The Studies On The Gene-engineered Strain Producing 5-O-avermectins

Avermectins are 16-membered macrocyclic polyketide antibiotics produced by Streptomyces avermitilis. They are known to be an excellent anthelmintic agents with a high efficiency and broad spectrum of activity against a variety of nematode and anthropod parasites, and with a low level of side-effects on the host. Furthermore, a series of semi-synthesized avermectins could be produced by chemical synthesis with avermectins as the raw materials.In this paper, the molecular biological methods were used and a gene-engineered strain, which was able to produce 5-O-avermectins, was obtained by the gene disruption of aveF which was involved in the biosynthesis of avermectins. Besides, the function of aveC in the gene cluster involved in the biosynthesis of avermectins was also explored.First, the PCR products of 1.8 kb DNA fragment containing aveF gene were obtained by PCR method with the primers designed according to the nucleotide sequence up and down stream of aveF gene reported by literature. Then the 1.5 kb DNA fragment of apramycin resistance gene was inserted into the SalI site in the aveF gene after the PCR product was ligated into the plasmid vector pMD18-T. Afterwards, the 3.3 kb DNA fragment was excided out, recovered and then ligated into the MCS site of plasmid pHJL401, a shuttle vector of Streptomyces-E. coli with the function of conjugal transfer. As a result, the recombinant disruption plasmid pPC5 was obtained.After the plasmid pPC5 was transferred into the Streptomyces avermitilis by conjugal transfer, the recombinant strain AV-F1 with a disrupted aveF gene was obtained by selecting strains which were resistance to apramycin and sensitive to thiostreptone.The analysis of fermentation products of strain AV-F1 showed that this strain produced mainly two components which were 5-O-avermectinl and 5-O-avermectin2 respectively. Moreover, it produced a small amount of avermectinB1a.In order to explore the function of aveC gene in the biosynthesis of avermectins, the aveC gene was interrupted by inserting a DNA fragment of antibiotic resistance gene. As a result, a recombinant strain AV-C24 was obtained which produced only small amount "2" components without "1"components. This indicated that aveC gene was related to the biosynthesis of "1"components, it may involved in the dehydration-cyclization reaction of the polyketide chain which was released from polyketide synthase after the thioester bond was hydrolyzed.