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Genome reduction boosts heterologous gene expression in Pseudomonas putida.

Lieder S, Nikel PI, de Lorenzo V, Takors R - Microb. Cell Fact. (2015)

Bottom Line: In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome).Under all the conditions tested the mutants also grew faster, had enhanced biomass yields and showed higher viability, and displayed increased plasmid stability than the parental strain.When the production of the green fluorescent protein (used as a model heterologous protein) was assessed in these cultures, the mutants reached a recombinant protein yield with respect to biomass up to 40% higher than that of P. putida KT2440.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany. lieder@ibvt.uni-stuttgart.de.

ABSTRACT

Background: The implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. Ongoing efforts include the adoption of Pseudomonas putida KT2440 variants with a reduced genome as the functional chassis for biotechnological purposes. In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome).

Results: In this study, two previously constructed multiple-deletion P. putida strains were systematically evaluated as microbial cell factories for heterologous protein production and compared to the parental bacterium (strain KT2440) with regards to several industrially-relevant physiological traits. Energetic parameters were quantified at different controlled growth rates in continuous cultivations and both strains had a higher adenosine triphosphate content, increased adenylate energy charges, and diminished maintenance demands than the wild-type strain. Under all the conditions tested the mutants also grew faster, had enhanced biomass yields and showed higher viability, and displayed increased plasmid stability than the parental strain. In addition to small-scale shaken-flask cultivations, the performance of the genome-streamlined strains was evaluated in larger scale bioreactor batch cultivations taking a step towards industrial growth conditions. When the production of the green fluorescent protein (used as a model heterologous protein) was assessed in these cultures, the mutants reached a recombinant protein yield with respect to biomass up to 40% higher than that of P. putida KT2440.

Conclusions: The two streamlined-genome derivatives of P. putida KT2440 outcompeted the parental strain in every industrially-relevant trait assessed, particularly under the working conditions of a bioreactor. Our results demonstrate that these genome-streamlined bacteria are not only robust microbial cell factories on their own, but also a promising foundation for further biotechnological applications.

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Related in: MedlinePlus

Summary of the growth parameters for the different strains under study in glucose-limited chemostat cultures. Shown are (A) the biomass yield coefficient (YX/S), calculated at three different dilution rates (D), and (B) the maintenance coefficient (mS). The growth parameters were calculated based on three independent biological experiments conducted in triplicate, and the bars represent the mean value of the corresponding parameter ± standard deviations.
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Fig2: Summary of the growth parameters for the different strains under study in glucose-limited chemostat cultures. Shown are (A) the biomass yield coefficient (YX/S), calculated at three different dilution rates (D), and (B) the maintenance coefficient (mS). The growth parameters were calculated based on three independent biological experiments conducted in triplicate, and the bars represent the mean value of the corresponding parameter ± standard deviations.

Mentions: The starting point in the characterization of the strains was the setup of continuous cultivations to explore the key kinetic and process parameters at different growth rates (Additional file 1: Figure S1). Yield coefficients, reflecting the efficiency in the conversion of the substrate into cell components, were calculated in glucose-limited continuous cultivations at steady-state conditions for various D values (Figure 2A). The mutant strains showed a higher YX/S value (statistically significant, P < 0.05) at all growth rates when compared to the wild-type strain. The highest difference (ca. 12%) was observed when comparing strain EM383 with wild-type KT2440 at D = 0.1 h−1. The differences between P. putida EM329 and EM383, on the contrary, were not statistically significant. The carbon emission rates (i.e., evolution of CO2) differed significantly between the strains. Averaging over all the tested D values, strains EM329 and EM383 had 9% and 16% lower CO2 formation, respectively, as compared to P. putida KT2440 (Additional file 1: Figure S2). This result suggests that the carbon substrate saved by-passing the synthesis of some cellular components (e.g., flagella) can be used for macromolecular biosynthesis, accompanied by a low evolution of CO2, an interesting trait for bioprocesses that depend on biomass formation. The next relevant question was whether these differences in biomass yields also correlate with energy maintenance.Figure 2


Genome reduction boosts heterologous gene expression in Pseudomonas putida.

Lieder S, Nikel PI, de Lorenzo V, Takors R - Microb. Cell Fact. (2015)

Summary of the growth parameters for the different strains under study in glucose-limited chemostat cultures. Shown are (A) the biomass yield coefficient (YX/S), calculated at three different dilution rates (D), and (B) the maintenance coefficient (mS). The growth parameters were calculated based on three independent biological experiments conducted in triplicate, and the bars represent the mean value of the corresponding parameter ± standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4352270&req=5

Fig2: Summary of the growth parameters for the different strains under study in glucose-limited chemostat cultures. Shown are (A) the biomass yield coefficient (YX/S), calculated at three different dilution rates (D), and (B) the maintenance coefficient (mS). The growth parameters were calculated based on three independent biological experiments conducted in triplicate, and the bars represent the mean value of the corresponding parameter ± standard deviations.
Mentions: The starting point in the characterization of the strains was the setup of continuous cultivations to explore the key kinetic and process parameters at different growth rates (Additional file 1: Figure S1). Yield coefficients, reflecting the efficiency in the conversion of the substrate into cell components, were calculated in glucose-limited continuous cultivations at steady-state conditions for various D values (Figure 2A). The mutant strains showed a higher YX/S value (statistically significant, P < 0.05) at all growth rates when compared to the wild-type strain. The highest difference (ca. 12%) was observed when comparing strain EM383 with wild-type KT2440 at D = 0.1 h−1. The differences between P. putida EM329 and EM383, on the contrary, were not statistically significant. The carbon emission rates (i.e., evolution of CO2) differed significantly between the strains. Averaging over all the tested D values, strains EM329 and EM383 had 9% and 16% lower CO2 formation, respectively, as compared to P. putida KT2440 (Additional file 1: Figure S2). This result suggests that the carbon substrate saved by-passing the synthesis of some cellular components (e.g., flagella) can be used for macromolecular biosynthesis, accompanied by a low evolution of CO2, an interesting trait for bioprocesses that depend on biomass formation. The next relevant question was whether these differences in biomass yields also correlate with energy maintenance.Figure 2

Bottom Line: In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome).Under all the conditions tested the mutants also grew faster, had enhanced biomass yields and showed higher viability, and displayed increased plasmid stability than the parental strain.When the production of the green fluorescent protein (used as a model heterologous protein) was assessed in these cultures, the mutants reached a recombinant protein yield with respect to biomass up to 40% higher than that of P. putida KT2440.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany. lieder@ibvt.uni-stuttgart.de.

ABSTRACT

Background: The implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. Ongoing efforts include the adoption of Pseudomonas putida KT2440 variants with a reduced genome as the functional chassis for biotechnological purposes. In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome).

Results: In this study, two previously constructed multiple-deletion P. putida strains were systematically evaluated as microbial cell factories for heterologous protein production and compared to the parental bacterium (strain KT2440) with regards to several industrially-relevant physiological traits. Energetic parameters were quantified at different controlled growth rates in continuous cultivations and both strains had a higher adenosine triphosphate content, increased adenylate energy charges, and diminished maintenance demands than the wild-type strain. Under all the conditions tested the mutants also grew faster, had enhanced biomass yields and showed higher viability, and displayed increased plasmid stability than the parental strain. In addition to small-scale shaken-flask cultivations, the performance of the genome-streamlined strains was evaluated in larger scale bioreactor batch cultivations taking a step towards industrial growth conditions. When the production of the green fluorescent protein (used as a model heterologous protein) was assessed in these cultures, the mutants reached a recombinant protein yield with respect to biomass up to 40% higher than that of P. putida KT2440.

Conclusions: The two streamlined-genome derivatives of P. putida KT2440 outcompeted the parental strain in every industrially-relevant trait assessed, particularly under the working conditions of a bioreactor. Our results demonstrate that these genome-streamlined bacteria are not only robust microbial cell factories on their own, but also a promising foundation for further biotechnological applications.

Show MeSH
Related in: MedlinePlus