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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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Cellular viability assay. The cellular viability of wild-type KT2440 (blue) and the streamlined strain EM383 (green) was compared in stationary-phase cultures. Cells grown overnight in LB medium or in M9 minimal medium added with 0.2% (w/v) of either citrate, glucose, or succinate were stained with propidium iodide (PI) and analysed by flow cytometry to estimate the percentage of dead cells (i.e., PI+ cells). The results of at least four biological independent experiments are represented as box plot charts (Tukey-style box plots). The median is marked as a grey line within the charts and the dark grey dots outside the box represent the data outliers. The results for P. putida KT2440 grown in M9 minimal medium with citrate and some of the replicas in LB and M9-glucose are taken from Martínez-García et al. [36]. The asterisks (**) indicate a significant difference for strain EM383 as compared to wild-type KT2440 according to the Mann–Whitney U test (P <0.01).
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Fig6: Cellular viability assay. The cellular viability of wild-type KT2440 (blue) and the streamlined strain EM383 (green) was compared in stationary-phase cultures. Cells grown overnight in LB medium or in M9 minimal medium added with 0.2% (w/v) of either citrate, glucose, or succinate were stained with propidium iodide (PI) and analysed by flow cytometry to estimate the percentage of dead cells (i.e., PI+ cells). The results of at least four biological independent experiments are represented as box plot charts (Tukey-style box plots). The median is marked as a grey line within the charts and the dark grey dots outside the box represent the data outliers. The results for P. putida KT2440 grown in M9 minimal medium with citrate and some of the replicas in LB and M9-glucose are taken from Martínez-García et al. [36]. The asterisks (**) indicate a significant difference for strain EM383 as compared to wild-type KT2440 according to the Mann–Whitney U test (P <0.01).

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)

Cellular viability assay. The cellular viability of wild-type KT2440 (blue) and the streamlined strain EM383 (green) was compared in stationary-phase cultures. Cells grown overnight in LB medium or in M9 minimal medium added with 0.2% (w/v) of either citrate, glucose, or succinate were stained with propidium iodide (PI) and analysed by flow cytometry to estimate the percentage of dead cells (i.e., PI+ cells). The results of at least four biological independent experiments are represented as box plot charts (Tukey-style box plots). The median is marked as a grey line within the charts and the dark grey dots outside the box represent the data outliers. The results for P. putida KT2440 grown in M9 minimal medium with citrate and some of the replicas in LB and M9-glucose are taken from Martínez-García et al. [36]. The asterisks (**) indicate a significant difference for strain EM383 as compared to wild-type KT2440 according to the Mann–Whitney U test (P <0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Cellular viability assay. The cellular viability of wild-type KT2440 (blue) and the streamlined strain EM383 (green) was compared in stationary-phase cultures. Cells grown overnight in LB medium or in M9 minimal medium added with 0.2% (w/v) of either citrate, glucose, or succinate were stained with propidium iodide (PI) and analysed by flow cytometry to estimate the percentage of dead cells (i.e., PI+ cells). The results of at least four biological independent experiments are represented as box plot charts (Tukey-style box plots). The median is marked as a grey line within the charts and the dark grey dots outside the box represent the data outliers. The results for P. putida KT2440 grown in M9 minimal medium with citrate and some of the replicas in LB and M9-glucose are taken from Martínez-García et al. [36]. The asterisks (**) indicate a significant difference for strain EM383 as compared to wild-type KT2440 according to the Mann–Whitney U test (P <0.01).
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