Breeding And Optimization Of Chlortetracycline Production Strain

Chlortetracycline,also known as chlortetracycline,is a tetracyclic antibiotic produced by the fermentation of Streptomyces aureus.It has broad-spectrum antibacterial activity against Gram-positive bacteria,Gram-negative bacteria,Rickettsia,and trachoma virus.And lymphogranuloma virus,etc.have strong inhibitory and killing effects.Chlortetracycline is clinically mainly used for pneumonia,trachoma,typhoid fever,lymphogranuloma and other bacterial infections.However,due to its large side effects,the current clinical application is mainly for external drugs and ointments.In addition,chlortetracycline is also one of the few antibiotics that can be used as feed additives.It can not only effectively prevent common animal intestinal diseases,but also has the effects of improving feed utilization,fattening,and weight gain.In recent years,with the rapid development of domestic and foreign animal husbandry,the demand for chlortetracycline is increasing.The current production method of chlortetracycline is the fermentation production method of Streptomyces aureus.However,in the actual production process,there are often problems such as poor genetic stability of the production strain,serious strain degradation,unstable production performance,and low yield of chlortetracycline.In order to solve these problems in the production process,we conducted a preliminary culture and fermentation performance study on a chlortetracycline production strain Streptomyces aureofaciens JH4.568 from Tianjin Jinhui Pharmaceutical Group Co.,Ltd.,and investigated the growth patterns of different colonies.The relationship between the characteristics and the yield of chlortetracycline has been preliminarily determined the morphological indicators for the selection of high-yielding strains in the production process,and through the natural selection of strains,a strain with good genetic stability and high fermentation yield of chlortetracycline has been obtained.The strain S.aureofaciens JH-1.Through the preliminary optimization of the fermentation medium,the highest yield of chlortetracycline in shake flask fermentation can reach 2720 U/m L,which is an increase of 14% compared with the initial strain.After 10 consecutive passages,a relatively stable production level can still be maintained without significant degradation.On this basis,using the chemical properties of chlortetracycline acid-base amphoteric compounds,the extraction method after fermentation of chlortetracycline was improved,the extraction efficiency was effectively improved,and an efficient extraction method of chlortetracycline was created.In order to further improve the fermentation yield of chlortetracycline by genetic engineering methods,the genetic transformation method of strain S.aureofaciens JH-1 was studied.By optimizing the conjugation culture conditions,the plasmids p KC1139 and p SET152 containing the apramycin resistance gene(amp R)were successfully transformed into S.aureofaciens JH-1,in order to further improve the chlortetracycline fermentation of this strain by means of genetic engineering.Production provides technical support.In order to quickly obtain high-yielding chlortetracycline strains,try a genetic modification method using kanamycin resistance gene(kana R)as a selection marker,and integrate the resistance genes into specific sites in the chlortetracycline synthesis gene cluster to achieve high-yielding strains Targeted screening.During the experiment,it was discovered that there is an unknown regulatory factor in front of a specific site that would be affected.Try to use molecular biology and other methods to analyze it to block it.After testing,blocking the bacterial yield did not affect it,which proved that this gene is not involved in chlortetracycline biology.The key enzyme gene of the synthetic pathway.On this basis,the function of some genes in the chlortetracycline biosynthesis gene cluster was further analyzed,and anti SMASH was used for gene prediction.In order to obtain higher yielding strains,the enzymes related to tetracycline expatriation and transport in the synthetic gene cluster were overexpressed.The results showed that The yield of the expression bacteria increased,but the yield was similar to that of the original strain that undergoes cell disruption.It can be obtained with a 12% increase on the original basis,reducing the difficulty of extraction,and obtaining a production strain with a maximum of3220 U/mL.