High‐cell‐density fermentation for production of L‐N‐carbamoylase using an expression system based on the Escherichia coli rhaBAD promoter

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 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.

Recombinant Expression of tatABC and tatAC Results in the Formation of Interacting Cytoplasmic TatA Tubes in Escherichia coli

“…To construct expression vectors for C-terminal green fluorescent protein (GFP) fusions of RR-(FG5) or KK-(FG5), gfp was amplified using pTB-DG (17) as template and the primers gfp-SacII-F (5Ј-AGG GCC CGC GGG TAA AGG AGA AGA ACT TTT CAC-3Ј) and gfp-XhoI-R (5Ј-GTC TGC TCG AGT TAT TTG TAT AGT TCA TCC ATG CC-3Ј), the fragment was cut with SacII and XhoI and cloned into the corresponding sites of pBW-R5 or pBW-R5-KK, resulting in pBW-R5-gfp and pBW-R5-gfp-KK, respectively. The tatA expression vector pBWtatA-H 6 was constructed by amplification of tatA with pABStatABC as template, using the primers tatA-NdeI-F (5Ј-GAA CAC ATA TGG GTG GTA TCA GTA TTT GGC-3Ј) and tatABamHI-R (5Ј-AAC ACG GAT CCC ACC TGC TCT TTA TCG TGG-3Ј), restriction with NdeI/BamHI and ligation into the corresponding sites of pBW22 (21). The vector pTB-DG-KK was derived from pTB-DG (17) by exchange of the two arginine codons in the signal sequence coding region by two lysine codons using the QuikChange method with the primer pair pTB-DG-gfp-KK-F (5Ј-GGC TGC TGA GGT GAG TAA GAA GGG TTT GGT AAA AAC GAC AGC-3Ј)/pTB-DG-gfp-KK-R (5Ј-GCT GTC GTT TTT ACC AAA CCC TTC TTA CTC ACC TCA GCA GCC-3Ј).…”

DnaK Plays a Pivotal Role in Tat Targeting of CueO and Functions beside SlyD as a General Tat Signal Binding Chaperone

“…For construction of pBW-hip-strep, hip was amplified by PCR with pEXH5 as template using the primer pair hip-NdeI-F (5Ј-GGA GAT ATA CAT ATG TCC GAT AAG CCA ATC AGC-3Ј) and hipBamHI-R (5Ј-AAC GGG GAT CCG CCG GCC TTC AGG GTC CAG-3Ј), and the PCR product was introduced into the NdeI and BamHI sites of a Strep tag derivative of pBW22 (16) to allow a rhamnose-inducible production of a C-terminal Strep tag II fusion of HiPIP. The vector pBW-mat-hip-strep was analogously constructed, but the signal sequence coding region was excluded by using the forward primer hip-mat-NdeI-F (5Ј-CCG CCC ATA TGT CCG CTC CCG CCA ATG CCG-3Ј) in the PCR.…”

Aspergillus niger β-galactosidase production by yeast in a continuous high cell density reactor

“…This can be achieved by increasing the amount of protein per cell per time and/or by increasing cell concentration per time [18]. In this work, the feasibility of using flocculent continuous high-cell-density systems for higher extracellular protein production has been shown, being achieved an increase in ␤-galactosidase productivity (4-11-fold increase) when compared to batch cultures for the same substrate concentration.…”