SummarySecondary structures and the short Shine–Dalgarno sequence in the 5′‐untranslated region of bacterial mRNAs (UTR) are known to affect gene expression at the level of translation. Here we report the use of random combinatorial DNA sequence libraries to study UTR function, applying the strong, σ32/σ38‐dependent, and positively regulated Pm promoter as a model. All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site. The libraries were screened using the ampicillin‐resistance gene (bla) as reporter, allowing easy identification of UTR mutants that display high levels of expression (up to 20‐fold increase relative to the wild‐type at the protein level). Studies of the two UTR mutants identified by a modified screening procedure showed that their expression is stimulated to a similar extent at both the transcript and protein product levels. For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18‐fold higher than the wild type), while there was no evidence of a difference in transcript stability. The two UTR sequences also stimulated expression from a constitutive σ70‐dependent promoter (P1/Panti‐tet), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription.

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...

The inducible Pm promoter integrated into broad-host-range plasmid RK2 replicons can be fine-tuned continuously between the uninduced and maximally induced levels by varying the inducer concentrations. To lower the uninduced background level while still maintaining the inducibility for applications in, for example, metabolic engineering and synthetic (systems) biology, we report here the use of mutations in the Pm DNA region corresponding to the 5 untranslated region of mRNA (UTR). Five UTR variants obtained by doped oligonucleotide mutagenesis and selection, apparently reducing the efficiency of translation, were all found to display strongly reduced uninduced expression of three different reporter genes (encoding ␤-lactamase, luciferase, and phosphoglucomutase) in Escherichia coli. The ratio between induced and uninduced expression remained the same or higher compared to cells containing a corresponding plasmid with the wild-type UTR. Interestingly, the UTR variants also displayed similar effects on expression when substituted for the native UTR in another and constitutive promoter, P1 (P antitet ), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C 50 carotenoid sarcinaxanthin in an engineered strain of E. coli that produces the precursor lycopene. Sarcinaxanthin is produced in this particular strain by expressing three Micrococcus luteus derived genes from the promoter Pm. The results indicated that UTR variants can be used to eliminate sarcinaxanthin production under uninduced conditions, whereas cells containing the corresponding plasmid with a wild-type UTR produced ca. 25% of the level observed under induced conditions.The initially used methods of deleting or overexpressing genes have been demonstrated to be inadequate for many applications in metabolic engineering (21,31,32). For example, when the goal is to optimize the expression level of a desired protein by engineering the relevant metabolic pathway, it might be necessary to change the expression of multiple enzymes simultaneously and to different levels (30,38). Also, reducing the formation of particular by-products can increase the flux of the desired product (29). In addition, low basal expression is critical for applications such as the expression of toxic genes, metabolic engineering, and control analysis (2, 14, 27, 34). This has led to an increased focus on development of genetic tools to fine-tune gene expression to the desired levels. A commonly used strategy is to make so-called promoter libraries of constitutive promoters (1,15,17). Such promoters seem to be preferred over the corresponding inducible ones for industrial scale productions because of factors such as inducer costs, sensitivity to inducer concentration, and heterogeneity of expression caused by an all-or-none effect of induction (1,19). However, the all-or-none induction effect may be eliminated if the inducer enters the cell interior by passive diffusion (20). Thus, regulatable promoter s...

Microbial promoters are important targets both for understanding the global gene expression and developing genetic tools for heterologous expression of proteins and complex biosynthetic pathways. Previously, we have developed and used combinatorial mutagenesis methods to analyse and improve bacterial expression systems. Here, we present for the first time an analogous strategy for yeast. Our model promoter is the strong and inducible P AOX1 promoter in methylotrophic Pichia pastoris. The Zeocin resistance gene was applied as a valuable reporter for mutant P AOX1 promoter activity, and we used an episomal plasmid vector to ensure a constant reporter gene dosage in the yeast host cells. This novel design enabled direct selection for colonies of recombinant cells with altered Zeocin tolerance levels originating solely from randomly introduced point mutations in the P AOX1 promoter DNA sequence. We demonstrate that this approach can be used to select for P AOX1 promoter variants with abolished glucose repression in large mutant libraries. We also selected P AOX1 promoter variants with elevated expression level under induced conditions. The properties of the selected P AOX1 promoter variants were confirmed by expressing luciferase as an alternative reporter gene. The tools developed here should be useful for effective screening, characterization, and improvement of any yeast promoters.

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