Pseudomonas Putida Research Articles

Engineering a phi15-based expression system for stringent gene expression in Pseudomonas putida

The T7 phage RNA polymerase (RNAP) is a widely used expression platform, but its implementation in non-model microbial hosts poses significant challenges due to cytotoxicity. We constructed an optimized phage phi15-based expression system as alternative to the T7 platform for a wide range of applications in Pseudomonas putida. The new system employs the small phi15 RNAP, driving expression from an orthogonal phi15 promoter. By finetuning expression levels of phi15rnap and introducing a phi15 lysozyme mutant that inhibits phi15 RNAP in uninduced conditions, a stringent system was created with 200-fold inducibility. Moreover, by successfully decoupling cell growth and protein production using phi15 gp16, a host RNAP inhibitor, expression levels could be enhanced further (20%). Apart from creating four optimized platform P. putida hosts and a set of Golden Gate-compatible vectors, we demonstrate the extensive flexibility of the phi15 system. A proof-of-concept expression for industrially relevant fluorinase resulted in 2.5- and 5-fold increased yield compared to other widely-adopted expression systems. The system functions well in combination with several inducer systems, and in a variety of vector-based and genomically integrated set-ups. In conclusion, the phi15 RNAP, promoter, lysozyme and growth-decoupler provide a valuable plug-and-play set of genetic parts for the P. putida toolbox.

Clustering Zwitterionic Amino Acids at the Upper Rim of Cone Calix[4]arene Triggers the Selective Recognition of Gram‐Negative Bacterial Envelope

Eleven calix[4]arene ligands, bearing zwitterionic α‐amino acids or charged ammonium or sulfonate/carboxylate groups, are synthesized and screened for the binding to the envelopes of three bacterial strain representatives of Gram‐positive, Gram‐negative, and mycobacteria. The binding is followed by on‐cell Saturation Transfer Difference Nuclear Magnetic Resonance (NMR) experiments directly on alive cells. While the anionic tetrasulfonatocalixarene does not bind to any bacterial strains significantly and the cationic calixarenes strongly bind to both Gram‐positive and Gram‐negative bacteria, the zwitterionic tetraprolino‐ and tetraphenylalaninocalix[4]arene show a remarkable selectivity for Gram‐negatives over Gram‐positives and mycobacteria. The tetraprolinocalixarene binds to the lipopolysaccharides extracted from two Gram‐negative bacteria (Pseudomonas putida or Escherichia coli), suggesting these biomacromolecules as possible targets in the recognition of their cell walls. The ligand binding epitope map demonstrates a deep involvement of the amino acids and calixarene aromatic nuclei in the interaction. In this study, for the first time, the ability of synthetic macrocycles to selectively recognize the envelope of Gram‐negative bacteria is highlighted, and the way to the chemical modifications of the ligand structure is paved to develop devices for the detection or treatment of bacterial infections, thus allowing to add another string to the bow for the fight against antimicrobial resistance.

Phenanthrene degradation by a flavoprotein monooxygenase from Phanerodontia chrysosporium.

Phenanthrene (PHEN), a polycyclic aromatic hydrocarbon (PAH), is degraded by white-rot fungi like Phanerochaete chrysosporium (the fungus has been renamed as Phanerodontia chrysosporium). PHEN is metabolized by P. chrysosporium and transformed into various monohydroxylated and dihydroxylated products. These intermediates are further degraded by cleavage of the aromatic ring. However, the enzymes involved in PHEN conversion in P. chrysosporium remain largely unidentified. We aimed to identify and characterize the P. chrysosporium enzymes involved in the degradation of PHEN and its intermediates. Recombinant P. chrysosporium flavoprotein monooxygenase 11 (FPMO11), a homolog of the salicylate 1-monooxygenase from the naphthalene-degrading bacterium Pseudomonas putida G7, was overexpressed in Escherichia coli. FPMO11 catalyzes the oxidative decarboxylation of 1-hydroxy-2-naphthoate (1H2N) and 2-hydroxy-1-naphthoate (2H1N) to 1,2-dihydroxynaphthalene (1,2DHN). To the best of our knowledge, this is the first study to identify and characterize enzymes with 1H2N and 2H1N monooxygenase activities in members of the FPMO superfamily. Additionally, our search for a dioxygenase with the ability to catalyze the aromatic ring cleavage of 1,2DHN led to the identification of intradiol dioxygenase (IDD) 1 and IDD2 from P. chrysosporium, which catalyzes the ring cleavage of 1,2DHN. Thus, this study also identified, for the first time, intradiol 1,2DHN dioxygenase activity in members of the IDD superfamily. The findings highlight the unique substrate spectra of FPMO11 and IDDs, rendering them attractive candidates for biotechnological applications, especially mitigation of environmental and health risks associated with PAH pollution.IMPORTANCEPhenanthrene (PHEN), a polycyclic aromatic hydrocarbon (PAH), is a widely studied pollutant in environmental science and toxicology due to its presence in fossil fuels, tobacco smoke, and as a byproduct of incomplete combustion processes. White-rot fungi like P. chrysosporium can degrade PHEN through the production of extracellular oxidative enzymes. We investigated the properties of PHEN-degrading enzymes in P. chrysosporium, specifically one flavoprotein monooxygenase (FPMO11) and two intradiol dioxygenases (IDD1 and IDD2). Our findings indicate that the enzymes catalyze the aromatic ring cleavage of PHEN, using the intermediates as substrates, transforming them into less harmful and more biodegradable compounds. This could help reduce environmental pollution and mitigate health risks associated with PAH exposure. The potential of these enzymes for biotechnological applications is also highlighted, emphasizing their critical role in understanding PAH degradation by white-rot fungi.

Impact of Bambusa vulgaris-supplemented diet on Nile tilapia challenged with Pseudomonas putida: Hematological, immune, and oxidative responses.

This study investigated the effects of bamboo shoot extract (Bambusa vulgaris) as a feed additive on the health profiles and infection resistance of Nile tilapia (Oreochromis niloticus) against Pseudomonas putida. Bamboo shoot extract was added at levels of 0g, 40g, and 60g per 1000g of diet over a 60-day period. The fish were then challenged with a pathogenic P. putida strain. Chemical analysis of the bamboo shoot extract identified 3,5-dinitrophenol and hydroquinone as the two most abundant compounds. Results showed that fish fed bamboo-enriched diets exhibited significantly enhanced levels of red blood cells, hemoglobin, hematocrit, white blood cells, and platelets, and improved erythrocyte cellular and nuclear morphologies, indicating improved health profiles after the challenge. Liver function indicators, including AST, ALT, and ALP, were notably balanced in fish receiving bamboo shoot extract post-challenge (p<0.05). Blood levels of K+ were lower in the bamboo-fed groups. Additionally, blood levels of Ca++ and Na+ were reduced in fish fed 40g and 60g of bamboo, respectively, compared to the control group (p<0.01). The bamboo extract also enhanced immune and oxidative capacities, as demonstrated by increased catalase, superoxide dismutase, lysozyme activity, and phagocytic activity, along with reduced malondialdehyde levels and elevated serum immunoglobulin M (p<0.01). Gene expression analysis revealed significant effects of Bambusa vulgaris extract, Pseudomonas infection, and their interaction on the expression of interleukin-1β, interleukin-10, and NK-lysin genes, with varying expression levels at 1, 3, and 7 days post-challenge (p<0.05). The liver bacterial load in fish exposed to P. putida significantly decreased in the bamboo-fed groups, with the lowest count observed in the 60g bamboo group. Additionally, survival rates were markedly higher in the bamboo-fed groups compared to the control, with no significant difference between the two bamboo-fed groups. In conclusion, dietary supplementation with bamboo shoot extract enhances hematological parameters, blood cell and nuclear morphology, and increases survival rates in Nile tilapia following infection.

Algal organic matter triggers re-assembly of bacterial community in plumbing system

Algal bloom outbreaks in upstream drinking water reservoirs inevitably lead to algal organic matter (AOM) pollution in downstream drinking water plants and distribution systems. However, the responses of indoor piped drinking water quality and microbial community to AOM remain to be well studied. In this study, we investigated the effects of low and high concentrations of Chlorella organic matters on pipe-based drinking water. We found that AOM introduced nitrogen and phosphorus contamination into drinking water and promoted massive regeneration of bacteria during stagnation, along with increased bacterial metabolic activity. Compared to the Control group, the utilization capacity of alcohols, acids, esters, and amino acids increased under the influence of AOM. In addition, AOM intrusion reduced the bacterial community diversity in drinking water. The bacterial communities became more saturated, interspecific relationships became more complex, and interspecific competition increased. Bacteria with the ability to denitrification, such as Pseudomonas putida, Sphingobium amiense, Delftia tsuruhatensis, and Acidovorax temperans, were the most abundant. Residual chlorine, ammonium, nitrite, and iron had notable effects on the bacterial community under the influence of AOM. The results help elucidate the response mechanism of microbial community to AOM contamination in indoor drinking water pipes and provide a scientific basis for drinking water safety risk management.

Bioremediation of petroleum hydrocarbon contaminated soil by xylanase enzyme

Background: The global spread of petrochemical and petroleum contamination, such as petroleum hydrocarbons (PHCs), is currently a significant environmental risk. The global biosphere is badly harmed by these pollutants, and biodiversity is significantly reduced. This study was to screen for xylanase synthesis in Pseudomonas spp. and evaluate its efficiency as a bioremediator in removal of hydrocarbons from hydrocarbon-contaminated soil.Methods: Soil samples from Al-Dora oil plant Baghdad, Iraq, were cultured in nutritional agar medium containing 0.5% of corn cob xylan for determination of xylanase producers and measuring of xylanase activity, after that xylanaseproducers were identified. The xylanase was purified with DEAE-cellulose chromatography and the percentage of hydrocarbon degradation was calculated after treatment of hydrocarbon-contaminated soil with purified xylanase and detection of hydrocarbon degradation percentage.Results: Pseudomonas putida had the highest productivity for xylanase in comparison with other Pseudomonas species such as Pseudomonas syringae and Pseudomonas aeruginosa, which revealed lower levels in xylanase production. Ammonium salt saturation and ion exchange chromatography were used to purify the xylanase enzyme on a DEAE-cellulose column with ultimate recovery of 43% and 4.3 fold of purification. With pure xylanase, hydrocarbons degraded over time, peaking after two weeks and then progressively diminishing.Conclusions: Pseudomonas putida is the best producer foe xylanase than other species. The purified xylanase led to removal of hydrocarbons from hydrocarbon-contaminated soil with time increasing manner until maximum removal after 15 days. Authors recommend using xylanase for cleaning up of oil-contaminated areas. Therefore, employing microorganisms as biological tools may be a more feasible way to handle one of the most serious issues in modern society which might be a more workable and affordable way to minimize waste and preserve natural resources.Keywords: Petroleum hydrocarbons; Pseudomonas putida; Xylanase; Bioremediation; Soil contamination

Assessing the speed of individual bacteria dispersing on mycelial networks

Abstract The movement of bacteria on the hyphae of fungi and other mycelial-forming organisms is an important process that determines their ability to actively disperse in water-unsaturated habitats. However, direct observation and characterization of bacterial cell movement on mycelial networks have been difficult to achieve. In this study, we developed a new method that uses high-speed video recording to track the dispersal of individual fluorescently tagged cells of two closely related strains of Pseudomonas putida (UWC1 and KT2440) over the mycelial network of the oomycete Pythium ultimum. We found high intra-population heterogeneity and between-population differences in dispersal speeds for the two bacterial strains. The fitting of the speed distribution functions led to the separation of speeds into two ranges (fast/slow) at an intersection of the fitted curves. In the lower speed range, the UWC1 strain dispersed faster, while the KT2440 strain moved faster in the higher speed range. This finding helps explain conflicting competition outcomes revealed in previous studies and suggests that population mean speed alone does not capture key aspects of bacterial dispersal in mycelial networks. Our new method opens the possibility of studying bacterial dispersal, competition, and other social interactions in spatially heterogeneous environments, such as soils.

The Pseudomonas ligninolytic catalytic network reveals the importance of auxiliary enzymes in lignin biocatalysts

Lignin degradation by biocatalysts is a key strategy to develop a plant-based sustainable carbon economy and thus alleviate global climate change. This process involves synergy between ligninases and auxiliary enzymes. However, auxiliary enzymes within secretomes, which are composed of thousands of enzymes, remain enigmatic, although several ligninolytic enzymes have been well characterized. Moreover, it is a challenge to understand synergistic lignin degradation via a diverse array of enzymes, especially in bacterial systems. In this study, the coexpression network of the periplasmic proteome uncovers potential accessory enzymes for B-type dye-decolorizing peroxidases (DypBs) in Pseudomonas putida A514. The catalytic network of the DypBs-based multienzyme complex is characterized. DypBs couple with quinone reductases and nitroreductase to participate in quinone redox cycling. They work with superoxide dismutase to induce Fenton reaction for lignin oxidation. A synthetic enzyme cocktail (SEC), recruiting 15 enzymes, was consequently designed with four functions. It overcomes the limitation of lignin repolymerization, exhibiting a capacity comparable to that of the native periplasmic secretome. Importantly, we reveal the synergistic mechanism of a SEC-A514 cell system, which incorporates the advantages of in vitro enzyme catalysis and in vivo microbial catabolism. Chemical analysis shows that this system significantly reduces the molecular weight of lignin, substantially extends the degradation spectra for lignin functional groups, and efficiently metabolizes lignin derivatives. As a result, 25% of lignin is utilized, and its average molecular weight is reduced by 27%. Our study advances the knowledge of bacterial lignin-degrading multienzymes and provides a viable lignin degradation strategy.

Pathway Elucidation and Key Enzymatic Processes in the Biodegradation of Difenoconazole by Pseudomonas putida A-3.

The extensive agricultural use of the fungicide difenoconazole (DIF) and its associated toxicity increasingly damage ecosystems and human health. Thus, an urgent need is to develop environmentally friendly technological approaches capable of effectively removing DIF residues. In this study, strain Pseudomonas putida A-3 was isolated for the first time which can degrade DIF efficiently. After optimization of the degradation conditions, the degradation rate reached 75.98%. Moreover, a new DIF degradation pathway, including hydroxylation, hydrolysis, dechlorination, and ether bond breaking. The acute and chronic toxicity of DIF degradation products assessed using ECOSAR software showed lower toxicity than the parent compound. Furthermore, strain A-3 remarkably accelerated the degradation of DIF in contaminated water-sediment systems. We successfully predicted six potential key enzymes for DIF degradation based on the results of whole genome sequencing, RT-qPCR, and molecular docking. Overall, the results revealed novel pathways for DIF biodegradation and provide a strong candidate for bioremediation of DIF residue-polluted environments.

Time-Dependent Growth of ZnO Nanowires: Unveiling Antibacterial and Photocatalytic Properties.

This study investigates the synthesis, characterization, and functional properties of well-aligned zinc oxide (ZnO) nanowires (NWs) obtained by a two-step hydrothermal method. ZnO NWs were grown on silicon substrates precoated with a ZnO seed layer. The growth process was conducted at 90 °C for different durations (2, 3, and 4 h) to examine the time-dependent evolution of the nanowire properties. A comprehensive characterization of the ZnO NWs was performed using several analytical techniques. Scanning electron microscopy (SEM) revealed the morphological progression, specifically tracking changes in length and diameter as a function of the growth time. Ultraviolet (UV)-visible spectroscopy was employed to determine the optical band gap, while photoluminescence (PL) analysis provided insight into the concentration of structural defects and its evolution as a function of nanowire growth. The photocatalytic efficiency of the ZnO NWs was evaluated through the degradation of the organic dye methylene blue (MB) under UV light irradiation (365 nm). The kinetic of MB degradation was monitored for each growth time, with non-purgeable organic carbon (NPOC) analysis providing a detailed perspective on the photocatalytic activity over time. The antibacterial properties were tested against Pseudomonas putida, a Gram-negative bacterium, to determine the efficiency of the synthesized ZnO NWs as antimicrobial agents. The release of zinc ions (Zn2+), a key factor in the antibacterial mechanism, was quantified using inductively coupled plasma (ICP) analysis for each sample. By exploration of the relationship between the growth time, nanostructure morphology, and functional properties, this study provides insights into optimizing the synthesis of ZnO NWs for enhanced photocatalytic and antibacterial applications. These findings contribute to the development of advanced materials for environmental and biomedical applications.

Insight into the phylogeny and antibiotic resistance of Pseudomonas spp. originating from soil of the Białowieża National Park in Northeastern Poland.

The Pseudomonas genus includes species present in various environments and known for antibiotic resistance. However, only hospital-associated Pseudomonas aeruginosa have been extensively studied regarding antibiotic resistance. Thus, to fill the gap in knowledge on antibiotic resistance among other Pseudomonas spp., we investigated 41 isolates from soil samples taken in the Białowieża National Park in Northeastern Poland. This unique forest without notable anthropogenic influence, provides excellent conditions for research of antibiotic resistance from the perspective of natural environments. The phylogeny trees obtained based on the nucleotide sequence of the 16S rRNA gene and gyrB gene grouped the isolates into clusters belonging to the Pseudomonas fluorescens, Pseudomonas koreensis, and Pseudomonas putida groups, originating from the P. fluorescens lineage. All isolates under study demonstrated resistance to at least 12 out of the 24 antibiotics tested. Resistance to colistin, cefotaxime, and imipenem was detected in 73, 73, and 17% of the isolates, respectively. Most isolates showing resistance to imipenem and colistin clustered within the P. fluorescens group. Seven isolates were highly multi-resistant, to up to 18 of the 24 antibiotics tested. The presence of resistance genes related to intrinsic resistance of P. aeruginosa has been confirmed in environmental isolates.

Structure, kinetics, and mechanism of Pseudomonas putida sulfoquinovose dehydrogenase, the first enzyme in the sulfoglycolytic Entner-Doudoroff pathway.

The sulfosugar sulfoquinovose (SQ) is catabolized through the sulfoglycolytic Entner-Doudoroff pathway, beginning with the oxidation of SQ to sulfogluconolactone by SQ dehydrogenase. We present a comprehensive structural and kinetic characterization of Pseudomonas putida SQ dehydrogenase (PpSQDH). PpSQDH is a tetrameric enzyme belonging to the short-chain dehydrogenase/reductase (SDR) superfamily with a strong preference for NAD+ over NADP+. Kinetic analysis revealed a rapid equilibrium ordered mechanism in which the NAD+ cofactor is the first substrate to bind, and NADH is the last product to dissociate. Structural studies revealed a homotetrameric structure in solution and crystals, involving cross-subunit interactions in which the C-terminus residue (Gln260) inserts into the diagonally opposite subunit to form part of the second shell of residues lining the active site. Complexes of PpSQDH with SQ or NAD+ provide insight into the recognition of SQ and together with the kinetic analysis allow the proposal of a catalytic reaction mechanism. Our findings illuminate the mechanism of SQ degradation and the evolution of the SDR superfamily for organosulfonate catabolism.

Engineering probiotic Escherichia coli for inflammation-responsive indoleacetic acid production using RiboJ-enhanced genetic circuits

BackgroundAs our understanding of gut microbiota’s metabolic impacts on health grows, the interest in engineered probiotics has intensified. This study aimed to engineer the probiotic Escherichia coli Nissle 1917 (EcN) to produce indoleacetic acid (IAA) in response to gut inflammatory biomarkers thiosulfate and nitrate.ResultsGenetic circuits were developed to initiate IAA synthesis upon detecting inflammatory signals, optimizing a heterologous IAA biosynthetic pathway, and incorporating a RiboJ insulator to enhance IAA production. The engineered EcN strains demonstrated increased IAA production in the presence of thiosulfate and nitrate. An IAA-responsive genetic circuit using the IacR transcription factor from Pseudomonas putida 1290 was also developed for real-time IAA monitoring.ConclusionsGiven IAA’s role in reducing gastrointestinal inflammation, further refinement of this strain could lead to effective, in situ IAA-based therapies. This proof-of-concept advances the field of live biotherapeutic products and offers a promising approach for targeted therapy in inflammatory bowel diseases.

Mixed bacterial infections in trout fish farms of the Leningrad region

In recent years, outbreaks of mixed bacterial infections, similar to bacterial hemorrhagic septicemia, have become more frequent in the cage trout farms of the Leningrad Region. Previously, a similar course of the infectious process was observed mainly in closed water supply installations, especially for the first time years of their operation. High water temperatures play an important role in the development of the disease process (more than 20 °C), which last for a month or more, as well as high planting densities. In the period from 2021 to 2024, in the summer, a bacterial infection, the causative agents of which are Yersenia ruckeri and Flexibacter columnaris, is dangerous in cages. In May, and later during the summer, the presence of mixed bacterial infections caused by the development of opportunistic microflora was revealed — aeromonas (Aeromonas hydrophila, Aeromonas caviae), pseudomonas (Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas chlororaphis) and myxobacteria (Flexibacter psyhrophila, Flexibacter columnaris). It should be noted the appearance of enterobacteria (Enterobacter spp.) as representatives of the secondary microflora, whose pathogenicity has been confirmed. The complex of aeromonas with flavobacteria is characterized by increased aggressiveness, which contributes to the development of serious septic infection. In one farm, two or three associations of pathogenic bacteria could be noted. A significant difficulty in detecting bacterial hemorrhagic septicemia is the selection of antimicrobial drugs. In this case, it is necessary to take into account the sensitivity to them of the main representatives of the association of pathogens. Otherwise, you can provoke anew outbreak of the disease. It was noted that mixed infections were often found in farms where fish purchased from three to four suppliers were grown on one fish farm. An extremely negative effect was given by stocking in the autumn with juveniles with low fatness and with a small amount of cavitary fat. Timely measures taken, including the normalization of conditions of detention and feeding, as well as medical and preventive measures, made it possible to suppress the development of infections and improve the epizootic state of kindergarten farms. In the future, it is recommended to avoid stocking one site with a large number of fish of different origin, and, accordingly, with different opportunistic microflora, the contact of which with each other leads to a deterioration in the epizootic state of farms. To normalize the in testinal microflora and increase the immune-physiological status, the use of probiotics can be proposed.

Relevance of plant growth-promoting bacteria in reducing the severity of tomato wilt caused by Fusarium oxysporum f. sp. lycopersici by altering metabolites and related genes.

Among the biotic stresses, wilt disease severely affects tomato quality and productivity globally. The causal organism of this disease is Fusarium oxysporum f. sp. lycopersici (Fol), which is very well known and has a significant impact on the productivity of other crops as well. Efforts have been made to investigate the effect of plant growth-promoting bacteria (PGPB) on alleviating tomato wilt disease. Four PGPB strains, such as Pseudomonas aeruginosa BHUPSB01 (T1), Pseudomonas putida BHUPSB04 (T2), Paenibacillus polymyxa BHUPSB16 (T3), and Bacillus cereus IESDJP-V4 (T4), were used as inocula to treat Fol-challenged plants. The results revealed that PGPB treatments T1, T2, T3, and T4 were able to decrease the severity of Fusarium wilt in the tomato plants at different levels. Among the treatments, T3 displayed the strongest protective effect, with the lowest disease frequency, which was 15.25%. There were no significant differences observed in parameters such as fruit yield and relative water content in the PGPB-inoculated plants, although T3 and T4 showed minimal electrolyte leakage. Significant changes in chlorophyll fluorescence were also recorded. A lower level of H2O2 and malondialdehyde (MDA) was observed in the T3 and T4 treatments. In addition, proline accumulation was highest in the T3-treated plants. Antioxidative enzyme activities, such as catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), significantly increased in the PGPB-treated plants. Furthermore, the highest phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) activity was reported in the T3 and T4 plants, respectively. The PGPB-treated plants showed elevated expression of the PAL, PPO, PR3, PR2, SOD, CAT, and PO genes. This study's results reveal that PGPB strains can be utilized as biocontrol agents (BCAs) to enhance tomato resistance against Fusarium wilt.

Comparing three emerging industrial cell factories: Pseudomonas putida KT2440, Halomonas bluephagenesis TD01, and Zymomonas mobilis ZM4.

Nonmodel microbes with unique advantages are emerging as industrial platforms, driven by advances in genetic engineering and omics technologies. Notable examples include the versatile soil bacterium Pseudomonas putida KT2440, the halophilic Halomonas bluephagenesis TD01, and the ethanologenic Zymomonas mobilis ZM4. While all three primarily use the Entner-Doudoroff pathway for glucose metabolism, they differ in various metabolic pathways and product synthesis. This review summarizes and compares their central carbon metabolism, advancements in genome engineering tools, and progress in scaling industrial applications from lab scale, to pilot scale, to full-scale commercial production. Understanding their similarities and differences informs future research on optimizing industrial applications and may guide the development of new microbial hosts.

Bioprocess exploitation of microaerobic auto-induction using the example of rhamnolipid biosynthesis in Pseudomonas putida KT2440

BackgroundIn biomanufacturing of surface-active agents, such as rhamnolipids, excessive foaming is a significant obstacle for the development of high-performing bioprocesses. The exploitation of the inherent tolerance of Pseudomonas putida KT2440, an obligate aerobic bacterium, to microaerobic conditions has received little attention so far. Here low-oxygen inducible promoters were characterized in biosensor strains and exploited for process control under reduction of foam formation by low aeration and stirring rates during biosynthesis of rhamnolipids.ResultsIn this study, homologous promoters of P. putida inducible under oxygen limitation were identified by non-targeted proteomic analyses and characterized by fluorometric methods. Proteomics indicated a remodeling of the respiratory chain and the regulation of stress-related proteins under oxygen limitation. Of the three promoters tested in fluorescent biosensor assays, the promoter of the oxygen-sensitive cbb3-type cytochrome c oxidase gene showed high oxygen-dependent controllability. It was used to control the gene expression of a heterologous di-rhamnolipid synthesis operon in an auto-inducing microaerobic two-phase bioprocess. By limiting the oxygen supply via low aeration and stirring rates, the bioprocess was clearly divided into a growth and a production phase, and sources of foam formation were reduced. Accordingly, rhamnolipid synthesis did not have to be controlled externally, as the oxygen-sensitive promoter was autonomously activated as soon as the oxygen level reached microaerobic conditions. A critical threshold of about 20% oxygen saturation was determined.ConclusionsUtilizing the inherent tolerance of P. putida to microaerobic conditions in combination with the application of homologous, low-oxygen inducible promoters is a novel and efficient strategy to control bioprocesses. Fermentation under microaerobic conditions enabled the induction of rhamnolipid production by low oxygen levels, while foam formation was limited by low aeration and stirring rates.

Addressing genome scale design tradeoffs in Pseudomonas putida for bioconversion of an aromatic carbon source

Genome-scale metabolic models (GSMM) are commonly used to identify gene deletion sets that result in growth coupling and pairing product formation with substrate utilization and can improve strain performance beyond levels typically accessible using traditional strain engineering approaches. However, sustainable feedstocks pose a challenge due to incomplete high-resolution metabolic data for non-canonical carbon sources required to curate GSMM and identify implementable designs. Here we address a four-gene deletion design in the Pseudomonas putida KT2440 strain for the lignin-derived non-sugar carbon source, p-coumarate (p-CA), that proved challenging to implement. We examine the performance of the fully implemented design for p-coumarate to glutamine, a useful biomanufacturing intermediate. In this study glutamine is then converted to indigoidine, an alternative sustainable pigment and a model heterologous product that is commonly used to colorimetrically quantify glutamine concentration. Through proteomics, promoter-variation, and growth characterization of a fully implemented gene deletion design, we provide evidence that aromatic catabolism in the completed design is rate-limited by fumarase hydratase (FUM) enzyme activity in the citrate cycle and requires careful optimization of another fumarate hydratase protein (PP_0897) expression to achieve growth and production. A double sensitivity analysis also confirmed a strict requirement for fumarate hydratase activity in the strain where all genes in the growth coupling design have been implemented. Metabolic cross-feeding experiments were used to examine the impact of complete removal of the fumarase hydratase reaction and revealed an unanticipated nutrient requirement, suggesting additional functions for this enzyme. While a complete implementation of the design was achieved, this study highlights the challenge of completely inactivating metabolic reactions encoded by under-characterized proteins, especially in the context of multi-gene edits.

Exploring 1-alkene biosynthesis in bacterial antagonists and Jeotgalicoccus sp. ATCC 8456.

Terminal olefins are important platform chemicals, drop-in compatible hydrocarbons and also play an important role as biocontrol agents of plant pathogens. Currently, 1-alkenes are derived from petroleum, although microbial biosynthetic routes are known. Jeotgalicoccus sp. ATCC 8456 produces 1-alkenes via the fatty acid decarboxylase OleTJE. UndA and UndB are recently identified non-heme iron oxidases converting medium-chain fatty acids into terminal alkenes. Our knowledge about the diversity and natural function of OleTJE, UndA, and UndB homologs is scarce. We applied a combined screening strategy-solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME GC-MS) and polymerase chain reaction (PCR)-based amplification-to survey an environmental strain collection for microbial 1-alkene producers and their corresponding enzymes. Our results reinforce the high level of conservation of UndA and UndB genes across the genus Pseudomonas. In vivo production of defined 1-alkenes (C9-C13; C15; C19) was directed by targeted feeding of fatty acids. Lauric acid feeding enabled 1-undecene production to a concentration of 3.05mg l-1 in Jeotgalicoccus sp. ATCC 8456 and enhanced its production by 105% in Pseudomonas putida 1T1 (1.10mg l-1). Besides, whole genome sequencing of Jeotgalicoccus sp. ATCC 8456 enabled reconstruction of the 1-alkene biosynthetic pathway. These results advance our understanding of microbial 1-alkene synthesis and the underlying genetic basis.

One-Pot lignin bioconversion to polyhydroxyalkanoates based on hierarchical utilization of heterogeneous compounds.

Pseudomonas putida degraded 35% of compounds in alkali-pretreated lignin liquor under nitrogen-replete conditions but with low polyhydroxyalkanoates (PHA) production, while limiting nitrogen supplement improved PHA content (PHA/dry cell weight) to 43% at the expense of decreased lignin degradation of 22%. Increase of initial cell biomass (0.1-1.5g/L) monotonically improved the lignin degradation from 22% to 33% under nitrogen-limited conditions. Hierarchical utilization of heterogenous compounds under cell growth restricted conditions has been unveiled - simple carbon sources were prioritized for valorization, followed by aromatic compounds bioconversion. Based on the results of hierarchy and leveraging the initial bacterial biomass, acetate was augmented to facilitate one-pot lignin bioconversion under nitrogen-limited conditions. This approach improved lignin bioconversion closer to its upper degradation limit of 35%, concomitant with PHA yield of 39mg/g-lignin. Anaerobic digestion of lignocellulose was redesigned to favor acetate-type fermentation, with acetate constituting 91wt%, providing an economic source of acetate.