教酒链霉菌中卡西霉素生物合成基因簇的calU3基因功能分析  

Functional Analysis of the calU3 Gene in Calcimycin Biosynthesis Gene Cluster from Streptomyces chartreusis

作　　者：苟丽霞 杨清浅 刘孟欣 汪志军[2] 韩铁生 GOU Lixia;YANG Qingqian;LIU Mengxin;WANG Zhijun;HAN Tiesheng(School of Life Sciences,North China University of Science and Technology,Tangshan,063210;State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai,200240;Hebei Province Key Laboratory of Occupational Health and Safety for Coal Industry,School of Public Health,North China University of Science and Technology,Tangshan,063210)

机构地区：[1]华北理工大学生命科学学院,唐山063210 [2]上海交通大学生命科学技术学院微生物代谢国家重点实验室,上海200240 [3]华北理工大学公共卫生学院,河北省煤矿卫生与安全重点实验室,唐山063210

出　　处：《基因组学与应用生物学》2023年第4期418-426,共9页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31700083);河北省自然科学基金项目(H2020209001)共同资助。

摘　　要：卡西霉素(calcimycin)是重要的离子载体抗生素,具有广泛的生物活性,其生物合成基因簇已从教酒链霉菌(Streptomyces chartreusis)NRRL3882的基因组DNA中成功克隆,但其生物合成途径尚未阐明。本研究拟分析卡西霉素生物合成途径中calU3基因的功能。采用PCR-targeting方法构建calU3基因中断质粒,通过接合转移和同源重组的方法构建得到教酒链霉菌NRRL3882的ΔcalU3突变株,并对突变株进行calU3基因的互补分析,通过高效液相色谱(HPLC)及液相色谱-质谱(LC-MS)分析突变株及互补菌株的代谢产物。构建calU3的表达载体,纯化得到重组CalU3蛋白,对其进行紫外/可见光的吸光谱分析。结果显示calU3基因中断的突变株丧失产生卡西霉素及N-脱甲基卡西霉素的能力,但有色唑霉素(cezomycin)的积累,互补菌株恢复野生菌株产卡西霉素的能力。CalU3蛋白具有明显的黄色,在377 nm和455 nm有特征性的吸收峰。以上结果表明calU3和卡西霉素苯并噁唑环3位取代基团的形成相关。CalU3具有FAD-NAD(P)的结合位点,可能是一个FAD-NAD(P)依赖的氧化还原酶,负责催化活化的色唑霉素中苯并噁唑环的3位羟化反应,生成3-羟基色唑霉素。本研究初步阐明了calU3基因对卡西霉素的苯并噁唑环3位的修饰功能,为全面解析卡西霉素的生物合成途径打下了坚实基础。Calcimycin is an essential ionophoric antibiotics exhibiting multiple biological effects.The gene cluster for calcimycin biosynthesis has been cloned from the genomic DNA of Streptomyces chartreusis NRRL3882,but the calcimycin biosynthetic pathway remains unclear.This study aims to analyze the function of the calU3 gene in calcimycin biosynthesis gene cluster.The calU3 gene disruption plasmid was constructed by PCR-targeting.The ΔcalU3 mutant of Streptomyces chartreusis NRRL3882 was generated through introducing the calU3 disruption vector into the S.chartreusis strains by conjugation transfer and homologous recombination.For complementary strain,the calU3 complemented plasmid was introduced into theΔcalU3 mutantvia conjugation.The metabolites ofΔcalU3 mutant and complementary strain were analyzed by high performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC-MS).The calU3 over-expression vector was constructed,and recombinant N-terminal His6-tagged CalU3 protein was over-expressed in E.coli and purified,and its UV/Vis absorbance spectra were analyzed.The result showed that theΔcalU3 mutant was unable to produce calcimycin and N-demethyl-calcimycin,whereas cezomycin was still produced,and the complementation strain restored the antibiotic production ability of the wild-type.The purified His6-tagged CalU3 has a distinctive yellow color,and the major absorption wavelengths of CalU3 were at 377 nm and 455 nm.The above results indicated thatcalU3 was related to the formation of the substituent group at the 3-position of the calcimycin benzoxazole ring.CalU3 possesses a binding site for FAD-NAD(P),and may be a FAD-NAD(P)-dependent oxidoreductase,which is responsible for catalyzing the 3-hydroxylation of the benzoxazole ring in activated cezomycin to generate 3-Hydroxycezomycin.This study preliminarily elucidated the modification function ofcalU3 gene on the 3-position of the benzoxazole ring of calcimycin,which laid a solid foundation for a comprehensive analysis of the biosynthetic

关 键 词：卡西霉素 生物合成 氧化还原酶 FAD依赖 calU3 

分 类 号：Q933[生物学—微生物学]