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Biomolecules Nov 2019Pseudomonas KT2440, one of the best characterized pseudomonads, is a metabolically versatile producer of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) that...
Pseudomonas KT2440, one of the best characterized pseudomonads, is a metabolically versatile producer of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) that serves as a model bacterium for molecular studies. The synthesis of mcl-PHAs is of great interest due to their commercial potential. Carbon and phosphorus are the essential nutrients for growth and their limitation can trigger mcl-PHAs' production in microorganisms. However, the specific molecular mechanisms that drive this synthesis in species under unfavorable growth conditions remain poorly understood. Therefore, the proteomic responses of KT2440 to the limited carbon and phosphorus levels in the different growth phases during mcl-PHAs synthesis were investigated. The data indicated that biopolymers' production was associated with the cell growth of KT2440 under carbon- and phosphorus-limiting conditions. The protein expression pattern changed during mcl-PHAs synthesis and accumulation, and during the different physiological states of the microorganism. The data suggested that the majority of metabolic activities ceased under carbon and phosphorus limitation. The abundance of polyhydroxyalkanoate granule-associated protein (PhaF) involved in PHA synthesis increased significantly at 24 and 48 h of the cultivations. The activation of proteins belonging to the phosphate regulon was also detected. Moreover, these results indicated changes in the protein profiles related to amino acids metabolism, replication, transcription, translation, stress response mechanisms, transport or signal transduction. The presented data allowed the investigation of time-course proteome alterations in response to carbon and phosphorus limitation, and PHAs synthesis. This study provided information about proteins that can be potential targets in improving the efficiency of mcl-PHAs synthesis.
Topics: Bacterial Proteins; Bioreactors; Carbon; Phosphorus; Polyhydroxyalkanoates; Proteomics; Pseudomonas putida; Stress, Physiological
PubMed: 31795154
DOI: 10.3390/biom9120796 -
Microbial Biotechnology Jan 2020Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour...
Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic-catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole-cell biocatalyst for funnelling lignin-derived compounds to value-added products, including its native carbon storage product, medium-chain-length polyhydroxyalkanoates (mcl-PHA). In this work, a series of metabolic engineering targets to improve mcl-PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p-coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in β-oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl-PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain - which contains all the genetic modifications detailed above - demonstrated a 53% and 200% increase in mcl-PHA titre (g l ) and a 20% and 100% increase in yield (g mcl-PHA per g cell dry weight) from p-coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl-PHA production from aromatic compounds and lignin.
Topics: Lignin; Metabolic Engineering; Polyhydroxyalkanoates; Pseudomonas putida
PubMed: 31468725
DOI: 10.1111/1751-7915.13481 -
Applied Microbiology and Biotechnology Jun 2020Monoterpenoids are widely used in industrial applications, e.g. as active ingredients in pharmaceuticals, in flavor and fragrance compositions, and in agriculture....
Monoterpenoids are widely used in industrial applications, e.g. as active ingredients in pharmaceuticals, in flavor and fragrance compositions, and in agriculture. Severe toxic effects are known for some monoterpenoids making them challenging compounds for biotechnological production processes. Some strains of the bacterium Pseudomonas putida show an inherent extraordinarily high tolerance towards solvents including monoterpenoids. An understanding of the underlying factors can help to create suitable strains for monoterpenoids de novo production or conversion. In addition, knowledge about tolerance mechanisms could allow a deeper insight into how bacteria can oppose monoterpenoid containing drugs, like tea tree oil. Within this work, the resistance mechanisms of P. putida GS1 were investigated using selected monoterpenoid-hypertolerant mutants. Most of the mutations were found in efflux pump promoter regions or associated transcription factors. Surprisingly, while for the tested monoterpenoid alcohols, ketone, and ether high efflux pump expression increased monoterpenoid tolerance, it reduced the tolerance against geranic acid. However, an increase of geranic acid tolerance could be gained by a mutation in an efflux pump component. It was also found that increased monoterpenoid tolerance can counteract efficient biotransformation ability, indicating the need for a fine-tuned and knowledge-based tolerance improvement for production strain development.Key points• Altered monoterpenoid tolerance mainly related to altered activity of efflux pumps.• Increased tolerance to geranic acid surprisingly caused by decreased export activity. • Reduction of export activity can be beneficial for biotechnological conversions.
Topics: Biotechnology; Biotransformation; Drug Resistance, Bacterial; Monoterpenes; Mutation; Pseudomonas putida; Terpenes; Transcription Factors
PubMed: 32296906
DOI: 10.1007/s00253-020-10566-3 -
Microbial Cell Factories Feb 2024Bio-upcycling of plastics is an emerging alternative process that focuses on extracting value from a wide range of plastic waste streams. Such streams are typically too...
Bio-upcycling of plastics is an emerging alternative process that focuses on extracting value from a wide range of plastic waste streams. Such streams are typically too contaminated to be effectively processed using traditional recycling technologies. Medium-chain-length (mcl) diols and dicarboxylates (DCA) are major products of chemically or enzymatically depolymerized plastics, such as polyesters or polyethers. In this study, we enabled the efficient metabolism of mcl-diols and -DCA in engineered Pseudomonas putida as a prerequisite for subsequent bio-upcycling. We identified the transcriptional regulator GcdR as target for enabling metabolism of uneven mcl-DCA such as pimelate, and uncovered amino acid substitutions that lead to an increased coupling between the heterologous β-oxidation of mcl-DCA and the native degradation of short-chain-length DCA. Adaptive laboratory evolution and subsequent reverse engineering unravelled two distinct pathways for mcl-diol metabolism in P. putida, namely via the hydroxy acid and subsequent native β-oxidation or via full oxidation to the dicarboxylic acid that is further metabolized by heterologous β-oxidation. Furthermore, we demonstrated the production of polyhydroxyalkanoates from mcl-diols and -DCA by a single strain combining all required metabolic features. Overall, this study provides a powerful platform strain for the bio-upcycling of complex plastic hydrolysates to polyhydroxyalkanoates and leads the path for future yield optimizations.
Topics: Pseudomonas putida; Polyhydroxyalkanoates; Polyesters; Carboxylic Acids; Oxidation-Reduction
PubMed: 38365718
DOI: 10.1186/s12934-024-02310-7 -
Scientific Reports Sep 2018Bacterial motility plays a crucial role in competitiveness and colonization in the rhizosphere. In this work, Chromatin ImmunoPrecipitation Sequencing (ChIP-seq)...
Bacterial motility plays a crucial role in competitiveness and colonization in the rhizosphere. In this work, Chromatin ImmunoPrecipitation Sequencing (ChIP-seq) analysis has been used to identify genes putatively regulated by the transcriptional regulatory protein FleQ in Pseudomonas fluorescens F113 and Pseudomonas putida KT2440. This protein was previously identified as a master regulator of flagella and biofilm formation in both strains. This work has demonstrated that FleQ from both bacteria are conserved and functionally equivalent for motility regulation. Furthermore, the ChIP-seq analysis has shown that FleQ is a global regulator with the identification of 121 and 103 FleQ putative binding sites in P. fluorescens F113 and P. putida KT2440 respectively. Putative genes regulated by FleQ included, as expected, flagellar and motility-related genes and others involved in adhesion and exopolysaccharide production. Surprisingly, the ChIP-seq analysis also identified iron homeostasis-related genes for which positive regulation was shown by RT-qPCR. The results also showed that FleQ from P. fluorescens F113 shares an important part of its direct regulon with AmrZ, a global regulator also implicated in environmental adaption. Although AmrZ also regulates motility and iron uptake, the overlap occurred mostly with the iron-related genes, since both regulators control a different set of motility-related genes.
Topics: Bacterial Proteins; Binding Sites; Biofilms; Flagella; Gene Expression Regulation, Bacterial; Gene Ontology; Genome, Bacterial; Iron; Iron-Regulatory Proteins; Molecular Sequence Annotation; Movement; Protein Binding; Pseudomonas fluorescens; Pseudomonas putida; Regulon; Trans-Activators
PubMed: 30177764
DOI: 10.1038/s41598-018-31371-z -
Metabolic Engineering Jul 2018Given its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, the platform strain Pseudomonas putida EM42, a genome-edited derivative of the...
Given its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, the platform strain Pseudomonas putida EM42, a genome-edited derivative of the soil bacterium P. putida KT2440, can efficiently host a suite of harsh reactions of biotechnological interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire of P. putida EM42 is centered largely on organic acids, aromatic compounds and some hexoses (glucose and fructose). To enlarge the biochemical network of P. putida EM42 to include disaccharides and pentoses, we implanted heterologous genetic modules for D-cellobiose and D-xylose metabolism into the enzymatic complement of this strain. Cellobiose was actively transported into the cells through the ABC complex formed by native proteins PP1015-PP1018. The knocked-in β-glucosidase BglC from Thermobifida fusca catalyzed intracellular cleavage of the disaccharide to D-glucose, which was then channelled to the default central metabolism. Xylose oxidation to the dead end product D-xylonate was prevented by deleting the gcd gene that encodes the broad substrate range quinone-dependent glucose dehydrogenase. Intracellular intake was then engineered by expressing the Escherichia coli proton-coupled symporter XylE. The sugar was further metabolized by the products of E. coli xylA (xylose isomerase) and xylB (xylulokinase) towards the pentose phosphate pathway. The resulting P. putida strain co-utilized xylose with glucose or cellobiose to complete depletion of the sugars. These results not only show the broadening of the metabolic capacity of a soil bacterium towards new substrates, but also promote P. putida EM42 as a platform for plug-in of new biochemical pathways for utilization and valorization of carbohydrate mixtures from lignocellulose processing.
Topics: Bacterial Proteins; Cellobiose; Gene Knock-In Techniques; Glucose; Oxidation-Reduction; Pseudomonas putida; Xylose; beta-Glucosidase
PubMed: 29864584
DOI: 10.1016/j.ymben.2018.05.019 -
Applied Microbiology and Biotechnology Jun 2017Starting from mature vegetable compost, four bacterial strains were selected using a lignin-rich medium. 16S ribosomal RNA identification of the isolates showed high...
Starting from mature vegetable compost, four bacterial strains were selected using a lignin-rich medium. 16S ribosomal RNA identification of the isolates showed high score similarity with Pseudomonas spp. for three out of four isolates. Further characterization of growth on mixtures of six selected lignin model compounds (vanillin, vanillate, 4-hydroxybenzoate, p-coumarate, benzoate, and ferulate) was carried out with three of the Pseudomonas isolates and in addition with the strain Pseudomonas putida KT2440 from a culture collection. The specific growth rates on benzoate, p-coumarate, and 4-hydroxybenzoate were considerably higher (0.26-0.27 h) than those on ferulate and vanillate (0.21 and 0.22 h), as were the uptake rates. There was no direct growth of P. putida KT2440 on vanillin, but instead, vanillin was rapidly converted into vanillate at a rate of 4.87 mmol (g h) after which the accumulated vanillate was taken up. The growth curve reflected a diauxic growth when mixtures of the model compounds were used as carbon source. Vanillin, 4-hydroxybenzoate, and benzoate were preferentially consumed first, whereas ferulate was always the last substrate to be taken in. These results contribute to a better understanding of the aromatic metabolism of P. putida in terms of growth and uptake rates, which will be helpful for the utilization of these bacteria as cell factories for upgrading lignin-derived mixtures of aromatic molecules.
Topics: Bacterial Proteins; Benzaldehydes; Benzoates; Composting; Coumaric Acids; Culture Media; Genes, Bacterial; Lignin; Parabens; Pseudomonas putida; RNA, Ribosomal, 16S
PubMed: 28299400
DOI: 10.1007/s00253-017-8211-y -
Journal of Bacteriology Sep 2020KT2440 retains three homologs (PplR1 to PplR3) of the LitR/CarH family, an adenosyl B-dependent light-sensitive MerR family transcriptional regulator. Transcriptome...
KT2440 retains three homologs (PplR1 to PplR3) of the LitR/CarH family, an adenosyl B-dependent light-sensitive MerR family transcriptional regulator. Transcriptome analysis revealed the existence of a number of photoinducible genes, including , (encoding DNA photolyase), (furan-containing fatty acid synthase), (GTP cyclohydrolase I), (cryptochrome-like protein), and multiple genes without annotated/known function. Transcriptional analysis by quantitative reverse transcription-PCR with knockout mutants of to showed that a triple knockout completely abolished the light-inducible transcription in , which indicates the occurrence of ternary regulation of PplR proteins. A DNase I footprint assay showed that PplR1 protein specifically binds to the promoter regions of light-inducible genes, suggesting a consensus PplR1-binding direct repeat, 5'-T(G/A)TACANTGTA(C/T)A-3'. The disruption of B biosynthesis cluster did not affect the light-inducible transcription; however, disruption of (where LOV indicates "light, oxygen, or voltage") and , encoding blue light photoreceptors adjacently located to and , respectively, led to the complete loss of light-inducible transcription. Overall, the results suggest that the three PplRs and two PpSB-LOVs cooperatively regulate the light-inducible gene expression. The wide distribution of the / cognate pair homologs in spp. and related bacteria suggests that the response and adaptation to light are similarly regulated in the group of nonphototrophic bacteria. The LitR/CarH family is a new group of photosensor homologous to MerR-type transcriptional regulators. Proteins of this family are distributed to various nonphototrophic bacteria and grouped into at least five classes (I to V). retaining three class II LitR proteins exhibited a genome-wide response to light. All three paralogs were functional and mediated photodependent activation of promoters directing the transcription of light-induced genes or operons. Two LOV (light, oxygen, or voltage) domain proteins, adjacently encoded by two genes, were also essential for the photodependent transcriptional control. Despite the difference in light-sensing mechanisms, the DNA binding consensus of class II LitR [T(G/A)TA(C/T)A] was the same as that of class I. This is the first study showing the actual involvement of class II LitR in light-induced transcription.
Topics: Bacterial Proteins; Binding Sites; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Light; Operon; Photoreceptors, Microbial; Promoter Regions, Genetic; Pseudomonas putida
PubMed: 32967908
DOI: 10.1128/JB.00146-20 -
Metabolic Engineering Sep 2022Anthranilate, an intermediate of the shikimate pathway, is a high-value aromatic compound widely used as a precursor in the production of dyes, fragrances, plastics and...
Anthranilate, an intermediate of the shikimate pathway, is a high-value aromatic compound widely used as a precursor in the production of dyes, fragrances, plastics and pharmaceuticals. Traditional strategies adopted for microbial anthranilate production rely on the implementation of auxotrophic strains-which requires aromatic amino acids or complex additives to be supplemented in the culture medium, negatively impacting production costs. In this work, we engineered the soil bacterium Pseudomonas putida for high-titer, glucose-dependent anthranilate production by repurposing elements of the Esa quorum sensing (QS) system of Pantoea stewartii. The P promoter mediated a self-regulated transcriptional response that effectively knocked-down the expression of the trpDC genes. Next, we harnessed the synthetic QS elements to engineer a growth-to-anthranilate production switch. The resulting plasmid-free P. putida strain produced the target compound at 3.8 ± 0.3 mM in shaken-flask cultures after 72 h-a titer >2-fold higher than anthranilate levels reported thus far. Our results highlight the value of dynamic flux regulation for the production of intermediate metabolites within highly-regulated routes (such as the shikimate pathway), thereby circumventing the need of expensive additives.
Topics: Glucose; Plasmids; Pseudomonas putida; Quorum Sensing; ortho-Aminobenzoates
PubMed: 35659519
DOI: 10.1016/j.ymben.2022.05.008 -
MBio Dec 2022In the soil bacterium Pseudomonas putida, the motor torque for flagellar rotation is generated by the two stators MotAB and MotCD. Here, we construct mutant strains in...
In the soil bacterium Pseudomonas putida, the motor torque for flagellar rotation is generated by the two stators MotAB and MotCD. Here, we construct mutant strains in which one or both stators are knocked out and investigate their swimming motility in fluids of different viscosity and in heterogeneous structured environments (semisolid agar). Besides phase-contrast imaging of single-cell trajectories and spreading cultures, dual-color fluorescence microscopy allows us to quantify the role of the stators in enabling P. putida's three different swimming modes, where the flagellar bundle pushes, pulls, or wraps around the cell body. The MotAB stator is essential for swimming motility in liquids, while spreading in semisolid agar is not affected. Moreover, if the MotAB stator is knocked out, wrapped mode formation under low-viscosity conditions is strongly impaired and only partly restored for increased viscosity and in semisolid agar. In contrast, when the MotCD stator is missing, cells are indistinguishable from the wild type in fluid experiments but spread much more slowly in semisolid agar. Analysis of the microscopic trajectories reveals that the MotCD knockout strain forms sessile clusters, thereby reducing the number of motile cells, while the swimming speed is unaffected. Together, both stators ensure a robust wild type that swims efficiently under different environmental conditions. Because of its heterogeneous habitat, the soil bacterium Pseudomonas putida needs to swim efficiently under very different environmental conditions. In this paper, we knocked out the stators MotAB and MotCD to investigate their impact on the swimming motility of P. putida. While the MotAB stator is crucial for swimming in fluids, in semisolid agar, both stators are sufficient to sustain a fast-swimming phenotype and increased frequencies of the wrapped mode, which is known to be beneficial for escaping mechanical traps. However, in contrast to the MotAB knockout, a culture of MotCD knockout cells spreads much more slowly in the agar, as it forms nonmotile clusters that reduce the number of motile cells.
Topics: Bacterial Proteins; Pseudomonas putida; Swimming; Agar; Flagella
PubMed: 36409076
DOI: 10.1128/mbio.02182-22