The Construction Of Saccharopolyspora Erythraea λC3-SRR Mutant And Identification Of Its Product

With the far-ranging use of antibiotics, the problem of antibiotics resistance became more and more serious. It was urgent for us to develop new antimicrobials, which could overcome some problems of antibiotics resistance. Erythromycin was a kind of macrolide antibiotics. The global increase in macrolide resistance among respiratory pathogens threatened erythromycin's future usefulness. In order to develop new erythromycin derivation that could treat erythromycin-resistant bacteria, we tried to modify the structure of erythromycin by genetic engineering while erythromycin was derivatized by chemical modification.The ketolides, of which telithromycin was the first to undergo clinical development, were named as the third generation of erythromycins that had been developed with a view to overcoming the problem of macrolide resistance. Until now, telithromycin was synthesized by chemical modification of erythromycin. L-cladinose at position C3 of macrolactone ring had been replaced by a ketone fuction in the ketolide. This modification enabled ketolides to bind to their targets without tripping the inducible resistance to macrolide-lincosamide-streptogramin B (MLSb) drugs, and could help other carbons of ketolides extend easily. The ketolides could treat some erythromycin-resistant bacteria. Whether C3 hydroxyl of macrolactone ring would be reduced depended on the ketoreductase (KR6) domain of the sixth module of polyketide synthase that catalyzed the synthesis of 6-deoxy-erythronolide B, so we could make KR6 domain activity killed by genetic engineering for the ketolides' biological synthesis.On the basis of Donadio's report, there were proposed high conserved NADPH binding sites with VSRRG five amino acids in the KR6 domain, the reason the ketoreductase (KR3) domain of the third module was nonfunctional may be that itlacked VSRRG. We used homologous recombination technique to knock out the nine nucleotides corresponding to SRR amino acids in the KR6 domain, and got the mutant S.erythraea ?oC3-SRR which could synthesize the compound 3-deoxy-3-oxo-erythronolide B (DOEB).Taking genomic DNA of S.erythraea as a template, KR6 domain DNA fragment without the nine nucleotides corresponding to SRR was amplified by overlapping PCR techniques and cloned into homologous recombinant vector pWHM3 to build the plasmid pWHM3-SRR. Plasmid pWHM3-SRR was introduced into protoplasts of S.erythraea X.C3 and integrated into the gene locus for erythromycin biosynthesis in the chromosome, and three integrates, XC3-A, A.C3-B and X.C3-C, were sorted out. After the integrate A.C3-A grew for at least two generations on R3M media without thiostrepton, the protoplasts were prepared and made to single colonies on R3M plates. One mutant, named as S.erythraea X.C3-SRR, which couldn't grow on the R3M media with thiostrepton and couldn't restrain B.subtilis PUBllO's growth, was picked out for DNA sequencing. DNA sequencing proved that the nine nucleotides corresponding to SRR amino acids in the chromosome of S.erythraea XC3-SRR had been knocked out. Mass spectrometry showed that S.erythraea XC3-SRR could synthesize the compound 3-deoxy-3-oxo-erythronolide B. Comparing mutant ^.C3-SRR's mass spectrometry and mutant M's, we found that mutant M had synthesized the outgrowth, 2-desmethyl-3-deoxy-3-oxo-erythronolide B, but mutant A.C3-SRR hadn't synthesized that outgrowth.We had constructed the mutant S.erythraea AO-SRR that could synthesize ketolide DOEB. Our research showed that SRR amino acids played an important role in the reductivity of KR6 domain. Comparing the KR6 domain's amino acid sequences with actinorhodin polyketide reductase's and human, E. coli, P. aeruginosa's glutathione reductases'. We found that two basic amino acids RR in the SRR amino acids were the binding sites for NADPH 2'-phosphate in the KR6 domain. There were many well-conserved motifs of SDR (short-chain dehydrogenase/reductase) family in the KR6 domain, and the active sites of KR6 domain were the same as those of complex SDR. We concluded that KR6 domain ofS.erythraea polyketide synthase belonged to complex SDR.