Enhancement Of Ansamitocin Production By Genetic Engineering And Mutation Breeding

Ansamitocin P-3?AP-3?is a macrolactam antibiotic that belongs to type I polyketide,which has been identified as a potent antitumor agent by binding to the?subunit of tubulin,blocking the assembly of tubulin.Nowadays,in spite of efforts made to enhance its production through fermentation medium optimization and metabolic engineering,the production of AP-3 remains limited.Given its significant pharmaceutical value and rising marketing demand,it is an urgent task to enhance the titer.Based on the previous investigations,combined genetic modification and random mutation breeding were performed to achieve increased AP-3 titer.Nocardiopsis ansamitocini EGI80425 is a newly isolated AP-3producer.The genome of N.ansamitocini EGI80425 was sequenced,general features of its genome and the distribution of secondary metabolic gene clusters in N.ansamitocini EGI8042.Prior to the manipulation of this strain for improving ansamitocin production,an efficient genetic system is required.A conjugation system was firstly established with mycelia and integrative plasmid pIB139,and an efficiency of 6.6×10^-3 was achieved by optimizing incubation time to 20 h for conjugation,4×10⁴ CFU recipient,1.2×10⁷ CFU donor,1:300 recipient/donor ratio and 40 mmol/L Mg²⁺in the ISP4 medium.In addition,given the different performance between integrative plasmid and replicating vector,a conjugation system using a replicating vector pJTU1278 for gene deletion,with 4×10⁵ CFU recipient,1.2×10⁷ CFU donor,1:30 recipient/donor ratio and an efficiency of 6.2×10^-4 was established after optimizing recipient/donor ratio.The establishment of this system laid the foundation for the genetic engineering and biosynthetic study of this strain.There are three PKS gene clusters in the genome of N.ansamitocini EGI80425,and only two of them were actively expressed?Cluster I and Cluster VII?.They might compete with AP3biosynthesis for the same precursors and therefore were deleted using the replicating vector.Compared to the wild-type,AP-3 productions in the Cluster I deleted mutant?CXY01?and Cluster VII deleted mutant?CXY02?were increased by 95.3%to 12.6 mg/L and by 15.2%to 7.4 mg/L,respectively.Besides genetic modification,another breeding strategy using atmospheric and room temperature plasma?ARTP?mutagenesis was applied to improve AP-3 production in A.pretiosum ATCC 31280.The ARTP has relative safety and high mutation rate,and the mutants have higher genetic stability.According to the lethal curve of A.pretiosum ATCC 31280,an ARTP mutation breeding system was established.Subsequently,fermentation conditions of 25oC,180 rpm for 5 days in 24deep-well plates were optimizing based on the conditions in shake-flask.A high-throughput screening method was established using a sensitive indicator yeast strains and different concentrations of AP-3 to select the ideal mutants with increased production.Finally,SNV and InDel mutated sites were found through resequencing,and the causes for the yield improvement were premlinarily analyzed.In conclusion,an efficient genetic manipulation system was established and applied for gene deletion,which resulted in improved ansamitocin productions.Besides,ARTP was used to obtain ansamitocin-overproducing strain and paved a way for further titer increase by genetic engineering and mutation breeding.