Fedbatch design for periplasmic product retention in Escherichia coli.
The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.3, 0.2 and 0.1 h(-1) where studied and resulted in 20, 9 and 6%, respectively, of the total ZZ-cutinase accumulating in the medium. It was also shown that leakage during fedbatch production of a Fab fragment was also influenced by the feed rate in a similar manner to ZZ-cutinase. If intracellular product accumulation is desired the advantage of a high productivity, resulting from a high substrate feed rate, is diminished because of a reduced product retention. Biochemical analysis revealed that the growth rate, resulting from a glucose limited feed, influenced the outer membrane protein compositions with respect to OmpF and LamB, whilst OmpA was largely unaffected. As the feed rate increased the amount of total outer membrane protein decreased. When ZZ-cutinase was produced there were further reductions in outer membrane protein accumulation, by 82, 100 and 22% for OmpF, LamB and OmpA, respectively, and the total reduction was almost 60% with a high product formation rate. We suggest that the reduced titre of the outer membrane proteins, OmpF and LamB, may have contributed to a reduced ability for the cell to retain recombinant protein secreted to the periplasm.
Bäcklund
Emma
E
School of Biotechnology, Royal Institute of Technology, Roslagstullsbacken 21, SE-10691 Stockholm, Sweden.
Reeks
Dominic
D
Markland
Katrin
K
Weir
Neil
N
Bowering
Leigh
L
Larsson
Gen
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D001709:Biotechnology / Q000379:methods*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003470:Culture Media; D004926:Escherichia coli / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D008969:Molecular Sequence Data; D019897:Periplasm / Q000378:metabolism*; D011994:Recombinant Proteins; D013997:Time Factors
D001426:Bacterial Proteins; D003470:Culture Media; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase