Molecular Screening Of Potential Ansamycin-producing Strains And Early Discrimination Of The Chemical Structural Types Produced By Them

The compound,3-amino-5-hydroxybenzoic acid(AHBA),is the specific and indispensible starter unit for the biosynthesis of ansamycins in microbes.AHBA synthase is the terminal enzyme catalyzing the final formation of AHBA in the aminoshikimate pathway for AHBA biosynthesis. Multi-alignment of known AHBA synthases revealed that they are highly similar.Therefore, degenerate oligonucleotides could be designed to amplify AHBA synthase genes from unkown microbial strains.The existence of AHBA synthase gene in a microbial strain could also be used as an indicator that the strain possess the potential for AHBA,and furtherly,ansamycin biosynthesis. Under such belief,a PCR screening method for potential ansamycin-producing actinomycete strains had been established and 33 strains from 2000 soil-isolates had been obtained as AHBA-synthase gene positive strains in our lab.In this paper,the anti-bacterial/anti-virus/anti-cancer cell activities of the fermentation products of 33 AHBA synthase gene positive strains obtained above were assayed.Six strains,that is,Streptomyces sp.8-21,2209,22-24,4088,3-27 and 4353,showed good biological activities in their fermentation products,which are worthy to study furtherly.Several methods including PEG-mediated protoplasts transformation and conjugal transfer were investigated for each of the six strians.Conjugal transfer was accomplished successfully in four of the six strains,that is Streptomyces sp.22-24,4088,3-27 and 4353.AHBA synthase gene disruption mutants for each of the four strains,by single cross-over,were obtained by conjugal transfer of pKC 1139,inserted with the coresponding AHBA synthase gene fragment in its multiple cloning sites,from E.coli ET12567(pUZ8002)to the Streptomyces sp..Mutants of Streptomyces sp.4353 produced no further a yellow compound,which was identified later as actinomycin D.Mutants of Streptomyces sp.3-27 showed no observed changes in its fermentation products.Mutants of Streptomyces sp.22-24 produced less amounts of an active compound in its fermentation production.Mutants of Streptomyces sp.4088 produced a new active compound in its fermentation production.In this paper,we established also a simple method for early preliminary discrimination of benzenic ansamycins and their derivatives.With the treatment of alkali such as NaOH,which break the amide bond formed between the benzenic amino-group and the ansa-bridge's terminal carboxy-group,benzenic ansamycins such as geldanamycin changed its color from yellow to purple. The minimal detection limit of this method on the silica gel plate for geldanamycin was 4μg if NaOH(2.0mol/L)was sprayed on the TLC plate.This method was used successfully for early preliminary discrimination of geldanamycin produced by two soil-isolated novel strains, Streptomyces sp.4-4 and Streptomyces sp.3-57,which were strains with ansamycin producing potential(s).To investigate the AHBA synthase gene and some other genes associated with AHBA biosynthesis in Streptomyces sp.4353,the AHBA synthase gene and its flanking regions were amplified by combinations of PCR,then cloned and sequenced.In the 2872bp DNA we obtained, the ORFs of AHBA synthase and AHBA oxidoreductase and AHBA phosphotase(partial)were discovered.The AHBA synthase gene from Streptomyces sp.4353 was expressed sucessfully in E. coli as inclusion bodies in our study.The AHBA synthase gene,AHBA oxidoreductase gene and, possibly,the AHBA phosphotase gene(partial)could be useful candidates for re-constitution of hybrid AHBA biosynthetic pathways heterologously(such as in E.coli),therefore providing a good starting point for combinatorial biosynthesis of ansamycins.