Strain Selection And Genetic Engineering Construction Of Staurosporin&Optimization Of Fermentation And Purification Technology

Midostaurin is a new drug approved by FDA for the treatment of acute myelogenous leukemia in 2017.The key intermediate of its synthesis is staurosporine,which is produced by microbial fermentation.At present,it is reported that the fermentation unit of staurosporine-producing bacteria is low and the production cost is high.In this study,strains with high yield of staurosporine were obtained by strain mutagenesis and resistance screening,and then the electrical transformation conditions were optimized and the construction of genetically engineered bacteria was carried out to further improve the production capacity of strains.Finally,the optimization of fermentation and purification process of high yield strain was completed.First,UV and NTG mutagenesis techniques combined with resistance screening methods of staurosporine,streptomycin,tryptophan and methionine were used to mutagen and screen the mutants of staurosporine-producing strain SIPI-ST-23.A high yield and stable mutant strain was obtained,which was named as SIPI-ST-07.The shaker fermentation unit reached 503 mg/L,which was 191%higher than that of SIPIST-23.Secondly,the electrical transformation conditions of strain SIPI-ST-07 were optimized and its genetically engineered bacteria were constructed.Taking strain SIPI-ST-07 as the starting strain,the optimal electrical transformation conditions were obtained:the glycerol/mannitol concentration ratio was 20:1.5,the glycine concentration was 2%,the electric field intensity was 9 kV/cm,the SIPI-ST-07 mycelium concentration was 150 μL,the plasmid concentration was 1 μg,the incubation time was 6 h and the temperature was 0～4℃.The electro-transformation efficiency was improved from the initial 4×106 to 9.6×108 CFU/μg DNA.Using PCR amplification to obtain corresponding gene fragments,constructing recombinant plasmids through homologous recombination,and screening to obtain transformants.The results showed that,five recombinant plasmids,pIB139-vgb,pIB139-fkbN,pSET152-staR,pSET152-staR-ermE*p and pSET152-staR-kasO*p were obtained.,then six engineering bacteria were constructed,such as SIPI-ST-A-1(pSET152),SIPI-ST-B-17(pIB139-vgb),SIPI-ST-C-6(pIB139-fkbN),SIPIST-D-5(pSET152-staR),SIPI-ST-E-9(pSET152-staR-ermE*p)and SIPI-ST-H23(pSET1 52-staR-kasO*p).Next,the shaking fermentation process of SIPI-ST-07 strain was optimized.The best carbon sources were glucose and corn starch,the best nitrogen sources were cotton seed cake powder,soybean cake powder and yeast powder,and the inorganic salt ions were ammonium dihydrogen phosphate,copper sulfate pentahydrate,magnesium sulfate heptahydrate and ferrous sulfate pentahydrate,and calcium carbonate and sodium sulfate were added.The optimum fermentation conditions were as follows:shaking speed 220 rpm,seed culture temperature 28℃,fermentation temperature 28℃,first seed inoculum quantity 7%,initial pH 6.3,shaking bottle quantity 30 mL,fermentation cycle 5 d.The fermentation titer of SIPI-ST-07 was increased by 67%to 835 mg/L by the optimization of shaking flask fermentation process.The optimized fermentation process was verified by engineering bacteria,in which the yield of engineering bacteria SIPI-ST-H-23(pSET152-staR-kasO*p)increased by 17%,compared with that of strain SIPI-ST-07,reaching 923 mg/L.Then,the process of 5L fermenter of SIPI-ST-H-23 was studied,and factors such as inoculation amount,glucose supplement and pH control were optimized.The optimal inoculation amount of secondary seeds was 10%,the reduced sugar of late fermentation was maintained at 10～20 g/L,and the pH was maintained at 6.5～6.9.Under the optimized 5L fermenter condition,SIPI-ST-H-23 strain with high yield showed that the unit of staurosporine was 1002mg/L,which increased by 53%compared with the original process.Finally,the purification process of staurosporine was studied,and the optimum purification process of staurosporine was established.The pH of staurosporine fermentation liquid was adjusted to 10～11,the fermentation liquid was centrifuged,and the bacterial residue was dried and crushed.With butyl acetate extraction for 3 times,the single step yield could reach about 90.1%.The extraction solution of ethyl acetate was added with hydrochloric acid for two times,and the single step yield reached 86.1%.Then,the extraction solution of acid water was added with ethyl acetate for two times,and the single step yield reached about 80.1%.The total yield was above 55%and the purity of staurosporine was above 97%by HPLC.In this study,the high yield engineering bacteria of synthesis intermediate staurosporine for midostaurin was constructed,and its fermentation and purification process was established,which laid a foundation for industrialization.