The issues in new antibiotic discovery are pressing, because the frequent re-discovery of antibiotic scaffolds leads to few novel antibiotics discovered, besides, with the widespread use of antibacterial agents, multi-resistant pathogens are emerging, which poses more huge challenge in antibiotic discovery. However, next generation sequencing technology and bioinformatics have revealed that many secondary metabolite biosynthesis gene clusters possess the potential of producing new bioactive secondary metabolites (BSMs), which were ignored previously. Among the microorganisms, actinomycetes species are the best sources for those gene clusters. Synthetic biology is the enabling technology to activate those silent gene clusters, which aims to engineer organisms for expected applications with combination of various biotechnologies. The project employs the reciprocal regulation system between jadomycin (Jd) and chloramphenicol (Cm) in S. venezuelae: JadR1 activates Jd synthesis while represses Cm synthesis with ethanol shock. This system can be used to rationally engineer S. venezuelaee for heterologous production of BSMs with re-factored gene clusters containing appropriate control elements: deletion of the jadR1 gene shall lead to down-regulation of Jd production, simultaneously induce overproduction of Cm due to the relieved repression of the Cm structural genes’ promoters. Besides, the cml gene cluster should be completely deleted to avoid interfering with the introduced gene cluster. The appropriate control element is an inducible promoter screened out with GUS assay among cmlFp, cmlIp, cmlXp, jadJp. The inducible promoter would be used to construct an inducible system for industrial scale production of BSMs, because constitutive heterologous expression of BSMs is harmful for producing hosts.The jadR1- cml- mutants were successfully generated with Gibson Assembly, transconjugation, double crossover and replica plating. The gene cluster MP112-09-Lac was cloned with native promoter and ermE* respectively and transconjugated to jadR1- cml- mutant, however, cloing of MPS05-B41-Lin was hindered by wrong PCR amplification. The four promoters were tested with GUS assay, based on MYM medium and cmlF is speculated to be the most desirable inducible promoter.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:ntnu-27325 |
| Date | January 2014 |
| Creators | Zhang, Jianhai |
| Publisher | Norges teknisk-naturvitenskapelige universitet, Institutt for bioteknologi, Institutt for bioteknologi |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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