High-cell-density fermentation for production of L-N-carbamoylase using an expression system based on the Escherichia coli rhaBAD promoter.
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 x L(-1) cell dry weight and 3.8 g x 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.
Institut für Industrielle Genetik Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
B
B
Wilms
A
A
Hauck
M
M
Reuss
C
C
Syldatk
R
R
Mattes
M
M
Siemann
J
J
Altenbuchner
D000581:Amidohydrolases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D000591:Amino Acid Isomerases; D001173:Arthrobacter / Q000201:enzymology; D002452:Cell Count / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012210:Rhamnose / Q000378:metabolism
D000581:Amidohydrolases; C104561:N-carbamoyl-L-amino-acid hydrolase; D000591:Amino Acid Isomerases; D012210:Rhamnose