title: Expression of two recombinant chloramphenicol acetyltransferase variants in highly reduced genome Escherichia coli strains.
abstract: Highly reduced E. coli strains, MDS40, MDS41, and MDS42, lacking approximately 15% of the genome, were grown to high cell densities to test their ability to produce a recombinant protein with high yields. These strains lack all transposons and insertion sequences, cryptic prophage and many genes of unknown function. In addition to improving genetic stability, these deletions may reduce the biosynthetic requirements of the cell potentially allowing more efficient production of recombinant protein. Basic growth parameters and the ability of the strains to produce chloramphenicol acetyltransferase (CAT) under high cell density, batch cultivation were assessed. Although growth rate and recombinant protein production of the reduced genome strains are comparable to the parental MG1655 strain, the reduced genome strains were found to accumulate significant amounts of acetate in the medium at the expense of additional biomass. A number of hypotheses were examined to explain the accumulation of acetate, including oxygen limitation, carbon flux imbalance, and metabolic activity of the recombinant protein. Use of a non-catalytic CAT variant identified the recombinant protein activity as the source of this phenomenon; implications for the metabolic efficiency of the reduced genome strains are discussed.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Clemson University, Clemson, South Carolina 29634, USA.
authors.forename: Shamik S
authors.initials: SS
authors.lastname: Sharma
authors.forename: John W
authors.initials: JW
authors.lastname: Campbell
authors.forename: David
authors.initials: D
authors.lastname: Frisch
authors.forename: Frederick R
authors.initials: FR
authors.lastname: Blattner
authors.forename: Sarah W
authors.initials: SW
authors.lastname: Harcum
mesh_terms: D000085:Acetates / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000378:metabolism; D018533:Biomass; D001709:Biotechnology / Q000379:methods; D002452:Cell Count; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression / Q000187:drug effects; D016680:Genome, Bacterial; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006019:Glycolysis / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D010709:Phosphate Acetyltransferase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism
chemical_list: D000085:Acetates; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D000105:Acetyl Coenzyme A; D015500:Chloramphenicol O-Acetyltransferase; D010709:Phosphate Acetyltransferase; D005947:Glucose; D005990:Glycerol; D010100:Oxygen