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

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.