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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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Characterization of the energy and redox status of wild-typeP.putidaKT2440 and the streamlined strain EM383. (A) The ATP content on biomass (YATP/X) and the adenylate energy charge (AEC) were calculated for exponentially-growing cells in shaken-flask cultures using M9 minimal medium containing 0.2% (w/v) glucose. Each bar represents the mean value and SD of the ATP content on biomass (CDW, cell dry weight) or the adenylate energy charge for duplicate measurements from at least three independent experiments. (B) Redox ratios were determined from the absolute intracellular concentrations of NAD+, NADH, NADP+, and NADPH. The pyridine nucleotide cofactors were enzymatically determined in exponentially-growing cells in shaken-flask cultures using M9 minimal medium containing 0.2% (w/v) glucose. Bars represent mean values along with SD of the corresponding parameter for duplicate measurements from at least three independent experiments. 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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Fig4: Characterization of the energy and redox status of wild-typeP.putidaKT2440 and the streamlined strain EM383. (A) The ATP content on biomass (YATP/X) and the adenylate energy charge (AEC) were calculated for exponentially-growing cells in shaken-flask cultures using M9 minimal medium containing 0.2% (w/v) glucose. Each bar represents the mean value and SD of the ATP content on biomass (CDW, cell dry weight) or the adenylate energy charge for duplicate measurements from at least three independent experiments. (B) Redox ratios were determined from the absolute intracellular concentrations of NAD+, NADH, NADP+, and NADPH. The pyridine nucleotide cofactors were enzymatically determined in exponentially-growing cells in shaken-flask cultures using M9 minimal medium containing 0.2% (w/v) glucose. Bars represent mean values along with SD of the corresponding parameter for duplicate measurements from at least three independent experiments. 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: The growth parameters described in the preceding section indicated that P. putida EM383 had an enhanced capacity to transform metabolic precursors into biomass. The biochemical reasons for this phenomenon were analyzed by exploring the energy and redox status of the cells at stake. Figure 4A shows the energy standing of the strains under examination in terms of both the ATP availability and the adenylate energy charge (AEC), a parameter which weights the ATP content against all the three possible phosphorylated forms of adenine. During exponential growth on glucose, EM383 cells had a 1.6-fold higher content of ATP per biomass unit [YATP/X, in μmol g cell dry weight (CDW)−1] than P. putida KT2440. This feature was translated into a 1.2-fold increase in the AEC. This surplus of high-energy phosphate in P. putida EM383 not only helps explaining the enhanced growth of this strain under different culture conditions, but also endows cells with an extra capacity to sustain extra ATP (and other NTPs) consuming functions. In close connection with the energy status, a significant increase in the NADPH/NADP+ ratio was observed in P. putida EM383 strain as compared to the wild-type (Figure 4B). It is remarkable that such redox charge was manifested mostly for anabolic processes, as the NADPH/NADP+ ratio was 1.3-fold higher in P. putida EM383 than in strain KT2440, but not in its catabolic counterpart (i.e., the NADH/NAD+ ratio, which remained more or less the same in the two strains). The larger split of NADPH signals this strain as more capable to sustain biotransformations that demand a higher redox charge [20].Figure 4


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)

Characterization of the energy and redox status of wild-typeP.putidaKT2440 and the streamlined strain EM383. (A) The ATP content on biomass (YATP/X) and the adenylate energy charge (AEC) were calculated for exponentially-growing cells in shaken-flask cultures using M9 minimal medium containing 0.2% (w/v) glucose. Each bar represents the mean value and SD of the ATP content on biomass (CDW, cell dry weight) or the adenylate energy charge for duplicate measurements from at least three independent experiments. (B) Redox ratios were determined from the absolute intracellular concentrations of NAD+, NADH, NADP+, and NADPH. The pyridine nucleotide cofactors were enzymatically determined in exponentially-growing cells in shaken-flask cultures using M9 minimal medium containing 0.2% (w/v) glucose. Bars represent mean values along with SD of the corresponding parameter for duplicate measurements from at least three independent experiments. 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: Characterization of the energy and redox status of wild-typeP.putidaKT2440 and the streamlined strain EM383. (A) The ATP content on biomass (YATP/X) and the adenylate energy charge (AEC) were calculated for exponentially-growing cells in shaken-flask cultures using M9 minimal medium containing 0.2% (w/v) glucose. Each bar represents the mean value and SD of the ATP content on biomass (CDW, cell dry weight) or the adenylate energy charge for duplicate measurements from at least three independent experiments. (B) Redox ratios were determined from the absolute intracellular concentrations of NAD+, NADH, NADP+, and NADPH. The pyridine nucleotide cofactors were enzymatically determined in exponentially-growing cells in shaken-flask cultures using M9 minimal medium containing 0.2% (w/v) glucose. Bars represent mean values along with SD of the corresponding parameter for duplicate measurements from at least three independent experiments. 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: The growth parameters described in the preceding section indicated that P. putida EM383 had an enhanced capacity to transform metabolic precursors into biomass. The biochemical reasons for this phenomenon were analyzed by exploring the energy and redox status of the cells at stake. Figure 4A shows the energy standing of the strains under examination in terms of both the ATP availability and the adenylate energy charge (AEC), a parameter which weights the ATP content against all the three possible phosphorylated forms of adenine. During exponential growth on glucose, EM383 cells had a 1.6-fold higher content of ATP per biomass unit [YATP/X, in μmol g cell dry weight (CDW)−1] than P. putida KT2440. This feature was translated into a 1.2-fold increase in the AEC. This surplus of high-energy phosphate in P. putida EM383 not only helps explaining the enhanced growth of this strain under different culture conditions, but also endows cells with an extra capacity to sustain extra ATP (and other NTPs) consuming functions. In close connection with the energy status, a significant increase in the NADPH/NADP+ ratio was observed in P. putida EM383 strain as compared to the wild-type (Figure 4B). It is remarkable that such redox charge was manifested mostly for anabolic processes, as the NADPH/NADP+ ratio was 1.3-fold higher in P. putida EM383 than in strain KT2440, but not in its catabolic counterpart (i.e., the NADH/NAD+ ratio, which remained more or less the same in the two strains). The larger split of NADPH signals this strain as more capable to sustain biotransformations that demand a higher redox charge [20].Figure 4

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