Breeding Of Medermycin-Overproducing Strain And Prokaryotic Expression Of Proteins Involved In Medermycin Biosynthesis

Medermycin is a polyketide antibiotic classified in the family of benzoisochromanequinones(BIQs),which shows strong antibacterial and antitumor activity by inhibiting the signal transduction pathway in varied tumor cells with a novel mechanism.In this study,in order to elucidate the mechanism of the regulation of medermycin biosynthesis and to conduct medermycin-overproducing strains,genetical,molecular biological,analytical chemistry and other approaches were used to investigate the mechanism of the novel regulatory gene med-ORF10 located on medermycin biosynthetic gene cluster and the involvement of the ribosome recycling factor(ecoded by frr)in the medermycin production.1.Construction of medermycin-overproducing strain by overexpression of ribosome recycling factorRibosome recycling factor is involved in the fourth step of protein biosynthesis(ribosome recycling),which can improve the efficiency of protein synthesis,so could act as a positive regulator of antibiotic biosynthesis.The yield of medermycin in the wild-type strain Streptomyces sp.AM-7161 has yet to be improved.In this study,overexpression experiment of ribosome recycling factor in Streptomyces sp.AM-7161 was performed to promote the production of medermycin.The degenerate primers were designed firstly based on the alignment of ribosome recycling factor genes from several streptomycetes in GenBank,and Streptomyces sp.AM-7161 genomic DNA was isolated as template.The target fragment amplified by PCR from the genome DNA of Streptomyces sp.AM-7161 was inserted into pT7Blue and sequenced.Blast analysis showed the maximal identity of the target PCR product to sequences in GenBank is 98%,suggesting that the cloned gene is frr gene of Streptomyces sp.AM-7161.The sequence of frr was submitted to GenBank and the given accession number is JF807993.The frr gene of Streptomyces sp.AM-7161 was inserted into pIJ8600,an expression vector used in streptomycetes,to gain an expression plasmid designated as pHSL56.2.Then pHSL56.2 was introduced into the wild-type strain Streptomyces sp.AM-7161.Solid fermentation showed that more red-brown pigmentation indicating medermycin production in the recombination strain AM-7161/pHSL56.2 could be observed than that in the wild-type strain Streptomyces sp.AM-7161.The broth of liquid cultivation was extracted and analyzed by LC-MS/MS,which showed that medermycin production was promoted at least by 3 fold of that in the wild-type strain,as well as the increased accumulation of medermycin-biosynthetic intermediates or other metabolites could be observed.2.Expression and purification of the proposed target genes regulated by med-ORF10med-ORF10 could play a regulatory role in medermycin biosynthesis with an unknown mechanism.Its homologous gene actVI-ORFA could target several genes and regulate the production of actinorhodin biosynthesis.So med-ORF10 was deduced to control the expression of more genes besides med-ORF12 and med-ORF1 revealed by previous transcription analysis.In this study,the deduced target products including Med-ORF30,Med-ORF29,Med-ORF28,Med-ORF27,Med-ORF23,Med-ORF11,Med-ORF6 and Med-ORF5 were expressed and purified for preparation of polyclonal antibodies which will be used to study whether the expression of these genes was controlled by med-ORF10 at the translational level.Firstly,the proposed target genes were amplified from plasmid pIK340 which contains the whole medermycin biosynthetic gene cluster.All of these fragments were inserted into pT7Blue respectively,to generate plasmids pHSL57.1(med-ORF30),pHSL58.1(med-ORF29),pHSL59.1(med-ORF28),pHSL60.1(med-ORF27),pHSL61.1(med-ORF23)and pHSL62.1(med-ORF6),respectively.After sequencing,these genes were subcloned into an expression vector pET28a(+)to gain expression plasmids pHSL57.2(med-ORF30),pHSL58.2(med-ORF29),pHSL59.2(med-ORF28),pHSL60.2(med-ORF27),pHSL61.2(med-ORF23)and pHSL62.2(med-ORF6),respectively.med-ORF11 and med-ORF5 were cloned into pET28a(+)directly and the resulting plasmids named as pHSL53.2 and pHSL54 respectively.All the expressing plasmids were transformed into E.coli BL21(DE3)to establish the prokaryotic expression systems for these proposed target genes.Five proteins of these target products,including Med-ORF30,Med-ORF29,Med-ORF28,Med-ORF27 and Med-ORF6,have been purified by optimizing the introduction and purification protocol.3.Investigation of the regulation of med-ORF10 on the expression of C-glycosyltransferase gene med-ORF8 in medermycin biosynthetic pathwayacrVI-ORFA,the homology of med-ORF10,was found to control the expression of structural genes mainly involved in middle steps of actinorhodin biosynthesis.Here,we intended to investigate whether the genes involved in the last step of medermycin biosynthesis could be regulated by med-ORF10.The previous study showed that med-ORF8 was involved in the last step of medermycin biosynthesis for binding a sugar moiety(angolasamine)to the polyketide skeleton(kalafungin)through C-C glycosidic bond to produce medermycin.The expression level of Med-ORF8 in med-ORF10 overexpression strain and the wild type strain will be detected by Med-ORF8 polyclonal antibody.med-ORF8 was subcloned into an expression vector pET28a(+),to get the recombinant expression vector pHSL52.pHSL52 was introduced into E.coli BL21(DE3)competent cells to establish the prokaryotic expression system.A high expression level of Med-ORF8 in BL21(DE3)could be gained under optimized conditions,but mainly in the form of inclusion bodies.So it was purified under 8 mol/L urea condition.The polyclonal antibodies were prepared after the rabbit was immunized with purified Med-ORF8 and showed a high specificity against Med-ORF8 by Western blot and a high titer of 1:64000 by ELISA analysis.The resultant polyconal antibody against Med-ORF8 was used to measure the expression level of Med-ORF8 in the med-ORF10-overexpression strain.Western blot showed that the expression level of Med-ORF8 in med-ORF10-overexpression strain AM-7161/pHSL98 was a slightly higher than that in wild type strain AM-7161,indicating med-ORF10 might have a weak influence on last step in the medermycin biosynthetic pathway.