放线菌含硫天然产物thiolactomycin（TLM）对革兰氏阳性和阴性细菌都具有显著抑菌活性，并且对多种病原虫具有杀灭作用，有望开发成为新一代高效低毒的抗感染或抗虫药物。TLM结构中独特的五元硫代内酯环是其发挥生物学功能的必需基团，然而目前结构中关键的硫原子引入及硫代内酯环的形成机制尚不明确。在前期研究中，申请人从一株南极深海放线菌的发酵产物中分离获得了TLM及系列衍生物；通过基因组测序和生物信息学分析捕获了TLM生物合成基因簇，并对簇内关键基因进行了功能预测。在此基础上，根据文献调研结果及关键酶的基本催化原理，提出了全新的硫代谢路径。为了验证这一假说，本项目拟通过体内基因敲除和体外酶活重建阐明硫代谢过程的关键步骤，并对相关酶学机制进行研究。项目的顺利实施将为构建基因工程/代谢工程TLM高产菌株，以及运用组合生物合成/合成生物学手段创制新型TLM衍生物提供理论基础和催化元件。

The sulfur-containing natural product thiolactomycin (TLM) derived from actinomycetes has great potential to be developed as an antibiotic or vermifuge drug, benefiting from its significant antibiotic activities against both Gram-positive and Gram-negative bacteria, as well as its protozoon-killing effects. The unique γ-thiolactone moiety in TLM is an essential group for its biological activities. However, the biosynthetic mechanisms of the sulfur atom incorporation and thiolactone formation remain unclear. In the applicant’s preliminary study, TLM and its analogues were isolated from an actinomycete strain derived from Antarctic deep-sea sponge, and the TLM biosynthetic gene cluster has been identified by genome sequencing of the producing strain and bioinformatics analysis. Here, a novel S-metabolism pathway was proposed based on the related literature and biocatalytic principles of the key enzymes involved in TLM biosynthesis. To elucidate the biosynthetic mechanisms of the sulfur atom incorporation and γ-thiolactone formation in TLM, in vivo gene knockout/complementation and in vitro enzymatic activity reconstitution based on our proposed mechanistic hypothesis will be carried out; and the catalytic mechanisms of the involved key enzymes will also be investigated. We anticipate that this project will provide theoretical foundations for the future construction of TLM high-producing strains by genetic/metabolic engineering, and for creating new bioactive TLM derivatives by combinatorial biosynthesis and synthetic biology approaches.