Antibiotic Overproduction Of Streptomyces Diastatochromogenes By Genome Shuffling

Streptomyces diastatochromogenes 1628,isolated from Tianmu Mountain in Zhejiang Province,has a high antifungal activity against various plant pathogenic fungi,and can biosynthesize at least 4 kinds of antibiotics,including toyocamycin,tetramycin A,tetrin B and tetramycin P.4 mutant strains with different phenotypes10(str~r),88(rif~r),99(par~r)and 143(str~r)were screened by ribosome engineering in our previous study.In this study,genome shuffling method was furthermore applied to increase the secondary metabolites of S.diastatochromogenes.The mutant sites of rsmG,rpsL and rpoB,growth characteristics of the high-yielding fused strains were also analyzed.The results are as follows:1.Conditions for protoplast fusion were optimized:The rate of protoplast fusion was 7.29%,fusion by 40%PEG 4000,under 20?,10 min.2.Two fused strains R3-33 and R3-37,were obtained by three rounds of genome shuffling and screened for resistance to three antibiotics,streptomycin,rifampicin and paromomycin.R3-33 had the higher capacity to produce toyocamycin(664.2 mg/L),5.9-fold higher than the parental strain 10(str~r).Another fused strain R3-37 could effectively enhance the productivity of tetramycin P(442.2 mg/L),tetrin B(155.5 mg/L)and tetramycin A(377.6 mg/L),5.4-,2.8-,4.2-fold higher than that of the parental strain 10(str~r),respectively.3.The mutation sites of high-yielding fused strains were detected.The results of gene sequencing showed that the high toyocamycin-producing strain R3-33 had a nonsense mutation in rsmG gene,the 48th codon encoding the cysteine became the stop codon;A point mutation was found in the rpsL gene,the 122th codon encoding the glycine to valine.The high tetraene macrolide antibiotics-producing strain R3-37had a nonsense mutation in rsmG gene,the 48th codon encoding the cysteine became the stop codon.Only rsmG mutation in parental strain 10(str~r)shuffled to the high-producing strain,but the mutation on rpoB gene was not detected.4.The growth characteristics of high-yielding strains were studied.The results showed that the R3-33 and R3-37 grew more slowly than 1628,and the time of s secondary metabolites synthesis was accelerated.The mycelia dry weight of R3-33and R3-37 could reach 6.1 mg/L and 6.5 mg/L,while the wild-type strain was only 2.6mg/L.Secondary metabolites production by the fused strains was found to be medium-dependent.The effects of different media(GYM,2×GYM,M3G,CP and TPM media)on secondary metabolites production by mutant strains were detected by HPLC.The high-yielding strain R3-33,produced 1173.6 mg/L toyocamycin in TPM medium,which were 10.6-fold higher than that of the parental strain 10(str~r).And the 3-37,produced 965.2 mg/L tetramycin A,475 mg/L tetramycin P,and 832 mg/L mg/L tetrin B in TPM medium,which were 11.7-,8.4-and 9.2-fold as much as the parental strain10(str~r).The results of this study provided new idea and method for the breeding of S.diastatochromogenes.The recursive protoplast fusion to S.diastatochromogenes by genomic shuffling could effectively enhance the capacity to produce toyocamycin,tetramycin A,tetramycin P,and tetrin B,and it was more economical and faster than traditional breeding methods.