Studies On A Gene Transfer System In Streptomyces Tenebrarius

To establish a gene transfer system in Streptomyces tenebrariu.s, a producer of apramycin, tobramycin and kanamycin B, several methods including PEG-mediated protoplasts transformation, conjugal transfer as well as electroporation were investigated. Parameters affecting protoplast formation and regeneration of S.tenebrarius 9904 were firstly studied in detail. Medium CP appeared to be more suitable than R2YE, YEME, GPYP, P3, SGGP for protoplast formation. A procedure of effective formation and reversion of the protoplasts was developed. The strain was shake-cultured at 37扖 for 48 h in medium CR The culture was then transferred to fresh medium CP supplemented with 2% glycine, and shake-cultured at 28 揅 for 20 h. The washed mycelia were incubated in buffer P containing 2 mg/mi lysozyme at 37 揅 for 1 to 1.5 h. Under the conditions, the amount of prepared protoplasts was 4.6 x 1091m1, estimated by microscopic counts. The protoplasts were plated on the R2YE regeneration agar medium, except that the concentration of sucrose and MgCl2~6H2O was 0.2 moIJL and 2OmmoIfL, respectively, instead of 0.3 mol/L and SOmmoIIL. The plates were incubated at 28 揅 for 5 d and the regeneration frequency was about 18%. Many attempts to transform S.tenebrarius 9904 protoplasts were unsuccessful by Streptomyces plasmid vectors pU702, p114083, pMT66O and Streptomyces-Escherichia co/i shuttle plasmids pKCL 140, pI{Z 132 and pCS5 etc. The activity of protoplastassociated DNase was so strong that plasmid DNA was completely degraded when protoplasts and plasmid DNA were mixed together and incubated for lh at 37 慍. No transformant was achieved even if the protoplast-associated DNase was inactivated by heat treatment or dsDNA was converted ssDNA before transformation. All the results suggested that S.tenebrarius expressed restriction endonuclease that blocked transformation by plasmid DNA.Restriction barriers inherent in some str鏿tomycetes could be3ctrcumvented by conjugation or electroporation. A recombinant E.coli ET12567(pUZ8002, pHZl32) was obtained by transforming E.coli ET12567(pUZ8002) with pHZl32, an E.coli-Streptomyces shuttle plasmid incorporating oriT from RK2. In mating experiments, E.coliETl2567 (pUZ8002, pHZ 132) was the donor, and the recipient was S.tenebrarius 9904 spores after pregerminating by heat shock. The donor and the recipient were mixed and spread on MS agar plate. After selection by nalidixic acid and thiostreptone, several hundred conjugants per plate were achieved. Initially, successful transformation of S.tenebrarius 9904 by electroporation was not observed. Parameters affecting electrotransformation were investigated in detail. Under optimal conditions, plasmid pHZ 132 could transfonn the pretreated mycelia by eletroporation at very low frequency. The restriction system could also be circumvented when S. tenebrarius 9904 protoplasts were transformed by plasmid DNA modified by the host itselL and the transformation frequency was about iO~ to io~ per ~.tg of pHZ132 DNA. The established gene transfer system by PEG-mediated protoplast transformation, conjugal transfer as well as electroporation would lay a good foundation for cloning and reforming the antibiotic biosynthesis genes in S. tenebrarius.