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

Abstract

Inducible promoters such as P_lac 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 (P_{R ox132} ) and PP_3332 (P_{R 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 P_{R ox306} to the σ⁷⁰ consensus sequence (P_{R 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 (P_{R ox132} , P_{R ox306} ) or 0.7 (P_{R ox3061} ) as determined by RT-qPCR. The QS dependency of P_{R 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 P_{R ox306} . This confirmed previous findings that a bacterial compound accumulates within the culture and induces protein expression.

Figure 1

A. Genetic origins of P_Rox promoters. Top panel: P_Rox132 is part of the upstream genetic region of ddcA (light grey). It starts with the RoxR recognition element (Rox RE), the regulatory regions (−35 and −10) and a ribosome binding site (RBS), which is located in the 5′ untranslated region (white). Bottom panel: P_Rox306 consists of the whole intergenic region between P. putida KT2440 genes PP_3332 and PP_3331. B. DNA sequences of P_Rox132, P_Rox306 and P_Rox3061. The −35 and −10 regions predicted by BPROM (Solovyev and Salamov, 2011) are depicted in bold. Experimentally determined transcriptional start sites are marked with + 1. In P_Rox3061, the −10 region was mutated to the consensus sequence of the major σ‐factor of the σ⁷⁰ family, TATAAT. The initiation codon of the regulated gene is underlined. The sequence of the Rox RE is shaded, the putative RBS sequence depicted in italics.

Figure 2

Expression of sfGFP by P. putida (A) under control of P_Rox132, P_Rox306 and P_Rox3061 and (B) under control of the constitutive promoter P_GAP and the inducible promoter P_BAD. C. Comparison of sfGFP expression under control of P_Rox3061 in LB and MOPS medium. D. Quantitative comparison of sfGFP accumulation within the cells with different promoters. P. putida cells containing plasmids for expression of sfGFP under control of the different promoters were cultivated in 500 ml shake flasks in LB or MOPS medium at 30°C, and OD ₅₇₈ and fluorescence intensity (FI, excitation: 485 nm, emission: 510 nm) were monitored. After reaching stationary phase, the cells were lysed, their proteins separated by SDS‐PAGE and sfGFP detected by means of anti‐sfGFP and secondary HRP‐conjugated antibodies. The detected band intensities were quantified and provided relative to the band intensity of P_Rox3061. The data are derived from one representative experiment, with the mean of three technical replicates shown. The error bars represent the standard deviation.

Figure 3

A. Scheme of the unprocessed MATE‐mCherry fusion protein. After cleavage of the signal peptide (SP), the protein consists of a 6xHis epitope, the passenger mCherry, OmpT and fXa cleavage sites, a PEYFK epitope and the EhaA linker and β‐barrel (Sichwart et al., 2015). B,C. Expression of MATE‐mCherry under control of P_Rox132, P_Rox306 and P_Rox3061. P. putida without plasmid and with plasmid pP_Rox132‐MATEmCherry (B), pP_Rox306‐MATE‐mCherry and pP_Rox3061‐MATE‐mCherry (C) were cultivated in LB medium in a 24‐well MTP at 30°C. OD ₅₇₈ and fluorescence intensity (FI, excitation: 580 nm, emission: 620 nm) were monitored. These data are mean values of biological triplicates. Error bars are not visible due to small standard deviations that are covered by the icons.

Figure 4

Analysis of P. putida pP_Rox3061‐sfGFP (A,B), and pP_Rox132‐MATE‐mCherry (C,D) via fluorescence microscopy. The strains were cultivated at 30°C, 200 rpm in LB medium for 8 h and 24 h respectively. 2.5 × 10⁶ cells were washed three times with PBS and fixed on a microscope slide with DABCO/Mowiol. The samples were analysed with the 100× oil immersion lens of a BZ‐9000 fluorescence microscope (Keyence, Neu‐Isenburg, Germany). A,C. Brightfield pictures. (B) GFP‐filter, excitation 472/30 nm, emmission 593/40 nm. D. TexasRed‐filter, excitation 560/40 nm, emmission 630/75 nm. The length of the scales corresponds to 5 μm.

Figure 5

RT‐qPCR analysis of MATE‐mCherry expression under control of P_BG35 (A), P_Rox132 (B), P_Rox306 (C) and P_Rox3061 (D). P. putida cells containing plasmids for expression of MATE‐mCherry under control of different promoters were cultivated in LB medium at 30°C, 200 rpm. At different time points, 2.5 × 10⁸ cells were removed from the cultures and the total amount of RNA was isolated. 1000 ng of total RNA was reversely transcribed to cDNA. 50 ng cDNA was amplified and analysed with specific primers in a qPCR cycler. The gene expression of MATE‐mCherry was determined relative to the gene expression of the reference gene rpoD (Fujita et al., 1995). The measured Cq values were analysed by applying a model of Pfaffl (2001). All RT‐qPCR analyses were performed as biological triplicates, each of them conducted as technical triplicates. Error bars indicate the standard deviation.

Figure 6

Influence of conditioned medium (CM) on expression of MATE‐mCherry. P. putida pP_Rox306‐MATE‐mCherry was cultivated in LB medium to an OD ₅₇₈ = 0.8. Five millilitres of the cell suspension were harvested and suspended in mixtures of 4 ml fresh LB and 1 ml of different CM. The cells were then cultivated in a 24‐well MTP for another 12 h while monitoring OD ₅₇₈ and fluorescence intensity (FI). A. 24 h CM, (B) 12 h CM, (C) 8 h CM, (D) 4 h CM. These data are mean values of biological triplicates. Error bars are not visible due to small standard deviations that are covered by the icons.