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We are interested in converting second generation feedstocks into styrene, a valuable chemical compound, using the solvent-tolerant Pseudomonas putida DOT-T1E as a chassis. Styrene biosynthesis takes place from L-phenylalanine in two steps: firstly, L-phenylalanine is converted into trans-cinnamic acid (tCA) by PAL enzymes and secondly, a decarboxylase yields styrene. This study focuses on designing and synthesizing a functional trans-cinnamic acid decarboxylase in Pseudomonas putida. To achieve this, we utilized the “wholesale” method, involving deriving two consensus sequences from multi-alignments of homologous yeast ferulate decarboxylase FDC1 sequences with > 60% and > 50% identity, respectively. These consensus sequences were used to design Pseudomonas codon-optimized genes named psc1 and psd1 and assays were conducted to test the activity in P. putida. Our results show that the PSC1 enzyme effectively decarboxylates tCA into styrene, whilst the PSD1 enzyme does not. The optimal conditions for the PSC1 enzyme, including pH and temperature were determined. The L-phenylalanine DOT-T1E derivative Pseudomonas putida CM12-5 that overproduces L-phenylalanine was used as the host for expression of pal/psc1 genes to efficiently convert L-phenylalanine into tCA, and the aromatic carboxylic acid into styrene. The highest styrene production was achieved when the pal and psc1 genes were co-expressed as an operon in P. putida CM12-5. This construction yielded styrene production exceeding 220 mg L. This study serves as a successful demonstration of our strategy to tailor functional enzymes for novel host organisms, thereby broadening their metabolic capabilities. This breakthrough opens the doors to the synthesis of aromatic hydrocarbons using Pseudomonas putida as a versatile biofactory.

Acid phosphatases are enzymes that play a crucial role in the hydrolysis of various organophosphorous molecules. A putative acid phosphatase called FS6 was identified using genetic profiles and sequences from different environments. FS6 showed high sequence similarity to type C acid phosphatases and retained more than 30% of consensus residues in its protein sequence. A histidine-tagged recombinant FS6 produced in Escherichia coli exhibited extremophile properties, functioning effectively in a broad pH range between 3.5 and 8.5. The enzyme demonstrated optimal activity at temperatures between 25 and 50°C, with a melting temperature of 51.6°C. Kinetic parameters were determined using various substrates, and the reaction catalysed by FS6 with physiological substrates was at least 100- fold more efficient than with p- nitrophenyl phosphate. Furthermore, FS6 was found to be a decamer in solution, unlike the dimeric forms of crystallized proteins in its family

Lignocellulosic residues are amongst the most abundant waste products on Earth. Therefore, there is an increasing interest in the utilization of these residues for bioethanol production and for biorefineries to produce compounds of industrial interest. Enzymes that breakdown cellulose and hemicellulose into oligomers and monosaccharides are required in these processes and cellulolytic enzymes with optimum activity at a low pH area are desirable for industrial processes. Here, we explore the fungal biodiversity of Rıo Tinto, the largest acidic ecosystem on Earth, as far as the secretion of cellulolytic enzymes is concerned. Using colorimetric and industrial substrates, we show that a high proportion of the fungi present in this extremophilic environment secrete a wide range of enzymes that are able to hydrolyze cellulose and hemicellulose at acidic pH (4.5–5). Shotgun proteomic analysis of the secretomes of some of these fungi has identified different cellulases and hemicellulolytic enzymes as well as a number of auxiliary enzymes. Supplementation of pre-industrial cocktails from Myceliophtora with Rio Tinto secretomes increased the amount of monosaccharides released from corn stover or sugar cane straw. We conclude that the Rio Tinto fungi display a good variety of hydrolytic enzymes with high industrial potential.

Lignocellulosic residues are amongst the most abundant waste products on Earth. Therefore, there is an increasing interest in the utilization of these residues for bioethanol production and for biorefineries to produce compounds of industrial interest. Enzymes that breakdown cellulose and hemicellulose into oligomers and monosaccharides are required in these processes and cellulolytic enzymes with optimum activity at a low pH area are desirable for industrial processes. Here, we explore the fungal biodiversity of Rıo Tinto, the largest acidic ecosystem on Earth, as far as the secretion of cellulolytic enzymes is concerned. Using colorimetric and industrial substrates, we show that a high proportion of the fungi present in this extremophilic environment secrete a wide range of enzymes that are able to hydrolyze cellulose and hemicellulose at acidic pH (4.5–5). Shotgun proteomic analysis of the secretomes of some of these fungi has identified different cellulases and hemicellulolytic enzymes as well as a number of auxiliary enzymes. Supplementation of pre-industrial cocktails from Myceliophtora with Rio Tinto secretomes increased the amount of monosaccharides released from corn stover or sugar cane straw. We conclude that the Rio Tinto fungi display a good variety of hydrolytic enzymes with high industrial potential.

Pseudomonas putida DOT-T1E is a highly solvent tolerant strain for which many genetic tools have been developed. The strain represents a promising candidate host for the synthesis of aromatic compounds—opening a path towards a green alternative to petrol-derived chemicals. We have engineered this strain to produce phenylalanine, which can then be used as a raw material for the synthesis of styrene via trans-cinnamic acid. To understand the response of this strain to the bioproducts of interest, we have analyzed the in-depth physiological and genetic response of the strain to these compounds. We found that in response to the exposure to the toxic compounds that the strain can produce, the cell launches a multifactorial response to enhance membrane impermeabilization. This process occurs via the activation of a cis to trans isomerase that converts cis unsaturated fatty acids to their corresponding trans isomers. In addition, the bacterial cells initiate a stress response program that involves the synthesis of a number of chaperones and ROS removing enzymes, such as peroxidases and superoxide dismutases. The strain also responds by enhancing the metabolism of glucose through the specific induction of the glucose phosphorylative pathway, Entner-Doudoroff enzymes, Krebs cycle enzymes and Nuo. In step with these changes, the cells induce two efflux pumps to extrude the toxic chemicals. Through analyzing a wide collection of efflux pump mutants, we found that the most relevant pump is TtgGHI, which is controlled by the TtgV regulator. Graphical Abstract: [Figure not available: see fulltext.]

The extensive use of petrochemicals has produced serious environmental pollution problems; fortunately, bioremediation is considered an efficient way to fight against pollution. In line with Synthetic Biology is that robust microbial chassis with an expanded ability to remove environmental pollutants are desirable. Pseudomonas putida KT2440 is a robust lab microbe that has preserved the ability to survive in the environment and is the natural host for the self-transmissible TOL plasmid, which allows metabolism of toluene and xylenes to central metabolism. We show that the P. putida KT2440 (pWW0) acquired the ability to use octane as the sole C-source after acquisition of an almost 62-kb ICE from a microbial community that harbours an incomplete set of octane metabolism genes. The ICE bears genes for an alkane monooxygenase, a PQQ-dependent alcohol dehydrogenase and aldehyde dehydrogenase but lacks the electron donor enzymes required for the monooxygenase to operate. Host rubredoxin and rubredoxin reductase allow metabolism of octane to octanol. Proteomic assays and mutants unable to grow on octane or octanoic acid revealed that metabolism of octane is mediated by redundant host and ICE enzymes. Octane is oxidized to octanol, octanal and octanoic acid, the latter is subsequently acylated and oxidized to yield acetyl-CoA that is assimilated via the glyoxylate shunt; in fact, a knockout mutant in the aceA gene, encoding isocitrate lyase was unable to grow on octane or octanoic acid.

Much of the energy being used to power our lives comes from fossil fuels such as coal, natural gas and petroleum. These energy sources are non-renewable, are being exhausted and also pollute the air, water and soil with toxic chemicals. Their mining, transportation, refining and use are associated with a large carbon footprint that contributes significantly to global warming. In addition, the geopolitical complexities surrounding the main fossil fuel producers create risks and uncertainties around the world. Replacing fossil fuels with clean, renewable forms of energy is paramount to creating a sustainable and healthy future, and for laying the foundations for global political stability and prosperity. Using biomass from plants, microbes can produce biofuels that are identical to or perform as well as fossil fuels. In addition of creating sustainable energy, advancing the biofuel industry will create new, high-quality rural jobs whilst improving energy security.

Pseudomonas putida BIRD-1 is a microorganism that inhabits the rhizosphere and solubilizes phosphate and iron and produces indolacetic acid [Roca, A., Pizarro-Tobías, P., Udaondo, Z., Fernández, M., Matilla, M.A., Molina-Henares, M.A., et al. (2013) Analysis of plant growth-promoting properties encoded by the genome of the rhizobacterium Pseudomonas putida BIRD-1. Environ Microbiol 15: 780–794]. In this study, we generated mutant strains that are capable of producing the plant growth stimulating compounds L-tryptophan and L-phenylalanine. We prepared clones that overproduce L-tryptophan by first mutagenizing P. putida BIRD-1, then by selecting for clones in the presence of inhibitory concentrations of 5-fluoro-D,L-tryptophan. The production of this aromatic amino acid was confirmed by chemical analysis and cross-feeding experiments with auxotrophs. One of the mutants, named P. putida BIRD-1-12, was mutagenized again to isolate clones that are also able to grow in the presence of inhibitory concentrations of p-fluoro-D,L-phenylalanine. One of these resulting clones was then isolated and named BIRD-1-12F. Our analysis revealed that the strains that either overproduce L-tryptophan, or L-tryptophan and L-phenylalanine, excel at promoting the growth of a number of plant crops of agricultural interest.

Genome "scale reconstructions of metabolism are computational species" specific knowledge bases able to compute systemic metabolic properties. We present a comprehensive and validated reconstruction of the biotechnologically relevant bacterium Pseudomonas putida KT2440 that greatly expands computable predictions of its metabolic states. The reconstruction represents a significant reactome expansion over available reconstructed bacterial metabolic networks. Specifically, iJN1462 (i) incorporates several hundred additional genes and associated reactions resulting in new predictive capabilities, including new nutrients supporting growth; (ii) was validated by in vivo growth screens that included previously untested carbon (48) and nitrogen (41) sources; (iii) yielded gene essentiality predictions showing large accuracy when compared with a knock¿out library and Bar¿seq data; and (iv) allowed mapping of its network to 82 P. putida sequenced strains revealing functional core that reflect the large metabolic versatility of this species, including aromatic compounds derived from lignin. Thus, this study provides a thoroughly updated metabolic reconstruction and new computable phenotypes for P. putida, which can be leveraged as a first step toward understanding the pan metabolic capabilities of Pseudomonas.

This article addresses the lifestyle of Pseudomonas and focuses on how Pseudomonas putida can be used as a model system for biotechnological processes in agriculture, and in the removal of pollutants from soils. In this chapter we aim to show how a deep analysis using genetic information and experimental tests has helped to reveal insights into the lifestyle of Pseudomonads. Pseudomonas putida is a Plant Growth Promoting Rhizobacteria (PGPR) that establishes commensal relationships with plants. The interaction involves a series of functions encoded by core genes which favor nutrient mobilization, prevention of pathogen development and efficient niche colonization. Certain Pseudomonas putida strains harbor accessory genes that confer specific biodegradative properties and because these microorganisms can thrive on the roots of plants they can be exploited to remove pollutants via rhizoremediation, making the consortium plant/Pseudomonas a useful tool to combat pollution.

Phylogenetic analysis of more than 4000 annotated bacterial acid phosphatases was carried out. Our analysis enabled us to sort these enzymes into the following three types: (1) class B acid phosphatases, which were distantly related to the other types, (2) class C acid phosphatases and (3) generic acid phosphatases (GAP). Although class B phosphatases are found in a limited number of bacterial families, which include known pathogens, class C acid phosphatases and GAP proteins are found in a variety of microbes that inhabit soil, fresh water and marine environments. As part of our analysis, we developed three profiles, named Pfr-B-Phos, Pfr-C-Phos and Pfr-GAP, to describe the three groups of acid phosphatases. These sequence-based profiles were then used to scan genomes and metagenomes to identify a large number of formerly unknown acid phosphatases. A number of proteins in databases annotated as hypothetical proteins were also identified by these profiles as putative acid phosphatases. To validate these in silico results, we cloned genes encoding candidate acid phosphatases from genomic DNA or recovered from metagenomic libraries or genes synthesized in vitro based on protein sequences recovered from metagenomic data. Expression of a number of these genes, followed by enzymatic analysis of the proteins, further confirmed that sequence similarity searches using our profiles could successfully identify previously unknown acid phosphatases.