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. 2015 Feb 21;14:23.
doi: 10.1186/s12934-015-0207-7.

Genome Reduction Boosts Heterologous Gene Expression in Pseudomonas Putida

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Free PMC article

Genome Reduction Boosts Heterologous Gene Expression in Pseudomonas Putida

Sarah Lieder et al. Microb Cell Fact. .
Free PMC article

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.

Figures

Figure 1
Figure 1
Rationale behind the design of reduced-genome derivatives of P . putida KT2440. Strains EM329 and EM383 were constructed using the seamless deletion system described by Martínez-García and de Lorenzo [20]. Note that while strain EM329 only lacks the genes encoding flagellar genes [23], the multiple deletions in strain EM383 were designed to endow the bacterium with the properties of a true microbial platform for a variety of applications. The relative physical location of the genes eliminated in the chromosome of P. putida KT2440 are indicated with slanted arrowheads and the percentage of the genome deleted is shown in each case. The red arrowhead represents the chromosomal location of the flagellar genes (deleted in strain EM329), while the orange arrowheads indicate the genes and gene clusters eliminated in strain EM383.
Figure 2
Figure 2
Summary of the growth parameters for the different strains under study in glucose-limited chemostat cultures. Shown are (A) the biomass yield coefficient (Y X/S), calculated at three different dilution rates (D), and (B) the maintenance coefficient (m S). 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.
Figure 3
Figure 3
Characterization of energy parameters for the different strains under study in glucose-limited chemostat cultures. Shown are (A) the yield of ATP on biomass (Y ATP/X), (B) the yield of total nucleosides phosphates on biomass (Y AXP/X), and (C) the adenylate energy charge (AEC) of the cells at three different dilution rates (D). The availability of phosphorylated adenine forms inside the cell and the AEC calculations are based on three independent biological experiments conducted in triplicate, and the bars represent the mean value of the corresponding parameter ± standard deviations.
Figure 4
Figure 4
Flow cytometry analysis of the green fluorescent protein accumulation in the strains under study. (A) Schematic representation of plasmid pS234G, carrying gfp under the transcriptional control of the IPTG-inducible P trc promoter. The activity of P trc is controlled by the transcriptional regulator LacIQ. The transcriptional terminators included in the plasmid backbone are depicted as T0 and T1. The elements in this outline are not drawn to scale. P. putida KT2440 (B), EM329 (C), and EM383 (D) carrying pS234G were grown on M12 minimal medium containing glucose and harvested in mid-exponential phase. Gray and green peaks represent non-induced and induced cells, respectively. The vertical dashed line indicates the background fluorescence of the corresponding strain carrying the empty pSEVA234 plasmid, used as a negative control. The results shown are from a representative experiment, and the fold change in fluorescence upon induction is indicated in each case. A.F.U., arbitrary fluorescence units.
Figure 5
Figure 5
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 (Y GFP/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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