二萜广布于植物和真菌中，具有多种生物活性。细菌来源的二萜类成分研究较少报道。基因组学研究结果预测很多细菌，特别是放线菌，含有二萜合成酶，应成为二萜的重要生物来源。前期工作中，以ent-copalyl diphosphate (ent-CPP) 合成酶的保守序列为引物，利用PCR技术从100株土壤放线菌中筛选到25株阳性菌。本项目拟从海绵内生放线菌中筛选二萜生产阳性菌株。结合化学研究和全基因组序列分析，开展以下研究工作：从PCR阳性菌株中发现二萜类成分；分析新型二萜类成分的生物合成基因簇，利用基因敲除等分子生物学手段揭示目标化合物的生物合成路径；异源 (E.coli) 表达合成路径中二萜合成酶，对其酶学特征进行研究。本项目研究结果为二萜类成分的发现提供可借鉴的研究思路和方法；丰富自然界中微生物来源二萜类成分的结构类型和数量；为二萜合成酶基因工程菌株的构建及新型二萜类化合物的制造奠定工作基础。

Diterpenoids (DTs) comprise the largest family of natural products and play important roles in all living organisms. DTs of plant and fungi origins have been extensively discovered, however, DTs of bacterial origin are known but rare. Recent advances in genomics have revealed that the biosynthetic potential for terpenoids in bacteria, particularly in actinomycetes, may be significantly underestimated. In our previous discovery of DTs from actinomycetes, 25 potential DTs producing strains were screened from 100 soil actinomycetes using a two-tiered PCR method. In this project, we continue to screen potential DTs producing strains from sponge endogenous actinomycetes, using DT synthases as the searching targets. On the basis of the PCR-based screening, chemical investigation and the whole genome sequence, we carry out the following research: 1.discovery of DTs from sponge endogenous actinomycetes based on the PCR screening; 2.sequencingand identification of the DT biosynthetic gene clusters and characterization of the genes’ functions by gene deletion; 3.heterologous expression of DT synthases in E.coli, and characterization of the enzyme activity in vitro. Significances: 1. developing new idea and methods for microbial natural products research; 2.Enriching thestructural diversity of DTs from bacteria; 3. Characterizing the biosynthetic pathway of DTs in sponge endogenous actinomycetes and laying the foundation for more DTs discovery.