基于化学遗传学筛选产萘醌类化合物内生放线菌

Chemical-genetics based screening for furanonaphthoquinone producing endophytic actinomycetes from seeds of Trewia nudiflora

【目的】为探究含具有抗肿瘤活性的美登素的滑桃(Trewia nudiflora)种子中内生放线菌的多样性,以及从内生放线菌中寻找萘醌类化合物产生菌。【方法】利用放线菌富集筛选培养基对经消毒处理的滑桃种子进行内生菌分离,根据菌落形态及16S rRNA基因序列分析进行菌种的分类鉴定。通过对所分离到的内生放线菌拮抗模式病原细菌(金黄色葡萄球菌、铜绿假单胞菌)、作物病原真菌(小麦赤霉菌、水稻纹枯病致病菌等)活性检测,以及萘醌类化合物合成关键基因为探针定向筛选萘醌类化合物产生菌。【结果】从分离到的100余株滑桃种子内生菌中鉴定出66株以链霉菌为主的放线菌,发现Streptomyces sp.HTZ27菌株含有目标基因,经固体发酵、化合物分离纯化、鉴定后,发现该菌发酵产物中有呋喃萘醌I,得率接近5 mg/L。【意义】本研究采用的化学遗传学方法可有效提高筛选目标化合物产生菌的效率,所筛选到FNQ I产生菌为深入研究呋喃萘醌类化合物生物合成与调控、抗肿瘤分子机理以及产业化应用等创建了有利条件。

[ Objetive] The seeds of Trewia nudiflora containing maytansine （an anticancer agent） , was investigated to explore the endophytic actinomycetes diversity and screen for naphthoquinones producing strain. [ Methods] The seeds of Trewia nudiflora were sliced and plated on different selective media after surface sterilization. Clones that looked like actinomycetes were selected, and classified according to the 16S rRNA sequences. Isolated strains were screened for furanonaphthoquinone biosynthesis gene by PCR, and tested for antibacterial and antifungal activity using Staphyloccocusaureus, Pseudomonasaeruginosa, Bacillus subtilis, Rhizoctoniasolani and Gibberellasaubinetii. LC-MS and NMR were used to determine the structure of candidate compounds. [ Results] More than 100 endophytic bacteria were isolated. Among them 66 were streptomycetes. FNQ6 （polyketide synthase Type III） and FNQ21 （carboxymuconate cycloisomerase） were only detected in Streptomyces sp. HTZ 27. We got 5 mg pure furanonaphthoquinone （FNQI） from 1 liter Streptomyces sp. HTZ 27 agar fermentation medium. [ Conclusion] The use of chemicalgenetics method increased the efficiency of screening for target compound producing bacteria.