Flask Fermentation Optimization Of Avermectin And Study On High Yield Metabolomics Mechanism

Avermectin,produced by Streptomyces avermitilis through secondary metabolism,is a kind of high efficiency,spectral and low toxicity antibiotic,which has been widely used in agriculture,forestry and medical fields.At present,China is the only producer of avermectin in the world,so the study of abamectin is of great significance.In this study,the medium composition and culture conditions of S.avermitilis were optimized,and the high-yield mechanism was preliminarily explored based on GC-MS metabomics methods.In this experiment,S.avermitilis 9-39 was used as the starting strain to optimize the composition of avermectin fermentation medium.Firstly,the composition of S.avermitilis fermentation medium was optimized by single factor addition experiment.Thirteen factors Al₂（SO₄）₃,Co Cl₂,shikimic acid,malonic acid,1,4-butyrolactone,polyethylene glycol-2000,sodium propionate,sodium nitroprusside,sodium butyrate,sodium-β-glycerophosphate,calcium,colchicine and Al₂O₃were selected for single factor addition test.Compared with the initial medium,the titer of avermectin increased by 4.15%,14.28%,5.12%,7.58%,18.65%,6.56%,10.26%,8.35%,7.86%,4.79%,6.12%,17.25%and 3.96%respectively.Co Cl₂,1,4-butyrolactone and colchicine,which had significant effects on avermectin yield,were selected by Plackett-Burman experiment,and the central point of Box-Behnken experiment design was determined by the steepest climbing experiment.The Box-Behnken experiment and the fitted regression equation obtained from the analysis were used to determine the optimal combination of media.,glucose 12.8g/L,maltose 19.2g/L,threonine2.0 g/L,K₂HPO₄0.3 g/L,Mg SO₄·7H₂O 0.5 g/L,Na Cl 0.5 g/L,Fe SO₄·7H₂O 0.01 g/L,Mn SO₄0.015g/L,MOPS 5.0 g/L,Co Cl₂4.25 mg/L,1,4-butyrolactone 0.85 mmol/L,colchicine 7.98 mg/L,through the verification experiment,avermectin titer reached 456.97mg/L,26.93%higher than the initial medium.And the results of the verification experiment and the optimal value of 450.21 mg/L predicted by the fitting regression equation were almost the same,indicating that the whole experiment design is reasonable and the results are reliable.After optimizing the medium components,the culture conditions were optimized.Firstly,single factor experiment was conducted to explore the effects of four factors,namely rotational speed,inoculum size,liquid volume and fermentation time,on the production of avermectin by S.avermitilis fermentation.Then the orthogonal experimental design was carried out and the experimental results were analyzed.The optimal combination of rotational speed of 300 r/min,inoculum size of 10%,liquid volume of 30m L（250 m L conical flask）and fermentation time of 11 d was obtained and verified by shaking flask fermentation.The non-targeted metabolomics method of GC-MS was used to sample the intracellular small molecule metabolites of the high-yield medium and the original medium fermented by the medium optimization at 2 d,4 d,6 d and 8 d during the fermentation process,respectively.The samples were processed for computer detection,and the instrument was used for online data collection.Detected 234 species of small molecule metabolites and were compared with the NIST database 17.0,52 kinds of metabolites identified.After statistical analysis of the original data,8 different metabolites related to avermectin synthesis were screened out,respectively,acetic acid,α-ketoglutaric acid,glucose,leucine,valine,lauric acid,stearyl acid,oxaloacetic acid.The key metabolic pathways of these 8 different metabolites were analyzed,and it was found that the high-yielding group had stronger glycolysis and tricarboxylic acid cycle,which could provide precursors for avermectin synthesis.