Improving the autotransporter-based surface display of enzymes in Pseudomonas putida KT2440.

Pseudomonas putida can be used as a host for the autotransporter-mediated surface display of enzymes (autodisplay), resulting in whole-cell biocatalysts with recombinant functionalities on their cell envelope. The efficiency of autotransporter-mediated secretion depends on the N-terminal signal peptide as well as on the C-terminal translocator domain of autotransporter fusion proteins. We set out to optimize autodisplay for P. putida as the host bacterium by comparing different signal peptides and translocator domains for the surface display of an esterase. The translocator domain did not have a considerable effect on the activity of the whole-cell catalysts. In contrast, by using the signal peptide of the P. putida outer membrane protein OprF, the activity was more than 12-fold enhanced to 638 mU ml-1  OD-1 compared with the signal peptide of V. cholerae CtxB (52 mU ml-1  OD-1 ). This positive effect was confirmed with a ß-glucosidase as a second example enzyme. Here, cells expressing the protein with N-terminal OprF signal peptide showed more than fourfold higher ß-glucosidase activity (181 mU ml-1  OD-1 ) than with the CtxB signal peptide (42 mU ml-1  OD-1 ). SDS-PAGE and flow cytometry analyses indicated that the increased activities correlated with an increased amount of recombinant protein in the outer membrane and a higher number of enzymes detectable on the cell surface.

Cell density-dependent auto-inducible promoters for expression of recombinant proteins in Pseudomonas putida.

Inducible promoters such as Plac are of limited usability for industrial protein production with Pseudomonas putida. We therefore utilized cell density-dependent auto-inducible promoters for recombinant gene expression in P. putida KT2440 based on the RoxS/RoxR Quorum Sensing (QS) system of the bacterium. To this end, genetic regions upstream of the RoxS/RoxR-regulated genes ddcA (PR ox132 ) and PP_3332 (PR ox306 ) were inserted into plasmids that mediated the expression of superfolder green fluorescent protein (sfGFP) and surface displayed mCherry, confirming their promoter functionalities. Mutation of the Pribnow box of PR ox306 to the σ70 consensus sequence (PR ox3061 ) resulted in a more than threefold increase of sfGFP production. All three promoters caused cell density-dependent expression, starting transcription at optical densities (OD578 ) of approximately 1.0 (PR ox132 , PR ox306 ) or 0.7 (PR ox3061 ) as determined by RT-qPCR. The QS dependency of PR ox306 was further shown by cultivating P. putida in media that had already been used for cultivation and thus contained bacterial signal molecules. The longer P. putida had grown in these media before, the earlier protein expression in freshly inoculated P. putida appeared with PR ox306 . This confirmed previous findings that a bacterial compound accumulates within the culture and induces protein expression.

Direct optical density determination of bacterial cultures in microplates for high-throughput screening applications.

A convenient and most abundantly applied method to determine the growth state of a bacterial cell culture is to determine the optical density (OD) spectrophotometrically. Dilution of the samples, which is necessary to measure within the linear range of the spectrophotometer, is time-consuming and not compatible with high-throughput applications. Here we present a direct approach to estimate the OD at 578 nm (OD578) of bacterial cultures in microplates without the need for sample dilution. This could be advantageous for high-throughput analysis of bacterial cells in microplates for example when optimizing growth conditions, screening for new substrates of a bacterial strain or monitoring enzymatic activity after enzyme evolution. Pseudomonas putida cells were grown in shake flasks. The OD578 was determined in parallel in a microplate directly without dilution and in a spectrophotometer cuvette after dilution. The resulting data set was used to identify a conversion formula, which enables direct and reliable transformation of OD measurements of undiluted samples into the corrected OD values as would have been obtained for diluted samples measured in a standard spectrophotometer. Subsequently we could show that just a few OD calibration points are required to adjust this conversion formula and make it suitable for other suspensions or cultures of bacterial strains different than P. putida. The OD calibration points can be obtained by any combination of microplate reader and cuvette spectrophotometer. For this purpose, conversion formulas for a formazine standard suspension and a suspension of Escherichia coli BL21(DE3) cells were successfully generated. The OD values calculated by both conversion formulas turned out to be identical with the values as obtained by the control measurements in the spectrophotometer. This indicates the general applicability of the conversion formula as described.