Screening Of High Avermectin B1a Producing Strain And Its Molecular Modification

Avermectin(AVM),produced by Streptomyces avermitilis,is a class of 16-membered macrolide antibiotics.Streptomyces avermitilis was initially discovered and isolated from Japan Shizuoka soil by Satoshi omura and Merck's research group.Then it was identified as a new species,the fermentation broth of streptomyces avermitilis was found having a strong effect against agricultural pests.The structure of AVM was composed of eight parts,including A1a,A2a,B1a,B2a,A1b,A2b,B1b and B2b.Among them,the B1a is the most efficient component.Currently,as people's understanding of biological pesticide,avermectin has attracted more.and more attention and increasing efforts have been exerted to achieve its application.However,the major production strains of avermectin Bla,which are used frequently at present,need to be improved urgently since the yield is very low.Therefore,in this study,for the elevated output of avermectin Bla,we used Streptomyces avermitilis A3 as original strain,attempting to obtain a high-yield strain through mutation breeding and genetic engineering techonology.In mutation breeding,diethylenetriamine was used as mutagen and streptomycin resistant was used as the selection marker.First,we rejuvenated the original strain A3 and got the strain A19 of which the Bla yield was 3050 ?g/mL.Then it was treated with diethylenetriamine,leading to the generation of variation group.Screening with streptomycin resistant,the highest-yield strain A270 with avermectin Bla yield of 4372?g/mL was screened out.When compared with A19,its production of Bla has increased by 43.3%.Then through protoplast mutagenesis,we gained mutant A1102,of which the yield of Bla was elevated to 4509 ?g/mL.Last,the genome shuffling was introduced.First,six strains that exhibited further improved yield of B1a including A1102,A1116,A1124,A1142,A1119 and A1022 were selected out and pooled as genome shuffling parents.Then they were treaed with:ultraviolet(UV)irradiation for 5 min,mutagenesis with 0.3%diethylenetriamine for 15 min and heating at 50 ? for 5 min.After that different kinds of inactivated protoplasts were mixed in a cell ratio of 1:1,and re-suspended in 5mL stabilizer solution containing 40%PEG(MW 6000).Then the mixture was incubated at 30? for 15 min and spread on regeneration broth containing B1a to obtain feedback-resistant strains.After three rounds of genome shuffling,the strain G310 with the yield of B1a about 5451 ?g/mL was obtained,showing yields of B1a 20.9%and 78.7%higher than that of the A1102 and A19 strains respectively.For the higher output of B1a,we also used the genetic engineering technology based on the strain G310.We knocked out the aveD gene by double exchange first in order to the disappearance of the A component and then screened the high-yield mutant strains based on streptomycin resistant.The mutant strain AK10 has the yield of B1a about 5950?g/mL,showing 9.1%and 95%increase over the original strain G310 and A19 respectively.In addition,we find component 1 and component 2 are the same compound.The reason is obvious since the second module is vital for the DH activity of PKS gene of aveAl and the downstream domains can not distinguish whether the dehydration has happened or not.Taken together,in this paper,by homologous recombination,we use the erythromycin DH which has been confirmed with full functionality to replace the original DH structure,making the complete dehydration come ture.Thus,the hydroxyl component will be reduced and the yield of B1a will be increased correspondingly.