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

Cell survival of wild-typeP.putidaKT2440 and the streamlined strain EM383 in the presence of different stressors. (A) Drop assays were used to compare the fitness of wild-type KT2440 and strain EM383 when exposed to different chemical stressors. Overnight cultures were diluted in PBS and spotted onto LB agar plates supplemented with the particular compound (250 μg ml−1 carbenicillin, 50 μg ml−1 ampicillin, or 10 μM paraquat). LB medium was used in the control plate. (B) Survival ratio plots of wild-type KT2440 (blue) and strain EM383 (green) when exposed to 10 μM paraquat during 24 h. The survival ratio was calculated by dividing the optical density at 600 nm (OD600) of cultures with the drug to the OD600 of control cultures without paraquat along the time, thus merging the intrinsic effect of the stressor with the growth capability of each strain. Cells were grown in M9 minimal medium amended with 0.2% (w/v) of either glucose or succinate. The mean survival ratio values are plotted along with the SD of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Cell survival of wild-typeP.putidaKT2440 and the streamlined strain EM383 in the presence of different stressors. (A) Drop assays were used to compare the fitness of wild-type KT2440 and strain EM383 when exposed to different chemical stressors. Overnight cultures were diluted in PBS and spotted onto LB agar plates supplemented with the particular compound (250 μg ml−1 carbenicillin, 50 μg ml−1 ampicillin, or 10 μM paraquat). LB medium was used in the control plate. (B) Survival ratio plots of wild-type KT2440 (blue) and strain EM383 (green) when exposed to 10 μM paraquat during 24 h. The survival ratio was calculated by dividing the optical density at 600 nm (OD600) of cultures with the drug to the OD600 of control cultures without paraquat along the time, thus merging the intrinsic effect of the stressor with the growth capability of each strain. Cells were grown in M9 minimal medium amended with 0.2% (w/v) of either glucose or succinate. The mean survival ratio values are plotted along with the SD of three independent experiments.

Mentions: As mentioned above, one of the inherent qualities of P. putida is its ability to host redox reactions that often result in a high level of intracellular ROS [21]. One way to mimic such oxidative stress is treating cells with the chemical paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride). This compound is reduced in vivo by NADPH and then oxidized by an electron receptor such as dioxygen to produce superoxide, a major contributor to the ROS pool [73]. Therefore, paraquat both decreases NADPH and causes endogenous ROS (which itself has to be counteracted with enzymes that are ultimately fuelled by NADPH). The simple spot dilution test shown in Figure 5A indicated that P. putida EM383 performs better in the presence of the oxidative agent than the wild-type strain – a trait that correlates with the high NADPH/NADP+ ratio reported above. To have a better quantification of this phenomenon, the survival ratio of either strain was examined (Figure 5B) when grown in M9 minimal media with either glucose or succinate as the C source and in the presence of paraquat as detailed in the Methods section. The resulting curves expose both the early sensitivity to the stressor and how well they recover after the insult, since the dimensionless survival ratio merges both the tolerance and the intrinsic growth ability of each strain. Figure 5B clearly shows a better response of the cell-factory strain in both situations: after 8 h, the survival ratio of the streamlined strain EM383 in glucose was 0.26 ± 0,10 (0.05 ± 0.02 for the wild-type strain), while in succinate the survival ratio of strain EM383 was 0.30 ± 0.04 (0.10 ± 0.01 for the wild-type strain). This dataset documents that the observed increase of the redox charge of P. putida EM383 is concomitant with (and perhaps the reason of) the superior tolerance of this strain to endogenous redox stress and ROS damage. Figure 5A also shows that strain EM383 was more sensitive to the β-lactam antibiotics carbenicillin and ampicillin. This characteristic is surely originated in the lack of flagella, as observed before [57].Figure 5


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)

Cell survival of wild-typeP.putidaKT2440 and the streamlined strain EM383 in the presence of different stressors. (A) Drop assays were used to compare the fitness of wild-type KT2440 and strain EM383 when exposed to different chemical stressors. Overnight cultures were diluted in PBS and spotted onto LB agar plates supplemented with the particular compound (250 μg ml−1 carbenicillin, 50 μg ml−1 ampicillin, or 10 μM paraquat). LB medium was used in the control plate. (B) Survival ratio plots of wild-type KT2440 (blue) and strain EM383 (green) when exposed to 10 μM paraquat during 24 h. The survival ratio was calculated by dividing the optical density at 600 nm (OD600) of cultures with the drug to the OD600 of control cultures without paraquat along the time, thus merging the intrinsic effect of the stressor with the growth capability of each strain. Cells were grown in M9 minimal medium amended with 0.2% (w/v) of either glucose or succinate. The mean survival ratio values are plotted along with the SD of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Cell survival of wild-typeP.putidaKT2440 and the streamlined strain EM383 in the presence of different stressors. (A) Drop assays were used to compare the fitness of wild-type KT2440 and strain EM383 when exposed to different chemical stressors. Overnight cultures were diluted in PBS and spotted onto LB agar plates supplemented with the particular compound (250 μg ml−1 carbenicillin, 50 μg ml−1 ampicillin, or 10 μM paraquat). LB medium was used in the control plate. (B) Survival ratio plots of wild-type KT2440 (blue) and strain EM383 (green) when exposed to 10 μM paraquat during 24 h. The survival ratio was calculated by dividing the optical density at 600 nm (OD600) of cultures with the drug to the OD600 of control cultures without paraquat along the time, thus merging the intrinsic effect of the stressor with the growth capability of each strain. Cells were grown in M9 minimal medium amended with 0.2% (w/v) of either glucose or succinate. The mean survival ratio values are plotted along with the SD of three independent experiments.
Mentions: As mentioned above, one of the inherent qualities of P. putida is its ability to host redox reactions that often result in a high level of intracellular ROS [21]. One way to mimic such oxidative stress is treating cells with the chemical paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride). This compound is reduced in vivo by NADPH and then oxidized by an electron receptor such as dioxygen to produce superoxide, a major contributor to the ROS pool [73]. Therefore, paraquat both decreases NADPH and causes endogenous ROS (which itself has to be counteracted with enzymes that are ultimately fuelled by NADPH). The simple spot dilution test shown in Figure 5A indicated that P. putida EM383 performs better in the presence of the oxidative agent than the wild-type strain – a trait that correlates with the high NADPH/NADP+ ratio reported above. To have a better quantification of this phenomenon, the survival ratio of either strain was examined (Figure 5B) when grown in M9 minimal media with either glucose or succinate as the C source and in the presence of paraquat as detailed in the Methods section. The resulting curves expose both the early sensitivity to the stressor and how well they recover after the insult, since the dimensionless survival ratio merges both the tolerance and the intrinsic growth ability of each strain. Figure 5B clearly shows a better response of the cell-factory strain in both situations: after 8 h, the survival ratio of the streamlined strain EM383 in glucose was 0.26 ± 0,10 (0.05 ± 0.02 for the wild-type strain), while in succinate the survival ratio of strain EM383 was 0.30 ± 0.04 (0.10 ± 0.01 for the wild-type strain). This dataset documents that the observed increase of the redox charge of P. putida EM383 is concomitant with (and perhaps the reason of) the superior tolerance of this strain to endogenous redox stress and ROS damage. Figure 5A also shows that strain EM383 was more sensitive to the β-lactam antibiotics carbenicillin and ampicillin. This characteristic is surely originated in the lack of flagella, as observed before [57].Figure 5

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