High-throughput Screening And Optimization Of Fermentation Conditions For The Improvement Of Ansamitocin P-3

Ansamitocin P-3(AP-3)is a polyketide antibiotic with high antitumor activity.At present,various antibody-drug conjugates of AP-3 are in clinical(pre-)research,such as TDM1,an antibody targeting drug made based on AP-3.However,AP-3 using microorganism has not only a low production,but also complicated fermentation conditions.In this paper,some strategies were used to increase AP-3 production including mutagenesis and high-throughput screening,optimization of fermentation conditions,and gene shuffling.The major work contained the followings:1.Improve AP-3 production in wild strains through combining normal temperature and pressure plasma(ARTP)breeding technology with high through-put screening(HTS)culture screening technology.The results indicated that when the ARTP treatment time was 90 s,mutagenesis effect on wild type strain(WT)was more suitable.The 703 mutant strains was carried out through a high-throughput screening with microplate reader,and a high AP-3 yield mutant strain was finally screened in shake flask fermentation,which named L-40.The AP-3 production was 27.3%higher than that of the wild strain.After 6 times subculture,the AP-3 production was still 22.5%higher than that of the wild strain,which proved that its genetic stability was better.2.Seed culture medium compositions and progressing of fermentation for culture about strain L-40 were optimized,the optimization results showed AP-3 production of strain L-40 reached a further enhancement and 26.8%higher than the wild strain.The composition of optimized seed medium as follows(g/L):glucose10,glycerol10,corn starch 5,yeast extract 5,calcium carbonate 2,pH 7.0,seed culture time shortened from 3 days,to 1 day second subculture was optimized for one time subculture.3.Addition of exogenous amino acids:the addition of leucine,valine and S-adenosine methionine(SAM)in the early stage of fermentation promoted the production of AP-3.When 0.5 g/L leucine and 0.5 g/L valine were added,the production of AP-3 increased by 8.8%and 19.5%compared with the control 205.3 mg/L,respectively.The production of AP-3 was 223 mg/L and 245 mg/L,respectively.When 0.045 g/L of S-adenosine methionine was added,the yield of AP-3 was 268.2 mg/L,which was 10.6%higher than that of control 242.5 mg/L.4.Several protoplasts-related techniques including conditions for preparation and fusion of protoplast were carried out to further improve the AP-3 productivity in Actinomycetes.The high-yielding strains after ARTP mutation was used as the parents of the first round of gene shuffling,and combining with high throughput screening method,after three rounds of genome shuffling,a mutant strain G3-96 was screened,its AP-3 yield in shaking flask fermentation reached to 335.9 mg/L.which was 44.5%higher than parents and a 93.8%increase over the wild strain.5.It was investigated that transcription levels of AP-3 synthesis-related genes by q-PCR analysis of in strain L-40 after ARTP mutagenesis.The results show that AP-3 synthesis-related genes are significantly up-regulated.At the same time,asm6663,a gene that controls cell division,its overexpression resulted in severe mycelial fragments.The gene asm6663 expression was significantly down-regulated in the L-40 strain,and the microscopic examination results showed that the mycelia of the mutagenized strain were compacted,which was positively correlated with the production of AP-3.