Mupirocin Producing Strains Mutation Breeding And Fermentation Conditions Optimization

Mupirocin is a monoacid compound with pyran ring synthesized by 35 groups of related genes in Pseudomonas fluorescen.It is mainly a broad-spectrum antibiotic for topical use.Negative bacteria and Staphylococcus aureus have good inhibitory effects,and their main site of action is tRNA synthetase(aminoacyl-tRNA synthesis,AaRS)inside the bacteria.Studies have shown that mupirocin is decomposed very quickly in human blood,has few side effects,and has no cross-resistance when combined with other antibiotics.It is safe and easy to use,economical and has the advantages of great development potential.In this article,we will use the laboratory-preserved mupirocin-producing Pseudomonas fluorescens strain PFMUMPA as the starting strain.In the process of strain selection,natural separation technology and ion implantation mutagenesis technology will be used for large-throughput screening and stable through passage.The sex test obtained stable and high-yielding mutant strains.Through the observation of colony morphology,and using mutant strains as the starting strain for fermentation process optimization,five aspects of slope culture conditions,seed culture conditions,fermentation culture conditions,fermentation medium and partial tank technology were optimized,and all levels were selected.Suitable culture conditions;through experimental design and analysis software,fifteen carbon sources such as glycerol,glucose and sorbitol,18 nitrogen sources such as urea,wheat germ powder,tryptone,etc.;iron citrate,sodium succinate,magnesium sulfate And other 17 kinds of inorganic salts;21 kinds of nutrients such as amino acids and vitamins.Screen new formulas of nutrient substance combinations that can increase fermentation potency,and finally scale up experiments on a 50L bioreactor to obtain the highest fermentation potency of the mutant strain and corresponding The best culture conditions and technological process.Experimental results are shown below;1.The stable and high-yielding mupirocin strain PFMUMP27 was selected from the mupirocin-producing strain PFMUMPA through natural isolation methods and strain stability test.2.Using PFMUMP27 as the starting strain for ion implantation mutagenesis,140 s ion mutagenesis was carried out at 20 keV energy,and the stable and high-yielding mupirocin mutant strain PFMUMP27-14(ie PFMUMPB)was obtained.The colony phenotype of this strain is that the colony is large,the surface is slightly rough,the middle is convex,and the transparent range is small.3.Obtained by optimizing the culture conditions,the best condition for slant culture of PFMUMPB is 30℃ for 36 hours;the best condition for seed culture is 32℃,shaker speed 240 r/min,and pH 6.6 for 48 hours;the best condition for fermentation culture is 28℃,Cultivate 72 hours under the condition of shaker rotation speed 240 r/min and pH 6.6.4.Through multiple single factor screening combined with Minitab 19 and Design-Expert 12 software analysis and fermentation test verification,a new process and new formula suitable for PFMUMPB were obtained.The fermentation test of the new formula on the 50 L fermenter is 32%higher than the original formula,and it can be increased by 10%after updating the fermentation process.The verification result of the 50 L fermentation test shows that the fermentation potency of the new technology and formula is 46%higher than that of the original technology and formula.