一株链霉菌菌株NCPC-1020中棘白霉素B脱酰基酶基因的克隆与功能分析

Molecular cloning and functional analysis of a gene encoding echinocandin B deacylase from Streptomyces sp. NCPC-1020

摘要:

【目的】从一株土壤放线菌来源的野生型链霉菌菌株NCPC-1020中克隆一个具有棘白霉素B脱酰基酶活性的新基因。【方法】采用Degenerate和TAIL PCR两种方法，从链霉菌菌株NCPC-1020基因组中快速克隆获得了该基因序列，然后将基因在变铅青链霉菌TK24中进行异源表达，并进行全细胞催化底物脱酰基反应，采用LC-MS检测反应产物。【结果】LC-MS检测证实，棘白霉素B结构中脂肪链被酶促水解，从而证实该基因具有脱酰基酶活性。【结论】采用Degenerate以及TAIL PCR的方法能够快速获得未知功能的新基因。此基因的克隆，奠定了进行半合成棘白霉素类药物的研发基础。

关键词:链霉菌，棘白霉素B，Degenerate PCR，TAIL PCR，脱酰基酶

Abstract:

[Objective] Cloning of a new gene possessing a function of Echinocandin B (ECB) side-chain deacylation from a wild type Streptomyces strain NCPC-1020 of soil source. [Methods] Using degenerate PCR and TAIL PCR strategy, we successfully cloned the target gene, which was heterologously expressed in S. lividans TK24, then a whole-cell catalysis method was developed to dectect the deacylation activity of ECB deacylase by LC-MS. [Results] The ECB deacylase gene was cloned and its function was confirmed. [Conclusion] Combined the two PCR techniques, degenerate and TAIL PCR, a new gene could be cloned quickly. The obtain of the new ECB deacylase gene lays a good foundation for the researches on semibiosynthetic of ECB related drugs.

Key words:Streptomyces, Echinocandin B, Degenerate PCR, Thermal asymmetric interlaced PCR, Deacylase