The Biosynthesis Research Of Antimuor Antibiotics Azinomycin B And Chlorothricin

Azinomycin B, also named Carzinophilin A, first isolated from Streptomyces sahachiroi by Japanese scientists in 1954, has potent antitumor activity in vivo and cytotoxicity. Then scientists isolated other Azinomycin B analogues-Azinomycins form the fermentation culture of Streptomyces griseofuscus. Azinomycins feature a set of unusual, densely assembled functionalities:a 3-methoxy-5-methyl naphthoic acid (NPA) moiety, a 2-amino-1,3-dicarbonyl group, an epoxide domain, and an unprecedented aziridino [1,2a] pyrrolidine (1-azabicyclo [3.1.0] hexane) ring system which has been the uniqueness structure in nature product. Azinomycin B has the best bioactivity among the Azinomycins. Its effect in vivo is comparable to that of the clinically used DNA ISC drug mitomycin C. Azinomycin B binds within the major groove of DNA, and forms covalent interstrand crosslinks (ISCs) with apparent sequence selectivity by electrophilic attacks of C10 and C21 onto N7 positions of purine bases. Because of the poor availability and extreme instability of Azinomycin B, extensive synthetic efforts have been carried out but very expensive to generate a number of azinomycin analogues for investigations into the structure-bioactivity relationship. Base on the research for the biosysthsis path way of Azinomycin B, Combinatorial biosynthesis of the Azinomycin B may be a economical way to produce new Azinomycin B analogues with better bioactivity and stability.A DNA fragment was got by PCR amplification with a general primers designed in accordance with the conservative amino acid of the iterative typeâ… polyketides(PKS). After screening the cosmid library of the genome of the Streptomyces sahachiroi NRR2485 with the PCR product as a probe, A 60kb DNA fragment was sequenced and deduced to be the biosysthesis gene cluster of the Azinomycin B which cotains 39 ORFS. Heterogeneous expression of aziB solo in the Streptomyces albus generated 5-methyl NPA, Co-heterogeneous expression of aziB, aziBl, aziB2 produced 3-methoxy-5-methyl NPA. These results confirmed the DNA sequence we cloned take responsibility for the biosysthesis of Azinomycin B. The mutant for ORF 36 knocked out still produced Azinomycin B, indicated that ORF36 was the boundary of Azinomycin B biosysthesis gene cluster.Chlorothricin is a spirotetronate antibiotics produced by Streptomyces antibioticus DSM 40725. The spirotetronate antibiotics have bioactivity including anti-infection,antitumer, antimalaria, and cholesterol biosynthesis. A 122kb DNA fragment encoding the biosynthetic gene cluster of Chlorothricin was cloned form the genome withâ… type PKS as probes, we found a iterative typeâ… polyketides ChlB1 which has the same organization and high sequence indensity with AziB form Azinomycin B gene cluster. Surprisedly, ChlB1 synthesis signal ring product-6-Methylsalicylic acid (6-MSA), AziB synthesis two rings product 5-methyl-NPA. what is the catalysis mechanism for them? we mutanted the dehydratase(DH) domain, ketereductase(KR) domain of ChlB1 and AziB. All mutants of AziB did not produce any intermediates, but DH mutant of ChlB1 (H947F) still produced 6-MSA and KR mutant of ChlB1 (produced orsellinic acid (OSA). All these information indicated that the DH domain of the iterative typeâ… PKS is very important but not the essential and the deletion of the function of KR domain did not affect the next extension reaction. Over-expression of the p450 hydroxylase aziB1, the O-methyltransferase aziB2, the non-ribosomal peptide synthetase aziA1 in Ecoli BL21(DE3). We characterized that AziB1 catalyzes a regiospecific hydroxylation at C3 position of 5-methyl-NPA, and the resulting hydroxyl group can be subsequently O-methylated by AziB2 to furnish the methoxy functionality to give 3-methoxy-5-methyl NPA, then 3-methoxy-5-methyl NPA was specifically recognize/activated by the NRPS AziAl in vitro, supporting its function as a loading module to employ 3-methoxy-5-methyl-naphthoyl as the starting unit for initiating the backbone formation of azinomycin B. These dates comfirmed the functions of the aziB 1, azi B2, aziA1 and that thepost-modification of product of iterative typeâ… polyketides azi B occurred on the free naphthoic acids.Because of good antibacterial and antitumor activity, Chlorothricin was a candidate for combinatorial biosynthesis. Post-modification of product of iterative typeâ… polyketides chlB1 occurred on the acyl carrier protein (ACP)-tethered 6-MSA.Former research work indicated that two acyltransferases(AT) chl B3,Chl B6 were less substrate specifically recognize, moreover, during study the mechanism of iterative typeâ… polyketides, we successfully changed the 6-Methylsalicylic acid synthase (6-MSAS) Chl B1 to orsellinic acid synthase (OSAS). This KR mutant chl B1 was introduced in the chl Bl knocked out mutant of Streptomyces antibioticus DSM 40725, surprisedly,this new mutant produced two new Chlorothricin analogues 7 and 8which have improved bioactivity.