Continuous control of the flow in biochemical pathways through 5' untranslated region sequence modifications in mRNA expressed from the broad-host-range promoter Pm.

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 ß-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.

Directed evolution of the transcription factor XylS for development of improved expression systems.

The inducible Pm promoter together with its cognate positive transcription regulator XylS has been shown to be useful for recombinant protein production under high cell density conditions. Here we report directed evolution of XylS resulting in mutant proteins with increased ability to stimulate transcription in Escherichia coli from Pm. A first round of mutagenesis using error-prone PCR on xylS was used to construct a library consisting of about 430,000 clones, and this library could be efficiently screened with respect to stimulation of expression from Pm due to a positive correlation between the level of expression of the reporter gene, bla (encoding ß-lactamase), and the ampicillin tolerance of the corresponding host cells. Fourteen different amino acid substitutions in XylS were found to separately lead to up to nearly a threefold stimulation of expression under induced conditions, relative to wild type. These mutations were all located in the part corresponding to the N-terminal half of the protein. Varying combinations of the mutations resulted in further stimulation, and the best results (about 10-fold stimulation under induced conditions) were obtained by using a random shuffling procedure followed by a new round of screening. The uninduced levels of expression for the same mutants also increased, but only about four times. Through in silico 3D modelling of the N-terminal domain of XylS, it was observed that the evolved mutant proteins contained substitutions that were positioned in different parts of the predicted structure, including a ß-barrel putatively responsible for effector binding and a coiled coil probably important for dimerization. The total production of the host-toxic antibody fragment scFv-phOx expressed from Pm with the evolved XylS mutant protein StEP-13 was about ninefold higher than with wild-type XylS, demonstrating that directed evolution of transcription factors can be an important new tool to achieve high-level recombinant protein production.