Development of genetic tools for heterologous protein expression in a pentose-utilizing environmental isolate of Pseudomonas putida

Abstract

Pseudomonas putida has emerged as a promising host for the conversion of biomass-derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non-native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass-derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non-native genes was achieved by using chassis-independent recombinase-assisted genome engineering (CRAGE) for single-step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.

FIGURE 1

Plasmid maps of dual‐inducible duet‐vectors pRGPDuo6 (A), pRGPDuo7 (B) and pRGPDuo8 (C). The recombinant P. putida KT2440 (D) and M2 (E) strains were investigated for GFP fluorescence in a minimal medium with glucose (0.5% w/v) as C‐source. Functionality of five promoters, P _tac (1 mM IPTG), P _tetR/tetA (1 μg/ml ATc), P _bad (0.2% arabinose), rhaP _bad (1 mM rhamnose), P _veg/Cuo (1 mM cumate) were assessed in KT2440 and M2. The inducers were added at t = 0 h. the graphs consist of relative fluorescence units normalized to OD₆₀₀ during the mid‐exponential phase for each recombinant strain. Levels of fluorescence for induced (green bar in graphs) and uninduced (no inducer was added, the blue bar in graphs) cultures are shown. The strains harbouring empty vectors were used as negative control and uninduced counterparts for determining background fluorescence. The (+) sign below each strain is indicative for inducer(s) added to induce reporter gene expression driven by corresponding promoter system and (−) sign is indicative of absence of inducer. The initial letters in recombinant strains name are representative of host background, PPK for ‘KT2440’ and PP2M for ‘M2’. For strain description and promoter‐RBS combination, refer to Table 1. Data represent mean values of triplicate assays from at least three individual cultivations, and error bars represent standard deviations.

FIGURE 2

Comparative strengths of ribosome binding sites (RBSs). The selected RBS sequences (1 to 11) were taken from literature (Wang et al., 2018) and previously designed to modulate gene expression in P. putida based on Salis RBS calculator to cover a wide spectrum of expression levels. GFP activity assay was performed to compare the translational efficiency of twelve different RBSs (0 to 11) in P. putida KT2440 (A, C, E) and M2 (B, D, F) in M9 minimal media supplemented with glucose (0.5% w/v). All recombinant strains (harbouring RBS plasmids) were induced with respective promoter systems, namely, P _tac (a, B), P _bad (C, D) and P _tetR/tetA (E, F) at t = 0 h. Levels of fluorescence for induced (green bar in graphs) and uninduced (no inducer was added, the blue bar in graphs) cultures are shown. The strains harbouring empty vectors were used as negative control and uninduced counterparts for determining background fluorescence. The number below each strain represents the corresponding RBS sequence under the control of a specific promoter system (P _tac , P _bad and P _tetR/tetA ). The (−) sign is indicative of empty vector, therefore, no RBS. For strain description and promoter‐RBS combination, refer to Table 1. Data represent mean values of triplicate assays from at least three individual cultivations, and error bars represent standard deviations.

FIGURE 3

Comparative strengths of three shortlisted RBSs with promoters rhaP _bad (1 mM rhamnose) and P _veg/Cuo (1 mM cumate) in M2 (A) and KT2440 (B). GFP levels were measured by growing strains in a minimal medium with glucose as C‐source with respective antibiotics. The inducers were added at t = 0 h. the graphs consist of relative fluorescence units normalized to OD₆₀₀ during the mid‐exponential phase for each recombinant strain. Levels of fluorescence for induced (green bar in graphs) and uninduced (no inducer was added, the blue bar in graphs) cultures are shown. The strains harbouring empty vectors were used as negative control and uninduced counterparts for determining background fluorescence. The number below each strain represents the corresponding RBS sequence (0, 8 and 10) under the control of a specific promoter system (P _veg/Cuo and rhaP _bad ). The (+) sign below each strain corresponds to inducer added to induce gene expression and (−) sign corresponds to either the empty vector (without any RBS) or absence of inducer. For strain description, a promoter‐RBS combination refers to Table 1. Data represent mean values of triplicate assays from at least three individual cultivations, and error bars represent standard deviations.

FIGURE 4

Checking the compatibility of pBBR1‐derived plasmids with pRO1600‐derived plasmids in M2 (A, B). GFP and RFP levels were investigated for recombinant strain, PP2M419 (M2 harbouring plasmids pRGPDuo1‐sfGFP_tet + pRGPDuo4‐RFP_bad). The GFP (a) and RFP (B) expression levels measured in the presence of a respective cognate inducer(s) are shown. The inducers were added at t = 0 h. The annotation indicates the presence or absence of inducers: Uninduced (no inducer added); induced (presence of both inducers: Combination of 1 μg/ml ATc and 0.2% w/v arabinose), +ATc (only ATc was added), +arabinose (only arabinose was added). GFP (C) and RFP (D) levels were also measured in strain PP2M419 with serial concentrations of specific inducer, namely, ATc (ranging from 0 to 4 μg/ml) and arabinose (ranging from 0 to 0.5% w/v). The graphs consist of relative fluorescence units normalized to OD₆₀₀ during the mid‐exponential phase for each construct. Data represent mean values of triplicate assays from at least two individual cultivations, and error bars represent standard deviations.

FIGURE 5

Indigoidine production in M2 (PP2M227, 525, 527) and KT2440 (PPK529, 531) in minimal medium with different C‐sources namely, glucose (A), xylose (B) and arabinose (C). The strain harbouring an empty vector (PP2M227) was used as negative control and uninduced counterparts for determining background fluorescence. The recombinant strains expressed plasmid‐encoded heterologous genes bpsA from S. lavendulae and sfp from B. subtilis for conversion of glutamine to indigoidine under the control of either ATc‐inducible (P _tetR/tetA ) or arabinose‐inducible (P _bad ) promoters. The samples were taken for each strain directly from 30 ml culture tubes with 5 ml of media at the late exponential phase (~16 h) to measure absorbance at OD_612nm. The inducers were added at t = 0 h. the (+) sign below each strain corresponds to inducer added to induce gene expression and (−) sign corresponds to absence of inducer. For strain description, refer to Table 1. Data represent mean values of triplicate assays from at least two individual cultivations, and error bars represent standard deviations.

FIGURE 6

Application of CRAGE technology for production of non‐native bioproduct flaviolin. The red‐coloured flaviolin pigment was quantified in M2 in a minimal medium with glucose and xylose. The graphs consist of relative OD_340nm normalized to OD₆₀₀ at the late exponential phase in induced and uninduced states. RppA protein from S. coelicolor catalyses malonyl‐CoA conversion to THN that spontaneously oxidizes to red‐coloured pigment flaviolin (Figure S6). Genes coding for rppA‐NT (S. coelicolor rppA gene with C‐terminal truncation of 25 amino acids) was cloned into accessory vector pW34 and conjugated to both KT2440 and M2 containing LP to insert rppA into the genome of the respective clone. For strain description, refer to Table 1. Data represent mean values of triplicate assays from at least two individual cultivations, and error bars represent standard deviations.