Thienamycin Heterologous Expression Of The Biosynthetic Gene Cluster And Its Transformation

Originated in the late 80s and classified as atypicalβ-lactam antibiotics, carbapenem antibiotics (carbapenems) show a broad spectrum of potent antibacterial activity, especially inhibit multidrug-resistant bacteria andβ-lactamase producing bacteria, and are now widely used in patients. Carbapenem antibiotics target on all types of penicillin-binding protein (PBPs) and show sustained antibiotic effect (PAE).Compared with other lactam antibiotics, carbapenem antibiotics have lower drug resistance and less side effects, thus becoming the main drug used in the treatment of serious nosocomial infection.Carbapenem antibiotics are structurally similar to penicillin, so it is difficult to isolate Carbapenem antibiotics from the fermentation solution containing both.Recombineering (recombination-mediated genetic engineering) is a powerful method for the fast and efficient construction of vectors for the subsequent manipulation of the mouse genome. It is also an efficient way of manipulating the bacterial genome directly. Recombineering is based on the homologous recombination in Escherichia coli using recombination proteins provided fromλphage. Exo creates a single stranded overhang, Bet protects these overhangs and assists in the subsequent recombination process, Gam prevents degradation of linear DNA by inhibiting E. coli RecBCD protein. Linear DNA (PCR product, oligo, etc.) with sufficient homology in the 5'and 3'ends to a target DNA molecule present in the bacteria (plasmid, BAC, or the bacterial genome itself) can be introduced into heat-shocked and electrocompetent bacteria, homologous recombination happens between the homology DNA through recombineering.In this study, recombineering technology was used to modify the thienamycin biosynthetic gene cluster by replacing with a strong ermEp* promoter, with the purpose to high level heterologous expression of the thienamycin biosynthetic gene cluster.Since 80s of the 20th century, with the number of cancer increasing the world's anti-cancer drug market were growth rapidly. Doxorubicin and daunorubicin are anthracyclines and also are the basic chemotherapy treatment drugs recommended by United Nations World Health Organization (WHO).In this study, recombineering technology was used to construste the doxorubicin expression vector and hoping to the methods of production by microbial fermentation of doxorubicin, so that we would get great social and economic benefits by simplifing the production process, reducing costs and decreasing pollution.