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Background  The 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.    Results  We 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 .    Conclusion  Our 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.

Strong stimulation of recombinant protein production in Escherichia coli by combining stimulatory control elements in an expression cassette