(A) study on transformation of plasmid RSF 1010 to brevibacterium lactofermentum

Abstract

As part of our endearvor to improve an amino-acid producing strain, transformation conditions of plasmid RSF1010 to Brevibacterium lactofermentum were first studied since plasmid RSF1010 isolated from E. coli was expressed in Corinebacterium sp. and Brevibacterium SP. has been widely used in amino-acid over-production. We tried two different methods to transformation: Y.K method, which includs the various salt treatment, and PEG induced protoplast transformation (PIPT), which includes the protoplasting and PEG treatment and regeneration of transformed protoplast. Among the conditions of YK method tested, stage of cell culture temperature and time of heat shock, and time of expression have significant effects on plasmid transformation efficiency. Under the optimized conditions, maximum transformation efficiency, $2.36\times 10^2$ transformants ug:DNA or $1.03\times10^{-7}$ transformants/cell could be obtained. The time necessary for the expression of streptomycin resistance in an antibiotic free medium was found to be 6 hr. The transformants were selection MMYE medium plate containing streptomycin (50ug/ml). Secondly, the PIPT method, which is very different to Y.K. method, was used to transform the plasmid RSF1010 into Brevibacterium lactofermentum. Among the conditions tested, protoplasting was very difficult step. But good protoplasting was obtained by glycine-penicilline G-lysozyme treatment. The regeneration frequency was 3.0\% on RNB plate. The transformation efficiency $4.05\times10^3$ transformantsug:DNA or $1.8\times10^{-2}$ transformants/cell could be obtained. The transformed regenerants were selected on RNB plate containing 100ug/ml streptomycin.