High-Throughput Screening Of Ansamitocin P-3 High-yield Strains Based On ARTP Mutagenesis

The ansamitocin,one of the bacterial maytansinoid,belongs to macrolactam antibiotic and exhibits potent antitumor activity.At present,many kinds of antibody conjugating molecules（ADC）related to ansamitocin have entered different stages of clinical trials.In view of the important medicinal value and broad market prospect of ansamitocin P-3（AP-3）,improving its production has become a key research project.However,the yield of ansamitocin by microbial fermentation is still very low,and it is difficult to meet the needs of clinical application.In order to fully understand the whole genome information of ansamitocin-producing strains and establish a gene network related to the high yield,our research strategy is to establish a high-throughput screening platform,including rapid and efficient ARTP mutagenesis,24-microtiter plate fermentation and a high-throughput bioassay screening.First of all,a high-throughput bioassay screening system was established using a highly sensitive yeast strain to ansamitocin,Filobasidium uniguttulatum.Through the optimization process,the best parameters of the high-throughput screening system could be determined.The indicator yeast reached mid-logarithmic growth stage at 15 h when cultured in liquid culture in shaking flask.Moreover,when the inoculation volume of seed culture from shake flask to the microtiter plate was 10%,and the incubation time of mixed yeast and ansamitocin was 30 h,a linear growth of the yeast was detected between 0.2-0.7μg/m L of AP-3.Under these conditions,the standard curve between the OD₆₀₀ value and the concentrations of AP-3 was determined with y=0.0228x+1.8377,R²=0.9906.With a 15%increase of the ansamitocin titer as the screening index,high-yield mutants of ansamitocin could be screened out.As for the establishment of rapid and efficient ARTP mutagenesis system,the mycelial fragmentation condition and ARTP exposure time were mainly optimized.When the starting strain NXJ24 was cultured in liquid YMG medium for 24 h,the mycelial fragmentation was most obvious,and cotton spore filters were used to ensure single cells for ARTP mutagenesis.When ARTP exposure time was 120 s,the mortality rate was85.6%.Mutants obtained with an ARTP exposure time of 120 s could be selected for later fermentation.By the high-throughput screening system,two mutants M13 and M144 with the highest yield were selected for the2nd-generation genome resequencing.The sequencing results showed that there are 58 mutated sites in M13 with a mutation rate of 6.35×10^-6 and51 mutated sites in mutant M144 with a mutation rate of 6.12×10^-6.In order to efficiently identify high-yield factors of ansamitocin by GWAS（Genome-Wide Association Studies）,the number of mutation sites in each mutant should be appropriately reduced to less than 10.Under the condition of 100 W electric power and an exposure time of 30 s,the lethal rate was 60.40%,and the positive mutation rate was 11.46%.When the exposure time was 60 s,the lethal rate was 81.45%,and the positive mutation rate was 17.71%.The lethal rate was 95.51%and the positive mutation rate was 9.78%with an exposure time of 120 s.Four mutants,30A1,30A6,120G8 and 120G10,were obtained with 21.2%,20.8%,23.3%and 34%titer increase of ansamitocin in shaking flasks,respectively.Besides that,under the condition of 120 W electric power,when the exposure time was 30 s,the lethal rate was 60.40%,and the positive mutation rate was 11.46%.When the exposure time was 60 s,the lethal rate was 81.45%and the positive mutation rate was 9.78%.The lethal rate was 95.51%and the positive mutation rate was 17.71%with an exposure time of 120 s.Four mutants,15H5,30G12,30H9 and 60D3,were obtained with titer increase of ansamitocin by 18.1%,23.3%,18.2%and 15.3%,respectively.In conclusion,a high efficiency and sensitivity high-throughput screening system was established based indicator yeast.And based on the mutation sites’results by 2nd-generation genome resequencing of the two high-yield mutants induced by ARTP at the initial stage,the positive mutation rate and lethality rate of the mutants under 100 W and 120 W electrical power were explored,and finally eight high-yield mutants were screened.In order to set up a large-scale mutant library in the later stage,the high-yield reasons of AP-3 would be analyzed from the whole genome level,and the metabolic network of Actinosynnema pretiosum would be investaged.