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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

Operons and genomic regions deleted inP.putidaKT2440 to construct a cell factory strain. (A) Position of the eleven gene(s)/regions deleted in wild-type P. putida KT2440 indicated in the physical map of the chromosome. (B) Roadmap for the construction of strains EM42 and EM383. Relevant genes are depicted in the order in which they were eliminated (see also Additional file 1: Table S1). (C) Electrophoresis of the diagnostic PCR amplifications to confirm the deletions. The flanking lanes (M) correspond to a DNA ladder [500-bp Molecular Ruler EZ Load™ (Bio-Rad Corp., Berkeley, CA, USA)], and lanes identified as ϕ are negative controls, i.e., samples without DNA template. The photograph shows the products resulting from PCR amplifications of [i] an internal gene within prophage 1, KT2440 (lane 1) and EM383 (lane 2); [ii] an internal gene of prophage 2, KT2440 (lane 3) and EM383 (lane 4); [iii] an internal gene of prophage 3, KT2440 (lane 5) and EM383 (lane 6); [iv] an internal gene of prophage 4, KT2440 (lane 7) and EM383 (lane 8); [v] an internal gene of the hsdRMS operon, KT2440 (lane 9) and EM383 (lane 10); [vi] the TS1-TS2 region of recA, KT2440 (lane 11) and EM383 (lane 12); [vii] an internal gene of the Tn7-like operon, KT2440 (lane 13) and EM383 (lane 14); [viii] the TS1-TS2 region of endA-1, KT2440 (lane 15) and EM383 (lane 16); [ix] the TS1-TS2 region of endA-2, KT2440 (lane 17) and EM383 (lane 18); [x] an internal gene of the flagellar operon, KT2440 (lane 19) and EM383 (lane 20); and [xi] an internal gene of the Tn4652 operon, KT2440 (lane 21) and EM383 (lane 22). The details of primers sequence used in these amplifications are given in Additional file 1: Table S2.
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Fig1: Operons and genomic regions deleted inP.putidaKT2440 to construct a cell factory strain. (A) Position of the eleven gene(s)/regions deleted in wild-type P. putida KT2440 indicated in the physical map of the chromosome. (B) Roadmap for the construction of strains EM42 and EM383. Relevant genes are depicted in the order in which they were eliminated (see also Additional file 1: Table S1). (C) Electrophoresis of the diagnostic PCR amplifications to confirm the deletions. The flanking lanes (M) correspond to a DNA ladder [500-bp Molecular Ruler EZ Load™ (Bio-Rad Corp., Berkeley, CA, USA)], and lanes identified as ϕ are negative controls, i.e., samples without DNA template. The photograph shows the products resulting from PCR amplifications of [i] an internal gene within prophage 1, KT2440 (lane 1) and EM383 (lane 2); [ii] an internal gene of prophage 2, KT2440 (lane 3) and EM383 (lane 4); [iii] an internal gene of prophage 3, KT2440 (lane 5) and EM383 (lane 6); [iv] an internal gene of prophage 4, KT2440 (lane 7) and EM383 (lane 8); [v] an internal gene of the hsdRMS operon, KT2440 (lane 9) and EM383 (lane 10); [vi] the TS1-TS2 region of recA, KT2440 (lane 11) and EM383 (lane 12); [vii] an internal gene of the Tn7-like operon, KT2440 (lane 13) and EM383 (lane 14); [viii] the TS1-TS2 region of endA-1, KT2440 (lane 15) and EM383 (lane 16); [ix] the TS1-TS2 region of endA-2, KT2440 (lane 17) and EM383 (lane 18); [x] an internal gene of the flagellar operon, KT2440 (lane 19) and EM383 (lane 20); and [xi] an internal gene of the Tn4652 operon, KT2440 (lane 21) and EM383 (lane 22). The details of primers sequence used in these amplifications are given in Additional file 1: Table S2.

Mentions: This study capitalizes on the intrinsic physiological and metabolic strength of P. putida KT2440 in the quest for an improved host of heterologous gene expression. One major constraint for such process is ensuring sufficient ATP availability to fuel the action of GroEL/ES in folding foreign polypeptides, which are often produced at high levels by the strong promoters of typical recombinant expression systems [32,33]. In fact, GroEL/ES seems to be the cell component that most avidly hydrolyzes ATP [34]. On the other hand, metabolic stress, which can cause ROS formation, is often accompanied by a higher consumption of reducing power [i.e., NAD(P)H] [10,22]. This situation indicates that engineering increased intracellular ATP and/or NAD(P)H levels is predicted to result in a better expression host. On the other hand, the implantation and performance of recombinant constructs is exposed to the many chromosomal elements that cause genetic instability and rejection of foreign genes, e.g., insertion sequences (IS), transposons, prophages, and DNA restriction systems. On this basis, the annotated genomic sequence of strain KT2440 (available on line in the Pseudomonas Genome Database [35]) was inspected to spot DNA segments encoding tasks which, while being non-essential, either grossly drain much metabolic currency or are likely to cause genomic instability. A tentative survey of such segments yield a minimum of 11 chromosomal sites determining a variety of functions (Figure 1A), the removal of which is justified as follows. First, there is a whole of 4 non-contiguous large segments (~170 kb in total, representing 2.6% of the genome of strain KT2440), encoding prophages known to display various degrees of activity [36]. These sequences are genuinely parasitic, and they make cells more sensitive to DNA damage and, when induced, they cause stochastic lysis in the bacterial population. Then it comes the 54 ISs (called ISPpu) and other mobile DNA elements borne by P. putida, which account for ~1% of the genome of P. putida KT2440 [37,38], and which are poised to counterselect knocked-in constructs that may burden the host [39,40]. While targeting all of them individually is beyond the scope of this work, two conspicuous cases were addressed. One instance is the 15.7-kb Tn4652 transposon [37,41], a member of the Tn3 transposon family which spans the open reading frames (ORFs) PP2964-PP2984 in the genome. Why is it relevant to focus on this transposon? While other mobile elements of the P. putida KT2440 are surely functional, Tn4652 is the only case in which its in vivo activity has been well accredited so far, especially when cells face C starvation [42–44]. A second genomic segment with the potential to cause instability of recombinant constructs (especially those assembled in Tn7 transposon vectors) spans ORFs PP5404-PP5407, and encodes a complete Tn7-like transposase cluster [37,41]. This genetic locus may interfere with inserts targeted at the specific Tn7 attachment site of the P. putida chromosome that is often used for stable introduction of foreign DNA [45,46], and was targeted as well as a potential cause of genetic instability.Figure 1


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)

Operons and genomic regions deleted inP.putidaKT2440 to construct a cell factory strain. (A) Position of the eleven gene(s)/regions deleted in wild-type P. putida KT2440 indicated in the physical map of the chromosome. (B) Roadmap for the construction of strains EM42 and EM383. Relevant genes are depicted in the order in which they were eliminated (see also Additional file 1: Table S1). (C) Electrophoresis of the diagnostic PCR amplifications to confirm the deletions. The flanking lanes (M) correspond to a DNA ladder [500-bp Molecular Ruler EZ Load™ (Bio-Rad Corp., Berkeley, CA, USA)], and lanes identified as ϕ are negative controls, i.e., samples without DNA template. The photograph shows the products resulting from PCR amplifications of [i] an internal gene within prophage 1, KT2440 (lane 1) and EM383 (lane 2); [ii] an internal gene of prophage 2, KT2440 (lane 3) and EM383 (lane 4); [iii] an internal gene of prophage 3, KT2440 (lane 5) and EM383 (lane 6); [iv] an internal gene of prophage 4, KT2440 (lane 7) and EM383 (lane 8); [v] an internal gene of the hsdRMS operon, KT2440 (lane 9) and EM383 (lane 10); [vi] the TS1-TS2 region of recA, KT2440 (lane 11) and EM383 (lane 12); [vii] an internal gene of the Tn7-like operon, KT2440 (lane 13) and EM383 (lane 14); [viii] the TS1-TS2 region of endA-1, KT2440 (lane 15) and EM383 (lane 16); [ix] the TS1-TS2 region of endA-2, KT2440 (lane 17) and EM383 (lane 18); [x] an internal gene of the flagellar operon, KT2440 (lane 19) and EM383 (lane 20); and [xi] an internal gene of the Tn4652 operon, KT2440 (lane 21) and EM383 (lane 22). The details of primers sequence used in these amplifications are given in Additional file 1: Table S2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Operons and genomic regions deleted inP.putidaKT2440 to construct a cell factory strain. (A) Position of the eleven gene(s)/regions deleted in wild-type P. putida KT2440 indicated in the physical map of the chromosome. (B) Roadmap for the construction of strains EM42 and EM383. Relevant genes are depicted in the order in which they were eliminated (see also Additional file 1: Table S1). (C) Electrophoresis of the diagnostic PCR amplifications to confirm the deletions. The flanking lanes (M) correspond to a DNA ladder [500-bp Molecular Ruler EZ Load™ (Bio-Rad Corp., Berkeley, CA, USA)], and lanes identified as ϕ are negative controls, i.e., samples without DNA template. The photograph shows the products resulting from PCR amplifications of [i] an internal gene within prophage 1, KT2440 (lane 1) and EM383 (lane 2); [ii] an internal gene of prophage 2, KT2440 (lane 3) and EM383 (lane 4); [iii] an internal gene of prophage 3, KT2440 (lane 5) and EM383 (lane 6); [iv] an internal gene of prophage 4, KT2440 (lane 7) and EM383 (lane 8); [v] an internal gene of the hsdRMS operon, KT2440 (lane 9) and EM383 (lane 10); [vi] the TS1-TS2 region of recA, KT2440 (lane 11) and EM383 (lane 12); [vii] an internal gene of the Tn7-like operon, KT2440 (lane 13) and EM383 (lane 14); [viii] the TS1-TS2 region of endA-1, KT2440 (lane 15) and EM383 (lane 16); [ix] the TS1-TS2 region of endA-2, KT2440 (lane 17) and EM383 (lane 18); [x] an internal gene of the flagellar operon, KT2440 (lane 19) and EM383 (lane 20); and [xi] an internal gene of the Tn4652 operon, KT2440 (lane 21) and EM383 (lane 22). The details of primers sequence used in these amplifications are given in Additional file 1: Table S2.
Mentions: This study capitalizes on the intrinsic physiological and metabolic strength of P. putida KT2440 in the quest for an improved host of heterologous gene expression. One major constraint for such process is ensuring sufficient ATP availability to fuel the action of GroEL/ES in folding foreign polypeptides, which are often produced at high levels by the strong promoters of typical recombinant expression systems [32,33]. In fact, GroEL/ES seems to be the cell component that most avidly hydrolyzes ATP [34]. On the other hand, metabolic stress, which can cause ROS formation, is often accompanied by a higher consumption of reducing power [i.e., NAD(P)H] [10,22]. This situation indicates that engineering increased intracellular ATP and/or NAD(P)H levels is predicted to result in a better expression host. On the other hand, the implantation and performance of recombinant constructs is exposed to the many chromosomal elements that cause genetic instability and rejection of foreign genes, e.g., insertion sequences (IS), transposons, prophages, and DNA restriction systems. On this basis, the annotated genomic sequence of strain KT2440 (available on line in the Pseudomonas Genome Database [35]) was inspected to spot DNA segments encoding tasks which, while being non-essential, either grossly drain much metabolic currency or are likely to cause genomic instability. A tentative survey of such segments yield a minimum of 11 chromosomal sites determining a variety of functions (Figure 1A), the removal of which is justified as follows. First, there is a whole of 4 non-contiguous large segments (~170 kb in total, representing 2.6% of the genome of strain KT2440), encoding prophages known to display various degrees of activity [36]. These sequences are genuinely parasitic, and they make cells more sensitive to DNA damage and, when induced, they cause stochastic lysis in the bacterial population. Then it comes the 54 ISs (called ISPpu) and other mobile DNA elements borne by P. putida, which account for ~1% of the genome of P. putida KT2440 [37,38], and which are poised to counterselect knocked-in constructs that may burden the host [39,40]. While targeting all of them individually is beyond the scope of this work, two conspicuous cases were addressed. One instance is the 15.7-kb Tn4652 transposon [37,41], a member of the Tn3 transposon family which spans the open reading frames (ORFs) PP2964-PP2984 in the genome. Why is it relevant to focus on this transposon? While other mobile elements of the P. putida KT2440 are surely functional, Tn4652 is the only case in which its in vivo activity has been well accredited so far, especially when cells face C starvation [42–44]. A second genomic segment with the potential to cause instability of recombinant constructs (especially those assembled in Tn7 transposon vectors) spans ORFs PP5404-PP5407, and encodes a complete Tn7-like transposase cluster [37,41]. This genetic locus may interfere with inserts targeted at the specific Tn7 attachment site of the P. putida chromosome that is often used for stable introduction of foreign DNA [45,46], and was targeted as well as a potential cause of genetic instability.Figure 1

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