Pristinamycin,produced by Streptomyces pristinaespiralis,which is a streptogramin-like antibiotic consisting of two chemically unrelated components:pristinamycin I(PI)and pristinamycin II(PII),shows potent activity ag...Pristinamycin,produced by Streptomyces pristinaespiralis,which is a streptogramin-like antibiotic consisting of two chemically unrelated components:pristinamycin I(PI)and pristinamycin II(PII),shows potent activity against many antibiotic-resistant pathogens.However,so far pristinamycin production titers are still quite low,particularly those of PI.In this study,we constructed a PI single component producing strain by deleting the PII biosynthetic genes(snaE1 and snaE2).Then,two metabolic engineering approaches,including deletion of the repressor gene papR3 and chromosomal integration of an extra copy of the PI biosynthetic gene cluster(BGC),were employed to improve PI production.The final engineered strain DPIIDpapR3/PI produced a maximum PI level of 132 mg/L,with an approximately 2.4-fold higher than that of the parental strain S.pristinaespiralis HCCB10218.Considering that the PI biosynthetic genes are clustered in two main regions in the 210 kb“supercluster”containing the PI and PII biosynthetic genes as well as a cryptic polyketide BGC,these two regions were cloned separately and then were successfully assembled into the PI BGC by the transformation-associated recombination(TAR)system.Collectively,the metabolic engineering approaches employed is very efficient for strain improvement in order to enhance PI titer.展开更多
以始旋链霉菌(S trep tomy ces p ristinaesp ira lis)HCCB 2003-8的染色体DNA为模板,通过优化PCR条件扩增出FM N还原酶、P IIA合酶的基因片段。与文献报道的基因序列做同源比较,snaA和snaB碱基序列分别只有一个碱基的差异,snaC碱基序...以始旋链霉菌(S trep tomy ces p ristinaesp ira lis)HCCB 2003-8的染色体DNA为模板,通过优化PCR条件扩增出FM N还原酶、P IIA合酶的基因片段。与文献报道的基因序列做同源比较,snaA和snaB碱基序列分别只有一个碱基的差异,snaC碱基序列完全匹配。将snaA、snaB和snaC的基因片段插入原核表达载体pET 30a中,并转化至大肠埃希菌BL 21(DE 3)。SDS-PAGE结果表明,经IPTG诱导后基因分别得到表达。展开更多
目的与方法本文主要采用均匀设计方法对普那霉素培养基进行优化,分别对糊精,蔗糖,葡萄糖,可溶性淀粉和甘油等碳源进行考察,使用软件Uniform Design Version 4.00分析发酵结果并优化配方,对软件给出的最优结果继续验证并优化。结果与结...目的与方法本文主要采用均匀设计方法对普那霉素培养基进行优化,分别对糊精,蔗糖,葡萄糖,可溶性淀粉和甘油等碳源进行考察,使用软件Uniform Design Version 4.00分析发酵结果并优化配方,对软件给出的最优结果继续验证并优化。结果与结论验证试验表明,含两种碳源即糊精3%葡萄糖3%的配方可使摇瓶培养的发酵单位提高48%,在两吨罐上验证该配方,连续3批进罐发酵单位提高30%。展开更多
基金This work was sponsored by the National Natural Science Foundation of China(31430004,31421061,31630003,31370081 and 31570072)the Science and Technology Commission of Shanghai Municipality(16490712100).
文摘Pristinamycin,produced by Streptomyces pristinaespiralis,which is a streptogramin-like antibiotic consisting of two chemically unrelated components:pristinamycin I(PI)and pristinamycin II(PII),shows potent activity against many antibiotic-resistant pathogens.However,so far pristinamycin production titers are still quite low,particularly those of PI.In this study,we constructed a PI single component producing strain by deleting the PII biosynthetic genes(snaE1 and snaE2).Then,two metabolic engineering approaches,including deletion of the repressor gene papR3 and chromosomal integration of an extra copy of the PI biosynthetic gene cluster(BGC),were employed to improve PI production.The final engineered strain DPIIDpapR3/PI produced a maximum PI level of 132 mg/L,with an approximately 2.4-fold higher than that of the parental strain S.pristinaespiralis HCCB10218.Considering that the PI biosynthetic genes are clustered in two main regions in the 210 kb“supercluster”containing the PI and PII biosynthetic genes as well as a cryptic polyketide BGC,these two regions were cloned separately and then were successfully assembled into the PI BGC by the transformation-associated recombination(TAR)system.Collectively,the metabolic engineering approaches employed is very efficient for strain improvement in order to enhance PI titer.
文摘以始旋链霉菌(S trep tomy ces p ristinaesp ira lis)HCCB 2003-8的染色体DNA为模板,通过优化PCR条件扩增出FM N还原酶、P IIA合酶的基因片段。与文献报道的基因序列做同源比较,snaA和snaB碱基序列分别只有一个碱基的差异,snaC碱基序列完全匹配。将snaA、snaB和snaC的基因片段插入原核表达载体pET 30a中,并转化至大肠埃希菌BL 21(DE 3)。SDS-PAGE结果表明,经IPTG诱导后基因分别得到表达。
文摘目的与方法本文主要采用均匀设计方法对普那霉素培养基进行优化,分别对糊精,蔗糖,葡萄糖,可溶性淀粉和甘油等碳源进行考察,使用软件Uniform Design Version 4.00分析发酵结果并优化配方,对软件给出的最优结果继续验证并优化。结果与结论验证试验表明,含两种碳源即糊精3%葡萄糖3%的配方可使摇瓶培养的发酵单位提高48%,在两吨罐上验证该配方,连续3批进罐发酵单位提高30%。
