Hyperproduction of aspartase of Escherichia coli K-12 by the use of a runaway plasmid vector

Plasmid pYT125 bearing the aspartase gene ( aspA) of Escherichia coli K-12 was constructed by the use of pSY343, runaway-replication plasmid vector. One of the transformants, strain MM294 (pYT125)-S maintained pYT125 stably at 37°C in a non-selective medium. This strain produced approx. 60-fold more aspartase than did the control strain after 30 cell generations. Polypeptide of the aspA product amounted to 25–30% of the total cellular protein. Plasmid DNA isolated from strain MM294 (pYT125)-S was very similar to that from unstable strain MM294 (pYT125) in length and restriction sites. The copy number was 83 in strain MM294 (pYT125)-S and was 380 in strain MM294 (pYT125). Vector pSY343 was also stabilized in cured strains, isolated from MM294 (pYT125)-S, accompanied by reduction of the copy number. Accordingly, stabilization of pYT125 was considered to be closely related to decrease of the copy number, owing to genetic alteration of MM294-S.