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Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression.

Martínez-García E, Nikel PI, Aparicio T, de Lorenzo V - Microb. Cell Fact. (2014)

Bottom Line: Since ATP and NAD(P)H availability - as well as genetic instability, are generally considered to be major bottlenecks for the performance of platform strains, a suite of functions that drain high-energy phosphate from the cells and/or consume NAD(P)H were targeted in particular, the whole flagellar machinery.Four prophages, two transposons, and three components of DNA restriction-modification systems were eliminated as well.Furthermore, it tolerated endogenous oxidative stress, acquired and replicated exogenous DNA, and survived better in stationary phase.

View Article: PubMed Central - PubMed

Affiliation: Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, 28049, Madrid, Spain. emartinez@cnb.csic.es.

ABSTRACT

Background: Because of its adaptability to sites polluted with toxic chemicals, the model soil bacterium Pseudomonas putida is naturally endowed with a number of metabolic and stress-endurance qualities which have considerable value for hosting energy-demanding and redox reactions thereof. The growing body of knowledge on P. putida strain KT2440 has been exploited for the rational design of a derivative strain in which the genome has been heavily edited in order to construct a robust microbial cell factory.

Results: Eleven non-adjacent genomic deletions, which span 300 genes (i.e., 4.3% of the entire P. putida KT2440 genome), were eliminated; thereby enhancing desirable traits and eliminating attributes which are detrimental in an expression host. Since ATP and NAD(P)H availability - as well as genetic instability, are generally considered to be major bottlenecks for the performance of platform strains, a suite of functions that drain high-energy phosphate from the cells and/or consume NAD(P)H were targeted in particular, the whole flagellar machinery. Four prophages, two transposons, and three components of DNA restriction-modification systems were eliminated as well. The resulting strain (P. putida EM383) displayed growth properties (i.e., lag times, biomass yield, and specific growth rates) clearly superior to the precursor wild-type strain KT2440. Furthermore, it tolerated endogenous oxidative stress, acquired and replicated exogenous DNA, and survived better in stationary phase. The performance of a bi-cistronic GFP-LuxCDABE reporter system as a proxy of combined metabolic vitality, revealed that the deletions in P. putida strain EM383 brought about an increase of >50% in the overall physiological vigour.

Conclusion: The rationally modified P. putida strain allowed for the better functional expression of implanted genes by directly improving the metabolic currency that sustains the gene expression flow, instead of resorting to the classical genetic approaches (e.g., increasing the promoter strength in the DNA constructs of interest).

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Evaluation ofP.putidaEM383 as achassisfor the heterologous expression ofgfpandluxCDABE. (A) Schematic representation of the bi-cistronic GFP-LuxCDABE reporter in which both gfp (GFP: green fluorescent protein) and luxCDABE (LuxC: fatty acid reductase, LuxD: acyl transferase, LuxE: acyl-protein synthase, LuxAB: luciferase) from Photorhabdus luminescens are placed under the control of the inducible Pm promoter. The activity of Pm is controlled by the transcriptional regulator XylS. The transcriptional terminator included in the plasmid backbone is depicted as T0. The elements in this outline, borne by plasmid pGL-XP, are not drawn to scale. The reporter plasmid pGL-XP was used to establish a comparison of the expression levels of gfp(B) and luxCDABE(C) in wild-type KT2440 and in the streamlined EM383 strain in response to 3-methylbenzoate. Overnight cultures in rich LB medium were diluted to an optical density at 600 nm (OD600) of 0.1, cells were further grown for 2 h, and then induced with 1 mM 3-methylbenzoate for 24 h. The reporter expression level was calculated by dividing either the arbitrary fluorescence units (AFU) or the arbitrary luminescence units (ALU) by the OD600. The bars represent the media and SD of three measurements from biological triplicates. Nil, no inducer; Ind, induced. The asterisk (*) indicates a significant difference for strain EM383 as compared to wild-type KT2440 according to the Student’s t test (P <0.05).
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Fig7: Evaluation ofP.putidaEM383 as achassisfor the heterologous expression ofgfpandluxCDABE. (A) Schematic representation of the bi-cistronic GFP-LuxCDABE reporter in which both gfp (GFP: green fluorescent protein) and luxCDABE (LuxC: fatty acid reductase, LuxD: acyl transferase, LuxE: acyl-protein synthase, LuxAB: luciferase) from Photorhabdus luminescens are placed under the control of the inducible Pm promoter. The activity of Pm is controlled by the transcriptional regulator XylS. The transcriptional terminator included in the plasmid backbone is depicted as T0. The elements in this outline, borne by plasmid pGL-XP, are not drawn to scale. The reporter plasmid pGL-XP was used to establish a comparison of the expression levels of gfp(B) and luxCDABE(C) in wild-type KT2440 and in the streamlined EM383 strain in response to 3-methylbenzoate. Overnight cultures in rich LB medium were diluted to an optical density at 600 nm (OD600) of 0.1, cells were further grown for 2 h, and then induced with 1 mM 3-methylbenzoate for 24 h. The reporter expression level was calculated by dividing either the arbitrary fluorescence units (AFU) or the arbitrary luminescence units (ALU) by the OD600. The bars represent the media and SD of three measurements from biological triplicates. Nil, no inducer; Ind, induced. The asterisk (*) indicates a significant difference for strain EM383 as compared to wild-type KT2440 according to the Student’s t test (P <0.05).

Mentions: Endogenous ROS stem not only from added-on redox reactions, but also from cell aging and nutrient starvation at the stationary phase [74]. The next obvious question was therefore whether P. putida EM383 can also deal better with such an inevitable physiological condition that all bacteria have to go through. To examine this issue propidium iodide was used along with cell cytometry as a reliable method to quantify cellular death, as this dye only stains bacteria with damaged membranes [75]. As shown in Figure 6, after overnight growth in LB medium, the cultures of the wild-type strain contained many more dead cells (8.2%) than in the P. putida EM383 counterpart (1.8%). A lower stationary-phase associated mortality of P. putida EM383 was also observed in M9 minimal medium with glucose or succinate, while no significant difference was observed when citrate was used as the C source (Figure 7). These results reflect the combination of the known effects of lacking flagella [57] and the prophage load [36], that in the present case seem to add to each other for increasing very significantly stationary phase survival.Figure 6


Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression.

Martínez-García E, Nikel PI, Aparicio T, de Lorenzo V - Microb. Cell Fact. (2014)

Evaluation ofP.putidaEM383 as achassisfor the heterologous expression ofgfpandluxCDABE. (A) Schematic representation of the bi-cistronic GFP-LuxCDABE reporter in which both gfp (GFP: green fluorescent protein) and luxCDABE (LuxC: fatty acid reductase, LuxD: acyl transferase, LuxE: acyl-protein synthase, LuxAB: luciferase) from Photorhabdus luminescens are placed under the control of the inducible Pm promoter. The activity of Pm is controlled by the transcriptional regulator XylS. The transcriptional terminator included in the plasmid backbone is depicted as T0. The elements in this outline, borne by plasmid pGL-XP, are not drawn to scale. The reporter plasmid pGL-XP was used to establish a comparison of the expression levels of gfp(B) and luxCDABE(C) in wild-type KT2440 and in the streamlined EM383 strain in response to 3-methylbenzoate. Overnight cultures in rich LB medium were diluted to an optical density at 600 nm (OD600) of 0.1, cells were further grown for 2 h, and then induced with 1 mM 3-methylbenzoate for 24 h. The reporter expression level was calculated by dividing either the arbitrary fluorescence units (AFU) or the arbitrary luminescence units (ALU) by the OD600. The bars represent the media and SD of three measurements from biological triplicates. Nil, no inducer; Ind, induced. The asterisk (*) indicates a significant difference for strain EM383 as compared to wild-type KT2440 according to the Student’s t test (P <0.05).
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Fig7: Evaluation ofP.putidaEM383 as achassisfor the heterologous expression ofgfpandluxCDABE. (A) Schematic representation of the bi-cistronic GFP-LuxCDABE reporter in which both gfp (GFP: green fluorescent protein) and luxCDABE (LuxC: fatty acid reductase, LuxD: acyl transferase, LuxE: acyl-protein synthase, LuxAB: luciferase) from Photorhabdus luminescens are placed under the control of the inducible Pm promoter. The activity of Pm is controlled by the transcriptional regulator XylS. The transcriptional terminator included in the plasmid backbone is depicted as T0. The elements in this outline, borne by plasmid pGL-XP, are not drawn to scale. The reporter plasmid pGL-XP was used to establish a comparison of the expression levels of gfp(B) and luxCDABE(C) in wild-type KT2440 and in the streamlined EM383 strain in response to 3-methylbenzoate. Overnight cultures in rich LB medium were diluted to an optical density at 600 nm (OD600) of 0.1, cells were further grown for 2 h, and then induced with 1 mM 3-methylbenzoate for 24 h. The reporter expression level was calculated by dividing either the arbitrary fluorescence units (AFU) or the arbitrary luminescence units (ALU) by the OD600. The bars represent the media and SD of three measurements from biological triplicates. Nil, no inducer; Ind, induced. The asterisk (*) indicates a significant difference for strain EM383 as compared to wild-type KT2440 according to the Student’s t test (P <0.05).
Mentions: Endogenous ROS stem not only from added-on redox reactions, but also from cell aging and nutrient starvation at the stationary phase [74]. The next obvious question was therefore whether P. putida EM383 can also deal better with such an inevitable physiological condition that all bacteria have to go through. To examine this issue propidium iodide was used along with cell cytometry as a reliable method to quantify cellular death, as this dye only stains bacteria with damaged membranes [75]. As shown in Figure 6, after overnight growth in LB medium, the cultures of the wild-type strain contained many more dead cells (8.2%) than in the P. putida EM383 counterpart (1.8%). A lower stationary-phase associated mortality of P. putida EM383 was also observed in M9 minimal medium with glucose or succinate, while no significant difference was observed when citrate was used as the C source (Figure 7). These results reflect the combination of the known effects of lacking flagella [57] and the prophage load [36], that in the present case seem to add to each other for increasing very significantly stationary phase survival.Figure 6

Bottom Line: Since ATP and NAD(P)H availability - as well as genetic instability, are generally considered to be major bottlenecks for the performance of platform strains, a suite of functions that drain high-energy phosphate from the cells and/or consume NAD(P)H were targeted in particular, the whole flagellar machinery.Four prophages, two transposons, and three components of DNA restriction-modification systems were eliminated as well.Furthermore, it tolerated endogenous oxidative stress, acquired and replicated exogenous DNA, and survived better in stationary phase.

View Article: PubMed Central - PubMed

Affiliation: Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, 28049, Madrid, Spain. emartinez@cnb.csic.es.

ABSTRACT

Background: Because of its adaptability to sites polluted with toxic chemicals, the model soil bacterium Pseudomonas putida is naturally endowed with a number of metabolic and stress-endurance qualities which have considerable value for hosting energy-demanding and redox reactions thereof. The growing body of knowledge on P. putida strain KT2440 has been exploited for the rational design of a derivative strain in which the genome has been heavily edited in order to construct a robust microbial cell factory.

Results: Eleven non-adjacent genomic deletions, which span 300 genes (i.e., 4.3% of the entire P. putida KT2440 genome), were eliminated; thereby enhancing desirable traits and eliminating attributes which are detrimental in an expression host. Since ATP and NAD(P)H availability - as well as genetic instability, are generally considered to be major bottlenecks for the performance of platform strains, a suite of functions that drain high-energy phosphate from the cells and/or consume NAD(P)H were targeted in particular, the whole flagellar machinery. Four prophages, two transposons, and three components of DNA restriction-modification systems were eliminated as well. The resulting strain (P. putida EM383) displayed growth properties (i.e., lag times, biomass yield, and specific growth rates) clearly superior to the precursor wild-type strain KT2440. Furthermore, it tolerated endogenous oxidative stress, acquired and replicated exogenous DNA, and survived better in stationary phase. The performance of a bi-cistronic GFP-LuxCDABE reporter system as a proxy of combined metabolic vitality, revealed that the deletions in P. putida strain EM383 brought about an increase of >50% in the overall physiological vigour.

Conclusion: The rationally modified P. putida strain allowed for the better functional expression of implanted genes by directly improving the metabolic currency that sustains the gene expression flow, instead of resorting to the classical genetic approaches (e.g., increasing the promoter strength in the DNA constructs of interest).

Show MeSH
Related in: MedlinePlus