Breeding Of High-yielding Sisomicin Strains And Optimization Of Fermentation Technology

Antibiotics,as the world’s largest class of anti-infective drugs,have a wide range of applications in the medical field.Sisomicin is an aminoglycoside broad-spectrum antibiotic.It is a secondary metabolite of Micromonospora.Its structure and antibacterial spectrum are similar to gentamicin,but its antibacterial effect is stronger.The semi-synthetic compounds netilmicin and plazomicin,which use sisomicin as the precursor,are more active against some drug-resistant bacteria,and have the advantages of less side effects such as ototoxicity and nephrotoxicity.However,compared with other antibiotics,the fermentation level of sisomicin is still low(about 1000 U·mL^-1),which restricts the application of sisomicin and its derivatives.At present,the molecular operation system of Micromonospora,the main strain of fermentative production of sisomicin,is still not perfect,and it is still very difficult to improve the fermentation level of sisomicin.In this paper,a strain of Micromonospora inyoensis OG-1 with a sisomicin fermentation unit of 1029 U·mL^-1 was used as the starting strain,and the protoplasts were mutagenized by atmospheric and room-temperature plasma（ARTP）.Afer obtain high-yield sisomicin mutant strains,we optimize the fermentation process of the high-yield mutant strains,and further improve the biological titer of sisomicin.Finally,through the transcription level analysis of the key enzymes in the metabolic pathway of sisomicin before and after the mutation of Micromonospora,the mechanism of the mutant strain’s high production of sisomicin was preliminarily analyzed.The main findings are as follows:（1）Protoplast mutagenesis of Micromonospora inyoensis which produces sisomicin.First,in order to improve the regeneration and breeding effects of the protoplast-mutated strains,the conditions such as the concentration of lysozyme,enzymatic hydrolysis temperature and time during the preparation of protoplasts were optimized.The results showed that the optimal concentration of lysozyme was 1.5 g·L^-1,the product of protoplast formation rate and regeneration rate was the highest when the enzymatic hydrolysis at 37℃ for 60 min,and the protoplast regeneration effect was the best.By ARTP mutagenesis,the protoplasts of Micromonospora inyoensis were mutagenized,and a mutant strain I4-10 with high yield and genetic stability of sisomicin was obtained.The biological titer of sisomicin reached1389 U·mL^-1,an increase of 35.4% compared with the original starting strain.（2）Optimizing the fermentation process of the high-yielding strain I4-10.Taking the high-yielding strain I4-10 obtained by mutagenesis as the research object,the carbon source and nitrogen source were optimized.The results show that soluble starch and beef meal can replace white dextrin and dry corn steep powder to become the most suitable carbon source and nitrogen source for the mutant strain I4-10.The biological titer of the optimized medium sisomicin reached 1597 U·mL^-1,which is 15% higher than before optimization.The response surface experimental design method is further used to optimize the complete components of the medium.The results of plackett-burman experiment showed that the concentration of soybean meal（hot）and DL-methionine had a significant effect on the biological potency of sisomicin.Using the central compound method design combined with experimental verification,it is found that when the soybean meal（hot）is 32.78 g·L^-1 and DL-methionine is 1.75 g·L^-1,the biological potency of sisomicin is the highest at 1849 U·mL^-1.Compared with the initial starting strain,the strain has increased by more than 80%,meeting the requirements of industrialized production of sisomicin.（3）Preliminary analysis of the mechanism of the mutant strain I4-10 producing sisomicin.A comparative analysis of the transcription level of the key enzymes related to the metabolic pathway of sisomicin and the growth of the strain in the starting bacteria and the high-yielding mutant strains,and preliminarily speculate that the weakening of the pentose phosphate pathway allows more glucose-6-phosphate to be used for the synthesis of the secondary metabolite sisomicin.The enhancement of sisomicin synthesis pathway and TCA cycle pathway may promote the high production of sisomicin in mutant strains.We also speculate on the key enzymes that affect the strain’s synthesis of sisomicin.