Limits...
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).

Show MeSH

Related in: MedlinePlus

Growth parameters ofP.putidaKT2440 and the streamlined strain EM383. (A) Duration of the lag phase of wild-type KT2440 cells (blue) and the streamlined strain EM383 (green) in rich LB medium or M9 minimal medium added with 0.2% (w/v) of either succinate (Suc), citrate (Cit), glucose (Glu), or fructose (Fru). The extent of the lag phase was calculated using data from growth curves as described by Dalgaard and Koutsoumanis [67]. (B) Final cell density (estimated as the optical density at 600 nm, OD600) of shaken-flask cultures of wild-type KT2440 (blue) and the streamlined strain EM383 (green) in rich LB medium. (C) Final cell density (estimated as the OD600) of shaken-flask cultures of wild-type KT2440 (blue) and the streamlined strain EM383 (green) in M9 minimal medium added with 0.2% (w/v) of either succinate (Suc), citrate (Cit), glucose (Glu), or fructose (Fru). In all cases, the mean values of the corresponding parameter are plotted along with the SD of three independent experiments. The asterisks indicate a significant difference in the corresponding parameter when comparing strain EM383 and wild-type KT2440 according to the Student’s t test (*, P <0.05; and **, P <0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4230525&req=5

Fig2: Growth parameters ofP.putidaKT2440 and the streamlined strain EM383. (A) Duration of the lag phase of wild-type KT2440 cells (blue) and the streamlined strain EM383 (green) in rich LB medium or M9 minimal medium added with 0.2% (w/v) of either succinate (Suc), citrate (Cit), glucose (Glu), or fructose (Fru). The extent of the lag phase was calculated using data from growth curves as described by Dalgaard and Koutsoumanis [67]. (B) Final cell density (estimated as the optical density at 600 nm, OD600) of shaken-flask cultures of wild-type KT2440 (blue) and the streamlined strain EM383 (green) in rich LB medium. (C) Final cell density (estimated as the OD600) of shaken-flask cultures of wild-type KT2440 (blue) and the streamlined strain EM383 (green) in M9 minimal medium added with 0.2% (w/v) of either succinate (Suc), citrate (Cit), glucose (Glu), or fructose (Fru). In all cases, the mean values of the corresponding parameter are plotted along with the SD of three independent experiments. The asterisks indicate a significant difference in the corresponding parameter when comparing strain EM383 and wild-type KT2440 according to the Student’s t test (*, P <0.05; and **, P <0.01).

Mentions: The first observable traits acquired by the multi-deleted strain P. putida EM383 were revealed by comparing its growth properties with those of the wild-type KT2440 strain. These tests were made both in rich LB medium and in M9 minimal medium supplemented with C sources that elicit different metabolic regimes, i.e., succinate and citrate for gluconeogenesis, or glucose and fructose for glycolysis. Three separate growth parameters were considered to this end. First, the duration of the lag phase before cells take off to grow exponentially was assessed. This parameter seems to be associated with the ability of cells to overcome the oxidative damage that is inherited from the stationary phase that they come from [66]. To examine this issue in our strains, growth curves were carried out in 96-well microtiter plates inoculated with an standard number of cells of each strain coming from overnight cultures, and then passed to the different culture media as explained in the Methods section. Inspection of the data of Figure 2A revealed that P. putida EM383 had a significantly shorter lag phase than wild-type cells in all instances, the effect being more evident when fructose was used as C source (2.8 ± 0.1 h for the wild-type strain vs. 0.8 ± 0.3 h for the EM383 streamlined strain). Since this early take off was observed before in P. putida cells lacking the flagella, it is likely that the property acquired by strain EM383 is due to the loss of the same genes (see below). The shorter lag phase phenomenon makes sense as cells without the flagellar operon have an increased level of NADPH that probably helps mitigating oxidative stress [57].Figure 2


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)

Growth parameters ofP.putidaKT2440 and the streamlined strain EM383. (A) Duration of the lag phase of wild-type KT2440 cells (blue) and the streamlined strain EM383 (green) in rich LB medium or M9 minimal medium added with 0.2% (w/v) of either succinate (Suc), citrate (Cit), glucose (Glu), or fructose (Fru). The extent of the lag phase was calculated using data from growth curves as described by Dalgaard and Koutsoumanis [67]. (B) Final cell density (estimated as the optical density at 600 nm, OD600) of shaken-flask cultures of wild-type KT2440 (blue) and the streamlined strain EM383 (green) in rich LB medium. (C) Final cell density (estimated as the OD600) of shaken-flask cultures of wild-type KT2440 (blue) and the streamlined strain EM383 (green) in M9 minimal medium added with 0.2% (w/v) of either succinate (Suc), citrate (Cit), glucose (Glu), or fructose (Fru). In all cases, the mean values of the corresponding parameter are plotted along with the SD of three independent experiments. The asterisks indicate a significant difference in the corresponding parameter when comparing strain EM383 and wild-type KT2440 according to the Student’s t test (*, P <0.05; and **, P <0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230525&req=5

Fig2: Growth parameters ofP.putidaKT2440 and the streamlined strain EM383. (A) Duration of the lag phase of wild-type KT2440 cells (blue) and the streamlined strain EM383 (green) in rich LB medium or M9 minimal medium added with 0.2% (w/v) of either succinate (Suc), citrate (Cit), glucose (Glu), or fructose (Fru). The extent of the lag phase was calculated using data from growth curves as described by Dalgaard and Koutsoumanis [67]. (B) Final cell density (estimated as the optical density at 600 nm, OD600) of shaken-flask cultures of wild-type KT2440 (blue) and the streamlined strain EM383 (green) in rich LB medium. (C) Final cell density (estimated as the OD600) of shaken-flask cultures of wild-type KT2440 (blue) and the streamlined strain EM383 (green) in M9 minimal medium added with 0.2% (w/v) of either succinate (Suc), citrate (Cit), glucose (Glu), or fructose (Fru). In all cases, the mean values of the corresponding parameter are plotted along with the SD of three independent experiments. The asterisks indicate a significant difference in the corresponding parameter when comparing strain EM383 and wild-type KT2440 according to the Student’s t test (*, P <0.05; and **, P <0.01).
Mentions: The first observable traits acquired by the multi-deleted strain P. putida EM383 were revealed by comparing its growth properties with those of the wild-type KT2440 strain. These tests were made both in rich LB medium and in M9 minimal medium supplemented with C sources that elicit different metabolic regimes, i.e., succinate and citrate for gluconeogenesis, or glucose and fructose for glycolysis. Three separate growth parameters were considered to this end. First, the duration of the lag phase before cells take off to grow exponentially was assessed. This parameter seems to be associated with the ability of cells to overcome the oxidative damage that is inherited from the stationary phase that they come from [66]. To examine this issue in our strains, growth curves were carried out in 96-well microtiter plates inoculated with an standard number of cells of each strain coming from overnight cultures, and then passed to the different culture media as explained in the Methods section. Inspection of the data of Figure 2A revealed that P. putida EM383 had a significantly shorter lag phase than wild-type cells in all instances, the effect being more evident when fructose was used as C source (2.8 ± 0.1 h for the wild-type strain vs. 0.8 ± 0.3 h for the EM383 streamlined strain). Since this early take off was observed before in P. putida cells lacking the flagella, it is likely that the property acquired by strain EM383 is due to the loss of the same genes (see below). The shorter lag phase phenomenon makes sense as cells without the flagellar operon have an increased level of NADPH that probably helps mitigating oxidative stress [57].Figure 2

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