Study On Factors Affecting The Stability Of The Seed And Fermentation Process Of The FK506 Production And Its Fermentation Process Optimization

FK506,a polyketide produced by fermentation of Streptomyces,is mainly used as an immunosuppressant for organ transplantation,systemic lupus erythematosus atopic dermatitis and so on,which still has insufficient production capacity compared to its vast market.Current research for the production of FK506 focuses on strain selection and fermentation process optimisation to increase yields.The instability of high-producing strains directly affects the level of secondary metabolite synthesis and production costs,while the instability of high yield traits greatly affects their application.This paper focuses on the screening of high-producing strains of FK506,investigating the factors of instability during seed and fermentation of high-producing strains and optimising the culture process of high-producing strains.In this paper,multiple assays including primary screening of inhibition circles,semi-quantification by TLC and quantification by HPLC were first established.The strain SIPI-FK-6-S-28 was obtained by natural screening from the FK506-producing strain SIPI-FK-006in the laboratory collection with an FK506 potency of 671 mg/L.The strain was initially identified as Streptomyces tsukubaensis by 16Sr RNA sequencing.The strain SIPI-FK-6-S-28was subjected to UV mutagenesis,NTG mutagenesis,high sugar plate screening,high salt plate screening,precursor resistance screening,ribosome engineering and a series of selections.a high-producing strain SIPI-FK-ST-18 was obtained with a 53.6%increase in yield and a potency of 1030.9 mg/L.This strain was selected as the subsequent experimental strain.To solve the problem of unstable FK506 synthesis during plate seeding and fermentation of high-producing strains,the factors affecting stability during plate seeding and fermentation culture were investigated based on literature research and preliminary experiments.The results found that,in terms of plate seed culture,the addition of high concentration of glucose to the plates had no effect on the stability of plate seeds;the addition of hydrogen peroxide to stimulate the strain to produce oxidative stress spores could improve the stability of plate seeds and promote the synthesis of FK506;the use of high concentration of coated plates for passaging could improve the uniformity and stability of plate spore distribution;the addition of 25μg/ml streptomycin to the plates could improve the the stability of plate seeds for passaging;the plates are more stable when stored at 28°C than at 4°C.In addition,in fermentation culture,the addition of defoamer to the fermentation medium could improve the stability and yield of FK506 produced by fermentation shake flasks.As defoamers shoued a significant effect on the fermentation stability of the strain,this factor was further investigated,including the type of defoamer,the amount of addition and the time of addition.The results showed that organosilicon,polyether and polyvinyl alcohol defoamers all had a beneficial effect on the yields of FK506,with organosilicon having the most significant effect;the optimum amount of defoamer was 2%;and the optimum time of addition was the 4th day of fermentation.To further improve the FK506 production of strain SIPI-FK-ST-18,the culture process was optimised,including plate,seed,fermentation medium and culture conditions.The results showed that the best plate medium was ISP4 medium;the optimal fermentation medium formulation was 4.5%maltose,1.0%corn starch,1.0%dextrin,1.0%soluble starch,2.0%yeast powder,0.75%yeast extract,0.8%dried corn pulp powder,0.12%lysine,0.12%leucine,0.04%copper sulphate pentahydrate,0.04%zinc sulphate heptahydrate,0.04%calcium carbonate 0.15%and antifoam agent 2.0%.The optimal plate culture time was 7～12d.For primary seed fermentation,the optimal culture conditions were 60 h of seed culture time,1×2 cm~2of inoculated plate spore area and 12%inoculum volume.For secondary seed fermentation,the optimal culture conditions were 48 h for primary seed culture,10%inoculum for primary seed,36 h for secondary seed culture and 5%inoculum for secondary seed.The optimum shake flask culture conditions were initial p H 7.1,shake flask loading of25 ml,incubation temperature of 25°C and shaker speed of 250rpm.After the fermentation process was optimized,the yield of FK506 was increased by 73.2%to 1785.3 mg/L.Scaled-up incubation of the optimised shake flask process in a 10L fermenter resulted in an increase in fermentation potency of 451 mg/L to 1152.4 mg/L compared to the pre-optimisation period;By optimising the replenishment,adjusting the speed,defoamer addition and stirring paddle,the fermentation efficiency of FK506 was finally increased to 1547.0mg/L,120.7%higher than that before the process optimisation.