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.

[1]  S. Luria,et al.  Transduction of lactose-utilizing ability among strains of E. coli and S. dysenteriae and the properties of the transducing phage particles. , 1960, Virology.

[2]  D. Summers.  Timing, self‐control and a sense of direction are the secrets of multicopy plasmid stability , 1998, Molecular microbiology.

[3]  Barry L. Wanner,et al.  Use of New Methods for Construction of Tightly Regulated Arabinose and Rhamnose Promoter Fusions in Studies of theEscherichia coli Phosphate Regulon , 1998, Journal of bacteriology.

[4]  D. Rawlings.  Proteic toxin-antitoxin, bacterial plasmid addiction systems and their evolution with special reference to the pas system of pTF-FC2. , 1999, FEMS microbiology letters.

[5]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[6]  U. Rinas,et al.  Simple fed-batch technique for high cell density cultivation of Escherichia coli. , 1995, Journal of biotechnology.

[7]  I. Pelletier,et al.  Positive selection vectors based on palindromic DNA sequences. , 1992, Methods in enzymology.

[8]  R. Schleif,et al.  A regulatory cascade in the induction of rhaBAD. , 1993, Journal of molecular biology.

[9]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[10]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[11]  W A Weigand,et al.  Monitoring GFP-operon fusion protein expression during high cell density cultivation of Escherichia coli using an on-line optical sensor. , 1999, Biotechnology and bioengineering.

[12]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[13]  C. Syldatk,et al.  Cloning, nucleotide sequence and expression of a new L-N-carbamoylase gene from Arthrobacter aurescens DSM 3747 in E. coli. , 1999 .

[14]  G. Church,et al.  Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization , 1997, Journal of bacteriology.

[15]  W. Deckwer,et al.  High cell density cultivation of Escherichia coli at controlled specific growth rate. , 1991, Journal of biotechnology.

[16]  C. Tate,et al.  Mapping, cloning, expression, and sequencing of the rhaT gene, which encodes a novel L-rhamnose-H+ transport protein in Salmonella typhimurium and Escherichia coli. , 1992, The Journal of biological chemistry.

[17]  U. Bornscheuer,et al.  Characterization and enantioselectivity of a recombinant esterase from Pseudomonas fluorescens , 1998 .

[18]  C W Hill,et al.  Inversions between ribosomal RNA genes of Escherichia coli. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[19]  J. Altenbuchner,et al.  Nucleotide sequence and role in DNA amplification of the direct repeats composing the amplifiable element AUD1 of Streptomyces lividans 66 , 1996, Molecular microbiology.

[20]  G. Sawers,et al.  Alternative regulation principles for the production of recombinant proteins in Escherichia coli , 1996, Applied Microbiology and Biotechnology.

[21]  D. Summers,et al.  Analysis of the CoIE1 stability determinant Rcd. , 1999, Microbiology.

[22]  S. Lee,et al.  High cell-density culture of Escherichia coli. , 1996, Trends in biotechnology.

[23]  H. Blanch,et al.  Recombinant Protein Expression in High Cell Density Fed-Batch Cultures of Escherichia Coli , 1992, Bio/Technology.

[24]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[25]  G W Luli,et al.  Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations , 1990, Applied and environmental microbiology.

[26]  C. Syldatk,et al.  Substrate-dependent enantioselectivity of a novel hydantoinase from Arthrobacter aurescens DSM 3745: purification and characterization as new member of cyclic amidases. , 1998, Journal of biotechnology.

[27]  D. Summers.  Derivatives of ColE1 cer show altered topological specificity in site‐specific recombination. , 1989, The EMBO journal.

[28]  J. Aguilar,et al.  Sequencing and characterization of a gene cluster encoding the enzymes for L-rhamnose metabolism in Escherichia coli , 1993, Journal of bacteriology.

[29]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[30]  D. Summers,et al.  Multicopy plasmid instability: the dimer catastrophe hypothesis , 1993, Molecular microbiology.