短密青霉NRRL 864异戊烯基转移酶PbPT的克隆表达及功能分析  

Functional Characterization of PbPT,a Prenyltransferase fromPenicillium Brevicompactum NRRL 864

作　　者：孙靖然[1,2] 刘长宁[3] 李荣贵[1] 张伟[2] 李盛英[2] 

机构地区：[1]青岛大学生命科学学院,山东青岛266071 [2]山东省合成生物学重点实验室中国科学院青岛生物能源与过程研究所,山东青岛266101 [3]中国科学院西双版纳热带植物园,云南勐伦666303

出　　处：《青岛大学学报（工程技术版）》2016年第2期107-115,共9页Journal of Qingdao University(Engineering & Technology Edition)

基　　金：国家自然科学基金资助项目(21472204)

摘　　要：在对丝状真菌短密青霉(penicillium brevicompactum NRRL 864)的全基因组进行分析时,发现该真菌可能表达一种异戊烯基转移酶PbPT。通过反转录RT-PCR(Reverse Transcription,RT-PCR)获得真菌cDNA,并以cDNA为模版,将编码PbPT的开放阅读框克隆到pET-28b构建表达载体pET28b-PbPT,将pET28b-PbPT转化Escherichia coli BL21(DE3)获得表达菌株,异丙基-β-D-硫代半乳糖苷(Isopropyl-β-D-thiogalactoside,IPTG)诱导高效表达PbPT,利用Ni-NTA树脂亲和纯化重组PbPT活性蛋白,并对该蛋白进行了底物特异性分析、产物结构鉴定以及酶促动力学分析。分析结果表明,以二甲基丙烯基二磷酸(dimethylallyl pyrophosphate,DMAPP)为供体,PbPT特异性催化异戊烯基转移至Brevianamide F的α位C上,生成Deoxybrevianamide E。Brevianamide F是合成真菌生物碱类杀虫抗生素brevianamides的重要中间体,真菌吲哚类异戊烯基转移酶家族新成员PbPT的发现。该研究为进一步寻找和阐明真菌生物碱类杀虫抗生素brevianamides的生物合成基因簇和合成途径奠定了基础。A novel prenyltransferase PbPT was mined out during the whole genome analysis of the filamen- tous fungal strain Penicilliurn brevicornpacturn NRRL 864. The open reading frame of PbPT was amplified by RT-PCR（Reverse Transcription PCR） using cDNA as template and cloned into pET28b to construct the expression vector pET28b-PbPT. The recombinant PbPT was overexpressed in Escherichia coli BL21 （DE3） and purified to homogeneity by one-step Ni-NTA affinity chromatography. The catalytic features of PbPT including substrate specificity, kinetic parameters, optimal pH and temperature, and preferred met- al ions were determined. The results showed that PbPT is able to specifically catalyze the prenylation of brevianamide F at the C-α position when dimethylallyl diphosphate （DMAPP） was used as the prenyl group donor. The product was structurally determined to be deoxybrevianamide E using MS and NMR analysis. Brevianamide F is an important intermediate for the biosynthesis of brevianamides, which belong to a fami- ly of fungal alkaloid antibiotics. The identification of PbPT as a new member of dimethylallyl tryptophan synthase, could set the foundation for searching thebiosynthetic gene cluster and illuminating the biosyn- thetic mechanism of this novel family of fungal alkaloid brevianamides.

关 键 词：短密青霉 异戊烯基转移酶 丝状真菌 DMAPP 功能分析 

分 类 号：Q78[生物学—分子生物学] Q936