 3882中钙霉素生物合成后修饰基因calD的功能分析

Functional analysis of calD involved in calcimycin postsynthetic modification by Streptomyces chartreusis NRRL 3882

摘要:

【目的】钙霉素是二价阳离子载体，是一类含有吡咯环的聚醚类抗生素，广泛用于细胞二价阳离子的生物学功能研究。本文以钙霉素生物合成基因簇中calD基因为研究对象，通过蛋白质同源序列比对、基因敲除、回补验证及HPLC/MS分析，对calD基因的功能进行表征。【方法】对calD基因功能进行生物信息学预测。选用钙霉素产生菌Streptomyces chartreusis NRRL 3882，通过PCR-targeting的方法对calD基因进行敲除获得突变株，再将calD基因克隆到链霉菌整合质粒上，通过接合转移技术将calD回补到缺失株中。使用HPLC/MS技术对菌株发酵产物进行分析。【结果】生物信息学预测CalD蛋白酶属于氧化还原酶。获得calD基因敲除突变株ΔcalD及基因回补菌株ΔcalD:calD。HPLC/MS检测到calD基因的缺失菌株大幅降低钙霉素产生能力，与野生型菌株相比，突变株中积累更多的3-hydroxylcezomycin和更少的氮-去甲基钙霉素。【结论】calD参与钙霉素的生物合成。calD的缺失导致3-hydroxylcezomycin的积累，推测calD负责钙霉素生物合成途径中苯并噁唑环3位上羟基转化成酮基的氧化反应。初步阐明了calD基因在钙霉素生物合成途径中的功能机制。

Abstract:

[objective] Calcimycin, a divalent cation specific ionophore, is a pyrrole moiety containing polyther antibiotic, widely used to study the biological function of divalent cation in eukaryotic cells. The biosynthetic mechanism of calcimycin remains to be fully elucidated. calD is a gene with unknown function in the calcimycin biosynthetic gene cluster. The aim of this study is to identify the function of calD in calcimycin biosynthesis pathway. [Methods] Bioinformatic analysis was used to predict the function of calD. PCR targeting was used to disrupt calD in Streptomyces chartreusis NRRL 3882. HPLC/MS was used to analyze the fermentation product. [Results] Bioinformatic analysis shows that CalD belongs to the family of alcohol dehydrogenase. The calD gene disruption mutant was obtained. The mutant was also complemented with calD. HPLC analysis shows that inactivating of calD decreases but not abolishes calcimycin production. The yield of N-demethylcalcimycin is also decreased. The calD mutant accumulates hydroxyl-cezomycin. [Conclusion] calD participates in calcimycin biosynthesis. It might be responsible for the oxidative conversion of the hydroxy-cezomycin to keto-cezomycin. This result paved way for the further analysis of the biochemical function of calD.