Breeding The Strain With High-Yield Of Pentostatin By Combined Mutation Strategy

Pentostatin(PNT)is a purine nucleoside antibiotic and a highly potent inhibitor of adenosine deaminase.PNT can be used to treat clinical diseases in a variety of tumors.The synthetic methods of PNT mainly include chemical synthesis and biosynthesis.Compared with biosynthesis,the chemical synthesis is costly,the reaction conditions are harsh and the route is complicated,which makes the implementation more difficult.The reaction conditions of the biosynthesis are mild and environmental friendly.Moreover,it has strong feasibility,but the biggest bottleneck in biosynthesis is the low yield of PNT.Based on this,this work mainly used metabolic regulation,atmospheric and room temperature plasma(ARTP)and ribosome engineering to mutate and breed high-yield PNT mutants.The knockout strains of key genes Ada E and Ada J in PNT synthesis and the overexpression strains of gene Ada A were constructed.The productivity of PNT was not detected by LC-MS.It may be that gene deletion and overexpression manipulation interfered with the regulation mechanism of the gene cluster itself,which affected the expression of PNT biosynthetic gene cluster.The high-yield PNT strains were selected by ARTP mutagenesis.Using Actinomadura sp.ATCC39365 as the starting strain,the lethality rate was used as the index,and the mutation conditions of ARTP were optimized to determine the optimum conditions for ARTP mutagenesis to be 130 w,12 SLM,200 s.The strain with good genetic stability was selected as Actinomadura sp.M-26(2.740 mg/L),and the yield of PNT in the shake flask fermentation was increased by 11.75% compared with the starting strain.The high-yield PNT strains with single resistance were selected using ribosomal engineering techniques.First,the minimum inhibitory concentration and optimal screening concentration of the spare antibiotic against wild type Actinomadura sp.ATCC39365 were determined.One PNT high-yield strain Actinomadura sp.M-65(3.298 mg/L)with good anti-penicillin and good genetic stability was obtained at the optimal screening concentration.Compared with the original strain Actinomadura sp.ATCC39365,the PNT yield increased by 34.5%.Actinomadura sp.M-26,which was selected by ARTP mutagenesis,was screened by streptomycin resistance to obtain a stable PNT high-yield strain Actinomadura sp.MM-10(5.188 mg/L).Compared with the Actinomadura sp.M-26,the PNT yield increased by 89.34% and the PNT yield in shake flask fermentation increased by 111.58% compared with the original strain.The double resistant PNT high-yielding strains were selected using ribosomal engineering.Based on the strains of Actinomadura sp.M-22,Actinomadura sp.M-26,Actinomadura sp.M-53 and Actinomadura sp.M-65 with high-yield PNT,six strains with high-yield PNT were obtained by resistance screening.The genetically stable and high yield mutant Actinomadura sp.MM-45(6.077 mg/L)was obtained,and the PNT yield increased by 84.15% based on the mutant strain Actinomadura sp.M-65.Compared with the starting strain,the PNT productivity in the shake flask fermentation increased by 148.44%.The key gene mutation sites in mutant strains were analysed: The analysis of mutation sites in key genes of 6 Str-resistant strains and 1 Gen-resistant strain by using Blast,Clustal X and Snap Gene Viewer software.Among the 6 Str-resistant strains,5 of the rsp L genes were deleted at base 28 of the rsp L gene,and only the rsp L gene of Actinomadura sp.MM-46 was not mutated.Compared with the reported literature,it may be that the rsp L mutation lead to Str-resistance.5 of the 6 Str-resistant strains had random mutations in the rpo B gene and had more mutation sites,while Actinomadura sp.MM-26 did not undergo mutation.It is hypothesized that the increase in PNT productivity is not related to rpo B.The main mutation positions of the rsm G gene mutation of 6 Str-resistant strains were 188 C deletion,193 C deletion and 467 T replacement C.It is possible that the mutation of the rsm G gene can also obtain Str-resistance.The mutation site of the rsl F gene in one Gen-resistant strain was 175 T replaced C and inserted base A after 462,and the mutation result may cause Gen-resistance of the strain.