High Frequency Transformation of Whole Cells of Amino Acid Producing Coryneform Bacteria Using High Voltage Electroporation

Abstract

We have investigated the use of high voltage electroporation to introduce plasmid vectors into several of the amino-acid producing corynebacteria. We report very successful transformation of plasmids into Corynebacterium glutamicum, Brevibacterium lactofermentum and several other species of these coryneform bacteria. Efficiencies of transformation of 10⁷/μg plasmid DNA were achieved with C. glutamicum using whole cells of this species. This is 100 to 1000 times higher than the efficiency reported for transformation of protoplasts. Using cell densities of 10¹⁰/ml of C. glutamicum and electrical pulses of 12.5 kV cm⁻¹ transformations were achieved with plasmid DNA levels as low as 10 picograms. A chimeric plasmid, restricted into its two components and religated, was transformed at a frequency of 10⁵/μg DNA. The advantage of the described technique is that it allows the construction of gene banks and the complementation of mutants directly in these industrially important bacteria by-passing conventional intermediate cloning systems involving E. coli.