Breeding Strains Of Streptomyces Avermitilis With High-Yield

The avermectins from Streptomyces avermitilis are the most effective agents to kill helminthes, nematode and arthropod parasites at present. They are eight components, and the Bl component has the most effective antiparasitic activity. Avermectins are the secondary metabolites and its biosynthesis is very complex. Now the biosynthetic pathway of the avermectins and the most important enzymes have been found. With the development of molecular biology, complete genome sequence has been determined and 30 gene clusters related with avermectins biosynthesis have been got and the key gene clusters have been achieved. Because of these, the biosynthesis of avermectins can be regulated in the point of molecular so that the high-yield strains are obtained. What is more, many new technologies have also been used to breed the strains of Streptomyces avermitilis. In our lab the ion implanting technology has been used and the high-yield strains have been got. More and more people realized that the environment of space is so complex that the mutation result can not be imagined on the earth. Therefore the technology of screening the high-yield strains carried by spaceship has attracted more and more people.In the experiment NH4+ with the ability of inhibition the biosynthesis of avermectins can increase the percent of A components of avermectins. So the strains of anti- NH4+ can enhance the yield of avermectins and the ratio of B components of avermectins. It has been confirmed that methionine offers C5-O methylation to A components of avermectins and can help to synthesize S-adenosylmethionine(SAM). So the strains that can use methionine to metabolism are hoped to be get so that the strains with high percent of B components are got. According to the mechanism, strains sensitive to sulfanilamide are bad at transferring methyl, so there are lower of SAM in the strains and the B components may increase. Because of these reasons, after mutation by ion implanting strains Z138 was mutated by NTG combined with the ways of chlorocaeaium tolerance, methionine tolerance and being sensitive to sulfanilamiderespectively. 5 high-yield strains with genetic stability, namely A22, D5,D6,77 and 73, were obtained. Their titers were 4974?g/mL, 4214?g/mL, 4930 ?g/mL, 4410?g/mL, 4369 u g/mL, respectively. Compared with the original strain, the titers were improved 51.58%, 28.48%, 64.87%, 16%, 15% respectively.After mutation of ion implanting and NTG strain HY1 was carried by spaceship. Then 1237 mutant strains were isolated and purified.24 high-yield strains with genetic stability were got by two rounds screening. Their titers all increased over 24% and the highest one reached 83%. By the thin layer chromatograph(TLC) and high pressure and liquid chromatograph(HPLC), 2strains with high ratio of Bla components were obtained. The liter of strain HY365 reached to 5924.67 ?g/mL and Bla component was 3892 ?g/mL,65.74% of the tiler. And the Bla component of strain HY516 was 3076 u g/mL,59.9%ofthetiter.An optimizing medium composition 7 was found via orthogonal experimenl and the resull was confirmed that corn starch and peanut influenced the liter of fermentation. Experiment on the influence of precursor was made and the liter can increase evidently. When ihe concenlralion of the precursor reached to 2.5mmol/L, the tiler of avermeclins was 6424 u g/mL.