Microbial Biotechnology

Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including from lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher ATP pool, in excess of demand. Pyruvate and acetate secretion in CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L-1 muconate at 1.1 g L-1 h-1. Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.

Muconic acid is an industrially valuable molecule that can be biologically produced from diverse biogenic and waste-derived feedstocks, including sugars and lignin- and plastic-derived aromatic compounds. However, accumulation of protocatechuate (PCA) has been observed in multiple microbes engineered for muconate production when the PCA decarboxylase, AroY, is used. This raises the question of whether PCA decarboxylation represents a rate-limiting step and how this bottleneck might be alleviated, especially given the toxicity and reactivity of PCA and catechol intermediates. To address this, we performed adaptive laboratory evolution (ALE) on a strain of Pseudomonas putida originally engineered for muconate production from aromatic compounds, but with catBC restored, to select for improved conversion of PCA and, in separate lineages, 4-hydroxybenzoate. Contrary to our expectations, the predominant beneficial mutations localized to the catA1 cassette encoding catechol 1,2-dioxygenase, rather than aroY or its associated cofactor biosynthesis genes. Transcriptomic analysis revealed elevated catA1 expression in evolved isolates from ALE, and introduction of these mutations improved productivity in strains designed for muconate production from both aromatic and sugar substrates. Quantitative proteomics and biochemical assays demonstrated that the mutations also led to increased CatA1 protein abundance and modest enhancements in catalytic efficiency, respectively, with strain phenotypes largely driven by high CatA1 levels and potentially synergistic kinetic improvements. Additional reverse-engineering studies identified variants with modest effects on muconate accumulation, including those with potential to enhance biosynthesis of the prenylated FMN cofactor of AroY. Collectively, these results indicate that catechol, not PCA, is the principal bottleneck in muconate production via the PCA decarboxylation route originally demonstrated by Draths et al., refining our understanding of pathway limitations and offering new strategies for improving rate, yield, and strain resilience in muconate bioproduction. HighlightsO_LIAccumulation of metabolic intermediates was alleviated by adaptive laboratory evolution C_LIO_LISequencing, proteomics, and enzyme kinetics revealed mechanisms for adaptation C_LIO_LIIncreased CatA1 expression reduced bottlenecks and improved muconate production C_LI

BackgroundThe corn smut fungus Ustilago maydis is an important microbial model organism representing a genetically amenable and readily cultivable basidiomycete. Research in this fungus addresses a broad range of fundamental questions and its biotechnological exploitation is on the rise. Although genetic engineering in principle is well established, efficient methodology for synthetic biology approaches such as metabolic engineering or pathway transplantation has remained limited. ResultsHere, we present a comprehensive toolbox for U. maydis based on modular cloning and the characterization of more than 20 promoters. Careful comparative evaluation of insertion loci and terminator as well as reporter effects was conducted and a novel color-based strategy for straightforward genome integration was implemented. Moreover, the cloning and subsequent one-step integration of four transcriptional units into U. maydis was demonstrated by creating a "rainbow" strain producing four fluorescent proteins. ConclusionOverall, this next generation toolkit strongly advances genetic engineering and systems biology approaches in U. maydis, fostering its development into a valuable and competitive fungal chassis and prime model, particularly in applied research.

Biocontainment systems designed to attenuate the spread of mobile DNA are challenging to evaluate within microbiomes of engineered environments. To better understand how toxin-based biocontainment systems affect horizontal gene transfer (HGT) in a microbiome, we evaluated the host range of pairs of plasmids using orthogonal catalytic RNA (cat-RNA) that amend distinct barcodes to 16S rRNA following HGT. We show that mobilizable (5 kb) and self-mobilizable (60 kb) plasmids, which use the same RP4 transfer machinery but different origins of replication, overlap in their host range when conjugated in parallel into a wastewater community, with 127 of the 143 amplicon sequence variants (ASVs) presenting barcoding signals from both plasmids (89%). We also find that mobilizable plasmids with or without the Escherichia coli CcdB toxin overlap in host range in a wastewater community. Among the two most abundant orders, CcdB attenuated the barcoding signal in Aeromonadales more consistently than Enterobacteriales, which have F plasmids containing the CcdB-CcdA toxin-antitoxin system used for biocontainment. Also, CcdB decreased the abundance of the mobilizable plasmid by >100-fold and yielded mutations in 85% of the reads. Together, these findings reveal how pairs of plasmids expressing orthogonal cat-RNA can be used to monitor the effects of plasmid-encoded traits on mobile DNA persistence following HGT. They also highlight challenges when using biocontainment systems containing genes related to those found in the microbiomes targeted for engineering.

Past few decades witnessed a boom in pharmaceutical and bioproduct industry with the help of bioprocess technology. Industrially important bioproducts can be produced in large scale for commercialization with the help of fermenters. Here in, pharmaceutically valuable bioproduct melanin, synthesized from Pseudomonas stutzeri strain BTC109 by using two different sized bioreactors. Under controlled conditions the bacteria were allowed to synthesis melanin nanoparticles. The important parameters to be monitored here are pH, dissolved oxygen, agitation, aeration, melanin production and cell biomass concentration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/737634v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@d92fe5org.highwire.dtl.DTLVardef@d76c07org.highwire.dtl.DTLVardef@f5516forg.highwire.dtl.DTLVardef@1b5864b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPharmaceutical and bioproduct development industries witnessed a shoot up due to bioprocess technology. C_LIO_LIIndustrially important bioproduct like melanin can be produced in large scale with the help of industrial fermentation technology. C_LIO_LIThe product thus obtained was found to be nano sized and it can be commercialized. C_LI

Plant growth-promoting bacteria are gaining significant attention as promising biofertilizers. However, the inconsistency between in vitro plant growth-promoting traits and actual field performance remains a challenge, driven partly by a limited understanding of in situ colonization. This study characterized the colonization patterns of Citrobacter sedlakii CESi7, a novel plant growth-promoting bacterium, isolated from oil palm waste compost, during Brassica rapa cultivation. The in situ behavior of CESi7 was observed in both sterilized medium and non-sterilized soil using fluorescence in situ hybridization with a strain-targeting probe. The results revealed that CESi7 can establish both epiphytic and endophytic populations that transiently colonize roots. In a sterilized medium, CESi7 was widely distributed throughout the root tissues. Conversely, in non-sterilized soil, the bacterium formed dense aggregates specifically at the root tips. This study provides direct microscopic evidence of the colonization strategy of CESi7, offering crucial insights for its development as an effective biofertilizer.

Nutrient-rich food-processing wastewaters represent valuable yet under-utilised side streams for sustainable protein production in the form of microbial biomass. Here we present an integrated dual-loop bioprocess that converts soybean-processing wastewater into microbial single-cell protein (SCP) while achieving substantial nutrient removal and product refinement. In the first loop, previously enriched microbial consortia were inoculated and cultivated in four parallel sequencing batch reactors (SBRs) for 44days at a hydraulic retention time (HRT) of 3days. This bioprocess configuration demonstrated features that support future scale-up while maintaining process stability, achieving a protein content of 33.3{+/-}3.2%, doubling the protein yield (15.32{+/-}3.49g dry weight per g soluble TKN) and quadrupling the production rate (0.29{+/-}0.06g dry weight L-1 d-1) compared to operating reactors without inoculation (HRT: 7.2days). Effluent treatment was stable, with 84% carbon and 78% nitrogen removal efficiencies, demonstrating efficient nutrient recovery. The SCP biomass was enriched in functional taxa, including Acidipropionibacterium, Lactococcus, Megasphaera, and Azospirillum, suggesting that reactor conditions and inoculum selection promoted a stable, protein-productive microbial community with potential probiotic benefits. In the second loop, bioreactor effluent was reused as aqueous matrix for heat treatment (60{degrees}C) of the SCP biomass, reducing the RNA content from 8.6% to 2.6%, with a 39% biomass loss accompanied by a 30% increase in total amino acid concentration. Hence, our valorisation approach integrates microbial biomass production, effluent reuse, and product refinement within a circular framework. The system provides a resource-efficient pathway for converting food-sector side streams into high-quality microbial community-based SCP, highlighting its potential scalability for sustainable nutrient and water management.

Municipal wastewater constitutes a major reservoir of unutilized reactive nitrogen, representing a significant opportunity for biological valorization. The biopolymer cyanophycin is promising as a means of nitrogen capture and recovery, but current production strategies are not optimized for the physicochemical constraints of municipal wastewater systems. Here, we engineered the naturally competent soil bacterium Acinetobacter baylyi ADP1 ISx to synthesize cyanophycin from carbon and nitrogen sources prevalent in municipal wastewater and over a range of wastewater-relevant temperatures. To overcome the recurring problem of arginine availability limiting cyanophycin synthesis, we engineered an arginine-producing strain (AP1) which accumulated cyanophycin when grown on acetate and ammonium (19% CDW), nitrate (9% CDW), or urea (29% CDW) and without arginine supplementation. During this work, we observed that conditions associated with reduced cell fitness correlated with increased intracellular cyanophycin content. As temperature strongly influences cell growth but cannot be realistically modulated in wastewater contexts, we investigated the potential of induced fructose-auxotrophy to modulate cell growth independently from temperature. This intervention, accomplished with a single knockout (gap), expanded the effective range of cyanophycin accumulation from 12 C up to 30 C. Collectively, these results establish the relevance of arginine-producing strains for cyanophycin biosynthesis and position A. baylyi as a promising chassis for continued development under real-world wastewater conditions.

Anaerocellum (formerly Caldicellulosiruptor) bescii, an anaerobic, extremely thermophilic (Topt [~]78 {degrees}C) lignocellulolytic bacterium, is a promising chassis for metabolic engineering and next-generation bioprocessing. Yet, a lack of well-characterized genetic parts in A. bescii has hampered metabolic engineering efforts. Here, using a previously developed hyperthermophilic {beta}-galactosidase reporter system, we screened a diverse panel of putative A. bescii promoter sequences, identifying promoters that drove reporter output across a broad range. For a select subset, we mapped their transcriptional start sites (TSSs) and evaluated ribosome binding site (RBS) regions using chimeric promoter constructs. By constructing truncated promoter variants, we defined functional regions within the widely used, high-expression S-layer protein promoter (Pslp) and engineered a compact 99 bp variant that retained substantial reporter activity. Finally, we demonstrated that these new promoters can be used for metabolic engineering by using two newly characterized promoters to express an established thermostable alcohol dehydrogenase from Thermoclostridium stercorarium to drive ethanol production in A. bescii. Together, this work expands and diversifies the A. bescii genetic toolkit, opening doors to future metabolic engineering efforts in this species.

Enzymatic depolymerization of polyethylene terephthalate (PET) has advanced rapidly, alongside a growing volume of publicly available metagenomic data from microbial communities under sustained selective pressure from plastic exposure. Reasoning that such environments may harbor underexplored polyester-active enzymes, we developed a targeted mining workflow that screens exclusively plastic-associated datasets through multi-step bioinformatic filtering--integrating catalytic-motif screening, disulfide-topology validation, structural-similarity scoring, and phylogenetic profiling--to recover high-confidence PETase candidates. Applied to 271 plastic-associated metagenomes, the pipeline yielded 21 non-redundant candidates, several of which combine the Type I catalytic motif (GHSMGGGG) with Type II-like extended loops and secondary disulfide bonds. Two candidates were experimentally confirmed as PET hydrolases; the more active, PET-KR1, is a thermostable enzyme (Tm = 66.5 {degrees}C) that depolymerizes PET across a broad temperature range, with markedly higher productivity on powdered than on film substrate. PET-KR1 achieved optimal depolymerization at 50 {degrees}C, yet at 60-65 {degrees}C, where total yields declined, the product pool was more strongly enriched in the terminal monomer TPA, suggesting that thermostability and substrate accessibility are the primary targets for further engineering. Molecular dynamics simulations revealed a conserved hydrophobic binding network around the catalytic serine, consistent with established PETase substrate-recognition modes, and rational disulfide engineering raised the melting temperature by 3.5 {degrees}C, confirming amenability to further optimization. Overall, PET-KR1 expands the scaffold space available for PETase engineering, while the discovery workflow, built entirely on publicly available tools and open-access data, provides a reproducible strategy for metagenomic mining of novel PET-degrading enzymes toward biocatalytic PET recycling.

Precision phage therapeutics provide a promising strategy to combat multidrug-resistant pathogens, including Staphylococcus aureus. Efficient, specific packaging of genetic cargoes remains challenging. Using modular design principles, we report a minimal phagemid packaging signal consisting of the phage terminase small subunit under its native promoter that significantly outperforms conventional packaging signals. The utility of this synthetic terS operon was demonstrated through production of highly concentrated and genetically pure CRISPR-Cas antimicrobials. To circumvent CRISPR-mediated self-targeting during antimicrobial generation, a terS-deficient strain was engineered to express the anti-CRISPR protein AcrIIA4, enabling titers above 1010 transducing units per milliliter (TRU/mL) with over 94% purity. With a high-copy origin of replication module, CRISPR-Cas phage-like particle titers could approach 1012 TRU/mL. We discovered that pure CRISPR-Cas antimicrobials are potent and can be amplified in hosts possessing prophages. Taken altogether, this study defines the minimal and optimal genetic requirements for efficient, specific creation of phage-based technologies. Technological ReadinessThe described system for engineering phage-like particles has reached a technological readiness level (TRL) of 4-5 based on the provided laboratory validation and strong literature support from other engineered phage therapies applied to in vivo models. Previous systems have demonstrated high-purity phage-like particle preparation, but always at the cost of severely reduced productivity. Therefore, our demarcation of a specific, efficient, and minimal system for packaging nucleic acid cargoes into phage vectors is a critical step toward real-world use. Despite this, low levels of contaminating host/phage DNA remain a key barrier to phage-based therapies. Protein and strain engineering efforts can help mitigate terminase nonspecificity, but care must be taken to not compromise productivity. More generally, widespread adoption will require deeper understanding of host-pathogen-phage interactions, development of scalable GMP manufacturing processes, and harmonized regulatory guidance that recognizes the dynamic nature of phage-derived technologies.

Nitrification inhibitors are applied to reduce nitrogen losses and greenhouse gas emissions from fertilized agricultural ecosystems. However, their characterization is typically focused on determining effective inhibitor concentrations from growth or substrate conversion assays that are time-intensive and provide limited mechanistic resolution. Here, we present a microrespirometry (MR)-based workflow for rapid mechanistic characterization of nitrification inhibitors using oxygen consumption as a real-time readout for metabolic activity. The workflow enables the simultaneous assessment of inhibitor efficacy, competitiveness, and enzyme specificity within a single experimental setup, as sequential substrate and inhibitor additions enable direct discrimination between competitive and non-competitive inhibition and between ammonia monooxygenase-specific and broader respiratory inhibition. As a proof of concept, we evaluated three known nitrification inhibitors phenylacetylene (PA), nitrapyrin (NP), and dicyandiamide (DCD) using the ammonia-oxidizing bacteria Nitrosomonas europaea and Nitrosospira multiformis, the complete ammonia oxidizer Nitrospira inopinata, and the nitrite oxidizer Nitrospira moscoviensis. We also compared the results from the MR-based inhibition workflow with those from a conventional growth-based approach and observed a poor correlation between results for inhibitors that are not fully enzyme specific. In conclusion, this work establishes MR as a rapid and versatile platform for the mechanistic screening of novel potential nitrification inhibitors. MR assays reproduce known inhibitory responses while substantially reducing experimental time and increasing mechanistic resolution compared to other assays types. Additionally, we provide the first pure-culture characterization of PA, NP, and DCD efficacy and inhibition mechanisms in a complete ammonia oxidizer, N. inopinata.

Caldimonas thermodepolymerans is a thermophilic polyhydroxyalkanoate (PHA)-producing bacterium with strong potential for sustainable bioplastic production. Besides serving as intracellular carbon and energy storage compounds, PHAs are increasingly associated with bacterial stress resistance and cellular robustness. This study aimed to investigate the physiological and transcriptomic response of C. thermodepolymerans to osmotic stress induced by elevated NaCl concentrations. Growth analysis demonstrated tolerance up to a supplementation of 2% NaCl, while moderate salt concentrations enhanced PHA accumulation, reaching 65% cell dry weight at 1.5% NaCl supplementation. To better understand the bacterial response to osmotic stress, RNA sequencing was performed under sublethal salt stress conditions. Differential expression analysis revealed major changes in genes related to osmoprotection, trehalose metabolism and type VI secretion systems, whereas motility and chemotaxis genes were strongly repressed. Phenotypic assays confirmed increased biofilm formation and reduced swarming motility under salt-induced osmotic stress. Although canonical PHA biosynthesis genes were not significantly differentially expressed, increased polymer accumulation suggests other underlying mechanisms linked to osmoadaptation. Together, these findings demonstrate that osmotic stress induces metabolic, physiological and regulatory responses in C. thermodepolymerans, highlighting the importance of PHA in stress adaptation besides its industrial applicability.

Hemp (Cannabis sativa L.) is an increasingly important crop with applications spanning fibre, seed oil and bioactive cannabinoid production, yet the development of reliable tissue culture systems for this species remains a significant challenge. The establishment of axenic seedling cultures is a prerequisite for hypocotyl-based regeneration and future genetic transformation pipelines, but hemp seeds harbour diverse endophytic microbial communities that frequently overwhelm standard surface sterilisation protocols. Here, we present a systematic comparative evaluation of seed sterilisation strategies across seven industrial hemp accessions, examining the effects of sterilisation chemistry, seed provenance and accession identity on both contamination outcome and the subsequent morphogenic competence of hypocotyl explants. Across all treatments and accessions, in-house glasshouse-harvested seeds achieved higher sterility rates than commercially sourced material regardless of protocol applied. This provenance effect, combined with considerable batch-to-batch variation within seed sources, indicates that contamination load is a primary determinant of successful hemp seed sterilisation. Among the sterilisation treatments evaluated, a baseline of 75% ethanol combined with sequential 1% hydrogen peroxide incubation performed consistently well for low-load seed batches, while supplementation with Plant Preservative Mixture (PPMTM) might be necessary to achieve acceptable rates of non-contaminated seedlings from high contamination load batches. Beyond their effect on contamination, sterilisation treatments influenced the morphogenic fate of hypocotyl explants independently of sterility outcomes. Notably, seedling treatment with the Prochloraz-based fungicide Octave promoted shoot and root co-regeneration in the absence of exogenous plant growth regulators in some cases. Hormone-free organogenesis from hypocotyl explants was achievable across multiple hemp accessions, demonstrating that this developmental capacity is broadly distributed within hemp, though its frequency and consistency varied with accession identity and protocol conditions. Together, these findings provide a practical framework for axenic hemp seed culture that can be used as starting point requiring local adaptation based on seed source, batch history and the intended downstream application.

Phage therapy offers a promising solution to the antimicrobial resistance crisis. However, a major concern preventing the adoption of phage therapy is the potential for unintended consequences of phage release; both in regard to preventing the spread of phage resistance, and the proliferation of a non-endemic virus into the microbial ecosystem. Conditional replication (biocontainment) of phages through bioengineering may address these concerns, but the impact on bactericidal efficacy is unknown. Here, we created a biocontained T7 phage (T7{Delta}capsid) lacking the major structural capsid gene, gp10AB, that can only replicate on Escherichia coli strains expressing gp10AB in trans, and assessed its bactericidal efficacy compared with wild-type T7. Congruent with model predictions, T7{Delta}capsid was only able to clear a well-mixed culture of E. coli at a multiplicity of infection (MOI) of 10 or higher, whereas wild-type T7 prohibited growth at an MOI of 0.1. The reduction in efficacy was more evident in a complex structured environment within a microfluidic device, where phage success depends on its ability to penetrate a microbial niche via propagation. In this environment, T7{Delta}capsid was unable to propagate into the bacterial population and unlike wild-type T7, had no impact on the population's growth. This study shows that whilst biocontainment of phages may improve the biosafety of phage therapy, it comes at the cost of its propagation efficacy and niche penetration in relevant environments.

Methanogenic archaea, such as Methanothermobacter marburgensis, represent a powerful biological platform for carbon capture and valorization, directly converting carbon dioxide (CO2) and molecular hydrogen (H2) into proteinogenic amino acids (AAs). In this study, we present a controlled and scalable strategy for tailoring AA production (biosynthesis and secretion) in continuous gas fermentation. By applying various Design of Experiments (DOE) techniques, we systematically identified and optimized key process parameters governing AA biosynthesis and shaping a targeted AA secretion profile. A hybrid modeling framework combining experimental data with scale-independent parameters derived from computational fluid dynamics (CFD) enabled robust performance prediction across bioreactor scales. This model-driven approach successfully translated the process from 120 mL glass bottles via 2 L to 150 L reactors, corresponding to a reaction-volume scale-up factor of 2000. These findings set the foundation for a robust and predictive platform for sustainable AA production, positioning archaea as a high-potential alternative in industrial biotechnology.

The tomato leafminer, Tuta absoluta, poses a severe global agricultural threat due to its rapid leaf-mining behavior and swift development of resistance to conventional chemical pesticides. While microbial chitinases are potent biopesticides, their field efficacy is limited by environmental degradation and the short exposure window before larvae penetrate leaf tissues. This study evaluates a stimuli-responsive, controlled-release nanobiopesticide system utilizing a novel chitinase from newly characterized Serratia marcescens GBS19. A 61.1 kDa chitinase (GBS19_ChiA) was heterologously expressed in Escherichia coli and purified to a specific activity of 215.01 U/mg. The enzyme was immobilized onto starch-coated silica nanoparticles designed for target-triggered release via host alpha-amylase. Genomic profiling and R-based kinetic modeling were integrated to evaluate the efficacy of purified and immobilized forms against T. absoluta. Immobilization enhanced thermal and pH stability, with the nanocarrier maintaining 85% activity over 10 weeks. In larval bioassays, immobilization increased mortality from 21.9% to 59.4% (5000 U/mL) by day 3, reaching 62.5% by day 6. Genomic analysis identified an expansive secretome and a Type VI Secretion System (T6SS), characterizing GBS19 as a multi-pronged pathogen. Kinetic modeling established that while immobilized enzymes are effective, the 2.5-hour exposure time on T. absoluta requires the synergistic action of chitinases (ChiA/B/C) to reach the lethal desiccation threshold before larvae establish protective mines. Starch-coated silica nanoparticles significantly improve chitinase stability and delivery. However, overcoming the rapid penetration of T. absoluta necessitates a whole-cell or multi-enzyme synergistic approach to outpace larval behavioural defences.

Recombinant proteins have transformative potential in biomedicine, but their production is often costly and carries contamination risks. Plants offer an attractive alternative, with low growth costs and reduced pathogen risk, yet their slow growth cycles limit their use for rapid protein engineering. Here, we establish Marchantia polymorpha, a genetically tractable liverwort with a short life cycle, as a new platform for recombinant protein production. Using stable Agrobacterium-mediated transformation, we expressed an anti-mCherry nanobody fused to the fluorescent protein mTurquoise2 with different purification tags. Expression levels reached up to [~]120 {micro}g/g fresh weight, and nanobody functionality was validated through a microscopy-based bead-binding assay. This yield rivals that of established systems such as Nicotiana benthamiana. Our results position M. polymorpha as a scalable, safe, and efficient chassis for protein engineering, with broad potential for applications in synthetic biology. This work opens the door to exploiting liverwort biotechnology for fast, cost-effective, and biosafe production of valuable recombinant proteins. Graphical Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/733889v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@aabc4aorg.highwire.dtl.DTLVardef@12f9fa4org.highwire.dtl.DTLVardef@199d3e0org.highwire.dtl.DTLVardef@2533a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chemically complex pharmaceutical mixtures in wastewater and sludge can affect microbial adaptation; however, the responses to different co-occurring compounds have not been elucidated well. In this study, the adaptation of a strain derived from hospital sludge, Klebsiella pneumoniae SS02, to 17-ethinylestradiol (EE2), warfarin sodium, and their combination has been studied. The organism grows under all three conditions, and pre-exposure experiments show induction and cross-induction to substrates. UHPLC MS/MS analyses demonstrated that there is conditional depletion of the parent compound EE2 by [~]15% at 36 h post-treatment compared to initial concentrations, but not for the abiotic and non-adapted controls. The rate of warfarin sodium depletion was approximately [~]30% within 36 h and was in accordance with first order kinetics (k = 0.0102 /h; t{square}/{square}= 67.9 h). Under the combined treatment regime, there was a delay in warfarin sodium depletion, suggesting staged substrate consumption. Growth inhibition with efflux inhibitors confirmed transport-driven tolerance. A genome-based study revealed the coordinated response strategy that involved a proposed flavin-dependent monooxygenase (RutA), an oxidative entry into the pathway; redox processing linked to Hpa; aromatic metabolism through {beta}-ketoadipate pathway; and RND efflux system. The structural study additionally supported ligand-mediated decrease in DNA binding affinity of RutR, which is in agreement with de-repression of the substrate-activated regulatory mechanism. All these findings lead to the development of a dual-strategy for adaptation model in which oxidative modification and efflux-mediated protection work together under the influence of a mixture of pharmaceuticals.

Engineering heterologous utilization of substrates requires selection of catabolic pathways that balance strain performance and product biosynthesis. Here, we compare the oxidative and isomerase arabinose utilization pathways in Pseudomonas putida strains engineered for cis,cis-muconic acid production from glucose and xylose. Based on the point of entry into central carbon metabolism, we hypothesized that the oxidative arabinose pathway would enable higher productivity while the arabinose isomerase pathway would enable higher muconate yield. In both strains, additional modifications were engineered to improve muconic acid production including sugar transporter tuning, catechol 1,2-dioxygenase overexpression, a feedback-resistant DAHP synthase, and a flux-stabilizing gltA variant. Consistent with our hypothesis, the oxidative arabinose pathway supported faster growth and higher productivity (0.58 g/L/h), whereas the arabinose isomerase pathway improved carbon efficiency, achieving muconate yields of up to 50 C-mol% in fed-batch bioreactors. Process modeling indicates that these performance metrics can reduce the minimum selling price of muconate-derived adipic acid to $2.74/kg and greenhouse gas emissions to 1.31 kg CO2e/kg, approaching cost parity and reducing emissions by 86% relative to fossil carbon-derived adipic acid. Overall, this study presents a systematic comparison of sugar catabolic pathways that enabled development of strains suited for the tradeoffs between rate and yield.