Characterization of the AlkS/PalkB-expression system as an efficient tool for the production of recombinant proteins in Escherichia coli fed-batch fermentations
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2007
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Journal Article
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Abstract
The availability of suitable, well‐characterized, and robust expression systems remains an essential requirement for successful metabolic engineering and recombinant protein production. We investigated the suitability of the Pseudomonas putida GPo1‐derived AlkS/PₐₗₖB expression system in strictly aqueous cultures. By applying the apolar inducer dicyclopropylketone (DCPK) to express green fluorescent protein (GFP) from this system in Escherichia coli and analyzing the resulting cultures on single‐cell level by flow cytometry, we found that this expression system gives rise to a homogeneous population of cells, even though the overall system is expected to have a positive feed‐back element in the expression of the regulatory gene alkS. Overexpressing E. coli's serine hydroxymethyltransferase gene glyA, we showed that the system was already fully turned on at inducer concentrations as low as 0.005% (v/v). This allows efficient mass production of recombinant enzymes even though DCPK concentrations decreased from 0.05% to 0.01% over the course of a fully aerated cultivation in aqueous medium. Therefore, we elaborated the optimum induction procedure for production of the biocatalytically promising serine hydroxymethyltransferase and found volumetric and specific productivity to increase with specific growth rate in glucose‐limited fed‐batch cultures. Acetate excretion as a result of recombinant protein production could be avoided in an optimized fermentation protocol by switching earlier to a linear feed. This protocol resulted in a production of a final cell dry weight (CDW) concentration of 52 g/L, producing recombinant GlyA with a maximum specific activity of 6.3 U/mg total protein. Biotechnol. Bioeng. 2007;96: 326–336. © 2006 Wiley Periodicals, Inc.
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96 (2)
Pages / Article No.
326 - 336
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Wiley
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Subject
expression system; alk system; biocatalysis; positive feed-back; growth rate-dependent protein production
Organisational unit
03602 - Panke, Sven / Panke, Sven
03713 - Sauer, Uwe / Sauer, Uwe
Notes
Received 13 January 2006, Accepted 29 June 2006, Published online 24 July 2006.