Amino acid stabilization for cell-free protein synthesis by modification of the Escherichia coli genome.
Cell-free biology provides a unique opportunity to assess and to manipulate microbial systems by inverse metabolic engineering. We have applied this approach to amino acid metabolism, one of the systems in cell-free biology that limits protein synthesis reactions. Four amino acids (arginine, tryptophan, serine and cysteine) are depleted during a 3-h batch cell-free protein synthesis reaction under various conditions. By modifying the genome of the Escherichia coli strain used to make the cell extract, we see significant stabilization of arginine, tryptophan and serine. Cysteine, however, continues to be degraded. Cell-free protein synthesis with the modified cell extract produces increased yields of the cysteine-free protein Outer Membrane Protein T (OmpT).
Michel-Reydellet
Nathalie
N
Department of Chemical Engineering, Stanford University, CA 94305-5025, USA.
Calhoun
Kara
K
Swartz
James
J
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins; D002474:Cell-Free System / Q000378:metabolism* / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods; D016680:Genome, Bacterial; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases; D018272:Porins / Q000096:biosynthesis* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D018272:Porins; D011994:Recombinant Proteins; C499588:ompT protein, E coli; D010447:Peptide Hydrolases