Gliotoxins are epipolythiodioxopiperazine toxins produced by the filamentous fungi, which show great potential in the treatment of liver and lung cancer because of its cytotoxicity. In this study, three novel genes related to gliotoxin biosynthesis, gliT, gliM and gliK encoding thioredoxin reductase, O-methyltransferase and gamma-glutamyl cyclotransferase, respectively, from the deep-sea-derived fungus Geosmithia pallida were cloned from G. pallida and expressed in Escherichia coli. The recombinant GliT, GliM and GliK proteins were expressed and purified by Ni affinity column, which was demonstrated by SDS-PAGE and Western blot analysis. The inclusion bodies of GliT were renatured and the corresponding enzymatic properties of the two enzymes were further investigated. Using DTNB as a substrate, GliT showed the highest enzymatic activity of 11041 mU/L at pH 7.0, and the optimal reaction temperature was 40 °C. Using EGCG as a substrate, GliM showed the highest enzymatic activity of 239.19 mU/mg at pH 7.0, the optimum temperature was 35 °C. GliK from G. pallida was firstly reported to show bi-function of glutymal cyclotransferase and acetyltransfearse actvity with highest enzymatic activity of 615.5 U/mg in this study. The results suggested the important enzymatic function of GliT, GliM and GliK in the gliotoxin biosynthesis in G. pallida, which would lay a foundation for the mechanism elucidation of the gliotoxin biosynthesis in G. pallida and the exploitation of novel gliotoxin derivaties.

Gliotoxins是由丝状真菌产生的表聚硫代二氧代哌嗪类毒素，由于其细胞毒性，在治疗肝癌和肺癌方面显示出巨大的潜力。在这项研究中，从G. pallida和在大肠杆菌中表达。重组 GliT、GliM 和 GliK 蛋白通过 Ni 亲和柱进行表达和纯化，SDS-PAGE 和蛋白质印迹分析证明了这一点。GliT 的包涵体被复性，并进一步研究了这两种酶的相应酶学性质。以 DTNB 为底物，GliT 在 pH 7.0 时表现出最高的酶活性为 11041 mU/L，最佳反应温度为 40 ℃。以EGCG为底物，GliM在pH 7.0时表现出最高的酶活性为239.19 mU/mg，最适温度为35°C。来自G. pallida的 GliK首次报道显示谷氨酸环转移酶和乙酰转移酶活性的双功能，在本研究中最高酶活性为 615.5 U/mg。研究结果表明GliT、GliM和GliK在G. pallida胶霉毒素生物合成中的重要酶促作用，为阐明胶叶胶毒素生物合成的机理和开发新的胶粘毒素衍生物奠定基础。