High‐level production of recombinant His‐tagged rhamnulose 1‐phosphate aldolase in Escherichia coli

Abstract

An expression system based on Escherichia coli and the T5 promoter allowed the overproduction of a his-tagged rhamnulose-1-phosphate aldolase (RhuA; EC 4.1.2.19), an enzyme with applications in the production of deoxyazasugars and deoxysugars compounds. Shake flask and bioreactor cultivation with E coli M15 (pQErham) were performed under different media and inducing conditions for RhuA expression. A Defined Medium (DM) with glucose as carbon source gave a high volumetric and enzyme productivity (3460 AU dm −3 and 288 AU dm −3 h −1 respectively) compared with Luria-Bertoni (LB) medium (2292 AU dm − 3 and 255 AU dm −3 h −1 ). The minimum quantity of (isopropyl-β-D-thiogalactoside) IPTG for optimal induction was estimated in 18-20 µmol IPTG gDCW −1 . The highest volumetric production of RhuA (8333 AU dm −3 ) was obtained when IPTG was added in the late log-phase. No significant differences were found in specific RhuA activity for induction temperatures of 30 and 37• C. An effective two-step purification process comprising affinity chromatography and gel permeation has been developed (overall recovery 66.5%). These studies provide the basis for the further development of an integrated process for recombinant RhuA production suitable for biotransformation applications.

Influence of secondary reactions on the synthetic efficiency of DHAP‐aldolases

“…Previous results using recombinant His-tagged FucA produced in our laboratory (Durany et al, 2004;Vidal et al, 2003), led to the synthesis product, (3R,4R,5S)-5-(benzyloxycarbonylamino)-5,6-dideoxy-1-O-phosphonohex-2-ulose, with a 100% diastereomeric excess (Espelt et al, 2005). (Fessner et al, 1993).…”

Influence of specific growth rate over the secretory expression of recombinant potato carboxypeptidase inhibitor in fed-batch cultures of Escherichia coli

“…The results are registered in Table 2. In the Production-Dominant zone (PD zone), there are two trends that apply for all three specific growth velocities: First, q p is proportional to , a fact already reported for the production of other proteins [29,32], and secondly, the secretion rates are around one order of magnitude lower than translocation rates, which explains why most of the active PCI is retained in the periplasmic space. For = 0.25 h −1 , q t /q p = 0.51, a ratio that indicates that the protein is being expressed in the cytosol at a higher rate than it can be exported by the SecYEG translocon, which can clarify the observed accumulation of the presecretory protein.…”

“…Thus, the first step to scale up the production of PCI from shake-flask to bioreactor scale was carrying out a non-induced fed-batch culture of BW 25113 (pIMAM3) according to this protocol, in order to validate its efficacy for this particular expression system. It is well known that control of growth rates is crucial to avoid accumulation of undesirable metabolites, mainly acetic acid [28,29], and also has a proven impact on the production yield of recombinant proteins [30].…”

Quantitative modeling of inducer transport in fed-batch cultures of Escherichia coli

“…coli M15 glyA derived from K-12 harboring the plasmids [pQE␣␤rham] [1] and [pREP4] was used for RhuA production. In order to ensure plasmid stability during the experiments, the expression system is glycine auxotrophic, which avoids antibiotic supplementation [24].…”

“…High recombinant protein productivities are usually obtained in high cell-density cultures, employing fed-batch operational mode [1,2]. Several induction strategies have been proposed, but pulse induction is the most commonly used due to its simplicity [3].…”