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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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Related in: MedlinePlus

Biomass yields of wild-typeP.putidaKT2440 and the streamlined strain EM383 in shaken-flask cultures. Biomass yield coefficients (YX/S) of wild-type P. putida KT2440 and P. putida EM383 were estimated in glucose and succinate shaken-flask cultures, as representative glycolytic and gluconeogenic C sources, respectively. Cells were grown as described in the Methods section, and the biomass yield coefficients were calculated during exponential growth by measuring the formation of biomass (CDW, cell dry weight) and the specific rate of substrate consumption. Growth parameters were based on three independent biological experiments conducted in triplicates, and the bars represent the mean value of the corresponding parameter and SD. 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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Fig3: Biomass yields of wild-typeP.putidaKT2440 and the streamlined strain EM383 in shaken-flask cultures. Biomass yield coefficients (YX/S) of wild-type P. putida KT2440 and P. putida EM383 were estimated in glucose and succinate shaken-flask cultures, as representative glycolytic and gluconeogenic C sources, respectively. Cells were grown as described in the Methods section, and the biomass yield coefficients were calculated during exponential growth by measuring the formation of biomass (CDW, cell dry weight) and the specific rate of substrate consumption. Growth parameters were based on three independent biological experiments conducted in triplicates, and the bars represent the mean value of the corresponding parameter and SD. 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 second important physiological parameter was the maximum growth rate. As shown in Table 2, the differences between strains in this case were not significant, except for LB cultures, where growth of the wild-type strain was slightly better. In all, these figures indicate that the multiple deletions introduced do not significantly affect the growth performance of strain EM383. However, it is to notice that faster growth also means more oxidative stress [68,69], which needs to be counteracted to the detriment of the NAD(P)H pool [70], thereby resulting in a reduced biomass yield. This prediction was confirmed when the final optical density at 600 nm (OD600) was assessed in shaken-flask cultures following 24 h of vigorous shaking (Figure 2B and C). Under these conditions, P. putida EM383 reached OD600 values both in LB medium (Figure 2B) and in M9 minimal medium amended with fructose (Figure 2C) that was remarkably higher than those for the wild-type strain. Note that fructose is the only C source that can be diverted through a standard glycolytic route in P. putida KT2440 [71]. These differences were exacerbated when the cultures were subject to a more intense aeration (data not shown), suggesting that the observed effect is connected to the way either strain deals with oxidative stress and ROS formation. Should that be the case, differences between the strains had to be noticed in cultures with glucose and succinate, which do not manifest in the data of Figure 2C. However, when the biomass yield coefficients (YX/S) of the wild-type P. putida KT2440 and P. putida EM383 on glucose and succinate cultures (Figure 3) were accurately calculated, a significant divergence between the two was observed in favour of the multi-deleted strain. Every growth parameter thus accredits a gross gain of physiological performance in P. putida EM383 cells.Table 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)

Biomass yields of wild-typeP.putidaKT2440 and the streamlined strain EM383 in shaken-flask cultures. Biomass yield coefficients (YX/S) of wild-type P. putida KT2440 and P. putida EM383 were estimated in glucose and succinate shaken-flask cultures, as representative glycolytic and gluconeogenic C sources, respectively. Cells were grown as described in the Methods section, and the biomass yield coefficients were calculated during exponential growth by measuring the formation of biomass (CDW, cell dry weight) and the specific rate of substrate consumption. Growth parameters were based on three independent biological experiments conducted in triplicates, and the bars represent the mean value of the corresponding parameter and SD. 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

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

Fig3: Biomass yields of wild-typeP.putidaKT2440 and the streamlined strain EM383 in shaken-flask cultures. Biomass yield coefficients (YX/S) of wild-type P. putida KT2440 and P. putida EM383 were estimated in glucose and succinate shaken-flask cultures, as representative glycolytic and gluconeogenic C sources, respectively. Cells were grown as described in the Methods section, and the biomass yield coefficients were calculated during exponential growth by measuring the formation of biomass (CDW, cell dry weight) and the specific rate of substrate consumption. Growth parameters were based on three independent biological experiments conducted in triplicates, and the bars represent the mean value of the corresponding parameter and SD. 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 second important physiological parameter was the maximum growth rate. As shown in Table 2, the differences between strains in this case were not significant, except for LB cultures, where growth of the wild-type strain was slightly better. In all, these figures indicate that the multiple deletions introduced do not significantly affect the growth performance of strain EM383. However, it is to notice that faster growth also means more oxidative stress [68,69], which needs to be counteracted to the detriment of the NAD(P)H pool [70], thereby resulting in a reduced biomass yield. This prediction was confirmed when the final optical density at 600 nm (OD600) was assessed in shaken-flask cultures following 24 h of vigorous shaking (Figure 2B and C). Under these conditions, P. putida EM383 reached OD600 values both in LB medium (Figure 2B) and in M9 minimal medium amended with fructose (Figure 2C) that was remarkably higher than those for the wild-type strain. Note that fructose is the only C source that can be diverted through a standard glycolytic route in P. putida KT2440 [71]. These differences were exacerbated when the cultures were subject to a more intense aeration (data not shown), suggesting that the observed effect is connected to the way either strain deals with oxidative stress and ROS formation. Should that be the case, differences between the strains had to be noticed in cultures with glucose and succinate, which do not manifest in the data of Figure 2C. However, when the biomass yield coefficients (YX/S) of the wild-type P. putida KT2440 and P. putida EM383 on glucose and succinate cultures (Figure 3) were accurately calculated, a significant divergence between the two was observed in favour of the multi-deleted strain. Every growth parameter thus accredits a gross gain of physiological performance in P. putida EM383 cells.Table 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