可利霉素产生菌中糖基转移酶基因ssp803的功能鉴定及酶学特性分析  

Functional characterization and enzymatic properties of glycosyltransferase gene ssp803 in carrimycin producing strain

作　　者：高晓杰 李可萌 赫卫清[1] GAO Xiao-jie;LI Ke-meng;HE Wei-qing(NHC Key Laboratory of Biotechnology of Antibiotics,CAMS Key Laboratory of Synthetic Biology for Drug Innovation,Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050,China)

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所卫健委抗生素生物工程重点实验室/医科院合成生物学重点实验室,北京100050

出　　处：《中国医药生物技术》2024年第5期432-442,共11页Chinese Medicinal Biotechnology

基　　金：国家重点研发计划(2018YFA0901800);中国医学科学院医学与健康科技创新工程(2021-1-I2M-028)。

摘　　要：目的 通过体内基因阻断和体外酶促反应鉴定可利霉素产生菌中糖基转移酶基因ssp803功能和酶学特性,以及与可利霉素生物合成的关系。方法 在可利霉素产生菌54-IA中利用CRISPR-Cas9基因编辑系统敲除ssp803基因,将组成型启动子kasOp*控制的ssp803及其同源基因oleD分别转化到54-IA和?803变株中进行回复和高表达实验,通过HPLC分析?803变株、回复株和高表达株中可利霉素主组分异戊酰螺旋霉素的产量变化。再将ssp803基因连接至冷休克表达载体pColdI上,利用大肠杆菌Transetta(DE3)中进行表达和纯化。以大环内酯类抗生素为糖苷配体和以UDP-葡萄糖为供体进行Ssp803催化反应,通过HPLC-MS检测酶催化后的产物,通过UDP-Glo试剂盒测定Ssp803的酶促动力学参数。结果 利用PCR筛选出了ssp803基因框内缺失突变株?803,在?803中异戊酰螺旋霉素产量与54-IA出发菌株相似,而在54-IA和?803中高表达ssp803完整基因时,异戊酰螺旋霉素的含量明显下降,导入高表达oleD基因的质粒后产量下降得更多。通过体外酶促反应证实Ssp803催化的最适温度是37℃,最适pH是9.06。Ssp803对所测的大环内酯类抗生素都具有糖基化失活作用,对异戊酰螺旋霉素I等16元环的大环内酯类抗生素的催化活性要低于14元环大环内酯类抗生素,对索利霉素的催化效率最高。结论 Ssp803对大环内酯类抗生素具有糖基化失活作用,对14元环大环内酯类抗生素的活性更高,最适底物是索利霉素;在可利霉素产生菌中高表达ssp803和oleD基因会抑制异戊酰螺旋霉素的生物合成。Objective To characterize the function and enzymatic properties of glycosyltransferase gene ssp803 in carrimycin producing bacteria and its effect on carrimycin biosynthesis through in vivo gene inactivation and in vitro enzymatic reaction.Methods The ssp803 gene was inactivated by the CRISPR-Cas9 gene editing system in the carrimycin producing strain 54-IA.Both ssp803 and its homologous gene oleD under the control of the constitutive promoter kasOp*were transformed into the 54-IA and∆803 mutant,respectively.The changes of isovalerylspiramycins(the major components of carrimycin)production in the∆803,the complementary strains and ssp803/oleD overexpression strains were detected by HPLC.The ssp803 gene was cloned into expression vector pColdI,expressed in Transetta(DE3).The purified Ssp803 glycosylation assays were performed in the enzymatic reaction with macrolide antibiotics(as aglycones)and uridine diphosphate-glucose(as a glycosyl donor).The glycosylation products were detected by HPLC-MS,and the enzymatic kinetic parameters of Ssp803 were determined using the UDP-Glo assay kit.Results The ssp803 in-frame deletion mutant∆803 was screened by PCR.The production of isovalerylspiramycins(the major components of carrimycin)in∆803 was similar to that of starting strain 54-IA.However,isovalerylspiramycins yield was significantly decreased in both∆803 and 54-IA strains containing highly expressed ssp803 gene,and the production was reduced even more in oleD overexpression in the two strains.Through in vitro enzymatic reaction,the optimal temperature and pH for Ssp803 catalysis were 37℃and 9.06,respectively.The Ssp803 could inactivated all tested macrolides by glycosylation.Its catalytic activity for 16-membered macrolide antibiotics,such as isovalerylspiramycin I,was lower than that of 14-membered macrolides,and the highest catalytic efficiency was achieved for solithromycin.Conclusions The Ssp803 is a glycosyltransferase for macrolides inactivation,being more specific for 14-membered macrolide antibiotics,

关 键 词：糖基转移酶 可利霉素 异戊酰螺旋霉素 大环内酯类抗生素 

分 类 号：Q78[生物学—分子生物学] R915[医药卫生—微生物与生化药学]