Feedstock variability impacts the bioconversion of sugar and lignin streams derived from corn stover by Clostridium tyrobutyricum and engineered Pseudomonas putida.

Feedstock variability represents a challenge in lignocellulosic biorefineries, as it can influence both lignocellulose deconstruction and microbial conversion processes for biofuels and biochemicals production. The impact of feedstock variability on microbial performance remains underexplored, and predictive tools for microbial behaviour are needed to mitigate risks in biorefinery scale-up. Here, twelve batches of corn stover were deconstructed via deacetylation, mechanical refining, and enzymatic hydrolysis to generate lignin-rich and sugar streams. These batches and their derived streams were characterised to identify their chemical components, and the streams were used as substrates for producing muconate and butyrate by engineered Pseudomonas putida and wildtype Clostridium tyrobutyricum, respectively. Bacterial performance (growth, product titers, yields, and productivities) differed among the batches, but no strong correlations were identified between feedstock composition and performance. To provide metabolic insights into the origin of these differences, we evaluated the effect of twenty-three isolated chemical components on these microbes, including three components in relevant bioprocess settings in bioreactors, and we found that growth-inhibitory concentrations were outside the ranges observed in the streams. Overall, this study generates a foundational dataset on P. putida and C. tyrobutyricum performance to enable future predictive models and underscores their resilience in effectively converting fluctuating lignocellulose-derived streams into bioproducts.

Synergistic effect of Pseudomonas putida and endomycorrhizal inoculation on the physiological response of onion (Allium cepa L.) to saline conditions.

Salinity stress negatively affects the growth and yield of crops worldwide. Onion (Allium cepa L.) is moderately sensitive to salinity. Beneficial microorganisms can potentially confer salinity tolerance. This study investigated the effects of endomycorrhizal fungi (M), Pseudomonas putida (Ps) and their combination (MPs) on onion growth under control (0 ppm), moderate (2000 ppm) and high (4000 ppm) NaCl salinity levels. A pot experiment was conducted with sandy loam soil and onion cultivar Giza 20. Results showed that salinity reduced growth attributes, leaf pigments, biomass and bulb yield while increasing oxidative stress markers. However, individual or combined inoculations significantly increased plant height, bulb diameter and biomass production compared to uninoculated plants under saline conditions. MPs treatment provided the highest stimulation, followed by Pseudomonas and mycorrhizae alone. Overall, dual microbial inoculation showed synergistic interaction, conferring maximum benefits for onion growth, bulbing through integrated physiological and biochemical processes under salinity. Bulb yield showed 3.5, 36 and 83% increase over control at 0, 2000 and 4000 ppm salinity, respectively. In conclusion, combined application of mycorrhizal-Pseudomonas inoculations (MPs) effectively mitigate salinity stress. This approach serves as a promising biotechnology for ensuring sustainable onion productivity under saline conditions.

Unraveling the effects of acrylonitrile butadiene styrene (ABS) microplastic ageing on the sorption and toxicity of ionic liquids with 2,4-D and glyphosate herbicides.

Microplastics represent a novel category of environmental pollutants, and understanding their interactions with typical xenobiotics is crucial. In this study, we investigated the impact of ionic liquids (ILs) containing herbicidal anions, namely glyphosate [Glyph] and 2,4-dichlorophenoxyacetate [2,4-D], and the surfactant cation - dodecyltrimethylammonium [C12TMA] on acrylonitrile butadiene styrene (ABS) microplastics. The aim of the study was to assess the sorption capacity of microplastics that were present in both untreated and aged form using standard and modified Fenton methods. In addition, impact on toxicity and stress adaptation of the model soil bacterium Pseudomonas putida KT2440 was measured. Upon ageing, ABS microplastics underwent a fivefold increase in BET surface area and total pore volume (from 0.001 to 0.004 cm3/g) which lead to a dramatic increase in adsorption of the cations on ABS microplastics from 40 to 45% for virgin ABS to 75-80% for aged ABS. Toxicity was mainly attributed to hydrophobic cations in ILs (EC50 ∼ 60-65 mg/dm3), which was also mitigated by sorption on ABS. Furthermore, both cations and anions behaved similarly across different ILs, corresponding chlorides, and substrates used in the ILs synthesis. These findings highlight microplastics potential as hazardous sorbents, contributing to the accumulation of xenobiotics in the environment.

Systems biology of electrogenic Pseudomonas putida - multi-omics insights and metabolic engineering for enhanced 2-ketogluconate production.

BACKGROUND: Pseudomonas putida KT2440 has emerged as a promising host for industrial bioproduction. However, its strictly aerobic nature limits the scope of applications. Remarkably, this microbe exhibits high bioconversion efficiency when cultured in an anoxic bio-electrochemical system (BES), where the anode serves as the terminal electron acceptor instead of oxygen. This environment facilitates the synthesis of commercially attractive chemicals, including 2-ketogluconate (2KG). To better understand this interesting electrogenic phenotype, we studied the BES-cultured strain on a systems level through multi-omics analysis. Inspired by our findings, we constructed novel mutants aimed at improving 2KG production. RESULTS: When incubated on glucose, P. putida KT2440 did not grow but produced significant amounts of 2KG, along with minor amounts of gluconate, acetate, pyruvate, succinate, and lactate. 13C tracer studies demonstrated that these products are partially derived from biomass carbon, involving proteins and lipids. Over time, the cells exhibited global changes on both the transcriptomic and proteomic levels, including the shutdown of translation and cell motility, likely to conserve energy. These adaptations enabled the cells to maintain significant metabolic activity for several weeks. Acetate formation was shown to contribute to energy supply. Mutants deficient in acetate production demonstrated superior 2KG production in terms of titer, yield, and productivity. The ∆aldBI ∆aldBII double deletion mutant performed best, accumulating 2KG at twice the rate of the wild type and with an increased yield (0.96 mol/mol). CONCLUSIONS: By integrating transcriptomic, proteomic, and metabolomic analyses, this work provides the first systems biology insight into the electrogenic phenotype of P. putida KT2440. Adaptation to anoxic-electrogenic conditions involved coordinated changes in energy metabolism, enabling cells to sustain metabolic activity for extended periods. The metabolically engineered mutants are promising for enhanced 2KG production under these conditions. The attenuation of acetate synthesis represents the first systems biology-informed metabolic engineering strategy for enhanced 2KG production in P. putida. This non-growth anoxic-electrogenic mode expands our understanding of the interplay between growth, glucose phosphorylation, and glucose oxidation into gluconate and 2KG in P. putida.

The biological activity of bacterial rhamnolipids on Arabidopsis thaliana and the cyst nematode Heterodera schachtii is linked to their molecular structure.

Rhamnolipids (RLs) are amphiphilic compounds of bacterial origin that offer a broad range of potential applications as biosurfactants in industry and agriculture. They are reported to be active against different plant pests and pathogens and thus are considered promising candidates for nature-derived plant protection agents. However, as these glycolipids are structurally diverse, little is known about their exact mode of action and, in particular, the relation between molecular structure and biological activity against plant pests and pathogens. Engineering the synthesis pathway in recombinant Pseudomonas putida strains in combination with advanced HPLC techniques allowed us to separately analyze the activities of mixtures of pure mono-RLs (mRLs) and of pure di-RL (dRLs), as well as the activity of single congeners. In a model system with the plant Arabidopsis thaliana and the plant-parasitic nematode (PPN) Heterodera schachtii we demonstrate that RLs can significantly reduce infection, whereas their impact on the host plant varied depending on their molecular structure. While mRLs reduced plant growth even at a low concentration, dRLs showed a neutral to beneficial impact on plant development. Treating plants with dRLs triggered an increased reactive oxygen species (ROS) production, indicating the activation of stress-response signaling and possibly plant defense. Pretreatment of plants with mRLs or dRLs prior to application of flagellin (flg22), a known ROS inducer, further increased the ROS response to flg22. While dRLs stimulated an elevated flg22-induced ROS peak, a pretreatment with mRLs resulted in a prolonged synthesis of ROS indicating a generally elevated stress level. Neither mRLs nor dRLs induced the expression of plant defense marker genes of salicylic acid, jasmonic acid, and ethylene pathways. Detailed studies on dRLs revealed that even high concentrations up to 755 ppm of these molecules have no lethal impact on H. schachtii infective juveniles. Infection assays with individual dRL congeners showed that the C10-C8 acyl chained dRL was the only congener without effect, while dRLs with C10-C12 and C10-C12:1 acyl chains were most efficient in reducing nematode infection even at concentrations below 2 ppm. As determined by phenotyping and ROS measurements, A. thaliana reacted more sensitive to long-chained dRLs in a concentration-dependent manner. Our experiments show a clear structure-activity relation for the effect of RLs on plants. In conclusion, functional assessment and analysis of the mode of action of RLs in plants and other organisms require careful consideration of their molecular structure and composition.

Effects and mechanisms of chlormequat on horizontal transfer of antibiotic resistance genes through plasmid-mediated conjugation in agro-ecosystems.

Chlormequat (CCC) is widely used in agricultural production to increase the crop yield. However, the effects of CCC on transfer of ARGs in agricultural system are still unclear. In this study, using E.coli DH5α (carrying RP4 plasmid with AmpR, TetR, KanR) as the donor bacterium, E.coli HB101, endophytic Pseudomonas sp. Ph6 or rhizosphere Pseudomonas putida KT2440 as the recipient strain, three conjugative systems were designed to investigate the effects of CCC on ARG transfer. Meanwhile, hydroponics experiments were designed to study the ARG spread in the rice-nutrient solution system after CCC application. The results showed that CCC significantly promoted the RP4 conjugation by expanding cell membrane permeability and improving the relative transcription levels of trfAp, trbBp, traA and traL genes in RP4. Furthermore, the conjugation frequency between E. coli and Pseudomonas was much higher than that between E. coli cells. Compared with spraying foliage with 2500 mg·L-1 of CCC, soaking seeds with 250 mg·L-1 of CCC was more beneficial to the colonization of ARB in rice, and also increased the abundance of ARGs in rice cultivation system. These results remind that the use of CCC in agricultural production might promote the ARG transmission in agro-ecosystems; however, foliage spraying with 2500 mg·L-1 of CCC could control its spread.

Optimized CRISPR Interference System for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly.

Pseudomonas alloputida KT2440 (formerly P. putida) has become both a well-known chassis organism for synthetic biology and a model organism for rhizosphere colonization. Here, we describe a CRISPR interference (CRISPRi) system in KT2440 for exploring microbe-microbe interactions in the rhizosphere and for use in industrial systems. Our CRISPRi system features three different promoter systems (XylS/Pm, LacI/Plac, and AraC/PBAD) and a dCas9 codon-optimized for Pseudomonads, all located on a mini-Tn7-based transposon that inserts into a neutral site in the genome. It also includes a suite of pSEVA-derived sgRNA expression vectors, where the expression is driven by synthetic promoters varying in strength. We compare the three promoter systems in terms of how well they can precisely modulate gene expression, and we discuss the impact of environmental factors, such as media choice, on the success of CRISPRi. We demonstrate that CRISPRi is functional in bacteria colonizing the rhizosphere, with repression of essential genes leading to a 10-100-fold reduction in P. alloputida cells per root. Finally, we show that CRISPRi can be used to modulate microbe-microbe interactions. When the gene pvdH is repressed and P. alloputida is unable to produce pyoverdine, it loses its ability to inhibit other microbes in vitro. Moreover, our design is amendable for future CRISPRi-seq studies and in multispecies microbial communities, with the different promoter systems providing a means to control the level of gene expression in many different environments.

Sustainable biocatalytic synthesis of substituted muconic acids.

The development of sustainable routes to organic building blocks is a critical endeavor for reducing the environmental impact of chemical synthesis. Biocatalysts are poised to play an important role in sustainable synthesis, as they perform highly selective reactions under mild conditions. The application of enzymes to organic synthesis requires an approach which is operationally simple, inexpensive to prepare, and reasonably scalable. In this work, we demonstrated the utility of a Type I ring-cleaving dioxygenase CatA (P. putida KT2440) for preparative-scale synthesis of muconic acid derivatives. Muconic acids are important precursors in the synthesis of polymers and commodity chemicals. In this work, we optimized the performance of CatA under millimolar substrate concentrations and characterized the activity of the enzyme with an array of catechol substrates. Furthermore, we developed a scalable platform using cellular lysates to produce diverse muconic acids, generating up to a gram of the desired product. A simple trituration procedure was utilized for the purification of these muconic acids that obviated the need for chromatographic purification and reduced overall solvent waste.

Bacterial surface display of PETase mutants and MHETase for an efficient dual-enzyme cascade catalysis.

Biological degradation of PET plastic holds great potential for plastic recycling. However, the high costs associated with preparing free enzymes for degrading PET make it unfeasible for industrial applications. Hence, we developed various cell catalysts by surface-displaying PETase mutants and MHETase using autotransporters in E. coli and P. putida. The efficiency of surface display was enhanced through modifying the host, co-expressing molecular chaperones, and evoluting the autotransporter. In strain EC9F, PET degradation rate was boosted to 3.85 mM/d, 51-fold and 23-fold increase compared to free enzyme and initial strain ED1, respectively. The reusability of cell catalyst EC9F was demonstrated with over 38 % and 30 % of its initial activity retained after 22 cycles of BHET degradation and 3 cycles of PET degradation. The highest reported PET degradation rate of 4.95 mM/d was achieved by the dual-enzyme cascade catalytic system EC9F+EM2+R, a mixture of cell catalyst EC9F and EM2 with surfactant rhamnolipid.

[Biomanufacturing of propionic acid].

Propionic acid as an important C3 platform chemical has been widely used in food, pharmaceutical, and chemical fields. The chemical synthesis of propionic acid from petroleum and other chemical products has serious environmental pollution and is not sustainable. In recent years, the production of propionic acid by microbial transformation of renewable resources has received extensive attention. Focusing on the biomanufacturing of propionic acid, this paper firstly reviews the studies about the metabolic engineering of Propionibacterium and the pathway reconstruction in heterogeneous hosts such as Escherichia coli and Saccharomyces cerevisiae. Secondly, this paper reviews the recent progress in the synthesis of high-purity propionic acid from L-threonine or bio-based 1, 2-propanediol by the design and modification of the pathway of Pseudomonas putida KT2440 based on synthetic biology.

Biosynthesis of Polyhydroalkanoates Doped with Silver Nanoparticles Using Pseudomonas putida and Pseudomonas aeruginosa for Antibacterial Polymer Applications.

In this study, the biosynthesis of polyhydroxyalkanoates (PHAs) was carried out using Pseudomonas putida and Pseudomonas aeruginosa. These PHAs were produced using reagent-grade glycerol and crude glycerol as the carbon sources. The objective was to compare the production of PHAs and to functionalize these polymers with silver nanoparticles to provide antibacterial properties for potential biomedical applications. The findings from the physical and chemical analyses confirmed the successful synthesis and extraction of PHAs, achieving comparable yields using both crude glycerol and reagent-grade glycerol as carbon sources across both strains. Approximately 16% higher PHAs production was obtained using Pseudomonas putida compared to Pseudomonas aeruginosa, and no significant difference was observed in the production rate of PHAs between the two carbon sources used, which means that crude glycerol could be utilized even though it has more impurities. Notably, PHAs functionalized with silver nanoparticles showed improved antibacterial effectiveness, especially those derived from reagent-grade glycerol and the Pseudomonas aeruginosa strain.

Quantum modeling simulates nutrient effect of bioplastic polyhydroxyalkanoate (PHA) production in Pseudomonas putida.

Polyhydroxyalkanoates (PHAs) could be used to make sustainable, biodegradable plastics. However, the precise and accurate mechanistic modeling of PHA biosynthesis, especially medium-chain-length PHA (mcl-PHA), for yield improvement remains a challenge to biology. PHA biosynthesis is typically triggered by nitrogen limitation and tends to peak at an optimal carbon-to-nitrogen (C/N) ratio. Specifically, simulation of the underlying dynamic regulation mechanisms for PHA bioprocess is a bottleneck owing to surfeit model complexity and current modeling philosophies for uncertainty. To address this issue, we proposed a quantum-like decision-making model to encode gene expression and regulation events as hidden layers by the general transformation of a density matrix, which uses the interference of probability amplitudes to provide an empirical-level description for PHA biosynthesis. We implemented our framework modeling the biosynthesis of mcl-PHA in Pseudomonas putida with respect to external C/N ratios, showing its optimization production at maximum PHA production of 13.81% cell dry mass (CDM) at the C/N ratio of 40:1. The results also suggest the degree of P. putida's preference in channeling carbon towards PHA production as part of the bacterium's adaptative behavior to nutrient stress using quantum formalism. Generic parameters (kD, kN and theta θ) obtained based on such quantum formulation, representing P. putida's PHA biosynthesis with respect to external C/N ratios, was discussed. This work offers a new perspective on the use of quantum theory for PHA production, demonstrating its application potential for other bioprocesses.

Taxis-enhanced mineralization and co-metabolism of PAHs by bacteria in micrometer-scale environments.

Polycyclic aromatic hydrocarbons (PAHs) are associated with micropores in sediments and soils. This limits the bioaccessibility of these compounds via existing bioremediation technologies, as biodegradation is strongly influenced by the ability of bacteria to access different sizes of pores. In this work, we employed naphthalene and pyrene as model contaminants to evaluate the transformation capacity of the soil bacterium Pseudomonas putida G7 (2 × 1 µm) via mineralization and co-metabolic activity, respectively. Under non-growing conditions and in the absence of hydraulic flow, we examined how the tactic behavior of this motile bacterium influenced biodegradation of these two PAHs when passing through membranes with micrometer-sized pores (3 and 5 µm). The bacteria were spontaneously retained by the membranes, which blocked the contaminants away from a passive dosing source. However, the cells were mobilized through 5 µm pores after the application plant root exudate components (γ-aminobutyric acid, citrate and fructose) as strong chemoeffectors, which enhanced the mineralization of naphthalene and co-metabolism of pyrene. The tactic-mediated biodegradation enhancement did not occur through 3 µm pores, possibly due a physical constrain to the gradient sensing mechanism. Our results suggest that bacterial transport by chemotaxis may enhance the biotransformation of poorly bioaccessible contaminants present in micro-meter scale environments.

Optimizing Systems for Robust Heterologous Production of Biosurfactants Rhamnolipid and Lyso-Ornithine Lipid in Pseudomonas putida KT2440.

Surfactants are amphiphilic molecules that are capable of mixing water and oil. Biosurfactants are eco-friendly, low-toxicity, and stable to a variety of environmental factors. Optimizing conditions for microorganisms to produce biosurfactants can lead to improved production suitable for scaling up. In this study, we compared heterologous expression levels of the luminescence system luxCDABE operon controlled by regulatable promoters araC-PBAD and its strong version araC-PBAD-SD in Escherichia coli K12, Pseudomonas aeruginosa PAO1, and P. putida KT2440. Real-time monitoring of luminescence levels in the three strains indicated that luxCDABE controlled by araC-PBAD-SD promoter with 0.2% arabinose supplementation in P. putida produced the highest level of luminescence. By using the araC-PBAD-SD promoter-controlled rhlAB expression in P. putida, we were able to produce mono-rhamnolipid at a level of 1.5 g L-1 when 0.02% arabinose was supplemented. With the same system to express olsB, lyso-ornithine lipid was produced at a level of 10 mg L-1 when 0.2% arabinose was supplemented. To our knowledge, this is the first report about optimizing conditions for lyso-ornithine lipid production at a level up to 10 mg L-1. Taken together, our results demonstrate that regulatable araC-PBAD-SD promoter in P. putida KT2440 is a useful system for heterologous production of biosurfactants.

Resistance of Nile tilapia fed with Padina boergesenii extract to Pseudomonas putida infection.

The aim of this research was to estimate the immunopotentiation effect of brown algae Padina boergesenii water extract on Nile tilapia, Oreochromis niloticus through resistance to Pseudomonas putida infection. Gas Chromatography Mass Spectrometry was utilized to characterize the seaweed phytoconstituents. One hundred and twenty-six fish were divided in triplicates into two equal groups corresponding to two diet variants that used to feed Nile tilapia for 20 successive days: a basal (control), and P. boergesenii water extract supplemented group. Fish samples were collected at 10-days intervals throughout the experiment. Serum biochemical constituents, total antioxidant capacity (TAC), and some immune related genes expression of the spleen and intestinal tissues of experimental fish were studied, as well as histological examination of fish immune tissues. Moreover, following 20 days of feeding, the susceptibility of Nile tilapia to P. putida infection was evaluated to assess the protective effect of the used extract. The findings indicated that the studied parameters were significantly increased, and the best immune response profiles were observed in fish fed P. boergesenii water extract for 20 successive days. A bacterial challenge experiment using P. putida resulted in higher survival within the supplemented fish group than the control. Thus, the lowered post-challenge mortality of the fish may be related to the protection provided by the stimulation of the innate immune system, reduced oxidative stress by higher activity of TAC, and elevated levels of expression of iterleukin-1beta (IL-1ß), beta-defensin (ß-defensin), and natural killer-lysin (NKl). Moreover, the constituents of the extract used showed potential protective activity for histological features of the supplemented fish group when compared to the control. Collectively, this study presents a great insight on the protective role of P. boergesenii water extract as an additive in Nile tilapia feed which suggests its potential for improving the immune response against P. putida infection.

Rhizobacterial syntrophy between a helper and a beneficiary promotes tomato plant health.

Microbial interactions impact the functioning of microbial communities. However, microbial interactions within host-associated communities remain poorly understood. Here, we report that the beneficiary rhizobacterium Niallia sp. RD1 requires the helper Pseudomonas putida H3 for bacterial growth and beneficial interactions with the plant host. In the absence of the helper H3 strain, the Niallia sp. RD1 strain exhibited weak respiration and elongated cell morphology without forming bacterial colonies. A transposon mutant of H3 in a gene encoding succinate-semialdehyde dehydrogenase displayed much attenuated support of RD1 colony formation. Through the subsequent addition of succinate to the media, we found that succinate serves as a public good that supports RD1 growth. Comparative genome analysis highlighted that RD1 lacked the gene for sufficient succinate, suggesting its evolution as a beneficiary of succinate biosynthesis. The syntrophic interaction between RD1 and H3 efficiently protected tomato plants from bacterial wilt and promoted tomato growth. The addition of succinate to the medium restored complex II-dependent respiration in RD1 and facilitated the cultivation of various bacterial isolates from the rhizosphere. Taken together, we delineate energy auxotrophic beneficiaries ubiquitous in the microbial community, and these beneficiaries could benefit host plants with the aid of helpers in the rhizosphere.

Pseudomonas putida KT2440: the long journey of a soil-dweller to become a synthetic biology chassis.

Although members of the genus Pseudomonas share specific morphological, metabolic, and genomic traits, the diversity of niches and lifestyles adopted by the family members is vast. One species of the group, Pseudomonas putida, thrives as a colonizer of plant roots and frequently inhabits soils polluted with various types of chemical waste. Owing to a combination of historical contingencies and inherent qualities, a particular strain, P. putida KT2440, emerged time ago as an archetype of an environmental microorganism amenable to recombinant DNA technologies, which was also capable of catabolizing chemical pollutants. Later, the same bacterium progressed as a reliable platform for programming traits and activities in various biotechnological applications. This article summarizes the stepwise upgrading of P. putida KT2440 from being a system for fundamental studies on the biodegradation of aromatic compounds (especially when harboring the TOL plasmid pWW0) to its adoption as a chassis of choice in metabolic engineering and synthetic biology. Although there are remaining uncertainties about the taxonomic classification of KT2440, advanced genome editing capabilities allow us to tailor its genetic makeup to meet specific needs. This makes its traditional categorization somewhat less important, while also increasing the strain's overall value for contemporary industrial and environmental uses.

Physiology, fast and slow: bacterial response to variable resource stoichiometry and dilution rate.

Microorganisms grow despite imbalances in the availability of nutrients and energy. The biochemical and elemental adjustments that bacteria employ to sustain growth when these resources are suboptimal are not well understood. We assessed how Pseudomonas putida KT2440 adjusts its physiology at differing dilution rates (to approximate growth rates) in response to carbon (C), nitrogen (N), and phosphorus (P) stress using chemostats. Cellular elemental and biomolecular pools were variable in response to different limiting resources at a slow dilution rate of 0.12 h-1, but these pools were more similar across treatments at a faster rate of 0.48 h-1. At slow dilution rates, limitation by P and C appeared to alter cell growth efficiencies as reflected by changes in cellular C quotas and rates of oxygen consumption, both of which were highest under P- and lowest under C- stress. Underlying these phenotypic changes was differential gene expression of terminal oxidases used for ATP generation that allows for increased energy generation efficiency. In all treatments under fast dilution rates, KT2440 formed aggregates and biofilms, a physiological response that hindered an accurate assessment of growth rate, but which could serve as a mechanism that allows cells to remain in conditions where growth is favorable. Our findings highlight the ways that microorganisms dynamically adjust their physiology under different resource supply conditions, with distinct mechanisms depending on the limiting resource at slow growth and convergence toward an aggregative phenotype with similar compositions under conditions that attempt to force fast growth. IMPORTANCE: All organisms experience suboptimal growth conditions due to low nutrient and energy availability. Their ability to survive and reproduce under such conditions determines their evolutionary fitness. By imposing suboptimal resource ratios under different dilution rates on the model organism Pseudomonas putida KT2440, we show that this bacterium dynamically adjusts its elemental composition, morphology, pools of biomolecules, and levels of gene expression. By examining the ability of bacteria to respond to C:N:P imbalance, we can begin to understand how stoichiometric flexibility manifests at the cellular level and impacts the flow of energy and elements through ecosystems.

Reliable Genomic Integration Sites in Pseudomonas putida Identified by Two-Dimensional Transcriptome Analysis.

Genomic integration is commonly used to engineer stable production hosts. However, so far, for many microbial workhorses, only a few integration sites have been characterized, thereby restraining advanced strain engineering that requires multiple insertions. Here, we report on the identification of novel genomic integration sites, so-called landing pads, for Pseudomonas putida KT2440. We identified genomic regions with constant expression patterns under diverse experimental conditions by using RNA-Seq data. Homologous recombination constructs were designed to insert heterologous genes into intergenic sites in these regions, allowing condition-independent gene expression. Ten potential landing pads were characterized using four different msfGFP expression cassettes. An insulated probe sensor was used to study locus-dependent effects on recombinant gene expression, excluding genomic read-through of flanking promoters under changing cultivation conditions. While the reproducibility of expression in the landing pads was very high, the msfGFP signals varied strongly between the different landing pads, confirming a strong influence of the genomic context. To showcase that the identified landing pads are also suitable candidates for heterologous gene expression in other Pseudomonads, four equivalent landing pads were identified and characterized in Pseudomonas taiwanensis VLB120. This study shows that genomic "hot" and "cold" spots exist, causing strong promoter-independent variations in gene expression. This highlights that the genomic context is an additional parameter to consider when designing integrable genomic cassettes for tailored heterologous expression. The set of characterized genomic landing pads presented here further increases the genetic toolbox for deep metabolic engineering in Pseudomonads.

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.