Breeding Of High Yield Strains Of Daptomycin And Optimization Of Fermentation Conditions

Daptomycin is a secondary metabolite produced by Streptomyces roseosporus,which is composed of 13 amino acids and belongs to a new type of cyclolipopeptide antibiotics.It is widely used in clinical practice due to the unique mechanism of action.The research and development of daptomycin in China started late,the yield by strain fermentation was low,and the drug price was expensive.Therefore,breeding of high yield mutant strains of daptomycin is very important to improve the yield of the target strains.In this study,the fermentation level of the strains producing daptomycin was improved by breeding and optimization of fermentation conditions.The contents and results of this study were as follows:1.Breeding of high yield strain of daptomycin by single factor mutagenesis.The original strain BNCC 342432 was mutated by four different mutagens,including atmospheric and room temperatureplasma（ARTP）,diethyl sulfate（DES）,dimethyl sulfate（DMS）,nitrosoguidine（NTG）.The mutagenesis time and the dose of four mutagens were determined.The mutagenesis effects of the four mutagens were compared according to the positive mutation rate of the strain and the yield of daptomycin.The results showed that DES had the best mutation effect,the positive mutation rate was 6%,and the highest yield of daptomycin was 12.52 mg/L,which was 21.55%higher than the original strain BNCC 342432.The mutation effects were followed by ARTP and NTG,the positive mutation rate was all 5%,the highest yield of daptomycin was 11.08 mg/L and 12.03 mg/L,respectively,which was14.56%and 16.80%higher than the original strain BNCC 342432.Therefore,two mutagenesis schemes of ARTP-DES continuous mutagenesis and ARTP-NTG continuous mutagenesis were determined.The original strain BNCC 342432 was mutated by ARTP,and screened by 5 antibiotics and precursor analogs decanoate sodium.The results showed that the positive mutation rate of streptomycin and decanoic acid sodium resistant mutants was all31%,the highest yield of daptomycin was 12.4 mg/L and 12.3 mg/L,respectively,which was14.56%and 16.80%higher than the original strain,so streptomycin and decanoate sodium were selected as the best resistance sieve.2.Breeding of high yield strains of daptomycin by continuous mutagenesis.The ARTP-DES was used in the first round of continuous mutagenesis,combined with streptomycin and decanoate sodium resistance screening,five high-yielding mutants were obtained,and the yield ranged from 14.71 mg/L to 15.80 mg/L,which was 41.81%to 53.40%higher than the original strain BNCC 342432.The second round of continuous was performed by ARTP-DES,and the mixed single spore suspension of five strains screened in the first round of mutagenesis was used as the original strain,five high-yielding mutants were obtained,and the yield ranged from 18.45 mg/L to 19.48 mg/L,which was 79.13%to 89.13%higher than the strain BNCC 342432.For the third round of continuous mutagenesis by ARTP-NTG,the mixed single spore suspension of 5 strains obtained in the second round of mutagenesis was used as the original strain,one strain AN-126 with the highest yield was obtained on the streptomycin resistance screening plate finally,the yield was 24.50 mg/L,which was 1.38 times higher than the strain BNCC 342432.3.The differences analyzed by the morphology and genetics between strain BNCC342432 and strain AN-126.From the morphological observation,it was found that the strain BNCC 342432 and the strain AN-126 was round,but compared with the strain BNCC 342432,the strain AN-126 raised in the middle,the sporulation ability were stronger and speed were more faster.When the seed liquid were cultured for 32 h,the mycelium pellets observed were different from that of the original strain BNCC 342432.The mycelium pellets of the strain BNCC 342432 is oval,and the surrounding mycelium distributed evenly.However,the mycelium pellets of the strain AN-126 was circular,the surrounding mycelium distributed dispersedly,and it can be seen that the mycelium wounded tightly from the color of mycelium pellets,and the stronger synthesis ability of daptomycin was detected.BOX-PCR was used to analyze the polymorphism of the starting strain BNCC 342432 and the high-yielding strain AN-126,the bands amplified by BOX primers ranged from 0.25 kb to 2.0 kb,and a specific band ranged from 0.75 kb to 1.0 kb for the high-yielding strain AN-126.4.Effects of vegetable oil,amino acids and growth factors on the yield of daptomycin.The high-yield strains AN-126 was used as the research object,the coconut oil,isoleucine,threonine,L-asparagine,VB₁₂and lipoic acid had significant effects on the yield of daptomycin through the single factor experiment.The optimal fermentation combination ratio were determined by orthogonal design experiments as follow:coconut oil 35 g/L,isoleucine 3g/L,threonine 3 g/L,L-asparagine 5 g/L,VB₁₂2 mg/L and lipoic acid 5 mg/L.The results of variance analysis showed that lipoic acid had extremely significant effect on the yield of daptomycin（P<0.01）,and coconut oil and VB₁₂had significant effects on the yield of daptomycin（P<0.05）.Under optimized fermentation conditions,the yield of higher yield mutant AN-126 was 41.58 mg/L,which was 69.71%higher than that under other conditions.