Large-scale Production of Antivirus Agent Based on Engineered Biosynthesis

Aphidicolin（ACL）是一种从甜菜中提取的抗病毒药物，具有抗肿瘤、植物毒性和特异性抑制DNA聚合酶α等多种生物活性。传统的真菌发酵方法虽然可以获得ACL，但其产量和发酵周期较长，阻碍了其大规模生产。近年来的研究进展促使我们探索与ACL生物合成有关的全生物合成基因的鉴定及其异源表达。为了鉴定催化通用二萜前体GGDP环化的基因，进行了同源PCR。利用P.betae的mRNA和基于其他二萜环化酶保守氨基酸序列的退化引物进行RT-PCR，我们扩增出了与真菌ent-kaurene合成酶显著相似的1100 bp的条带。利用基因特异性引物，用5′和3′-RACE测定全长cDNA的核苷酸序列。其中包含预测的2997-dp开放阅读框，编码998个氨基酸的产物，命名为aphidicolan-16 β-ol合成酶（ACS）。将全长cDNA连接到pGEX 4T-3载体上，在大肠杆菌JM109中表达谷胱甘肽s -转移酶(GST)-ACS融合蛋白。GGDP与rACS孵育得到的反应混合物的己烷提取物有三种产物。通过与合成标准品的保留时间和质谱比较，鉴定了这些产物为aphidicolan-16 β-ol、aphidicol16 -ene和aphidicol15 -ene。由于所有的产物都是在槟榔的菌丝提取物中发现的，这表明这些产物是由一种酶产生的。

Aphidicolin(ACL), an antivirus agent from Phoma betae shows a various biological activity such as antitumor, phytotoxic and specific inhibition of DNA polymerase α. Although ACL can be obtained by traditional fermentation method of the fungus, its productivity and longer fermentation period hamper its large-scale production. Recent progress of prompted us to explore identification of entire biosynthetic genes responsible for ACL biosynthesis, and heterologous expression of the genes.In order to identify the gene catalyzes cyclization of universal diterpene precusor GGDP, homology based PCR was conducted. RT-PCR with mRNA from P.betae and degenerate primers based on the conserved amino acid sequences of other diterpene cyclases allowed us to amplify 1100-bp band that showed a significant similarity to fungal ent-kaurene synthases. The nucleotide sequence of the full-length cDNA was determined by 5'-and 3'-RACE by using gene-specific primers. This contained the predicted 2997-dp open reading frame, encoding a product of 998 amino acids which was named aphidicolan-16 β-ol synthase(ACS). A full-length cDNA was ligated into a pGEX 4T-3 vector and the glutathione S-transferase(GST)-ACS fusion protein was expressed in Escherichia coli JM109. The hexane extracts of the reaction mixture obtained by incubation of GGDP with rACS afforded three products. These products of the rACS were identified, by comparison of retention time and mass spctra to those of synthetic standards, as aphidicolan-16 β-ol, aphidicol-16-ene and aphidicol-15-ene. Since all products are found in the mycelial extracts of P.betae, it suggests that these products are produced by a single enzyme.