产表阿霉素重组波赛链霉菌的构建（英文）

Construction of Epirubicin-producing Streptomyces peucetius by Genetic Engineering

表阿霉素是一种重要的蒽环类抗肿瘤抗菌药,主要通过化学半合成生产。本研究通过定点突变敲除波赛链霉菌的酮基还原酶dnmV基因,随后导入表异构化的酮基还原酶基因aveBIV,构建基因工程菌EPI-1,其表阿霉素产量达到47mg/L。在此基础上,探讨了柔红糖胺合成及糖基化相关基因过表达对表阿霉素合成的影响。最后,将质粒pAve-desSQ导入dnmV敲除菌株中,得到了工程菌EPI-2,表阿霉素产量提高了102%,达到95mg/L。在5L发酵罐上进行发酵小试研究,表阿霉素产量为110 mg/L,具有产业化应用的潜力。

Epirubicin,an important anthracycline-type antitumor drug,is industrially produced through a chemical semisynthetic process.The production of epirubicin by genetic engineering microorganism directly is a more sustainable alternative.But its yield was below 1mg/L in previous studies.Here the epirubicin was formed by introducing heterologous Streptomyces avermitilis aveBIV gene into high-doxorubicin producing S.peucetius SIPI-11 dnrn V mutant blocked in the biosynthesis of daunosamine,the deoxysugar component of doxorubicin,and a recombinant strain EPI- 1 was constructed.The recombinant strain EPI-1 produced 47mg/L epirubicin,which was more than 47-fold higher than the engineered strain ever reported,indicating using overproduction strains as hosts is an efficient way to enhance epirubicin yield.Furthermore,to increase the yield of epirubicin in recombinant S.peucetius,we overexpressed the related genes of daunosamine formation and gtycosylation in the epirubicin biosynthesis pathway.The engineered strain EPI-2 produced 95mg/L epirubicin,which increased by 102% compared with EPI-1,which also demonstrated that daunosamine formation and glycosylation vcere the rate-limiting steps in the biosynthesis of epirubicin.Finally,the EPI-2 was cultured in a 5L fermenter,and the maximum titer of epirubicin reached 110mg/L at 168h.The results showed that S.peucetius EPI-2 has potential industrial application in epirubicin production by one-step fermentation.