Transformation of Saccharopolyspora erythraea by electroporation of germinating spores: construction of propionyl Co-A carboxylase mutants

The introduction of plasmid DNA into germinating spores of an industrially improved strain of Saccharopolyspora erythraea was accomplished by electrop oration. Various parameters affecting the efficiency of electroporation wer e examined. The most critical factor was the extent of spore germination. E lectrocompetence was limited to a 4-h period following the initial emergenc e of the germ tube, Electroporation efficiencies as high as 2 x 10(5) CFU m u g(-1) Of plasmid DNA were obtained using electrocompetent germlings. The optimal field strength was 12-14 kV cm(-1) with a pulse duration of 15-20 m s. Electrocompetent germlings were stored at -80 degrees C without a signif icant decrease in transformation efficiency. The utility of this protocol w as demonstrated by isolating a propionyl-CoA carboxylase mutant through tar geted gene disruption and replacement.