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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.

The aldehyde 5-(hydroxymethyl)furfural (HMF) is of great importance for a circular bioeconomy. It is a renewable platform chemical that can be converted into a range of useful compounds to replace petroleum-based products such as the green plastic monomer 2,5-furandicarboxylic acid (FDCA). However, it also exhibits microbial toxicity for example hindering the efficient biotechnological valorization of lignocellulosic hydrolysates. Thus, there is an urgent need for tolerance-improved organisms applicable to whole-cell biocatalysis. Here, we engineer an oxidation-deficient derivative of the naturally robust and emerging biotechnological workhorse P. taiwanensis VLB120 by robotics-assisted adaptive laboratory evolution (ALE). The deletion of HMF-oxidizing enzymes enabled for the first time evolution under constant selection pressure by the aldehyde, yielding strains with consistently improved growth characteristics in presence of the toxicant. Genome sequencing of evolved clones revealed loss-of function mutations in the LysR-type transcriptional regulator-encoding mexT preventing expression of the associated efflux pump mexEF - oprN . This knowledge allowed reverse engineering of strains with enhanced aldehyde tolerance, even in a background of active or overexpressed HMF oxidation machinery, demonstrating a synergistic effect of two distinct tolerance mechanisms., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Bio-upcycling of plastics is an emerging alternative process that focuses on extracting value from a wide range of plastic waste streams. Such streams are typically too contaminated to be effectively processed using traditional recycling technologies. Medium-chain-length (mcl) diols and dicarboxylates (DCA) are major products of chemically or enzymatically depolymerized plastics, such as polyesters or polyethers. In this study, we enabled the efficient metabolism of mcl-diols and -DCA in engineered Pseudomonas putida as a prerequisite for subsequent bio-upcycling. We identified the transcriptional regulator GcdR as target for enabling metabolism of uneven mcl-DCA such as pimelate, and uncovered amino acid substitutions that lead to an increased coupling between the heterologous β-oxidation of mcl-DCA and the native degradation of short-chain-length DCA. Adaptive laboratory evolution and subsequent reverse engineering unravelled two distinct pathways for mcl-diol metabolism in P. putida, namely via the hydroxy acid and subsequent native β-oxidation or via full oxidation to the dicarboxylic acid that is further metabolized by heterologous β-oxidation. Furthermore, we demonstrated the production of polyhydroxyalkanoates from mcl-diols and -DCA by a single strain combining all required metabolic features. Overall, this study provides a powerful platform strain for the bio-upcycling of complex plastic hydrolysates to polyhydroxyalkanoates and leads the path for future yield optimizations., (© 2024. The Author(s).)

Ustilago maydis and Ustilago cynodontis are natural producers of a broad range of valuable molecules including itaconate, malate, glycolipids, and triacylglycerols. Both Ustilago species are insensitive toward medium impurities, and have previously been engineered for efficient itaconate production and stabilized yeast-like growth. Due to these features, these strains were already successfully used for the production of itaconate from different alternative feedstocks such as molasses, thick juice, and crude glycerol. Here, we analyzed the amylolytic capabilities of Ustilago species for metabolization of starch, a highly abundant and low-cost polymeric carbohydrate widely utilized as a substrate in several biotechnological processes. Ustilago cynodontis was found to utilize gelatinized potato starch for both growth and itaconate production, confirming the presence of extracellular amylolytic enzymes in Ustilago species. Starch was rapidly degraded by U. cynodontis, even though no α-amylase was detected. Further experiments indicate that starch hydrolysis is caused by the synergistic action of glucoamylase and α-glucosidase enzymes. The enzymes showed a maximum activity of around 0.5 U ml-1 at the fifth day after inoculation, and also released glucose from additional substrates, highlighting potential broader applications. In contrast to U. cynodontis, U. maydis showed no growth on starch accompanied with no detectable amylolytic activity., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Due to its tolerance properties, Pseudomonas has gained particular interest as host for oxidative upgrading of the toxic aldehyde 5-hydroxymethylfurfural (HMF) into 2,5-furandicarboxylic acid (FDCA), a promising biobased alternative to terephthalate in polyesters. However, until now, the native enzymes responsible for aldehyde oxidation are unknown. Here, we report the identification of the primary HMF-converting enzymes of P. taiwanensis VLB120 and P. putida KT2440 by extended gene deletions. The key players in HMF oxidation are a molybdenum-dependent periplasmic oxidoreductase and a cytoplasmic dehydrogenase. Deletion of the corresponding genes almost completely abolished HMF oxidation, leading instead to aldehyde reduction. In this context, two HMF-reducing dehydrogenases were also revealed. These discoveries enabled enhancement of Pseudomonas' furanic aldehyde oxidation machinery by genomic overexpression of the respective genes. The resulting BOX strains (Boosted OXidation) represent superior hosts for biotechnological synthesis of FDCA from HMF. The increased oxidation rates provide greatly elevated HMF tolerance, thus tackling one of the major drawbacks of whole-cell catalysis with this aldehyde. Furthermore, the ROX (Reduced OXidation) and ROAR (Reduced Oxidation And Reduction) deletion mutants offer a solid foundation for future development of Pseudomonads as biotechnological chassis notably for scenarios where rapid HMF conversion is undesirable., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Impranil ® DLN-SD is a poly(ester-urethane) (PEU) that is widely used as coating material for textiles to fine-tune and improve their properties. Since coatings increase the complexity of such plastic materials, they can pose a hindrance for sustainable end-of-life solutions of plastics using enzymes or microorganisms. In this study, we isolated Halopseudomonas formosensis FZJ due to its ability to grow on Impranil DLN-SD and other PEUs as sole carbon sources. The isolated strain was exceptionally thermotolerant as it could degrade Impranil DLN-SD at up to 50°C. We identified several putative extracellular hydrolases of which the polyester hydrolase Hfor_PE-H showed substrate degradation of Impranil DLN-SD and thus was purified and characterized in detail. Hfor_PE-H showed moderate temperature stability (T m = 53.9°C) and exhibited activity towards Impranil DLN-SD as well as polyethylene terephthalate. Moreover, we revealed the enzymatic release of monomers from Impranil DLN-SD by Hfor_PE-H using GC-ToF-MS and could decipher the associated metabolic pathways in H. formosensis FZJ. Overall, this study provides detailed insights into the microbial and enzymatic degradation of PEU coatings, thereby deepening our understanding of microbial coating degradation in both contained and natural environments. Moreover, the study highlights the relevance of the genus Halopseudomonas and especially the novel isolate and its enzymes for future bio-upcycling processes of coated plastic materials., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Polyketides from (hydroxy)benzoates are an interesting group of plant polyphenolic compounds, whose biotechnological production is so far underrepresented due to their challenging heterologous biosynthesis. Efficient heterologous production of 2,4,6-tri- and 2,3',4,6-tetrahydroxybenzophenone, 3,5-dihydroxybiphenyl, and 4-hydroxycoumarin by whole-cell biocatalysis in combination with in situ product extraction with an organic solvent was demonstrated. Production was highly dependent on the used CoA ligase and polyketide synthase type III. Therefore, different combinations of polyketide synthases and benzoate-CoA ligases were evaluated for their biosynthesis performance in the solvent-tolerant Pseudomonas taiwanensis VLB120. A solvent screening yielded 2-undecanone as biocompatible, extraction-efficient solvent with good phase separation. In aqueous-organic two-phase cultivations, this solvent extraction circumvents product instability in the aqueous cultivation medium, and it increases yields by reducing inhibitory effects. Complete de novo synthesis from glucose of all (hydroxy)benzoate-derived polyketides was achieved in two-phase cultivations with metabolically engineered strains. Additionally, mutasynthesis was applied to obtain fluorinated benzophenone derivatives., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Phenylpropanoids such as p-coumaric acid represent important precursors for the synthesis of a broad range of plant secondary metabolites including stilbenoids, flavonoids, and lignans, which are of pharmacological interest due to their health-promoting properties. Although extraction from plant material or chemical synthesis is possible, microbial synthesis of p-coumaric acid from glucose has the advantage of being less expensive and more resource efficient. In this study, Corynebacterium glutamicum was engineered for the production of the plant polyphenol precursor p-coumaric acid from glucose., Results: Heterologous expression of the tyrosine ammonia-lyase encoding gene from Flavobacterium johnsoniae enabled the conversion of endogenously provided tyrosine to p-coumaric acid. Product consumption was avoided by abolishing essential reactions of the phenylpropanoid degradation pathway. Accumulation of anthranilate as a major byproduct was eliminated by reducing the activity of anthranilate synthase through targeted mutagenesis to avoid tryptophan auxotrophy. Subsequently, the carbon flux into the shikimate pathway was increased, phenylalanine biosynthesis was reduced, and phosphoenolpyruvate availability was improved to boost p-coumaric acid accumulation. A maximum titer of 661 mg/L p-coumaric acid (4 mM) in defined mineral medium was reached. Finally, the production strain was utilized in co-cultivations with a C. glutamicum strain previously engineered for the conversion of p-coumaric acid into the polyphenol resveratrol. These co-cultivations enabled the synthesis of 31.2 mg/L (0.14 mM) resveratrol from glucose without any p-coumaric acid supplementation., Conclusions: The utilization of a heterologous tyrosine ammonia-lyase in combination with optimization of the shikimate pathway enabled the efficient production of p-coumaric acid with C. glutamicum. Reducing the carbon flux into the phenylalanine and tryptophan branches was the key to success along with the introduction of feedback-resistant enzyme variants., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Aromatics are valuable bulk or fine chemicals with a myriad of important applications. Currently, their vast majority is produced from petroleum associated with many negative aspects. The bio-based synthesis of aromatics contributes to the much-required shift towards a sustainable economy. To this end, microbial whole-cell catalysis is a promising strategy allowing the valorization of abundant feedstocks derived from biomass to yield de novo-synthesized aromatics. Here, we engineered tyrosine-overproducing derivatives of the streamlined chassis strain Pseudomonas taiwanensis GRC3 for efficient and specific production of 4-coumarate and derived aromatics. This required pathway optimization to avoid the accumulation of tyrosine or trans-cinnamate as byproducts. Although application of tyrosine-specific ammonia-lyases prevented the formation of trans-cinnamate, they did not completely convert tyrosine to 4-coumarate, thereby displaying a significant bottleneck. The use of a fast but unspecific phenylalanine/tyrosine ammonia-lyase from Rhodosporidium toruloides (RtPAL) alleviated this bottleneck, but caused phenylalanine conversion to trans-cinnamate. This byproduct formation was greatly reduced through the reverse engineering of a point mutation in prephenate dehydratase domain-encoding pheA. This upstream pathway engineering enabled efficient 4-coumarate production with a specificity of >95% despite using an unspecific ammonia-lyase, without creating an auxotrophy. In shake flask batch cultivations, 4-coumarate yields of up to 21.5% (Cmol/Cmol) from glucose and 32.4% (Cmol/Cmol) from glycerol were achieved. Additionally, the product spectrum was diversified by extending the 4-coumarate biosynthetic pathway to enable the production of 4-vinylphenol, 4-hydroxyphenylacetate, and 4-hydroxybenzoate with yields of 32.0, 23.0, and 34.8% (Cmol/Cmol) from glycerol, respectively., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Malonyl-CoA is a central precursor for biosynthesis of a wide range of complex secondary metabolites. The development of platform strains with increased malonyl-CoA supply can contribute to the efficient production of secondary metabolites, especially if such strains exhibit high tolerance towards these chemicals. In this study, Pseudomonas taiwanensis VLB120 was engineered for increased malonyl-CoA availability to produce bacterial and plant-derived polyketides. A multi-target metabolic engineering strategy focusing on decreasing the malonyl-CoA drain and increasing malonyl-CoA precursor availability, led to an increased production of various malonyl-CoA-derived products, including pinosylvin, resveratrol and flaviolin. The production of flaviolin, a molecule deriving from five malonyl-CoA molecules, was doubled compared to the parental strain by this malonyl-CoA increasing strategy. Additionally, the engineered platform strain enabled production of up to 84 mg L -1 resveratrol from supplemented p-coumarate. One key finding of this study was that acetyl-CoA carboxylase overexpression majorly contributed to an increased malonyl-CoA availability for polyketide production in dependence on the used strain-background and whether downstream fatty acid synthesis was impaired, reflecting its complexity in metabolism. Hence, malonyl-CoA availability is primarily determined by competition of the production pathway with downstream fatty acid synthesis, while supply reactions are of secondary importance for compounds that derive directly from malonyl-CoA in Pseudomonas., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Organic olvent-tolerant strains of the Gram-negative bacterial genus Pseudomonas are discussed as potential biocatalysts for the biotechnological production of various chemicals. However, many current strains with the highest tolerance are belonging to the species P. putida and are classified as biosafety level 2 strains, which makes them uninteresting for the biotechnological industry. Therefore, it is necessary to identify other biosafety level 1 Pseudomonas strains with high tolerance towards solvents and other forms of stress, which are suitable for establishing production platforms of biotechnological processes. In order to exploit the native potential of Pseudomonas as a microbial cell factory, the biosafety level 1 strain P. taiwanensis VLB120 and its genome-reduced chassis (GRC) variants as well as the plastic-degrading strain P. capeferrum TDA1 were assessed regarding their tolerance towards different n -alkanols (1-butanol, 1-hexanol, 1-octanol, 1-decanol). Toxicity of the solvents was investigated by their effects on bacterial growth rates given as the EC50 concentrations. Hereby, both toxicities as well as the adaptive responses of P. taiwanensis GRC3 and P. capeferrum TDA1 showed EC50 values up to two-fold higher than those previously detected for P. putida DOT-T1E (biosafety level 2), one of the best described solvent-tolerant bacteria. Furthermore, in two-phase solvent systems, all the evaluated strains were adapted to 1-decanol as a second organic phase (i.e., OD 560 was at least 0.5 after 24 h of incubation with 1% ( v/v ) 1-decanol), which shows the potential use of these strains as platforms for the bio-production of a wide variety of chemicals at industrial level.

In recent years branched short-chain dicarboxylates (BSCD) such as itaconic acid gained increasing interest in both medicine and biotechnology. Their use as building blocks for plastics urges for developing microbial upcycling strategies to provide sustainable end-of-life solutions. Furthermore, many BSCD exhibit anti-bacterial properties or exert immunomodulatory effects in macrophages, indicating a medical relevance for this group of molecules. For both of these applications, a detailed understanding of the microbial metabolism of these compounds is essential. In this study, the metabolic pathway of BSCD degradation from Pseudomonas aeruginosa PAO1 was studied in detail by heterologously transferring it to Pseudomonas putida. Heterologous expression of the PA0878-0886 itaconate metabolism gene cluster enabled P. putida KT2440 to metabolize itaconate, (S)- and (R)-methylsuccinate, (S)-citramalate, and mesaconate. The functions of the so far uncharacterized genes PA0879 and PA0881 were revealed and proven to extend the substrate range of the core degradation pathway. Furthermore, the uncharacterized gene PA0880 was discovered to encode a 2-hydroxyparaconate (2-HP) lactonase that catalyzes the cleavage of the itaconate derivative 2-HP to itatartarate. Interestingly, 2-HP was found to inhibit growth of the engineered P. putida on itaconate. All in all, this study extends the substrate range of P. putida to include BSCD for bio-upcycling of high-performance polymers, and also identifies 2-HP as promising candidate for anti-microbial applications., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In humans, plantar cutaneous mechanoreceptors provide critical input signals for postural control during walking and running. Because these receptors are located within the dermis, the mechanical properties of the overlying epidermis likely affect the transmission of external stimuli. Epidermal layers are highly adaptable and can form hard and thick protective calluses, but their effects on plantar sensitivity are currently disputed. Some research has shown no effect of epidermal properties on sensitivity to vibrations, whereas other research suggests that vibration and touch sensitivity diminishes with a thicker and harder epidermis. To address this conflict, we conducted an intervention study where 26 participants underwent a callus abrasion while an age-matched control group (n = 16) received no treatment. Skin hardness and thickness as well as vibration perception thresholds and touch sensitivity thresholds were collected before and after the intervention. The Callus abrasion significantly decreased skin properties. The intervention group exhibited no change in vibration sensitivity but had significantly better touch sensitivity. We argue that touch sensitivity was impeded by calluses because hard skin disperses the monofilament's standardized pressure used to stimulate the mechanoreceptors over a larger area, decreasing indentation depth and therefore stimulus intensity. However, vibration sensitivity was unaffected because the vibrating probe was adjusted to reach specific indentation depths, and thus stimulus intensity was not affected by skin properties. Since objects underfoot necessarily indent plantar skin during weight-bearing, calluses should not affect mechanosensation during standing, walking, or running., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Bio-upcycling of plastics is an upcoming alternative approach for the valorization of diverse polymer waste streams that are too contaminated for traditional recycling technologies. Adipic acid and other medium-chain-length dicarboxylates are key components of many plastics including polyamides, polyesters, and polyurethanes. This study endows Pseudomonas putida KT2440 with efficient metabolism of these dicarboxylates. The dcaAKIJP genes from Acinetobacter baylyi, encoding initial uptake and activation steps for dicarboxylates, were heterologously expressed. Genomic integration of these dca genes proved to be a key factor in efficient and reliable expression. In spite of this, adaptive laboratory evolution was needed to connect these initial steps to the native metabolism of P. putida, thereby enabling growth on adipate as sole carbon source. Genome sequencing of evolved strains revealed a central role of a paa gene cluster, which encodes parts of the phenylacetate metabolic degradation pathway with parallels to adipate metabolism. Fast growth required the additional disruption of the regulator-encoding psrA, which upregulates redundant β-oxidation genes. This knowledge enabled the rational reverse engineering of a strain that can not only use adipate, but also other medium-chain-length dicarboxylates like suberate and sebacate. The reverse engineered strain grows on adipate with a rate of 0.35 ± 0.01 h -1 , reaching a final biomass yield of 0.27 ± 0.00 g CDW g adipate -1 . In a nitrogen-limited medium this strain produced polyhydroxyalkanoates from adipate up to 25% of its CDW. This proves its applicability for the upcycling of mixtures of polymers made from fossile resources into biodegradable counterparts., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts. Species of the Pseudomonas clade thus represent especially valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chemicals. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compounds. Finally, toxic chemicals such as reactive aldehydes can be eliminated by oxidation and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective production of natural compounds and new chemicals. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnological context. We further highlight strategies for the identification of yet unknown tolerance-associated genes and their utilisation for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective production of natural compounds and novel biochemicals., (© 2021 The Author(s).)

Determining vibration perception thresholds (VPT) is a central concern of clinical research and science to assess the somatosensory capacity of humans. The response of different mechanoreceptors to an increasing contact force has rarely been studied. We hypothesize that increasing contact force leads to a decrease in VPTs of fast-adapting mechanoreceptors in the sole of the human foot. VPTs of 10 healthy subjects were measured at 30 Hz and 200 Hz at the heel of the right foot using a vibration exciter. Contact forces were adjusted precisely between 0.3 N-9.6 N through an integrated force sensor. Significant main effects were found for frequency and contact force. Furthermore, there was a significant interaction for frequency and contact force, meaning that the influence of an increasing contact force was more obvious for the 30 Hz condition. We presume that the principles of contrast enhancement and spatial summation are valid in Meissner and Pacinian corpuscles, respectively. In addition to spatial summation, we presume an effect on Pacinian corpuscles due to their presence in the periosteum or interosseous membrane.

A diverse group of Canadian experts was convened for a focused 2-d discussion on potential health and welfare problems associated with the marketing (i.e., transportation and sale) of male dairy calves. Written notes and audio recording were used to summarize the information provided on transport times and marketing practices. Content analysis was used to develop a consensus statement on concerns, possible solutions, and recommendations to improve male dairy calf marketing. The group noted that calves across all Canadian regions are commonly transported at 3 to 7 d of age and undergo transport for 12 to 24 h or longer depending on the location of their dairy farm of origin. Calves in some regions are marketed almost exclusively through auction markets, whereas others have more direct sales. A need was identified for better criteria for calf fitness for transport, maintaining farm biosecurity, reducing the use of antimicrobial therapy in calf production, and improving education for farmers and veterinarians on the importance of neonatal care for male dairy calves before transportation. Experts noted that major changes in male dairy calf marketing will be required to comply with amendments to the federal Health of Animals Regulations (Part XII) on animal transportation; collaborative effort will be needed to safeguard animal health and welfare as this transition is made., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Benzoic acid is one of the most commonly used food preservatives, but currently exclusively produced in petrochemical processes. In this study, a bio-based production pathway using an engineered strain of Pseudomonas taiwanensis is described. In a phenylalanine-overproducing strain, bacterial and plant genes are heterologously expressed to achieve production of benzoate via a β-oxidation pathway. Strategic disruption of the native Pseudomonas benzoate degradation pathway further allows the production of catechol and cis,cis-muconate. Taken together, this work demonstrates new routes for the microbial production of these industrially relevant chemicals from renewable resources., (© 2020 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Aromatics and their derivatives are valuable chemicals with a plethora of important applications and thus play an integral role in modern society. Their current production relies mostly on the exploitation of petroleum resources. Independency from dwindling fossil resources and rising environmental concerns are major driving forces for the transition towards the production of sustainable aromatics from renewable feedstocks or waste streams. Whole-cell biocatalysis is a promising strategy that allows the valorization of highly abundant, low-cost substrates. In the last decades, extensive efforts are undertaken to allow the production of a wide spectrum of different aromatics and derivatives using microbes as biocatalysts. Pseudomonads are intriguing hosts for biocatalysis, as they display unique characteristics beneficial for the production of aromatics, including a distinct tolerance and versatile metabolism. This review highlights biotechnological applications of Pseudomonas as host for the production of aromatics and derived compounds. This includes their de novo biosynthesis from renewable resources, biotransformations in single- and biphasic fermentation setups, metabolic funneling of lignin-derived aromatics, and the upcycling of aromatic monomers from plastic waste streams. Additionally, this review provides insights into unique features of Pseudomonads that make them exceptional hosts for aromatics biotechnology and discusses engineering strategies., (© 2020 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

The nitrogen-related phosphotransferase system (PTS Ntr ) of Rhizobium leguminosarum bv. viciae 3841 transfers phosphate from PEP via PtsP and NPr to two output regulators, ManX and PtsN. ManX controls central carbon metabolism via the tricarboxylic acid (TCA) cycle, while PtsN controls nitrogen uptake, exopolysaccharide production, and potassium homeostasis, each of which is critical for cellular adaptation and survival. Cellular nitrogen status modulates phosphorylation when glutamine, an abundant amino acid when nitrogen is available, binds to the GAF sensory domain of PtsP, preventing PtsP phosphorylation and subsequent modification of ManX and PtsN. Under nitrogen-rich, carbon-limiting conditions, unphosphorylated ManX stimulates the TCA cycle and carbon oxidation, while unphosphorylated PtsN stimulates potassium uptake. The effects are reversed with the phosphorylation of ManX and PtsN, occurring under nitrogen-limiting, carbon-rich conditions; phosphorylated PtsN triggers uptake and nitrogen metabolism, the TCA cycle and carbon oxidation are decreased, while carbon-storage polymers such as surface polysaccharide are increased. Deleting the GAF domain from PtsP makes cells "blind" to the cellular nitrogen status. PTS Ntr constitutes a switch through which carbon and nitrogen metabolism are rapidly, and reversibly, regulated by protein:protein interactions. PTS Ntr is widely conserved in proteobacteria, highlighting its global importance., Competing Interests: The authors declare no competing interest.

Microbial biocatalysis represents a promising alternative for the production of a variety of aromatic chemicals, where microorganisms are engineered to convert a renewable feedstock under mild production conditions into a valuable chemical building block. This study describes the rational engineering of the solvent-tolerant bacterium Pseudomonas taiwanensis VLB120 toward accumulation of L-phenylalanine and its conversion into the chemical building block t -cinnamate. We recently reported rational engineering of Pseudomonas toward L-tyrosine accumulation by the insertion of genetic modifications that allow both enhanced flux and prevent aromatics degradation. Building on this knowledge, three genes encoding for enzymes involved in the degradation of L-phenylalanine were deleted to allow accumulation of 2.6 mM of L-phenylalanine from 20 mM glucose. The amino acid was subsequently converted into the aromatic model compound t -cinnamate by the expression of a phenylalanine ammonia-lyase (PAL) from Arabidopsis thaliana . The engineered strains produced t -cinnamate with yields of 23 and 39% Cmol Cmol -1 from glucose and glycerol, respectively. Yields were improved up to 48% Cmol Cmol -1 from glycerol when two enzymes involved in the shikimate pathway were additionally overexpressed, however with negative impact on strain performance and reproducibility. Production titers were increased in fed-batch fermentations, in which 33.5 mM t -cinnamate were produced solely from glycerol, in a mineral medium without additional complex supplements. The aspect of product toxicity was targeted by the utilization of a streamlined, genome-reduced strain, which improves upon the already high tolerance of P. taiwanensis VLB120 toward t -cinnamate., (Copyright © 2019 Otto, Wynands, Lenzen, Filbig, Blank and Wierckx.)

Microbes harbor many traits that are dispensable or even unfavorable under industrial and laboratory settings. The elimination of such traits could improve the host's efficiency, genetic stability, and robustness, thereby increasing the predictability and boosting its performance as a microbial cell factory. We engineered solvent-tolerant Pseudomonas taiwanensis VLB120 to yield streamlined chassis strains with higher growth rates and biomass yields, enhanced solvent tolerance, and improved process performance. In total, the genome was reduced by up to 10%. This was achieved by the elimination of genes that enable the cell to swim and form biofilms and by the deletion of the megaplasmid pSTY and large proviral segments. The resulting strain GRC1 had a 15% higher growth rate and biomass yield than the wildtype. However, this strain lacks the pSTY-encoded efflux pump TtgGHI, rendering it solvent-sensitive. Through reintegration of ttgGHI by chromosomal insertion without (GRC2) and with (GRC3) the corresponding regulator genes, the solvent-tolerant phenotype was enhanced. The generated P. taiwanensis GRC strains enlarge the repertoire of streamlined chassis with enhanced key performance indicators, making them attractive hosts for biotechnological applications. The different solvent tolerance levels of GRC1, GRC2, and GRC3 enable the selection of a fitting host platform in relation to the desired process requirements in a chassis à la carte principle. This was demonstrated in a metabolic engineering approach for the production of phenol from glycerol. The streamlined producer GRC1Δ5-TPL38 outperformed the equivalent nonstreamlined producer VLB120Δ5-TPL38 concerning phenol titer, rate, and yield, thereby highlighting the added value of the streamlined chassis.

Ribosomal RNA (rRNA) operons have recently been identified as promising sites for chromosomal integration of genetic elements in Pseudomonas putida , a bacterium that has gained considerable popularity as a microbial cell factory. We have developed a tool for targeted integration of recombinant genes into the rRNA operons of various Pseudomonas strains, where the native context of the rRNA clusters enables effective transcription of heterologous genes. However, a sufficient translation of foreign mRNA  transcriptionally fused to rRNA required optimization of RNA secondary structures, which was achieved utilizing synthetic ribozymes and a bicistronic design. The generated tool further enabled the characterization of the six rRNA promoter units of P. putida S12 under different growth conditions. The presence of multiple, almost identical rRNA operons in Pseudomonas also allowed the integration of multiple copies of heterologous genetic elements. The integration of two expression cassettes and the resulting disruption of rRNA units only moderately affects growth rates, and the constructs were highly stable over more than 160 generations.

Until relatively recently, humans, similar to other animals, were habitually barefoot. Therefore, the soles of our feet were the only direct contact between the body and the ground when walking. There is indirect evidence that footwear such as sandals and moccasins were first invented within the past 40 thousand years 1 , the oldest recovered footwear dates to eight thousand years ago 2 and inexpensive shoes with cushioned heels were not developed until the Industrial Revolution 3 . Because calluses-thickened and hardened areas of the epidermal layer of the skin-are the evolutionary solution to protecting the foot, we wondered whether they differ from shoes in maintaining tactile sensitivity during walking, especially at initial foot contact, to improve safety on surfaces that can be slippery, abrasive or otherwise injurious or uncomfortable. Here we show that, as expected, people from Kenya and the United States who frequently walk barefoot have thicker and harder calluses than those who typically use footwear. However, in contrast to shoes, callus thickness does not trade-off protection, measured as hardness and stiffness, for the ability to perceive tactile stimuli at frequencies experienced during walking. Additionally, unlike cushioned footwear, callus thickness does not affect how hard the feet strike the ground during walking, as indicated by impact forces. Along with providing protection and comfort at the cost of tactile sensitivity, cushioned footwear also lowers rates of loading at impact but increases force impulses, with unknown effects on the skeleton that merit future study.

Aromatic compounds such as 4-hydroxybenzoic acid are broadly applied in industry for a myriad of applications used in everyday life. However, their industrial production currently relies heavily on fossil resources and involves environmentally unfriendly production conditions, thus creating the need for more sustainable biotechnological alternatives. In this study, synthetic biology was applied to metabolically engineer Pseudomonas taiwanensis VLB120 to produce 4-hydroxybenzoate from glucose, xylose, or glycerol as sole carbon sources. Genes encoding a 4-hydroxybenzoate production pathway were integrated into the host genome and the flux toward the central precursor tyrosine was enhanced by overexpressing genes encoding key enzymes of the shikimate pathway. The flux toward tryptophan biosynthesis was decreased by introducing a P290S point mutation in the trpE gene, and degradation pathways for 4-hydroxybenzoate, 4-hydroxyphenylpyruvate and 3-dehydroshikimate were knocked out. The resulting production strains were tailored for the utilization of glucose and glycerol through the rational modification of central carbon metabolism. In batch cultivations with a completely mineral medium, the best strain produced 1.37 mM 4-hydroxybenzoate from xylose with a C-mol yield of 8% and 3.3 mM from glucose with a C-mol yield of 19.0%. Using glycerol as a sole carbon source, the C-mol yield increased to 29.6%. To our knowledge, this is the highest yield achieved by any species in a fully mineral medium. In all, the efficient conversion of bio-based substrates into 4-hydroxybenzoate by these deeply engineered P. taiwanensis strains brings the renewable production of aromatics one step closer.

Aromatic chemicals are important building blocks for the production of a multitude of everyday commodities. Currently, aromatics production relies almost exclusively on petrochemical processes. To achieve sustainability, alternative synthesis methods need to be developed. Here, we strived for an efficient production of phenol, a model aromatic compound of industrial relevance, from renewable carbon sources using the solvent-tolerant biocatalyst Pseudomonas taiwanensis VLB120. First, multiple catabolic routes for the degradation of aromatics and related compounds were inactivated, thereby obtaining the chassis strain P. taiwanensis VLB120Δ5 incapable of growing on 4-hydroxybenzoate (ΔpobA), tyrosine (Δhpd), and quinate (ΔquiC, ΔquiC1, ΔquiC2). In this context, a novel gene contributing to the quinate catabolism was identified (quiC2). Second, we employed a combination of reverse- and forward engineering to increase metabolic flux towards the product, using leads obtained from the analysis of aromatics producing Pseudomonas putida strains previously generated by mutagenesis. Phenol production was enabled by the heterologous expression of a codon-optimized and chromosomally integrated tyrosine phenol-lyase encoding gene from Pantoea agglomerans AJ2985 (PaTPL2). The genomic modification of endogenous genes encoding TrpE P290S , AroF-1 P148L , and PheA T310I , and the deletion of pykA improved phenol production 17-fold, while also minimizing the burden caused by plasmids and auxotrophies. The additional overexpression of known bottleneck enzymes (AroG fbr , TyrA fbr ) derived from Escherichia coli further enhanced phenol titers. The best producing strain P. taiwanensis VLB120Δ5-TPL36 reached yields of 15.8% and 18.5% (Cmol/Cmol) phenol from glucose and glycerol, respectively, in a mineral medium without addition of complex nutrients. This is the highest yield ever reported for microbially produced phenol., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)