Random Mutagenesis of the Pm Promoter as a Powerful Strategy for Improvement of Recombinant-Gene Expression

Abstract

The inducible Pm-xylS promoter system has proven useful for production of recombinant proteins in several gram-negative species and in high-cell-density cultivations of Escherichia coli. In this study we subjected a 24-bp region of Pm (including the ؊10 element) to random mutagenesis, leading to large mutant libraries in E. coli. Low-frequency-occurring Pm mutants displaying strongly increased promoter activity (up-mutants) could be efficiently identified by using ␤-lactamase as a reporter. The up-mutants typically carried multiple point mutations positioned throughout the mutagenized region, combined with deletions around the transcription start site. Mutants displaying up to about a 14-fold increase in ␤-lactamase expression (relative to wild-type Pm) were identified without loss of the inducible phenotype. The mutants also strongly stimulated the expression of two other reporter genes, luc (encoding firefly luciferase) and celB (encoding phosphoglucomutase), and were found to significantly improve (twofold) a previously optimized process for high-level recombinant production of the medically important granulocyte-macrophage colony-stimulating factor in E. coli under high-cell-density conditions. These results demonstrate the potential of using random mutagenesis of promoters to improve protein expression at industrial levels and indicate that targeted modifications of individual functional elements are not sufficient to obtain optimized promoter sequences.The Pm-xylS promoter system drives the expression of the meta-cleavage operon carried by the Pseudomonas putida TOL plasmid pWW0. The gene products of this operon are involved in the catabolism of alkylbenzoates and are expressed in response to meta-pathway substrates (25). XylS positively regulates Pm when forming an activated complex with effectors like benzoate or its derivatives (13,26). Transcriptional activation occurs through binding of the activated XylS to two direct imperfect repeats located directly upstream of the Ϫ35 region of Pm (12).Pm-xylS has been shown to be useful for high-level expression of recombinant proteins in a wide range of gram-negative bacterial species (3,4,20,24). The uninduced expression level is low, and the use of different effector compounds at various concentrations can be used to regulate the level of induced expression (35). Many of the inducers are low-cost compounds that enter the cell by passive diffusion. Previously, we reported the use of this system in the construction of broad-host-range expression vectors based on the RK2 minimal replicon (2, 3). One of these vectors, pJB658, has proven useful for tightly regulated recombinant-gene expression in several gram-negative species (2,3,5,31,35). Industrial levels of production of human proteins under high-cell-density conditions (HCDC) of Escherichia coli has also been demonstrated with the Pm-xylS system coupled to the RK2 replicon (30,31). By studying the functionality of different secretion signal sequences, the effects of various vector copy numbers, and the condit...

Combinatorial Mutagenesis and Selection of Improved Signal Sequences and Their Application for High-Level Production of Translocated Heterologous Proteins in Escherichia coli

“…The plasmid pCSP1bla, used for construction of mutant libraries (Fig. 1A), is based on the vector pIB11 (32) in which the endogenous secretion signal sequence of the bla gene was replaced by the CSP signal sequence from pGM29CSP (17). A series of plasmids were made as intermediates of the final vectors pCSP1bla and pNSP1bla (expressing the bla gene without any signal sequence).…”

“…To introduce mutations in the CSP coding sequence, a strategy involving degenerated, synthetic oligonucleotides, similar to the protocol previously described for the Pm promoter and the untranslated region, was used (32,33). Synthetic oligonucleotides were designed to constitute a double-stranded DNA fragment with the CSP sequence and NdeI and NcoI compatible ends when annealed for subsequent easy cloning into the pCSP1bla vector.…”

“…Among the many techniques used to create random mutant libraries the most commonly used are error-prone PCR (for a review, see reference 27), DNA shuffling (28), and chemical mutagenesis (29,30). We have previously applied a combinatorial mutagenesis approach involving the use of degenerated oligonucleotides to improve the bacterial expression system PmxylS (31)(32)(33). By using synthetic oligonucleotide mixtures, the mutagenesis can be focused to specific genetic regions (20 to 100 bp in length), a method that allows for multiple point mutations to be introduced simultaneously.…”

Combinatorial engineering for heterologous gene expression

“…Most likely it is the step of transcription initiation which is influenced. The best highexpression variant (ML2-5, 3 here termed comP), increased protein expression of the reporter gene bla (coding for β-lactamase, conferring ampicillin resistance to the host cell) about 5 times. Also a high-expression variant chosen for the activator protein gene (StEP-13, 4 termed comX) seemed to mainly act at the level of transcription and led to a similar final protein expression as variant (comU) stimulated translation of bla about 15 times, while transcript levels were similar to those for comP and comX (Fig.…”

“…ComP and comX have been shown to act relatively gene-independent and led to increased expression also of other genes than bla. 3,4 For variants of the 5'-UTR there seems to exist a certain degree of gene-dependency, especially for those variants that mainly act on translation. 5 This might also have an influence on the outcome of combination of variants, and thus it might be a better approach to directly use 5'-UTR variants that have been identified as stimulating for the gene of interest.…”