Characterization And Manipulation Of The Jadomycin Effulux Gene And Construction Of A Biosensor-based Method To Screen For Novel Regulators

Antibiotics are important for their wide applications and commercial potential.Rational manipulate strains and improve antibiotic yield are the focus.The thesis includes two parts.Part I:Major Facilitator Superfamily(MFS)are membrane transporters amongst microorganisms and are usually used to export the antibiotics.We first took the sole MFS transporter JadL in jadomycin(Jd)biosynthetic gene cluster of Streptomyces venezuelae ISP5230 as an example to investigate its role through genetic engineering methods of gene knockout,complement and overexpression et al.The results suggest:the extracellular Jd B and the growth of jadL deletion strain LDM decreased,complemented strain LDCOM recover the production and the JadL overexpression strain LOE resulted the increase of Jd B by 37.87%compared to WT.This indicate that Jad L may increase the extracellular yield by exporting jadomycin;The strain WVR2006 which deleted Jd biosynthetic gene cluster(including jadL gene)regain its viability by exporting the Jd B or its Intermediate product;when added Jd B to culture of WVR2006 and jadL complemented strain WVR2006(WVR2006L),we found WVR2006L accumulated less intracellular Jd than WVR2006,which further verified JadL's role to efflux Jd B;When added Jd B to the culture of WT,LDM and LOE,LOE strain has the strongest tolerance.Likewise,overexpressing another intra-cluster MFS transporter CmcT also improve cephamycin C production in Streptomyces clavutligerius NRRL 3585.Part II:In the Escherichia coli which has clear background,base on Streptomyces coelicolor and the screen method of double-report-guided gene,we design and construct a set of effective and sensitive inducing-promoter biosensor through genetic engineering methods,such as constructing vector,protein expression and purification and EMSA et al.,to capture repressors which can bind to the target promoter.When we use the biosensor and set PscbA as target promoter,we obtained binding proteins CrpA?CrpB?Sco6323?Sco6792?Sco6784 from the constructed repressor library.Those proteins were verified by EMSA experiments,which also demonstrated the biosensor's sensitivity.The strategy of engineering intra-cluster MFS transporters and constructing effecive and sensitive method to capture repressor are of great significance to rational manipulation and antibiotic production improvement.