-
Current Opinion in Biotechnology Feb 2024The soil bacterium Pseudomonas putida, especially the KT2440 strain, is increasingly being utilized as a host for biotransformations of both industrial and environmental... (Review)
Review
The soil bacterium Pseudomonas putida, especially the KT2440 strain, is increasingly being utilized as a host for biotransformations of both industrial and environmental interest. The foundations of such performance include its robust redox metabolism, ability to tolerate a wide range of physicochemical stresses, rapid growth, versatile metabolism, nonpathogenic nature, and the availability of molecular tools for advanced genetic programming. These attributes have been leveraged for hosting engineered pathways for production of valuable chemicals or degradation/valorization of environmental pollutants. This has in turn pushed the boundaries of conventional enzymology toward previously unexplored reactions in nature. Furthermore, modifications to the physical properties of the cells have been made to enhance their catalytic performance. These advancements establish P. putida as bona fide chassis for synthetic biology, on par with more traditional metabolic engineering platforms.
Topics: Metabolic Engineering; Pseudomonas putida; Synthetic Biology; Biotransformation; Oxidation-Reduction
PubMed: 38061264
DOI: 10.1016/j.copbio.2023.103025 -
Molecules (Basel, Switzerland) Sep 2022Natural coumarins contribute to the aroma of licorice, and they are often used as a flavoring and stabilizing agents. However, coumarins usage in food has been banned by...
Natural coumarins contribute to the aroma of licorice, and they are often used as a flavoring and stabilizing agents. However, coumarins usage in food has been banned by various countries due to its toxic effect. In this study, a strain of HSM-C2 that can biodegrade coumarin with high efficiency was isolated from soil and identified as through performing 16S rDNA sequence analysis. The HSM-C2 catalyzed the biodegradation up to 99.83% of 1 mg/mL coumarin within 24 h under optimal culture conditions, such as 30 °C and pH 7, which highlights the strong coumarin biodegrading potential of this strain. The product, such as dihydrocoumarin, generated after the biodegradation of coumarin was identified by performing GC-MS analysis. The present study provides a theoretical basis and microbial resource for further research on coumarin biodegradation.
Topics: Biodegradation, Environmental; Coumarins; DNA, Ribosomal; Excipients; Pseudomonas putida; Soil; Soil Microbiology
PubMed: 36144743
DOI: 10.3390/molecules27186007 -
Microbial Biotechnology Oct 2022The extracellular 373-kDa PehA heme peroxidase of Pseudomonas putida KT2440 has two enzymatic domains which depend on heme cofactor for their peroxidase activity. A null...
The extracellular 373-kDa PehA heme peroxidase of Pseudomonas putida KT2440 has two enzymatic domains which depend on heme cofactor for their peroxidase activity. A null pehA mutant was generated to examine the impact of PehA in rhizosphere colonization competence and the induction of plant systemic resistance (ISR). This mutant was not markedly hampered in colonization efficiency. However, increase in pehA dosage enhanced colonization fitness about 30 fold in the root and 900 fold in the root apex. In vitro assays with purified His-tagged enzymatic domains of PehA indicated that heme-dependent peroxidase activity was required for the enhancement of root tip colonization. Evaluation of live/dead cells confirmed that overexpression of pehA had a positive effect on bacterial cell viability. Following root colonization of rice plants by KT2440 strain, the incidence of rice blast caused by Magnaporthe oryzae was reduced by 65% and the severity of this disease was also diminished in comparison to non-treated plants. An increase in the pehA dosage was also beneficial for the control of rice blast as compared with gene inactivation. The results suggest that PehA helps P. putida to cope with the plant-imposed oxidative stress leading to enhanced colonization ability and concomitant ISR-elicitation.
Topics: Antioxidants; Heme; Peroxidases; Plant Diseases; Plant Roots; Pseudomonas putida
PubMed: 35986900
DOI: 10.1111/1751-7915.14123 -
Metabolic Engineering Sep 2023Pseudomonas putida, a microbial host widely adopted for metabolic engineering, processes glucose through convergent peripheral pathways that ultimately yield...
Pseudomonas putida, a microbial host widely adopted for metabolic engineering, processes glucose through convergent peripheral pathways that ultimately yield 6-phosphogluconate. The periplasmic gluconate shunt (PGS), composed by glucose and gluconate dehydrogenases, sequentially transforms glucose into gluconate and 2-ketogluconate. Although the secretion of these organic acids by P. putida has been extensively recognized, the mechanism and spatiotemporal regulation of the PGS remained elusive thus far. To address this challenge, we adopted a dynamic C- and H-metabolic flux analysis strategy, termed D-fluxomics. D-fluxomics demonstrated that the PGS underscores a highly dynamic metabolic architecture in glucose-dependent batch cultures of P. putida, characterized by hierarchical carbon uptake by the PGS throughout the cultivation. Additionally, we show that gluconate and 2-ketogluconate accumulation and consumption can be solely explained as a result of the interplay between growth rate-coupled and decoupled metabolic fluxes. As a consequence, the formation of these acids in the PGS is inversely correlated to the bacterial growth rate-unlike the widely studied overflow metabolism of Escherichia coli and yeast. Our findings, which underline survival strategies of soil bacteria thriving in their natural environments, open new avenues for engineering P. putida towards efficient, sugar-based bioprocesses.
Topics: Pseudomonas putida; Sugars; Deuterium; Gluconates; Glucose
PubMed: 37454792
DOI: 10.1016/j.ymben.2023.07.004 -
Metabolic Engineering Nov 2022Formate is a promising, water-soluble C1 feedstock for biotechnology that can be efficiently produced from CO-but formatotrophy has been engineered in only a few...
Formate is a promising, water-soluble C1 feedstock for biotechnology that can be efficiently produced from CO-but formatotrophy has been engineered in only a few industrially-relevant microbial hosts. We addressed the challenge of expanding the feedstock range of bacterial hosts by adopting Pseudomonas putida as a robust platform for synthetic formate assimilation. Here, the metabolism of a genome-reduced variant of P. putida was radically rewired to establish synthetic auxotrophies that could be functionally complemented by expressing components of the reductive glycine (rGly) pathway. We adopted a modular engineering approach, dividing C1 assimilation in segments composed of both heterologous activities (sourced from Methylobacterium extorquens) and native biochemical reactions. Modular expression of rGly pathway elements enabled growth on formate as carbon source and acetate (predominantly for energy supply), and adaptive laboratory evolution of two lineages of engineered P. putida formatotrophs lead to doubling times of ca. 15 h. We likewise identified emergent metabolic features for assimilation of C1 units in these evolved P. putida populations. Taken together, our results consolidate the landscape of useful microbial platforms that can be implemented for C1-based biotechnological production towards a formate bioeconomy.
Topics: Pseudomonas putida; Metabolic Engineering; Formates; Methylobacterium extorquens; Glycine
PubMed: 36328297
DOI: 10.1016/j.ymben.2022.10.008 -
Folia Microbiologica Jun 2023Diethyl phthalate (DEP) is one of the extensively used plasticizers which has been considered a priority hazardous pollutant due to its carcinogenic, endocrine...
Diethyl phthalate (DEP) is one of the extensively used plasticizers which has been considered a priority hazardous pollutant due to its carcinogenic, endocrine disrupter, and multi-toxic effects on humans. The identification of DEP in different parts of the ecosphere has increased the global community's attention to the elimination of this pollutant in a bio-eco-friendly way. In this research, a novel aerobic bacterial strain nominates as ShA (GenBank accession number: MN298858) capable of consuming DEP as carbon and energy sources, was isolated from the upper phase (0-10 cm) of Anzali international wetland sediments by enrichment culture method. Morphological characteristics and 16S rRNA gene sequence analysis demonstrated that strain ShA belonged to Pseudomonas putida. The substrate utilization test demonstrated that strain ShA was able to grow in mineral salt medium containing dimethyl phthalate (DMP) and phthalic acid (PA) isomers including terephthalic and isophthalic acid. Degradation assay showed strain ShA completely degraded 200 mg/L DEP within 22 h (pH 7.0, 30 °C). Surprisingly, PA as the main intermediate of DEP biodegradation was identified by GC-FID. Moreover, the rapid degradation of 2000 mg/L PA to CO and HO was viewed in 22 h by strain ShA. The possible route of DEP degradation was DEP directly to PA and then PA consumption for growth. This study obtained results that provide a great contribution to applying strain ShA in the biodegradation of low molecular weight of PAEs and PA isomers in natural ecosystems. This is the first report of a P. putida strain able to degrade DEP and PA.
Topics: Humans; Pseudomonas putida; RNA, Ribosomal, 16S; Ecosystem; Biodegradation, Environmental; Environmental Pollutants
PubMed: 36635520
DOI: 10.1007/s12223-022-01022-y -
Microbial Cell Factories May 2023Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and...
BACKGROUND
Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of 2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida, previously grown in a culture medium containing ammonium mandelate, are a source of native benzoylformate decarboxylase (BFD) activity. BFD produced by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified enzyme preparations. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD-catalyzed enantioselective cross-coupling reaction.
RESULTS
The reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 mL of 200 mM phosphate buffer (pH 7) for 3 h. The optimal biomass concentration was assessed to be 0.006 g dry cell weight (DCW) mL. 2-HPP titer, yield and productivity using the free cells were 1.2 g L, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP g DCW h, respectively, under optimized biotransformation conditions (30 °C, 200 rpm). Calcium alginate (CA)-polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable beads degradation. Moreover, there was no production of benzyl alcohol as an unwanted by-product.
CONCLUSIONS
Bioconversion by whole P. putida resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones.
Topics: Pseudomonas putida; Carboxy-Lyases; Benzaldehydes; Hydroxypropiophenone; Stereoisomerism; Ketones; Acetaldehyde
PubMed: 37131175
DOI: 10.1186/s12934-023-02073-7 -
Scientific Reports Dec 2023Pseudomonads are ubiquitous bacteria with importance in medicine, soil, agriculture, and biomanufacturing. We report a novel Pseudomonas putida phage, MiCath, which is...
Pseudomonads are ubiquitous bacteria with importance in medicine, soil, agriculture, and biomanufacturing. We report a novel Pseudomonas putida phage, MiCath, which is the first known phage infecting P. putida S12, a strain increasingly used as a synthetic biology chassis. MiCath was isolated from garden soil under a tomato plant using P. putida S12 as a host and was also found to infect four other P. putida strains. MiCath has a ~ 61 kbp double-stranded DNA genome which encodes 97 predicted open reading frames (ORFs); functions could only be predicted for 48 ORFs using comparative genomics. Functions include structural phage proteins, other common phage proteins (e.g., terminase), a queuosine gene cassette, a cas4 exonuclease, and an endosialidase. Restriction digestion analysis suggests the queuosine gene cassette encodes a pathway capable of modification of guanine residues. When compared to other phage genomes, MiCath shares at most 74% nucleotide identity over 2% of the genome with any sequenced phage. Overall, MiCath is a novel phage with no close relatives, encoding many unique gene products.
Topics: Bacteriophages; Genome, Viral; Pseudomonas putida; DNA, Viral; Nucleoside Q; Sequence Analysis, DNA; Soil; Open Reading Frames; Phylogeny
PubMed: 38071193
DOI: 10.1038/s41598-023-48634-z -
Applied and Environmental Microbiology Oct 2020With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, KT2440 is emerging as an important chassis organism for metabolic...
With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields. To engineer novel metabolic pathways into , a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.
Topics: Alcohols; DNA Transposable Elements; DNA, Bacterial; Fatty Acids; Metabolic Networks and Pathways; Pseudomonas putida; Sequence Analysis, DNA
PubMed: 32826213
DOI: 10.1128/AEM.01665-20 -
Biotechnology Advances 2021Pseudomonas putida is a microbial chassis of huge potential for industrial and environmental biotechnology, owing to its remarkable metabolic versatility and ability to... (Review)
Review
Pseudomonas putida is a microbial chassis of huge potential for industrial and environmental biotechnology, owing to its remarkable metabolic versatility and ability to sustain difficult redox reactions and operational stresses, among other attractive characteristics. A wealth of genetic and in silico tools have been developed to enable the unravelling of its physiology and improvement of its performance. However, the rise of this microbe as a promising platform for biotechnological applications has resulted in diversification of tools and methods rather than standardization and convergence. As a consequence, multiple tools for the same purpose have been generated, whilst most of them have not been embraced by the scientific community, which has led to compartmentalization and inefficient use of resources. Inspired by this and by the substantial increase in popularity of P. putida, we aim herein to bring together and assess all currently available (wet and dry) synthetic biology tools specific for this microbe, focusing on the last 5 years. We provide information on the principles, functionality, advantages and limitations, with special focus on their use in metabolic engineering. Additionally, we compare the tool portfolio for P. putida with those for other bacterial chassis and discuss potential future directions for tool development. Therefore, this review is intended as a reference guide for experts and new 'users' of this promising chassis.
Topics: Biotechnology; Metabolic Engineering; Pseudomonas putida; Synthetic Biology
PubMed: 33785373
DOI: 10.1016/j.biotechadv.2021.107732