Abstract
A high-cell-density fed-batch fermentation for the production of
heterologous proteins in Escherichia coli was developed using the
positively regulated Escherichia coli rhaBAD promoter. The expression
system was improved by reducing of the amount of expensive L-rhamnose
necessary for induction of the rhamnose promoter and by increasing the
vector stability. Consumption of the inducer L-rhamnose was inhibited by
inactivation of L-rhamnulose kinase encoding gene rhaB of Escherichia
coli W3110, responsible for the first irreversible step in rhamnose
catabolism. Plasmid instability caused by multimerization of the
expression vector in the recombination-proficient W3110 was prevented by
insertion of the multimer resolution site cer from the ColE1 plasmid
into the vector. Fermentation experiments with the optimized system
resulted in the production of 100 g L-1 cell dry weight and 3.8 g L-1 of
recombinant L-N-carbamoylase, an enzyme, which is needed for the
production of enantiomeric pure amino acids in a two-step reaction from
hydantoins, (C) 2001 John Wiley & Sons, Inc.
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