Abstract
BackgroundThe XylS/Pm expression system has been used to produce recombinant proteins at industrial levels in Escherichia coli. Activation of transcription from the Pm promoter takes place in the presence of benzoic acid or derivatives of it. Previous mutagenesis studies resulted in identification of several variants of the expression control elements xylS (X), Pm (P) and the 5'-untranslated region (U) that individually gave rise to strongly stimulated expression. The goal of this study was to test if combination of such stimulatory mutations in the same expression vectors would lead to further increase of expression levels.ResultsWe combined X, P and U variants that were originally identified due to their ability to strongly stimulate expression of the reporter gene bla (resistance to penicillin). Combination of optimized elements stimulated bla expression up to 75-fold (X, P and U combined) relative to the wild-type system, while accumulated transcript levels increased about 50-fold. This is much more than for the elements individually. We also tested combination of the variant elements on two other and unrelated genes, celB (encoding phosphoglucomutase) and the human growth factor gene gm-csf. Protein production from these genes is much more efficient than from bla in the wild-type system, but expression was still significantly stimulated by the combination of X, P and U variants, although not to the same extent as for bla.We also integrated a single copy of the expression cassette with each gene into the E. coli chromosome and found that the expression level from this single copy was higher for bla than for the wild-type plasmid system, while it was lower for celB and gm-csf.ConclusionOur results show that combination of stimulatory expression control elements can be used to further increase production of different proteins in E. coli. For one reporter gene (bla) this allowed for more protein production from a single gene copy integrated on the chromosome, compared to the wild-type plasmid system. The approach described here should in principle be applicable for improvement of any expression cassette.
Highlights
The XylS/Pm expression system has been used to produce recombinant proteins at industrial levels in Escherichia coli
We have previously demonstrated that the strong and positively regulated xylS/Pm expression cassette in its wild-type form, combined with a mini-RK2 replicon, can serve as a tool to achieve industrial level production of recombinant proteins in Escherichia coli [13,14]
The expression level from Pm can be strongly stimulated by combining mutated DNA elements previously shown to individually enhance expression Initial studies involving combinations of previously isolated stimulatory control element variants (Pm promoter, its UTR and xylS) indicated that they at least to some extent acted additively, and based on these observations cells containing eight different plasmids were subjected to more detailed analyses
Summary
The XylS/Pm expression system has been used to produce recombinant proteins at industrial levels in Escherichia coli. Increasing the number of copies of the gene of interest on the chromosome could potentially eliminate this problem, but this requires further modification of the host genome in order to ensure the stability of the multiple times integrated DNA [11]. A recent study demonstrates an approach to ensure high levels of transcript formation, by placing the desired gene together with a tandem tac promoter cluster into the chromosome [12]. This method eliminates the problem of having multiple copies of the gene, the promoter utilized is constitutive. For metabolic engineering type applications it is desirable to have time-dependent expression
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