Laila Berg

The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5-untranslated part of mRNA

Secondary 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.

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 β-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 β-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.

Continuous Control of the Flow in Biochemical Pathways through 5 Untranslated Region Sequence Modifications in mRNA Expressed from the Broad-Host-Range Promoter Pm†

ABSTRACT 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 β-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 (Pantitet ), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C50 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.

Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters

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.

Ralstonia sp. U2 naphthalene dioxygenase and Comamonas sp. JS765 nitrobenzene dioxygenase show differences in activity towards methylated naphthalenes

Methylsubstituted naphthalenes constitute a significant part of light gas oil fractions (LGO). These are toxic compounds with low fuel value, and can potentially be enzymatically modified to increase the fuel value and at the same time reduce toxicity. The first step in the biodegradation of naphthalene involves dioxygenation of the aromatic ring catalysed by naphthalene dioxygenase (NDO). Here we show that recombinantly produced NDO from Ralstonia sp. U2 and the related nitrobenzene dioxygenase (NBDO) from Comamonas sp. JS765 can use several mono-, di-, tri-, and tetramethylated naphthalenes as substrates. For the majority of the substrates both enzymes catalyse the formation of a mixture of mono- and dioxygenated products, and it is only dioxygenated products that are likely to be processed further, leading to ring cleavage. In some cases, like for 1-methylnaphthalene, NDO mainly generates the monooxygenated form, while with NBDO, the dioxygenated form dominates. In other cases, as for 1,4-dimethylnaphthalene, the monooxygenated product dominates with NDO, whereas NBDO generates similar amounts of both forms. Presumably, the best future strategy for bioconversion of methylated naphthalenes in LGO is to develop engineered enzyme that are optimised with respect to the specific composition of naphthalene derivatives found in a given product.