scholarly journals Adaptive laboratory evolution restores solvent tolerance in plasmid-cured Pseudomonas putida S12; a molecular analysis

2020 ◽  
Author(s):  
Hadiastri Kusumawardhani ◽  
Benjamin Furtwängler ◽  
Matthijs Blommestijn ◽  
Adelė Kaltenytė ◽  
Jaap van der Poel ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Atp Synthase ◽  
Efflux Pump ◽  
Constitutive Expression ◽  
Regulatory System ◽  
Gene Clusters ◽  
Solvent Tolerance ◽  
Nucleotide Polymorphisms ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution

AbstractPseudomonas putida S12 is intrinsically solvent-tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The genome of P. putida S12 consists of a 5.8 Mbp chromosome, and a 580 kbp megaplasmid pTTS12 that carries several gene clusters involved in solvent tolerance. Removal of pTTS12 caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in plasmid-cured P. putida S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent-tolerance of this strain.Whole genome sequencing revealed several single nucleotide polymorphisms (SNPs) and a mobile element insertion, enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% v/v). Mutations were identified in an RND efflux pump regulator arpR, resulting in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit β’, global two-component regulatory system (GacA/GacS) and a putative AraC-family transcriptional regulator Afr. RNA-seq analysis further revealed a constitutive down-regulation of energy consuming activities in ALE-derived strains, including flagellar assembly, F0F1 ATP synthase, and membrane transport proteins. Out results indicate that constitutive expression of an alternative solvent extrusion pump in combination with high metabolic flexibility ensures restoration of solvent-tolerance in P. putida S12 lacking its megaplasmid.

scholarly journals Adaptive laboratory evolution restores solvent tolerance in plasmid-cured Pseudomonas putida S12; a molecular analysis

2021 ◽  
Author(s):  
Hadiastri Kusumawardhani ◽  
Benjamin Furtwängler ◽  
Matthijs Blommestijn ◽  
Adelė Kaltenytė ◽  
Jaap van der Poel ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Atp Synthase ◽  
Aromatic Compounds ◽  
Efflux Pump ◽  
Constitutive Expression ◽  
Solvent Tolerance ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Trade Offs ◽  
Biobased Production

Pseudomonas putida S12 is inherently solvent-tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The megaplasmid pTTS12 of P. putida S12 carries several gene clusters involved in solvent tolerance and the removal of this megaplasmid caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in the plasmid-cured P. putida S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent-tolerance of this strain. Whole genome sequencing identified several single nucleotide polymorphisms (SNPs) and a mobile element insertion enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% (vol/vol)). Mutations were identified in an RND efflux pump regulator arpR, resulting in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit β’, global two-component regulatory system (GacA/GacS) and a putative AraC-family transcriptional regulator Afr. Transcriptomic analysis further revealed a constitutive down-regulation of energy consuming activities in ALE-derived strains, such as flagellar assembly, F0F1 ATP synthase, and membrane transport proteins. In summary, constitutive expression of a solvent extrusion pump in combination with high metabolic flexibility enabled the restoration of solvent-tolerance trait in P. putida S12 lacking its megaplasmid. Importance: Sustainable production of high-value chemicals can be achieved by bacterial biocatalysis. However, bioproduction of biopolymers and aromatic compounds may exert stress on the microbial production host and limit the resulting yield. Having a solvent tolerance trait is highly advantageous for microbial hosts used in the biobased production of aromatics. The presence of a megaplasmid has been linked to the solvent tolerance trait of Pseudomonas putida, however, the extent of innate, intrinsic solvent tolerance in this bacterium remained unclear. Using adaptive laboratory evolution, we successfully adapted the plasmid-cured P. putida S12 strain to regain its solvent tolerance. Through these adapted strains, we begin to clarify the causes, origins, limitations, and trade-offs of the intrinsic solvent tolerance in P. putida. This work sheds a light on the possible genetic engineering targets to enhance solvent tolerance in Pseudomonas putida as well as other bacteria.

scholarly journals Novel Toxin-Antitoxin Module SlvT-SlvA Regulates Megaplasmid Stability and Incites Solvent Tolerance in Pseudomonas putida S12

2020 ◽  
Vol 86 (13) ◽  
Author(s):  
Hadiastri Kusumawardhani ◽  
David van Dijk ◽  
Rohola Hosseini ◽  
Johannes H. de Winde
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Aromatic Compounds ◽  
Efflux Pump ◽  
Gene Clusters ◽  
Host Cells ◽  
Solvent Tolerance ◽  
Content Type ◽  
Solvent Tolerant

ABSTRACT Pseudomonas putida S12 is highly tolerant of organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds. Previous studies revealed that P. putida S12 contains the single-copy 583-kbp megaplasmid pTTS12. pTTS12 carries several important operons and gene clusters facilitating P. putida S12 survival and growth in the presence of toxic compounds or other environmental stresses. We wished to revisit and further scrutinize the role of pTTS12 in conferring solvent tolerance. To this end, we cured the megaplasmid from P. putida S12 and conclusively confirmed that the SrpABC efflux pump is the major determinant of solvent tolerance on the megaplasmid pTTS12. In addition, we identified a novel toxin-antitoxin module (proposed gene names slvT and slvA, respectively) encoded on pTTS12 which contributes to the solvent tolerance phenotype and is important for conferring stability to the megaplasmid. Chromosomal introduction of the srp operon in combination with the slvAT gene pair created a solvent tolerance phenotype in non-solvent-tolerant strains, such as P. putida KT2440, Escherichia coli TG1, and E. coli BL21(DE3). IMPORTANCE Sustainable alternatives for high-value chemicals can be achieved by using renewable feedstocks in bacterial biocatalysis. However, during the bioproduction of such chemicals and biopolymers, aromatic compounds that function as products, substrates, or intermediates in the production process may exert toxicity to microbial host cells and limit the production yield. Therefore, solvent tolerance is a highly preferable trait for microbial hosts in the biobased production of aromatic chemicals and biopolymers. In this study, we revisit the essential role of megaplasmid pTTS12 from solvent-tolerant Pseudomonas putida S12 for molecular adaptation to an organic solvent. In addition to the solvent extrusion pump (SrpABC), we identified a novel toxin-antitoxin module (SlvAT) which contributes to short-term tolerance in moderate solvent concentrations, as well as to the stability of pTTS12. These two gene clusters were successfully expressed in non-solvent-tolerant strains of P. putida and Escherichia coli strains to confer and enhance solvent tolerance.

scholarly journals A novel toxin-antitoxin module SlvT–SlvA governs megaplasmid stability and incites solvent tolerance in Pseudomonas putida S12

2020 ◽  
Author(s):  
Hadiastri Kusumawardhani ◽  
David van Dijk ◽  
Rohola Hosseini ◽  
Johannes H. de Winde
Keyword(s):  
Pseudomonas Putida ◽  
Genetic Stability ◽  
Aromatic Compounds ◽  
Efflux Pump ◽  
Gene Clusters ◽  
Host Cells ◽  
Solvent Tolerance ◽  
E Coli ◽  
Solvent Tolerant

AbstractPseudomonas putida S12 is highly tolerant towards organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds. Previous studies reveal that P. putida S12 contains a single-copy 583 kbp megaplasmid pTTS12. This pTTS12 encodes several important operons and gene clusters facilitating P. putida S12 to survive and grow in the presence of toxic compounds or other environmental stresses. We wished to revisit and further scrutinize the role of pTTS12 in conferring solvent tolerance. To this end, we cured the megaplasmid from P. putida S12 and conclusively confirmed that the SrpABC efflux pump is the major contributor of solvent tolerance on the megaplasmid pTTS12. Importantly, we identified a novel toxin-antitoxin module (proposed gene names slvT and slvA respectively) encoded on pTTS12 which contributes to the solvent tolerant phenotype and is essential in conferring genetic stability to the megaplasmid. Chromosomal introduction of the srp operon in combination with slvAT gene pair created a solvent tolerance phenotype in non-solvent tolerant strains such as P. putida KT2440, E. coli TG1, and E. coli BL21(DE3).ImportanceSustainable alternatives for high-value chemicals can be achieved by using renewable feedstocks in bacterial biocatalysis. However, during bioproduction of such chemicals and biopolymers, aromatic compounds that function as products, substrates or intermediates in the production process may exert toxicity to microbial host cells and limit the production yield. Therefore, solvent-tolerance is a highly preferable trait for microbial hosts in the biobased production of aromatic chemicals and biopolymers. In this study, we revisit the essential role of megaplasmid pTTS12 from solvent-tolerant P. putida S12 for molecular adaptation to organic solvent. In addition to the RND efflux pump (SrpABC), we identified a novel toxin-antitoxin module (SlvAT) which contributes to tolerance in low solvent concentration as well as to genetic stability of pTTS12. These two gene clusters were successfully transferred to non-solvent tolerant strains of P. putida and to E. coli strains to confer and enhance solvent tolerance.

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

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Linde F. C. Kampers ◽  
Ruben G. A. van Heck ◽  
Stefano Donati ◽  
Edoardo Saccenti ◽  
Rita J. M. Volkers ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
In Silico ◽  
Redox Balance ◽  
Industrial Applications ◽  
Strictly Aerobic ◽  
Class Iii ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Atp Production ◽  
Lactobacillus Lactis

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 $$\text {O}_{2}$$ O 2 -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.

scholarly journals A Set of Genes Encoding a Second Toluene Efflux System in Pseudomonas putida DOT-T1E Is Linked to the tod Genes for Toluene Metabolism

2000 ◽  
Vol 182 (4) ◽  
pp. 937-943 ◽  
Author(s):  
Gilberto Mosqueda ◽  
Juan-Luis Ramos
Keyword(s):  
Pseudomonas Putida ◽  
Culture Medium ◽  
Efflux Pump ◽  
Site Directed Mutagenesis ◽  
Solvent Tolerance ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Efflux System ◽  
Host Chromosome ◽  
Knock Out

ABSTRACT Sequence analysis in Pseudomonas putida DOT-T1E revealed a second toluene efflux system for toluene metabolism encoded by the ttgDEF genes, which are adjacent to thetod genes. The ttgDEF genes were expressed in response to the presence of aromatic hydrocarbons such as toluene and styrene in the culture medium. To characterize the contribution of the TtgDEF system to toluene tolerance in P. putida, site-directed mutagenesis was used to knock out the gene in the wild-type DOT-T1E strain and in a mutant derivative, DOT-T1E-18. This mutant carried a Tn5 insertion in the ttgABCgene cluster, which encodes a toluene efflux pump that is synthesized constitutively. For site-directed mutagenesis, a cassette to knock out the ttgD gene and encoding resistance to tellurite was constructed in vitro and transferred to the corresponding host chromosome via the suicide plasmid pKNG101. Successful replacement of the wild-type sequences with the mutant cassette was confirmed by Southern hybridization. A single ttgD mutant, DOT-T1E-1, and a double mutant with knock outs in the ttgD andttgA genes, DOT-T1E-82, were obtained and characterized for toluene tolerance. This was assayed by the sudden addition of toluene (0.3% [vol/vol]) to the liquid culture medium of cells growing on Luria-Bertani (LB) medium (noninduced) or on LB medium with toluene supplied via the gas phase (induced). Induced cells of the singlettgD mutant were more sensitive to sudden toluene shock than were the wild-type cells; however, noninduced wild-type andttgD mutant cells were equally tolerant to toluene shock. Noninduced cells of the double DOT-T1E-82 mutant did not survive upon sudden toluene shock; however, they still remained viable upon sudden toluene shock if they had been previously induced. These results are discussed in the context of the use of multiple efflux pumps involved in solvent tolerance in P. putida DOT-T1E.

scholarly journals Adaptive laboratory evolution of Pseudomonas putida KT2440 improves p-coumaric and ferulic acid catabolism and tolerance

2020 ◽  
Vol 11 ◽  
pp. e00143 ◽  
Author(s):  
Elsayed T. Mohamed ◽  
Allison Z. Werner ◽  
Davinia Salvachúa ◽  
Christine A. Singer ◽  
Kiki Szostkiewicz ◽  
...  
Keyword(s):  
Ferulic Acid ◽  
Pseudomonas Putida ◽  
Adaptive Laboratory Evolution ◽  
Pseudomonas Putida Kt2440 ◽  
Laboratory Evolution

scholarly journals Efflux Pumps Involved in Toluene Tolerance in Pseudomonas putida DOT-T1E

1998 ◽  
Vol 180 (13) ◽  
pp. 3323-3329 ◽  
Author(s):  
Juan L. Ramos ◽  
Estrella Duque ◽  
Patricia Godoy ◽  
Ana Segura
Keyword(s):  
Pseudomonas Putida ◽  
Gas Phase ◽  
Cell Membranes ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Cell Number ◽  
Luria Bertani ◽  
Solvent Tolerance ◽  
Wild Type ◽  
Whole Cells

ABSTRACT The basic mechanisms underlying solvent tolerance inPseudomonas putida DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerantP. putida DOT-T1E grows in the presence of high concentrations (e.g., 1% [vol/vol]) of toluene and octanol. Growth of P. putida DOT-T1E cells in LB in the presence of toluene supplied via the gas phase has a clear effect on cell survival: the sudden addition of 0.3% (vol/vol) toluene toP. putida DOT-T1E pregrown with toluene in the gas phase resulted in survival of almost 100% of the initial cell number, whereas only 0.01% of cells pregrown in the absence of toluene tolerated exposure to this aromatic hydrocarbon. One class of toluene-sensitive octanol-tolerant mutant was isolated after Tn5-′phoA mutagenesis of wild-typeP. putida DOT-T1E cells. The mutant, calledP. putida DOT-T1E-18, was extremely sensitive to 0.3% (vol/vol) toluene added when cells were pregrown in the absence of toluene, whereas pregrowth on toluene supplied via the gas phase resulted in survival of about 0.0001% of the initial number. Solvent exclusion was tested with 1,2,4-[14C]trichlorobenzene. The levels of radiochemical accumulated in wild-type cells grown in the absence and in the presence of toluene were not significantly different. In contrast, the mutant was unable to remove 1,2,4-[14C]trichlorobenzene from the cell membranes when grown on Luria-Bertani (LB) medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase. The amount of 14C-labeled substrate in whole cells increased in competition assays in which toluene and xylenes were the unlabeled competitors, whereas this was not the case when benzene was the competitor. This finding suggests that the exclusion system works specifically with certain aromatic substrates. The mutation in P. putida DOT-T1E-18 was cloned, and the knockedout gene was sequenced and found to be homologous to the drug exclusion gene mexB, which belongs to the efflux pump family of the resistant nodulator division type.

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