The Screening Of Demethylchlortetracycline Producing Strain With High Yield And The Regulating Of The Metabolic Pathway

This topic reported the mutagenesis by UV、EMS、UV+LiCl、ARTP on Streptomyces aureofaciens.Several mutagenesis methods were made a comparison of the advantages and disadvantages. And ARTP60s was chosen as the best mutagenesis method. On the basis of analysing the existent metabolic pathways of demethylchlortetracycline (for short,DMCTC), we selected malonic acid and DMCTC as the screening materials.Finally,six high yield strains were got, and the mutant strain A6-6-9had the better stability and higher potency, the potency of A6-6-9was4900u/ml which was22.55%higher than the original strain.A preliminary study was carried out to find the reason which resulted in the increase of the potency in A6-6-9. Through the substrate metabolism and strains growth trends, we can found that, compared to the original strain, A6-6-9did not significantly improve the growth of strains, only to maintain a longer stable period and delay autolysis, make the anabolic were still active in the last phase. Organic acids were also studied during fermentation process, compared with the original strain, the A6-6-9mutant accumulated more precursor organic acid such as:oxaloacetic acid, malonic acid entered into the compound of Malonyl-CoA, finally enhanced the DMCTC productivity.2-DE and protein points by MS showed that malate dehydrogenase (MDH), glutamine synthetase (GS) and6-hydroxylation enzyme expressed more in the mutant strain, so that enhanced the DMCTC productivity.During the fermentation of Streptomyces aureofaciens, adding a certain amount of MgCl2、ZnCl2、CoCl2and MnCl2in difficent time of fermentation, the yield of DMCTC was respectively increased by13.6%、14%、12.5%and18%. By measuring several key enzymatic activities and amounts of organic acid, it was found that the adding of0.1%MnCl2at72h could promote the enzymatic activity of PEP、CAC、ACC and MCM. So that the strains could accumulate high concentrations of oxaloacetic acid and malonic acid, made the metabolic pathway flow more to DMCTC synthesis pathways.Finally, the potency of DMCTC was increased.On the basis of metabolic pathway of DMCTC and the catabolic pathway of branched chain amino acid, valine isoleucine and leucine were added to the fermentation medium.The results indicated that the potency incease about13.78%by adding leucine,but no provious of valine and isoleucine. By analyzing the change of strains growth and the total sugar, we found that the addition of0.5g/l leucine at the beginning of fermentation could promote the growth of strains and provide energy and precursors to the synthesis of DMCTC.Finally, by measuring ACC enzymatic activities and amounts of acetic acid, found that leucine could promote the enzymatic activity of ACC, make the acetyl CoA flow more to malonyl Co A synthesis, eventually increased the yield.