In fed-batch cultivation of Escherichia coli harboring a runaway-replication plasmid pCP3, runaway replication could not be induced at all by shifting the cultivation temperature from 30 to 42°C, which has normally been used in small-scale experiments. Therefore, cultivation conditions were reexamined by shake-flask cultivations. It was found that the plasmid copy number was restricted to a low level at 25°C but increased gradually at 30°C, and that full runaway replication could be induced at 37°C.
Thus, the recombinant E. coli was cultivated at 25°C in fed-batch culture using a minifermenter, and then the temperature was shifted to 37°C. When runaway replication was induced in the middle logarithmic growth phase (2.0kg dry cell/m³), the gene product was amplified almost 40-fold. Although overproduction of the gene product could be induced in the late logarithmic growth phase (10.4 kg dry cell/m³), the extent of amplification decreased.