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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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Growth parameters and protein production kinetics for the strains under study in batch bioreactor cultures. Shown are the specific growth rate (μmax) for cells grown on (A) glucose and (B) citrate, as well as the effect of plasmid maintenance and heterologous protein production under these growth conditions. The accumulation of the green fluorescent protein (GFP) in cultures of the strains carrying pS234G was assessed during exponential growth on M12 minimal medium containing either glucose or citrate through (C) the maximum specific rate of GFP formation (πmax) and (D) the yield of GFP on biomass (YGFP/X). The growth parameters and protein production kinetics 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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Fig5: Growth parameters and protein production kinetics for the strains under study in batch bioreactor cultures. Shown are the specific growth rate (μmax) for cells grown on (A) glucose and (B) citrate, as well as the effect of plasmid maintenance and heterologous protein production under these growth conditions. The accumulation of the green fluorescent protein (GFP) in cultures of the strains carrying pS234G was assessed during exponential growth on M12 minimal medium containing either glucose or citrate through (C) the maximum specific rate of GFP formation (πmax) and (D) the yield of GFP on biomass (YGFP/X). The growth parameters and protein production kinetics 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 derivative SG strains reached statistically significant higher μmax values than the wild-type KT2440 strain in all the cultivations performed (Figure 5). When grown on glucose as the sole carbon source, EM329 showed a 7% and EM383 a 10% increase in μmax (Figure 5A, P < 0.05). When using citrate as the carbon source, EM329 showed 4% and EM383 showed 11% faster growth (Figure 5B, P < 0.05). Mutant EM383 also reached statistically significant higher μmax compared to strain EM329. Besides, both EM329 and EM383 attained higher final CDW concentrations when grown on glucose as the sole carbon source (9% and 13%, respectively when compared to P. putida KT2440; P < 0.05) (Additional file 1: Figure S4), mirroring the results already observed in shaken-flask cultures (Table 2). This difference was not observed on citrate, as all the strains reached a similar final biomass density (Additional file 1: Figure S5). In all, these results show the importance of adequate aeration and mixing within the bioreactor. In the first place, all the strains attained higher μmax values and final cell densities in bioreactor cultivations as compared to the same traits in shaken-flask cultures. On the other hand, as both P. putida EM329 and EM383 are devoid of the flagellar machinery that would enable the cells to explore different microenvironments within the bioreactor, they tend to sediment and, if not properly stirred, the cells will likely become limited in O2 transfer, as previously hinted by Martínez-García et al. [23]. The same stirring speed and air bubbling applied to the bioreactor to grow P. putida KT2440 enabled a much better growth profile of the SG strains.Figure 5


Genome reduction boosts heterologous gene expression in Pseudomonas putida.

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

Growth parameters and protein production kinetics for the strains under study in batch bioreactor cultures. Shown are the specific growth rate (μmax) for cells grown on (A) glucose and (B) citrate, as well as the effect of plasmid maintenance and heterologous protein production under these growth conditions. The accumulation of the green fluorescent protein (GFP) in cultures of the strains carrying pS234G was assessed during exponential growth on M12 minimal medium containing either glucose or citrate through (C) the maximum specific rate of GFP formation (πmax) and (D) the yield of GFP on biomass (YGFP/X). The growth parameters and protein production kinetics 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

Fig5: Growth parameters and protein production kinetics for the strains under study in batch bioreactor cultures. Shown are the specific growth rate (μmax) for cells grown on (A) glucose and (B) citrate, as well as the effect of plasmid maintenance and heterologous protein production under these growth conditions. The accumulation of the green fluorescent protein (GFP) in cultures of the strains carrying pS234G was assessed during exponential growth on M12 minimal medium containing either glucose or citrate through (C) the maximum specific rate of GFP formation (πmax) and (D) the yield of GFP on biomass (YGFP/X). The growth parameters and protein production kinetics 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 derivative SG strains reached statistically significant higher μmax values than the wild-type KT2440 strain in all the cultivations performed (Figure 5). When grown on glucose as the sole carbon source, EM329 showed a 7% and EM383 a 10% increase in μmax (Figure 5A, P < 0.05). When using citrate as the carbon source, EM329 showed 4% and EM383 showed 11% faster growth (Figure 5B, P < 0.05). Mutant EM383 also reached statistically significant higher μmax compared to strain EM329. Besides, both EM329 and EM383 attained higher final CDW concentrations when grown on glucose as the sole carbon source (9% and 13%, respectively when compared to P. putida KT2440; P < 0.05) (Additional file 1: Figure S4), mirroring the results already observed in shaken-flask cultures (Table 2). This difference was not observed on citrate, as all the strains reached a similar final biomass density (Additional file 1: Figure S5). In all, these results show the importance of adequate aeration and mixing within the bioreactor. In the first place, all the strains attained higher μmax values and final cell densities in bioreactor cultivations as compared to the same traits in shaken-flask cultures. On the other hand, as both P. putida EM329 and EM383 are devoid of the flagellar machinery that would enable the cells to explore different microenvironments within the bioreactor, they tend to sediment and, if not properly stirred, the cells will likely become limited in O2 transfer, as previously hinted by Martínez-García et al. [23]. The same stirring speed and air bubbling applied to the bioreactor to grow P. putida KT2440 enabled a much better growth profile of the SG strains.Figure 5

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