| AnsamitocinP-3(AP-3)is an active and potential anti-tumor maytansinoid,which is usually produced by Actinosynnema spp.The microorganism has much attention on clinical trials of tumor therapy,due to the highest activity and the efficiency of immunoconjugates as target-specific chemotherapeutic agents.At present,the low yield of AP-3 has impeded its application inmedicine.In recent years,many approaches have been developed for the improvement of AP-3 production,such as genetic modification and medium optimization.However,traditional bioassay-guided discovery strategy do not meet the requirement of industrial production,due to higher cost,more labour,and longer time,which have been restricted the current production..In this study,protoplast regeneration,chemical mutation,the modern industrial microbial breeding techniques with a fast,efficient and high-throughput screening method were applied to obtain a strain with high-yield of AP-3.The results revealed in this study could be helpful to further improve the production of AP-3.The results are listed as follows.1.The strain can produce high yield of AP-3.According to the data of its 16S rDNA gene sequence and some morphological characteristics,the strain was belong to Actinosynnema mirum.2.The quality and quantity of AP-3 in fermentation broth were determined by the methods of Antibacterial activity experimental and thin-layer chromatography(TLC)Besides,a simple and highly effective half quantitative detection method was established to distinguish and discard those unimproved strains,which was suitable for screening the strains with high yield of AP-3.To remain only some best performance strains for the further screening program by ultra-high performance liquid chromatography(UPLC),it can greatly improve the efficiency of selection.3.Using Actinosynnema mirum APS1(13.4mg/L)as the initial strain,the spore suspension was prepared for screening the strain with high yield of AP-3 using the binary ethyleneimine(BEI)treatment for five rounds.The dose of mutagenesis concentration of binary ethyleneimine(BEI)was 0.075%.All colonies from the plates were primarily screened by microbe standardization of antibiotic and Thin-layer Chromatography(TLC)after primary fermentation and through UPLC detection when the secondary fermentation was finished inthe second round screening.About 1000 strains were selected for screening in our study.The results showed that more than half of the mutants with high yield of AP-3 were found.However,the best strain was A5-283(38.11mg/L).4.Using Actinosynnema mirum A5-283(38.11mg/L)as the initial strain,the protoplasts were prepared,and further screened using the ultraviolet(UV)and binary ethyleneimine(BEI)treatment for two rounds.The dose of ultraviolet irradiation was 30s and the mutagenesis concentration of binary ethyleneimine(BEI)was 0.04%.All colonies from the plates were primarily screened by microbe standardization of antibiotic after primary fermentation and through UPLC detection when the secondary fermentation was finished in the second round screening.About 1000 strains were screened in our study.The results showed that more than half of the mutants with high yield of AP-3 were found.However,The best 6 strains labeled as P2-102(48.74mg/L),P2-323(49.52mg/),P2-356(48.88mg/L),P2-389(50.31mg/L),P2-423(49.96 mg/L),P2-488(49.86 mg/L)were selected for the candidate library and prepared their protoplast,respectively.The protoplasts from different parent strains were inactivated in the following conditions:UV irradiation for 1 min,60? heat treatment for 30min,and 0.1%binary ethyleneimine(BEI)treat for 1h.After that,35%of PEG 6000 was used to fuse the protoplasts for 30 min at 28?.Those three successive rounds of genome shuffling were carried out to screen and collect more than 900 strains.One shuffled strain producing high yield of AP-3 with good genetic stability was obtained and labeled as strain G3-368.The yield of AP-3 in strain G3-368 reached 86.02mg/L after 5-day fermentation,which was 6.4 times higher than that of the initial strain Actinosynnema pretiosum APS1(13.4mg/L).5.The fermentation culture conditions in shake flask were further optimized for strain G3-368.Based on single-factor experiment,the optimal culture conditions were listed as follows:seed age 48 hours,8%inoculation,initial pH 7.2,45ml in 250ml shake flask,stirring speed 220r/min and temperature 28?.Under the optimal condition,the strain G3-368 could produce AP-3 112.46mg/L after fermentation for 7 days.The conditions offed batch fermentation were optimized by the uniform design experiment.The optimal loading conditions for AP-3 yield were determined,showing:300?L isobutanol was added a day after fermentation,and then 0.4g/L valine and 10%fresh fermented liquid was added after two days of fermentation.Under the optimal conditions of shaking flask fermentation for 7 days,the yield of AP-3 produced by strain G3-368 maximumly reached 132.12mg/L... |
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