Identification and florfenicol-treatment of pseudomonas putida infection in gilthead seabream (Sparus aurata) fed on tilapia-trash-feed.

The present study aimed to determine the major cause of the high mortality affecting farmed gilthead seabream (Sparus aurata) and controlling this disease condition. Fifteen diseased S. aurata were sampled from a private fish farm located at Eldeba Triangle, Damietta, fish showed external skin hemorrhages, and ulceration. Bacterial isolates retrieved from the diseased fish were identified biochemically as Pseudomonas putida and then confirmed by phylogenetic analysis of the 16 S rRNA gene sequence. P. putida was also isolated from three batches of tilapia-trash feed given to S. aurata. Biofilm and hemolytic assay indicated that all P. putida isolates produced biofilm, but 61.11% can haemolyse red blood cells. Based on the antibiotic susceptibility test results, P. putida was sensitive to florfenicol with minimum inhibitory concentrations ranging between 0.25 and 1.0 µg mL- 1, but all isolates were resistant to ampicillin and sulfamethoxazole-trimethoprim. Pathogenicity test revealed that P. putida isolate (recovered from the tilapia-trash feed) was virulent for S. aurata with LD50 equal to 4.67 × 107 colony forming unit (CFU) fish- 1. After intraperitoneal (IP) challenge, fish treated with 10 mg kg- 1 of florfenicol showed 16.7% mortality, while no mortality was recorded for the fish group that received 20 mg kg- 1. The non-treated fish group showed 46.7% mortality after bacterial challenge. HPLC analysis of serum florfenicol levels reached 1.07 and 2.52 µg mL- 1 at the 5th -day post-drug administration in the fish groups received 10 and 20 mg kg- 1, respectively. In conclusion, P. putida was responsible for the high mortality affecting cultured S. aurata, in-feed administration of florfenicol (20 mg kg- 1) effectively protected the challenged fish.

Pseudomonas putida infection induces immune-antioxidant, hepato-renal, ethological, and histopathological/immunohistochemical disruptions in Oreochromis niloticus: the palliative role of titanium dioxide nanogel.

BACKGROUND: Pseudomonas putida is a pathogenic bacterium that induces great losses in fishes, including Nile tilapia (Oreochromis niloticus). Currently, the application of nanomaterials in aquaculture practices has gained more success as it endows promising results in therapies compared to traditional protocols. OBJECTIVE: Therefore, the current perspective is considered the first report to assess the anti-bacterial efficacy of titanium dioxide nanogel (TDNG) against Pseudomonas putida (P. putida) in Nile tilapia. METHODS: The fish (n = 200; average body weight: 47.50±1.32 g) were allocated into four random groups (control, TDNG, P. putida, and TDNG + P. putida), where 0.9 mg/L of TDNG was applied as bath treatment for ten days. RESULTS: Outcomes revealed that P. putida infection caused ethological alterations (surfacing, abnormal movement, and aggression) and depression of immune-antioxidant variables (complement 3, lysozyme activity, total antioxidant capacity, superoxide dismutase, and reduced glutathione content). Additionally, a substantial elevation in hepatorenal biomarkers (aspartate and alanine aminotransferases and creatinine) with clear histopathological changes and immuno-histochemical alterations (very weak BCL-2 and potent caspase-3 immuno-expressions) were seen. Surprisingly, treating P. putida-infected fish with TDNG improved these variables and obvious restoration of the tissue architectures. CONCLUSION: Overall, this report encompasses the key role of TDNG as an anti-bacterial agent for controlling P. putida infection and improving the health status of Nile tilapia.

Synthesis of fluorinated amino acids by low-specificity, promiscuous aldolases coupled to in situ fluorodonor generation.

Fluorine (F) is an important element in the synthesis of molecules broadly used in medicine, agriculture, and materials. F addition to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to produce fluorometabolites (such as fluorinated amino acids, key building blocks for synthesis) are relatively scarce. This chapter discusses the use of L-threonine aldolase enzymes (LTAs), a class of enzymes that catalyze reversible aldol addition to the α-carbon of glycine. The C-C bond formation ability of LTAs, together with their known substrate promiscuity, make them ideal for in vitro F biocatalysis. Here, we describe protocols to harness the activity of the low-specificity LTAs isolated from Escherichia coli and Pseudomonas putida on 2-fluoroacetaldehyde to efficiently synthesize 4-fluoro-L-threonine in vitro. This chapter also provides a comprehensive account of experimental protocols to implement these activities in vivo. These methods are illustrative and can be adapted to produce other fluorometabolites of interest.

Functional Genome Analysis of a Conditionally Pathogenic Rhizobacterial Strain, Pseudomonas putida AKMP7.

This manuscript reports the whole genome sequence of a conditionally pathogenic rhizobacterial strain, Pseudomonas putida AKMP7, which has been previously reported by us to be beneficial to Arabidopsis thaliana under well-watered conditions and pathogenic to the plant under water stress. As part of a study to understand this unique behavior, the whole genome sequence of this strain was analyzed. Based on the results, it was identified that the total length of the AKMP7 genome is 5,764,016 base pairs, and the total GC content of the genome is 62.93% (typical of P. putida). Using RAST annotation pipeline, it was identified that the genome has 5605 coding sequences, 80 repeat regions, 71 tRNA genes, and 22 rRNA genes. A total of 4487 functional proteins and 1118 hypothetical proteins were identified. Phylogenetic analysis has classified it as P. putida species, with a P value of 0.03. In order to identify close relatives of this strain, comparative genomics was performed with 30 other P. putida strains, taken from publicly available genome databases, using Average Nucleotide Identity (ANI) analysis. Whole genome comparison with these strains reveals that AKMP7 possesses Type-IV Secretion System (T4SS) with conjugative transfer functionality. Interestingly, the T4SS feature is absent in all the beneficial/harmless strains of P. putida that we analyzed. All the plant pathogenic bacteria that were analyzed had the T4SS feature in their genome, indicating its role in pathogenesis. This study aims to address important gaps in understanding the molecular mechanisms involved in the conditional/opportunistic pathogenesis of plant-associated, beneficial soil bacteria, using genomics approaches.

Pseudomonas putida as saviour for troubled Synechococcus elongatus in a synthetic co-culture - interaction studies based on a multi-OMICs approach.

In their natural habitats, microbes rarely exist in isolation; instead, they thrive in consortia, where various interactions occur. In this study, a defined synthetic co-culture of the cyanobacterium S. elongatus cscB, which supplies sucrose to the heterotrophic P. putida cscRABY, is investigated to identify potential interactions. Initial experiments reveal a remarkable growth-promoting effect of the heterotrophic partner on the cyanobacterium, resulting in an up to 80% increase in the growth rate and enhanced photosynthetic capacity. Vice versa, the presence of the cyanobacterium has a neutral effect on P. putida cscRABY, highlighting the resilience of pseudomonads against stress and their potential as co-culture partners. Next, a suitable reference process reinforcing the growth-promoting effect is established in a parallel photobioreactor system, which sets the basis for the analysis of the co-culture at the transcriptome, proteome, and metabolome levels. In addition to several moderate changes, including alterations in the metabolism and stress response in both microbes, this comprehensive multi-OMICs approach strongly hints towards the exchange of further molecules beyond the unidirectional feeding with sucrose. Taken together, these findings provide valuable insights into the complex dynamics between both co-culture partners, indicating multi-level interactions, which can be employed for further streamlining of the co-cultivation system.

Enhancing Botrytis disease management in tomato plants: insights from a Pseudomonas putida strain with biocontrol activity.

AIMS: This study explores the biocontrol potential of Pseudomonas putida Z13 against Botrytis cinerea in tomato plants, addressing challenges posed by the pathogen's fungicide resistance. The aims of the study were to investigate the in vitro and in silico biocontrol traits of Z13, identify its plant-colonizing efficacy, evaluate the efficacy of different application strategies against B. cinerea in planta, and assess the capacity of Z13 to trigger induced systemic resistance (ISR) in plants. METHODS AND RESULTS: The in vitro experiments revealed that Z13 inhibits the growth of B. cinerea, produces siderophores, and exhibits swimming and swarming activity. Additionally, the Z13 genome harbors genes that encode compounds triggering ISR, such as pyoverdine and pyrroloquinoline quinone. The in planta experiments demonstrated Z13's efficacy in effectively colonizing the rhizosphere and leaves of tomato plants. Therefore, three application strategies of Z13 were evaluated against B. cinerea: root drenching, foliar spray, and the combination of root drenching and foliar spray. It was demonstrated that the most effective treatment of Z13 against B. cinerea was the combination of root drenching and foliar spray. Transcriptomic analysis showed that Z13 upregulates the expression of the plant defense-related genes PR1 and PIN2 upon B. cinerea inoculation. CONCLUSION: The results of the study demonstrated that Z13 possesses significant biocontrol traits, such as the production of siderophores, resulting in significant plant protection against B. cinerea when applied as a single treatment to the rhizosphere or in combination with leaf spraying. Additionally, it was shown that Z13 root colonization primes plant defenses against the pathogen.

Biological Valorization of Lignin-Derived Aromatics in Hydrolysate to Protocatechuic Acid by Engineered Pseudomonas putida KT2440.

Alongside fermentable sugars, weak acids, and furan derivatives, lignocellulosic hydrolysates contain non-negligible amounts of lignin-derived aromatic compounds. The biological funnel of lignin offers a new strategy for the "natural" production of protocatechuic acid (PCA). Herein, Pseudomonas putida KT2440 was engineered to produce PCA from lignin-derived monomers in hydrolysates by knocking out protocatechuate 3,4-dioxygenase and overexpressing vanillate-O-demethylase endogenously, while acetic acid was used for cell growth. The sugar catabolism was further blocked to prevent the loss of fermentable sugar. Using the engineered strain, a total of 253.88 mg/L of PCA was obtained with a yield of 70.85% from corncob hydrolysate 1. The highest titer of 433.72 mg/L of PCA was achieved using corncob hydrolysate 2 without any additional nutrients. This study highlights the potential ability of engineered strains to address the challenges of PCA production from lignocellulosic hydrolysate, providing novel insights into the utilization of hydrolysates.

Computational analysis into the potential of azo dyes as a feedstock for actinorhodin biosynthesis in Pseudomonas putida.

Fermentation-based biosynthesis in synthetic biology relies heavily on sugar-derived feedstocks, a limited and carbon-intensive commodity. Unconventional feedstocks from less-noble sources such as waste are being utilized to produce high-value chemical products. Azo dyes, a major pollutant commonly discharged by food, textile, and pharmaceutical industries, present significant health and environmental risks. We explore the potential of engineering Pseudomonas putida KT2440 to utilize azo dyes as a substrate to produce a polyketide, actinorhodin (ACT). Using the constrained minimal cut sets (cMCS) approach, we identified metabolic interventions that optimize ACT biosynthesis and compare the growth-coupling solutions attainable on an azo dye compared to glucose. Our results predicted that azo dyes could perform better as a feedstock for ACT biosynthesis than glucose as it allowed growth-coupling regimes that are unfeasible with glucose and generated an 18.28% higher maximum ACT flux. By examining the flux distributions enabled in different carbon sources, we observed that carbon fluxes from aromatic compounds like azo dyes have a unique capability to leverage gluconeogenesis to support both growth and production of secondary metabolites that produce excess NADH. Carbon sources are commonly chosen based on the host organism, availability, cost, and environmental implications. We demonstrated that careful selection of carbon sources is also crucial to ensure that the resulting flux distribution is suitable for further metabolic engineering of microbial cell factories.

Genetic Code Expansion in Pseudomonas putida KT2440.

Emerging microorganism Pseudomonas putida KT2440 is utilized for the synthesis of biobased chemicals from renewable feedstocks and for bioremediation. However, the methods for analyzing, engineering, and regulating the biosynthetic enzymes and protein complexes in this organism remain underdeveloped.Such attempts can be advanced by the genetic code expansion-enabled incorporation of noncanonical amino acids (ncAAs) into proteins, which also enables further controls over the strain's biological processes. Here, we give a step-by-step account of the incorporation of two ncAAs into any protein of interest (POI) in response to a UAG stop codon by two commonly used orthogonal archaeal tRNA synthetase and tRNA pairs. Using superfolder green fluorescent protein (sfGFP) as an example, this method lays down a solid foundation for future work to study and enhance the biological functions of KT2440.

Multi-Omics integration can be used to rescue metabolic information for some of the dark region of the Pseudomonas putida proteome.

In every omics experiment, genes or their products are identified for which even state of the art tools are unable to assign a function. In the biotechnology chassis organism Pseudomonas putida, these proteins of unknown function make up 14% of the proteome. This missing information can bias analyses since these proteins can carry out functions which impact the engineering of organisms. As a consequence of predicting protein function across all organisms, function prediction tools generally fail to use all of the types of data available for any specific organism, including protein and transcript expression information. Additionally, the release of Alphafold predictions for all Uniprot proteins provides a novel opportunity for leveraging structural information. We constructed a bespoke machine learning model to predict the function of recalcitrant proteins of unknown function in Pseudomonas putida based on these sources of data, which annotated 1079 terms to 213 proteins. Among the predicted functions supplied by the model, we found evidence for a significant overrepresentation of nitrogen metabolism and macromolecule processing proteins. These findings were corroborated by manual analyses of selected proteins which identified, among others, a functionally unannotated operon that likely encodes a branch of the shikimate pathway.

A model-driven approach to upcycling recalcitrant feedstocks in Pseudomonas putida by decoupling PHA production from nutrient limitation.

Bacterial polyhydroxyalkanoates (PHAs) have emerged as promising eco-friendly alternatives to petroleum-based plastics since they are synthesized from renewable resources and offer exceptional properties. However, their production is limited to the stationary growth phase under nutrient-limited conditions, requiring customized strategies and costly two-phase bioprocesses. In this study, we tackle these challenges by employing a model-driven approach to reroute carbon flux and remove regulatory constraints using synthetic biology. We construct a collection of Pseudomonas putida-overproducing strains at the expense of plastics and lignin-related compounds using growth-coupling approaches. PHA production was successfully achieved during growth phase, resulting in the production of up to 46% PHA/cell dry weight while maintaining a balanced carbon-to-nitrogen ratio. Our strains are additionally validated under an upcycling scenario using enzymatically hydrolyzed polyethylene terephthalate as a feedstock. These findings have the potential to revolutionize PHA production and address the global plastic crisis by overcoming the complexities of traditional PHA production bioprocesses.

Synthetically-primed adaptation of Pseudomonas putida to a non-native substrate D-xylose.

To broaden the substrate scope of microbial cell factories towards renewable substrates, rational genetic interventions are often combined with adaptive laboratory evolution (ALE). However, comprehensive studies enabling a holistic understanding of adaptation processes primed by rational metabolic engineering remain scarce. The industrial workhorse Pseudomonas putida was engineered to utilize the non-native sugar D-xylose, but its assimilation into the bacterial biochemical network via the exogenous xylose isomerase pathway remained unresolved. Here, we elucidate the xylose metabolism and establish a foundation for further engineering followed by ALE. First, native glycolysis is derepressed by deleting the local transcriptional regulator gene hexR. We then enhance the pentose phosphate pathway by implanting exogenous transketolase and transaldolase into two lag-shortened strains and allow ALE to finetune the rewired metabolism. Subsequent multilevel analysis and reverse engineering provide detailed insights into the parallel paths of bacterial adaptation to the non-native carbon source, highlighting the enhanced expression of transaldolase and xylose isomerase along with derepressed glycolysis as key events during the process.

Engineering Pseudomonas putida KT2440 for Dipicolinate Production via the Entner-Doudoroff Pathway.

Dipicolinic acid (DPA), a cyclic diacid, has garnered significant interest due to its potential applications in antimicrobial agents, antioxidants, chelating reagents, and polymer precursors. However, its natural bioproduction is limited since DPA is only accumulated in Bacillus and Clostridium species during sporulation. Thus, heterologous production by engineered strains is of paramount importance for developing a sustainable biological route for DPA production. Pseudomonas putida KT2440 has emerged as a promising host for the production of various chemicals thanks to its robustness, metabolic versatility, and genetic tractability. The dominant Entner-Doudoroff (ED) pathway for glucose metabolism in this strain offers an ideal route for DPA production due to the advantage of NADPH generation and the naturally balanced flux between glyceraldehyde-3-phosphate and pyruvate, which are both precursors for DPA synthesis. In this study, DPA production via the ED pathway was in silico designed in P. putida KT2440. The systematically engineered strain produced dipicolinate with a titer of 11.72 g/L from glucose in a 5 L fermentor. This approach not only provides a sustainable green route for DPA production but also expands our understanding of the metabolic potential of the ED pathway in P. putida KT2440.

Enhanced chaotrope tolerance and (S)-2-hydroxypropiophenone production by recombinant Pseudomonas putida engineered with Pprl from Deinococcus radiodurans.

Pseudomonas putida is a soil bacterium with multiple uses in fermentation and biotransformation processes. P. putida ATCC 12633 can biotransform benzaldehyde and other aldehydes into valuable α-hydroxyketones, such as (S)-2-hydroxypropiophenone. However, poor tolerance of this strain toward chaotropic aldehydes hampers efficient biotransformation processes. To circumvent this problem, we expressed the gene encoding the global regulator PprI from Deinococcus radiodurans, an inducer of pleiotropic proteins promoting DNA repair, in P. putida. Fine-tuned gene expression was achieved using an expression plasmid under the control of the LacIQ /Ptrc system, and the cross-protective role of PprI was assessed against multiple stress treatments. Moreover, the stress-tolerant P. putida strain was tested for 2-hydroxypropiophenone production using whole resting cells in the presence of relevant aldehyde substrates. P. putida cells harbouring the global transcriptional regulator exhibited high tolerance toward benzaldehyde, acetaldehyde, ethanol, butanol, NaCl, H2 O2 and thermal stress, thereby reflecting the multistress protection profile conferred by PprI. Additionally, the engineered cells converted aldehydes to 2-hydroxypropiophenone more efficiently than the parental P. putida strain. 2-Hydroxypropiophenone concentration reached 1.6 g L-1 upon a 3-h incubation under optimized conditions, at a cell concentration of 0.033 g wet cell weight mL-1 in the presence of 20 mM benzaldehyde and 600 mM acetaldehyde. Product yield and productivity were 0.74 g 2-HPP g-1 benzaldehyde and 0.089 g 2-HPP g cell dry weight-1 h-1 , respectively, 35% higher than the control experiments. Taken together, these results demonstrate that introducing PprI from D. radiodurans enhances chaotrope tolerance and 2-HPP production in P. putida ATCC 12633.

Pseudomonas putida as a platform for medium-chain length α,ω-diol production: Opportunities and challenges.

Medium-chain-length α,ω-diols (mcl-diols) play an important role in polymer production, traditionally depending on energy-intensive chemical processes. Microbial cell factories offer an alternative, but conventional strains like Escherichia coli and Saccharomyces cerevisiae face challenges in mcl-diol production due to the toxicity of intermediates such as alcohols and acids. Metabolic engineering and synthetic biology enable the engineering of non-model strains for such purposes with P. putida emerging as a promising microbial platform. This study reviews the advancement in diol production using P. putida and proposes a four-module approach for the sustainable production of diols. Despite progress, challenges persist, and this study discusses current obstacles and future opportunities for leveraging P. putida as a microbial cell factory for mcl-diol production. Furthermore, this study highlights the potential of using P. putida as an efficient chassis for diol synthesis.

Diversity in fatty acid elongation enzymes: The FabB long-chain ß-ketoacyl-ACP synthase I initiates fatty acid synthesis in Pseudomonas putida F1.

The condensation of acetyl-CoA with malonyl-acyl carrier protein (ACP) by ß-ketoacyl-ACP synthase III (KAS III, FabH) and decarboxylation of malonyl-ACP by malonyl-ACP decarboxylase are the two pathways that initiate bacterial fatty acid synthesis (FAS) in Escherichia coli. In addition to these two routes, we report that Pseudomonas putida F1 ß-ketoacyl-ACP synthase I (FabB), in addition to playing a key role in fatty acid elongation, also initiates FAS in vivo. We report that although two P. putida F1 fabH genes (PpfabH1 and PpfabH2) both encode functional KAS III enzymes, neither is essential for growth. PpFabH1 is a canonical KAS III similar to E. coli FabH whereas PpFabH2 catalyzes condensation of malonyl-ACP with short- and medium-chain length acyl-CoAs. Since these two KAS III enzymes are not essential for FAS in P. putida F1, we sought the P. putida initiation enzyme and unexpectedly found that it was FabB, the elongation enzyme of the oxygen-independent unsaturated fatty acid pathway. P. putida FabB decarboxylates malonyl-ACP and condenses the acetyl-ACP product with malonyl-ACP for initiation of FAS. These data show that P. putida FabB, unlike the paradigm E. coli FabB, can catalyze the initiation reaction in FAS.

Machine learning analysis of RB-TnSeq fitness data predicts functional gene modules in Pseudomonas putida KT2440.

There is growing interest in engineering Pseudomonas putida KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of P. putida relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of P. putida KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules ("functional modules"), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of P. putida. Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets. IMPORTANCE: This study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While Pseudomonas putida randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.

Pseudomonas putida configures Arabidopsis root architecture through modulating the sensing systems for phosphate and iron acquisition.

Iron (Fe) and phosphate (Pi) are two essential nutrients that are poorly available in the soil and should be supplemented either as fertilizers or organic amendments to sustain crop production. Currently, determining how rhizosphere bacteria contribute to plant mineral nutrient acquisition is an area of growing interest regarding its potential application in agriculture. The aim of this study was to investigate the influence of root colonization by Pseudomonas putida for Arabidopsis growth through Fe and Pi nutritional signaling. We found that root colonization by the bacterium inhibits primary root elongation and promotes the formation of lateral roots. These effects could be related to higher expression of two Pi starvation-induced genes and AtPT1, the major Pi transporter in root tips. In addition, P. putida influenced the accumulation of Fe in the root and the expression of different elements of the Fe uptake pathway. The loss of function of the protein ligase BRUTUS (BTS), and the bHLH transcription factors POPEYE (PYE) and IAA-LEUCINE RESISTANT3 (ILR3) compromised the root branching stimulation triggered by bacterial inoculation while the leaf chlorosis in the fit1 and irt1-1 mutant plants grown under standard conditions could be bypassed by P. putida inoculation. The WT and both mutant lines showed similar Fe accumulation in roots. P. putida repressed the expression of the IRON-REGULATED TRANSPORTER 1 (IRT1) gene suggesting that the bacterium promotes an alternative Fe uptake mechanism. These results open the door for the use of P. putida to enhance nutrient uptake and optimize fertilizer usage by plants.

Ecotoxicity studies reveal that organic cations in dicamba-derived ionic liquids can pose a greater environmental risk than the herbicide itself.

The following research provides novel and relevant insights into potential environmental consequences of combination of various organic cations with commercial systemic herbicide (dicamba), in accordance with a 'herbicidal ionic liquids' (HILs) strategy. Toxicity assays of five dicamba-based HILs comprising different hydrophobic and hydrophilic cations, namely choline [CHOL][DIC], ethyl betainate [BETC2][DIC], decyl betainate [BETC10][DIC], hexadecyl betainate [BETC16][DIC] and didecyldimethylammonium [DDA][DIC]), have been tested towards bacteria (Pseudomonas putida, Escherichia coli, Bacillus subtilis), algae (Chlorella vulgaris), fresh and marine water crustaceans (Daphnia magna, Artemia franciscana). The structure of respective substituents in the cation emerged as a decisive determinant of toxicity in the case of tested species. In consequence, small ions of natural origin ([CHOL] and [BETC2]) demonstrated toxicity numerous orders of magnitude lower compared to fully synthetic [DDA]. These results emphasize the role of cations' hydrophobicity, as well as origin, in the observed acute toxic effect. Time-dependent toxicity assays also indicated that betaine-type cations comprising an ester bond can rapidly transform into less harmful substances, which can generally result in a reduction in toxicity by even several orders of magnitude. Nonetheless, these findings challenge the concept of ionic liquids with herbicidal activity and give apparent parallels to adjuvant-dependent toxicity issues recently noted in typical herbicidal formulations.

Genome-scale and pathway engineering for the sustainable aviation fuel precursor isoprenol production in Pseudomonas putida.

Sustainable aviation fuel (SAF) will significantly impact global warming in the aviation sector, and important SAF targets are emerging. Isoprenol is a precursor for a promising SAF compound DMCO (1,4-dimethylcyclooctane) and has been produced in several engineered microorganisms. Recently, Pseudomonas putida has gained interest as a future host for isoprenol bioproduction as it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile host P. putida for isoprenol production. We employ two computational modeling approaches (Bilevel optimization and Constrained Minimal Cut Sets) to predict gene knockout targets and optimize the "IPP-bypass" pathway in P. putida to maximize isoprenol production. Altogether, the highest isoprenol production titer from P. putida was achieved at 3.5 g/L under fed-batch conditions. This combination of computational modeling and strain engineering on P. putida for an advanced biofuels production has vital significance in enabling a bioproduction process that can use renewable carbon streams.