Pseudomonas Putida Research Articles

Establishing a straightforward I-SceI-mediated recombination one-plasmid system for efficient genome editing in P. putida KT2440.

Pseudomonas putida has become an increasingly important chassis for producing valuable bioproducts. This development is not least due to the ever-improving genetic toolbox, including gene and genome editing techniques. Here, we present a novel, one-plasmid design of a critical genetic tool, the pEMG/pSW system, guaranteeing one engineering cycle to be finalized in 3 days. The pEMG/pSW system proved in the last decade to be valuable for targeted genome engineering in Pseudomonas, as it enables the deletion of large regions of the genome, the integration of heterologous gene clusters or the targeted generation of point mutations. Here, to expedite genetic engineering, two alternative plasmids were constructed: (1) The sacB gene from Bacillus subtilis was integrated into the I-SceI expressing plasmid pSW-2 as a counterselection marker to accelerated plasmid curing; (2) double-strand break introducing gene I-sceI and sacB counterselection marker were integrated into the backbone of the original pEMG vector, named pEMG-RIS. The single plasmid of pEMG-RIS allows rapid genome editing despite the low transcriptional activity of a single copy of the I-SceI encoding gene. Here, the usability of the pEMG-RIS is shown in P. putida KT2440 by integrating an expression cassette including an msfGFP gene in 3 days. In addition, a large fragment of 12.1 kb was also integrated. In summary, we present an updated pEMG/pSW genome editing system that allows efficient and rapid genome editing in P. putida. All plasmids designed in this study will be available via the Addgene platform.

Tailored antimicrobial PHBV-based packaging for extended shelf life of processed cheese

A coupled experimental and modelling approach was used to develop an adapted PHBV active film with Lauroyl Arginate Ethyl (LAE®) as an antimicrobial compound incorporated in the bulk or as a coating to prolong the shelf life of cheese. The minimum inhibitory concentration (MIC) of LAE® was evaluated in the range of 25–100 ppm against spoilage and pathogenic microorganisms, such as Micrococcus luteus, Penicillium roqueforti, Salmonella enteritidis, and Pseudomonas putida. Film production induced a loss of 50 % of LAE® when introduced into the bulk, whereas only 4 % was lost in the case of incorporation by coating. When in contact with food simulant D1 (50 % ethanol), a release of 43 % and 34 % of LAE® was achieved in 10 days at 20 °C for LAE® incorporated as a coating and in the bulk, respectively, while a lower release level of 34 % and 14 % respectively was observed in food simulant C (20 % ethanol), confirming the interest to use active coating for fatty foods. The developed packaging was well adapted to reach the MIC while remaining below the admissible daily intake. The results also showed that the PHBV film activated with LAE® incorporated as a coating was as effective as modified atmosphere packaging (MAP) to preserve cheese against microorganism growth.

Rhizoengineering with biofilm producing rhizobacteria ameliorates oxidative stress and enhances bioactive compounds in tomato under nitrogen-deficient field conditions

Nitrogen (N) deficiency limits crop productivity. In this study, rhizoengineering with biofilm producing rhizobacteria (BPR) contributing to productivity, physiology, and bioactive contents in tomato was examined under N-deficient field conditions. Here, different BPR including Leclercia adecarboxylata ESK12, Enterobacter ludwigii ESK17, Glutamicibacter arilaitensis ESM4, E. cloacae ESM12, Bacillus subtilis ESM14, Pseudomonas putida ESM17 and Exiguobacterium acetylicum ESM24 were used for the rhizoengineering of tomato plants. Rhizoengineered plants showed significant increase in growth attributes (15.73%–150.13 %) compared to the control plants. However, production of hydrogen peroxide (21.49–59.38 %), electrolyte leakage (19.5–38.07 %) and malondialdehyde accumulation (36.27–46.31 %) were increased remarkably more in the control plants than the rhizoengineered plants, thus N deficiency induced the oxidative stress. Compared to the control, photosynthetic rate, leaf temperature, stomatal conductance, intrinsic and instantaneous water use efficiency, relative water content, proline and catalase activity were incredibly enhanced in the rhizoengineered plants, suggesting both non-enzymatic and enzymatic antioxidant systems might protect tomato plants from oxidative stress under N-deficient field conditions. Yield (10.24–66.21 %), lycopene (4.8–7.94 times), flavonoids (52.32–110.46 %), phenolics (9.79–23.5 %), antioxidant activity (34.09–86.36 %) and certain minerals were significantly increased in the tomatoes from rhizoengineered plants. The principal component analysis (PCA) revealed that tomato plants treated with BPR induced distinct profiles compared to the control. Among all the applied BPR strains, ESM4 and ESM14 performed better in terms of biomass production, while ESK12 and ESK17 showed better results for reducing oxidative stress and increasing bioactive compounds in tomato, respectively. Thus, rhizoengineering with BPR can be utilized to mitigate the oxidative damage and increase the productivity and bioactive compounds in tomato under N-deficient field conditions.

Chemical and Microbiological Quality of Municipal Supply Water in Dhaka South Area, Bangladesh

This project aimed to find out the microbiological quality of municipal supply water in Dhaka South area and if the current regime of microbiological quality testing can determine the risk to public health from supply water satisfactorily. Water samples were collected from six different points of the water distribution network of Dhaka’s south locality, starting from the source (Shitalakhyariver, Narayanganj) to Jagannath University, Dhaka. The dominant taxonomic units were isolated using standard culture techniques, followed by biochemical identification, VITEK2 identification, and biofilm-forming potential of each isolate individually and co-cultured with other isolates. Presumptive identification of isolates from the Shitalakhya River revealed the presence of Citrobacter sp., Alcaligenes sp., and Pseudomonas putida. The sample from Saydabad Water Treatment Plant yielded Vibrio mimicus, Pseudomonas fluorescence, and Alcalimonas. The major aerobic bacteria in the water specimen from the community overhead tank included S aureus, Bacillus cereus, Micrococcus sp., Enterococcus sp., Lactococcus sp. Tap water revealed the presence of Escherichia coli, Lactococcus, Enterobacter, Shigella, Klebsiella, Pseudomonas, and Staphylococcus aureus, as well as anaerobic bacteria. Biochemical tests and VITEK2 yielded different results. Potential biofilm formers such as Klebsiella, E. coli, Enterococcus, Aerococcus, Enterobacter, and Cronobacter indicated quality deterioration potential inside the piped water supply system. Alteration in the major taxonomic units throughout the municipal water distribution network and the presence of opportunistic pathogens and biofilm-formers indicate the necessity for an updated water treatment, quality control, and in-line maintenance system.

Characterization and Abundance of Plasmid-Dependent Alphatectivirus Bacteriophages

Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis.

In vitro antagonistic activity of Bacillus spp. and Pseudomonas putida M5 isolated from feces of the violet-crowned hummingbird (Ramosomyia violiceps) from an urban environment

In vitro antagonistic activity of Bacillus spp. and Pseudomonas putida M5 isolated from feces of the violet-crowned hummingbird (Ramosomyia violiceps) from an urban environment

Evolution and engineering of pathways for aromatic O-demethylation in Pseudomonas putida KT2440

Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and O-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O-demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O-demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida, and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ∼1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate O-demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA, involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms, yielding a platform strain for efficient O-demethylation of lignin-related aromatic compounds to value-added products.

Computer-aided design and implementation of efficient biosynthetic pathways to produce high added-value products derived from tyrosine in Escherichia coli.

Developing efficient bioprocesses requires selecting the best biosynthetic pathways, which can be challenging and time-consuming due to the vast amount of data available in databases and literature. The extension of the shikimate pathway for the biosynthesis of commercially attractive molecules often involves promiscuous enzymes or lacks well-established routes. To address these challenges, we developed a computational workflow integrating enumeration/retrosynthesis algorithms, a toolbox for pathway analysis, enzyme selection tools, and a gene discovery pipeline, supported by manual curation and literature review. Our focus has been on implementing biosynthetic pathways for tyrosine-derived compounds, specifically L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine, with significant applications in health and nutrition. We selected one pathway to produce L-DOPA and two different pathways for dopamine-one already described in the literature and a novel pathway. Our goal was either to identify the most suitable gene candidates for expression in Escherichia coli for the known pathways or to discover innovative pathways. Although not all implemented pathways resulted in the accumulation of target compounds, in our shake-flask experiments we achieved a maximum L-DOPA titer of 0.71g/L and dopamine titers of 0.29 and 0.21g/L for known and novel pathways, respectively. In the case of L-DOPA, we utilized, for the first time, a mutant version of tyrosinase from Ralstonia solanacearum. Production of dopamine via the known biosynthesis route was accomplished by coupling the L-DOPA pathway with the expression of DOPA decarboxylase from Pseudomonas putida, resulting in a unique biosynthetic pathway never reported in literature before. In the context of the novel pathway, dopamine was produced using tyramine as the intermediate compound. To achieve this, tyrosine was initially converted into tyramine by expressing TDC from Levilactobacillus brevis, which, in turn, was converted into dopamine through the action of the enzyme encoded by ppoMP from Mucuna pruriens. This marks the first time that an alternative biosynthetic pathway for dopamine has been validated in microbes. These findings underscore the effectiveness of our computational workflow in facilitating pathway enumeration and selection, offering the potential to uncover novel biosynthetic routes, thus paving the way for other target compounds of biotechnological interest.

Integrating genomics, molecular docking, and protein expression to explore new perspectives on polystyrene biodegradation

Faced with the escalating challenge of global plastic pollution, this study specifically addresses the research gap in the biodegradation of polystyrene (PS). A PS-degrading bacterial strain was isolated from the gut of Tenebrio molitor, and genomics, molecular docking, and proteomics were employed to thoroughly investigate the biodegradation mechanisms of Pseudomonas putida H-01 against PS. Using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (ATR-FTIR), and contact angle analysis, significant morphological and structural changes in the PS films under the influence of the H-01 strain were observed. The study revealed several potential degradation genes and ten enzymes that were specifically upregulated in the PS degradation environment. Additionally, a novel protein with laccase-like activity, LacQ1, was purified from this strain for the first time, and its crucial role in the PS degradation process was confirmed. Through molecular docking and molecular dynamics (MD) simulations, the interactions between the enzymes and PS were detailed, elucidating the binding and catalytic mechanisms of the degradative enzymes with the substrate. These findings have deepened our understanding of PS degradation.

Evaluation of the benefits of plant growth-promoting rhizobacteria and mycorrhizal fungi on biochemical and morphophysiological traits of Aloe barbadensis Mill under water deficit stress

Aloe barbadensis is a drought-tolerant perennial medicinal plant with both nutritional and cosmetic uses. Drought is one of the main abiotic stresses limiting plant growth and development. However, the use of drought-resistant plants combined with beneficial soil micro-organisms could improve the effectiveness of biological methods to mitigate drought damage. This research aims to evaluate the effects of Funneliformis mosseae (MF), plant growth-promoting rhizobacteria (PGPR) (including Pseudomonas putida and Pantoea agglomerans), and their co-inoculation on the macronutrient status, antioxidant enzyme activities, and other morphophysiological traits of A. barbadensis under four irrigation regimes [25%, 50%, 75% and 100% of water requirement (WR)]. Three harvests were conducted, revealing that inoculation enhanced the survival rate and shoot fresh weight (SFW) compared to the control plants. However, at 25% WR, the SFW was reduced by 43% more than the control. across all harvests, while the PGPR + MF treatment showed increases of more than 19%, 11%, and 17% compared to the control, MF, and PGPR treatments, respectively. The results also showed that A. barbadensis exhibited innate drought tolerance up to a 50% WR level by enhancing physiological defenses, such as antioxidant enzyme activity. Inoculation increased the macronutrient status of the plant at all levels of irrigation regimes especially under severe drought conditions. The highest levels of nitrogen (N) (16.24 mg g−1 DW) and phosphorus (P) (11.29 mg g−1 DW) were observed in the PGPR + MF treatment at 100% WR. The maximum relative water content under MF inoculation and 75% WR (98.24%) (98.24%) was reached. PGPR + MF treatment alleviated drought-induced osmotic stress, as indicated by reduced antioxidant enzyme activities and electrolyte leakage. However, P. putida and P. agglomerans strains alone or in combination with F. mosseae increased plant yield, macronutrient uptake and antioxidant enzyme activity. This study underscores the potential of these PGPR and MF strains as invaluable biological tools for the cultivation of A. barbadensis in regions with severe drought stress.

Enhancing Pseudomonas cell growth for the production of medium-chain-length polyhydroxyalkanoates from Antarctic krill shell waste

Antarctic krill shell waste (AKSW), a byproduct of Antarctic krill processing, has substantial quantity but low utilization. Utilizing microbial-based cell factories, with Pseudomonas putida as a promising candidate, offers an ecofriendly and sustainable approach to producing valuable bioproducts from renewable sources. However, the high fluoride content in AKSW impedes the cell growth of P. putida. This study aims to investigate the transcriptional response of P. putida to fluoride stress from AKSW and subsequently conduct genetic modification of the strain based on insights gained from transcriptomic analysis. Notably, the engineered strain KT+16840+03100 exhibited a remarkable 33.7-fold increase in cell growth, capable of fermenting AKSW for medium-chain-length-polyhydroxyalkanoates (mcl-PHA) biosynthesis, achieving a 40.3-fold increase in mcl-PHA yield compared to the control strain. This research advances our understanding of how P. putida responds to fluoride stress from AKSW and provides engineered strains that serve as excellent platforms for producing mcl-PHA through AKSW.

Pseudomonas putida Facilitates Pearl Millet Growth in Cold Environments and Alleviates Drought Stress by Modulating Phytohormone, Antioxidant, and Secondary Metabolite Pathways

Pseudomonas putida Facilitates Pearl Millet Growth in Cold Environments and Alleviates Drought Stress by Modulating Phytohormone, Antioxidant, and Secondary Metabolite Pathways

Integrated transcriptomic and physio-molecular studies unveil the melatonin and PGPR induced protection to photosynthetic attributes in Brassica juncea L. under cadmium toxicity

Cd is highly mobile, non-essential trace element, that has become serious environmental issue due to its elevated concentration in soil. The present study was taken up to work out salutary effect of melatonin (Mlt) and PGPR ((Pseudomonas putida (Pp), Pseudomonas fluorescens (Pf) in 10 days old Cd stressed (0.3 mM) Brassica juncea L. seedlings. The present work investigated growth characteristics, photosynthetic pigments, secondary metabolites in melatonin-PGPR inoculated B. juncea seedlings. It was backed by molecular studies entailing RT-PCR and transcriptomic analyses. Our results revealed, substantial increase in photosynthetic pigments and secondary metabolites, after treatment with melatonin, P.putida, P. fluorescens in Cd stressed B. juncea seedlings, further validated with transcriptome analysis. Comparative transcriptome analyses identified 455, 5953, 3368, 2238 upregulated and 4921, 430, 137, 27 down regulated DEGs, Cn-vs-Cd, Cd-vs-Mlt, Cd-vs-Mlt-Pp-Pf, Cd-vs-Mlt-Pp-Pf-Cd comparative groups respectively. In depth exploration of genome analyses (Gene ontology, Kyoto encyclopaedia of genes), revealed that Cd modifies the expression patterns of most DEGs mainly associated to photosystem and chlorophyll synthesis. Also, gene expression studies for key photosynthetic genes (psb A, psb B, CHS, PAL, and PSY) suggested enhanced expression in melatonin-rhizobacteria treated Cd stressed B. juncea seedlings. Overall, results provide new insights into probable mechanism of Mlt-PGPR induced protection to photosynthesis in Cd stressed B. juncea plants.

Protein 1619 of Pseudomonas putida WBC-3 participates in para-nitrophenol degradation by converting p-benzoquinone to hydroquinone

It is necessary to develop appropriate approaches to eliminate para-nitrophenol (PNP) in our environment, because the pollutant is highly toxic and also able to persist in the environment. Previously, Pseudomonas sp. strain WBC-3 isolated from polluted soil was found to be able to use PNP as the sole carbon and nitrogen source, but not very efficiently. In this study, WBC-3 was shown to belong to Pseudomonas putida through de novo genome sequencing. To enhance its efficiency of PNP utilization, a mutant strain (PM1-33) with a significantly increased PNP degradation rate was obtained. Although no increase in the expression levels of known PNP catabolizing genes/proteins were observed between WBC-3 and PM1-33, the expression level of protein 1619 significantly increased in PM1-33. Deleting GM1619 in WBC-3 and PM1-33 caused decreased PNP degradation rates in both strains and eliminated the difference in PNP degradation between the two strains. Functional prediction using AlphaFold2 showed that protein1619 might bind to p-benzoquinone (BQ). Consequently, protein 1619 was biochemically characterized, confirming its ability to convert BQ into hydroquinone (HQ). Thus, a new protein involved in PNP degradation was identified, thereby adding new knowledge to bacterial PNP degradation pathways.

Nitrogen removal characteristics and Cr(VI) tolerance mechanisms of heterotrophic nitrifying bacterium Pseudomonas putida strain LX1

To solve the severe inhibition effect of nitrification process by heavy metals, necessitating the discovery of heavy metals-resistant nitrifying bacteria. In this study, a highly efficient bacterium, Pseudomonas putida strain LX1, capable of heterotrophic nitrification–aerobic denitrification (HN-AD) and resistant to hexavalent chromium (Cr(VI)) was isolated from activated sludge. Strain LX1 exhibited excellent growth activity and achieved efficient NH4+-N removal rates (94.07%–83.36 %) under 0–25 mg/L Cr(VI) stress. The heterotrophic nitrification ability of strain LX1 was superior to that of aerobic denitrification, and there was no accumulation of nitrite or nitrate during HN-AD process. Moreover, Three-dimensional excitation-emission matrix and Fourier-transform infrared spectroscopy analysis indicated that strain LX1 secreted more extracellular polymeric substances with negatively charged functional groups to bind Cr(VI) and alleviate its toxicity. Enzymatic analysis showed that strain LX1 countered the oxidative damage of reactive oxygen species by promoting the activities of oxidative dismutase and catalase. Furthermore, transcriptomic analyses revealed that the upregulation of genes associated with heavy metal substrate transporter proteins and antioxidant systems in strain LX1 contributed to the resistance against the high biotoxicity caused by Cr(VI). This study is important for understanding the heavy metal response mechanisms of HN-AD strains and its potential application in practical wastewater treatment.

Funneling lignin hydrolysates into β-ketoadipic acid by engineered Pseudomonas putida KT2440

Bioconversion of lignin into value-added chemicals could promote lignin valorization and the lignin-based bioeconomy. The heterogeneity nature of lignin and the lack of efficient conversion pathways pose limitations on the efficiency of lignin conversion. In this study, Pseudomonas putida KT2440 was successfully engineered for converting lignin to β-ketoadipic acid, a C6 keto-diacid that functions as building block for high performance polymers. Blocking the degradation pathway of β-ketoadipic acid enabled its biosynthesis from both H- and G-type lignin-derived monomers. The aromatic hydroxylation and O-demethylation was identified as the rate-limiting step for producing β-ketoadipic acid from H- and G- type lignin-derived monomers, respectively. Overexpressing pobA and vanAB successfully vanished the accumulation of 4-hydroxybenzoic acid and vanillic acid within 36 h and 30 h, respectively. The beneficial gene operations were integrated into the chromosome to create the strain HE65. Employing this strain, fed-batch strategies were used to achieve a record β-ketoadipic acid production of 14.80 g/L from mixed lignin-derived aromatics in shake-flask experiments. Remarkably, the strain could also thrive in actual lignin hydrolysates from corn stover, yielding a record β-ketoadipic acid output of 2.48 g/L without the need for separation or detoxification processes. Overall, these results underscore the significant potential of engineering P. putida for converting lignin into β-ketoadipic acid, offering a promising pathway for feasible lignin valorization.

Microbial consortium formulation in liquid organic fertilizer for managing bacterial leaf blight (Xanthomonas oryzae pv. oryzae), rice blast (Pyricularia oryzae), and enhancing rice productivity

Abstract. Nurcahyanti SD, Masnilah R, Budiman SA, Tanzil AI, Nurdika AAH. 2024. Microbial consortium formulation in liquid organic fertilizer for managing bacterial leaf blight (Xanthomonas oryzae pv. oryzae), rice blast (Pyricularia oryzae), and enhancing rice productivity. Biodiversitas 25: 2208-2220. The productivity of rice is significantly influenced by soil fertility and plant diseases as limiting factors. The overreliance on synthetic fertilizers and pesticides in rice cultivation has led to declining land quality. We propose the utilization of Liquid Organic Fertilizers (LOF) enriched with beneficial microbes as a sustainable solution to maintain soil fertility and protect plants from pathogens. Our research specifically focuses on the formulation of LOF enriched with the microbial consortium to enhance rice productivity and suppress bacterial leaf blight (Xanthomonas oryzae pv. oryzae), and rice blast (Pyricularia oryzae) diseases. The three LOF treatments were formulated from vegetable and fruit waste (FV), Rice Straw (RS), and Cattle Manure (CM) with a microbial consortium consisting of Pseudomonas putida Pf10, Bacillus subtilis JB12, and Trichoderma sp. The results showed that the LOF from the three materials is suitable for the growth of the microbial consortium with a population of 104-109 CFU/mL, has macro-micronutrient content, pH at 6.5-6.8, and C-organic to support plant growth. The applications of the three types of LOF were able to suppress the severity of bacterial leaf blight by up to 21% and rice blast by 4% at eight weeks after inoculation. All LOF treatments are capable of enhancing the growth and production of rice. There were no significant differences in LOF materials for disease suppression, growth, and rice production.

Pseudomonas putida Dynamics of Adaptation under Prolonged Resource Exhaustion.

Many nonsporulating bacterial species survive prolonged resource exhaustion, by entering a state termed long-term stationary phase. Here, we performed long-term stationary phase evolutionary experiments on the bacterium Pseudomonas putida, followed by whole-genome sequencing of evolved clones. We show that P. putida is able to persist and adapt genetically under long-term stationary phase. We observed an accumulation of mutations within the evolving P. putida populations. Within each population, independently evolving lineages are established early on and persist throughout the 4-month-long experiment. Mutations accumulate in a highly convergent manner, with similar loci being mutated across independently evolving populations. Across populations, mutators emerge, that due to mutations within mismatch repair genes developed a much higher rate of mutation than other clones with which they coexisted within their respective populations. While these general dynamics of the adaptive process are quite similar to those we previously observed in the model bacterium Escherichia coli, the specific loci that are involved in adaptation only partially overlap between P. putida and E. coli.

Systematic engineering for production of anti-aging sunscreen compound in Pseudomonas putida

Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO2 has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of 13C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production.

New Insight into the Degradation of Sunscreen Agents in Water Treatment Using UV-Driven Advanced Oxidation Processes

This study evaluates, for the first time, the effects of UV/PMS and UV/H2O2/PMS processes on the degradation of sunscreen agents in synthetic and natural water matrices and compares their effectiveness with the more conventional UV/H2O2. Investigations were conducted using a mixture of organic UV filters containing 4-methylbenzylidene camphor (4-MBC) and 2-ethylhexyl-4-methoxycinnamate. Among the investigated UV-driven AOPs, UV/PMS/H2O2 was the most effective in synthetic water, while in natural water, the highest degradation rate was observed during the degradation of EHMC by UV/PMS. The degradation of UV filters in the UV/PMS system was promoted by sulfate radical (68% of the degradation), with hydroxyl radical contributing approximately 32%, while both radical species contributed approximately equally to the degradation in the UV/H2O2/PMS system. The Vibrio fischeri assay showed an increase in inhibition (up to 70%) at specific stages of UV/H2O2 treatment when applied to natural water, which further decreased to 30%, along with an increase in UV fluence and progressive degradation. The Pseudomonas putida test recorded minor toxicity (<15%) after treatments. Magnetic biochar utilized in conjunction with UV-driven AOPs exhibited superior performance in eliminating residual contaminants, providing an efficient and sustainable approach to mitigate sunscreen agents in water treatment.