Construction Of A Genetically Engineered Strain And Optimization Of Its Fermentation Medium For High-Level Production Of BDSF

In the past decades,abuse of antibiotics has led to the production and prevalence of super-bacteria,which are resistant to multidrug.Research work targeting at pathogens,rather than antibiotics themselves,has been getting more and more urgent.Quorum sensing(QS)is a cell-cell signalling pathway widely used by microorganism to fit into environment and infect host.QS inhibitors has been shown to be able to down-regulate the production of microbial pathogenic factors,such as biofilm and type III secretion system,thereby weakening the fitness and pathogenicity of pathogen.BDSF is a Xanthomonas QS signal,showing inhibitory activity on the pathogenicity of a group of micro-organisms,including Candida albicans,Pseudomonas aeruginosa and Burkholderia cepacia.Such inhibition effect dose not cause cell damage,and therefore it is likely to reduce the risks of resistant mutations.The effect of BDSF mixed with other antibiotics has also been tested,which also gave rise to significant inhibitory effect on many pathogenic bacteria.In conclusion,BDSF has a great potential to be developed as a new drug,and the aim of this research is to improve the production of BDSF with genetics and fermentology tools.In order to obtain the genetically engineered strain which over-produces BDSF,the rpfF gene fragment from Xanthomonas campestris pv.cmpestris(Xcc)8004 was coloned into the Escherichia coli strain BL21(DE3)p Lys S,which gives BLSF strain.With BLSF strain,fermentation process was optimized by screening initial medium,carbon source and nitrogen source using single factor experiment,Placket-Burman test and central composite design.The optimal medium BBM(tryptone 12.15?13.32 g/L,yeast powder 15.12?15.35 g/L,glycerol 5.7 m L/L,sugar 20 g/L,soybean powder 40 g/L,K₂HPO₄12.54 g/L,KH₂PO₄ 2.31 g/L)was obtained and verified.BDSF production by BLSF strain in BBM reaches 1937.75±72.25 M after 60hours fermentation,which is about 12 times higher than that in the initial medium,and about 200 times higher than that of the Xcc mutant?rpf C?rpf B in BBM.