Patent
General methods and strains of bacteria are described that dramatically simplify and streamline plasmid DNA production. In one preferred embodiment, endolysin mediated plasmid extraction combined with flocculation mediated removal of cell debris and host nucleic acids achieves increased yield and purity with simplified downstream purification and reduced waste streams, thus reducing production costs.
1. A fed-batch fermentation method for production of covalently closed, super-coiled plasmid DNA, comprising the steps of:
a. growing bacterial cells containing a plasmid at a reduced temperature during a fed-batch phase, the reduced temperature during the fed-batch phase being approximately 30° C.;
b. restricting growth rate of the bacterial cells to approximately μ=0.12 h −1 during the fed-batch phase by nutrient limitation;
c. inducing plasmid production by increasing temperature to approximately 42° C.;
d. continuing growth at elevated temperature for up to 15 hours to accumulate plasmid product; and
e. holding cells for approximately 0.5 to 3 hrs at a reduced temperature prior to cell harvest, the reduced temperature prior to cell harvest being approximately 25 to 30° C.;
whereby said holding cells at a reduced temperature of approximately 25-30° C. prior to cell harvest increases plasmid yield and/or purity above yield and/or purity that are achieved when growing the bacterial cells either without holding cells at a reduced temperature of approximately 25-30° C. prior to cell harvest or holding cells at a reduced temperature of approximately 15° C. prior to cell harvest.