SUMMARY: An efficient host-vector system has been developed for an industrial strain of sp. (NRRL B3728) used for glucose isomerase production. Protoplasts of were generated by treating the cells with 0.5 mg lysozyme ml for 60 min in a solution containing 0.5 -sucrose. Around 30% of the protoplasts regenerated on agar containing 0.5 -sodium succinate as osmotic stabilizer. Three hybrid vectors, pBL2100, pCG1100 and pCG2100, were constructed by combining the plasmid pBR322, a kanamycin-resistance gene from pNCAT4 and a cryptic plasmid from either NCIB 9567 or NCIB 10026. These vectors transformed the protoplasts and expressed the kanamycin-resistance gene for screening. They contain a number of unique restriction sites for cloning of foreign DNA. The transformation frequency of this system was 10-10 transformants per μg of input plasmid and was constant up to 5 μg of DNA. The probability of a plasmid transforming a protoplast was in the range 10-10. The copy number of pBL2100 was around 5 per cell and those of pCG1100 and pCG2100 were around 33 per cell. Deletion mutants were generated from pCG2100. One of them, pCG2120, was able to transform protoplasts of strain NRRL B3728. Plasmids pBL2100 and pCG2100 were structurally stable in cells of NRRL B3728 but could not be maintained in non-selective medium. They segregated at a rate of 12.2 and 2.3% per generation respectively.


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