In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions

Linde F.C. Kampers, Ruben G.A. van Heck, Stefano Donati, Edoardo Saccenti, Rita J.M. Volkers, Peter J. Schaap, Maria Suarez-Diez, Pablo I. Nikel, Vitor A.P. Martins Dos Santos*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

BACKGROUND: Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. RESULTS: Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential [Formula: see text]-dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. CONCLUSIONS: We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.

Original languageEnglish
Number of pages1
JournalMicrobial Cell Factories
Volume18
Issue number1
DOIs
Publication statusPublished - 22 Oct 2019

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Pseudomonas putida
Adenosinetriphosphate
Computer Simulation
Adenosine Triphosphate
Metabolites
Acetate Kinase
Growth
Ribonucleotides
Molecular oxygen
Biotechnology
Escherichia coli
Industrial applications
Oxidoreductases
Lactobacillus
Genomics
Genes
Oxidation-Reduction
Industry
Genome
Oxygen

Keywords

  • Anaerobiosis
  • Comparative genomics
  • Constraint-based metabolic modelling
  • Domainome analysis
  • Microbial physiology
  • Synthetic biology

Cite this

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title = "In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions",
abstract = "BACKGROUND: Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. RESULTS: Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential [Formula: see text]-dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. CONCLUSIONS: We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.",
keywords = "Anaerobiosis, Comparative genomics, Constraint-based metabolic modelling, Domainome analysis, Microbial physiology, Synthetic biology",
author = "Kampers, {Linde F.C.} and {van Heck}, {Ruben G.A.} and Stefano Donati and Edoardo Saccenti and Volkers, {Rita J.M.} and Schaap, {Peter J.} and Maria Suarez-Diez and Nikel, {Pablo I.} and {Martins Dos Santos}, {Vitor A.P.}",
year = "2019",
month = "10",
day = "22",
doi = "10.1186/s12934-019-1227-5",
language = "English",
volume = "18",
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In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions. / Kampers, Linde F.C.; van Heck, Ruben G.A.; Donati, Stefano; Saccenti, Edoardo; Volkers, Rita J.M.; Schaap, Peter J.; Suarez-Diez, Maria; Nikel, Pablo I.; Martins Dos Santos, Vitor A.P.

In: Microbial Cell Factories, Vol. 18, No. 1, 22.10.2019.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions

AU - Kampers, Linde F.C.

AU - van Heck, Ruben G.A.

AU - Donati, Stefano

AU - Saccenti, Edoardo

AU - Volkers, Rita J.M.

AU - Schaap, Peter J.

AU - Suarez-Diez, Maria

AU - Nikel, Pablo I.

AU - Martins Dos Santos, Vitor A.P.

PY - 2019/10/22

Y1 - 2019/10/22

N2 - BACKGROUND: Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. RESULTS: Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential [Formula: see text]-dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. CONCLUSIONS: We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.

AB - BACKGROUND: Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. RESULTS: Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential [Formula: see text]-dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. CONCLUSIONS: We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.

KW - Anaerobiosis

KW - Comparative genomics

KW - Constraint-based metabolic modelling

KW - Domainome analysis

KW - Microbial physiology

KW - Synthetic biology

U2 - 10.1186/s12934-019-1227-5

DO - 10.1186/s12934-019-1227-5

M3 - Article

VL - 18

JO - Microbial Cell Factories

JF - Microbial Cell Factories

SN - 1475-2859

IS - 1

ER -