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Novel synthetic strategies for the production of high-value chemicals based on the 12 principles of green chemistry are highly desired. Herein, we present a proof of concept for two novel chemo-enzymatic one-pot cascades allowing for the production of valuable fragrance and flavor aldehydes. We utilized renewable phenylpropenes, such as eugenol from cloves or estragole from estragon, as starting materials. For the first strategy, Pd-catalyzed isomerization of the allylic double bond and subsequent enzyme-mediated (aromatic dioxygenase, ADO) alkene cleavage were performed to obtain the desired aldehydes. In the second route, the double bond was oxidized to the corresponding ketone via a copper-free Wacker oxidation protocol followed by enzymatic Baeyer–Villiger oxidation (phenylacetone monooxygenase from Thermobifida fusca), esterase-mediated (esterase from Pseudomonas fluorescens, PfeI) hydrolysis and subsequent oxidation of the primary alcohol (alcohol dehydrogenase from Pseudomonas putida, AlkJ) to the respective aldehyde products. Eight different phenylpropene derivatives were subjected to these reaction sequences, allowing for the synthesis of seven aldehydes in up to 55% yield after 4 reaction steps (86% for each step). [ABSTRACT FROM AUTHOR]

2-Hydroxy-3-pentanone and 3-hydroxy-2-pentanone are flavor molecules present in various foods, such as cheese, wine, durian, and honey, where they impart buttery, hay-like, and caramel-sweet aromas. However, their utilization as flavoring agents is constrained by a lack of developed synthesis methods. In this study, we present their synthesis from simple starting compounds available in natural quality, catalyzed by previously characterized ThDP-dependent carboligases. Additionally, we demonstrate that newly discovered homologues of pyruvate dehydrogenase from E. coli (Ec PDH E1), namely La PDH from Leclercia adecarboxylata , Cn PDH from Cupriavidus necator , and Tc PDH from Tanacetum cinerariifolium , exhibit promising potential for α-hydroxy pentanone synthesis in form of whole-cell biocatalysts. Enzyme stability at varying pH levels, kinetic parameters, and reaction intensification were investigated. Cn PDH, for example, exhibits superior stability across different pH levels compared to Ec PDH E1. Both α-hydroxy pentanones can be produced with Cn PDH in satisfactory yields (74% and 59%, respectively). • Synthesis of aroma constituents of wine, cheese and durian by ThDP-dependent carboligases from simple starting compounds. • Heterologous production and functional characterization of homologues of pyruvate dehydrogenase from E. coli (Ec PDH E1). • Cupriavidus necator PDH is more stable across different pH levels compared to Ec PDH E1. • Product distribution of carboligations can be tuned by choice of enzyme and also by choice of donor and acceptor substrate. [ABSTRACT FROM AUTHOR]

Thiamine diphosphate (ThDP) dependent carboligases catalyze the formation of carbon‐carbon bonds. These enzymes are prominent biocatalysts for the production of valuable α‐hydroxy carbonyl compounds, which serve as key building blocks in the synthesis of various pharmaceuticals and fine chemicals. Carboligases act in selective manner to afford regio‐ and stereochemically defined products from simple starting materials. This review explores the catalytic prowess of carboligases for synthetic purposes and is aimed at providing a selection tool of enzymes for particular sets of substrates or desired products. The comprehensive overview encompasses structural insights, relationships of the currently known sequence space and practical applications with a focus on recent literature, showcasing carboligases as potent tools for sustainable and efficient synthesis. [ABSTRACT FROM AUTHOR]

Nitriles have a wide range of uses as building blocks, solvents, and alternative fuels, but also as intermediates and components of flavors and fragrances. The enzymatic synthesis of nitriles by aldoxime dehydratase (Oxd) is an emerging process with significant advantages over conventional approaches. Here we focus on the immobilization of His-tagged Oxds on metal affinity resins, an approach that has not been used previously for these enzymes. The potential of the immobilized Oxd was demonstrated for the synthesis of phenylacetonitrile (PAN) and E -cinnamonitrile, compounds applicable in the fragrance industry. A comparison of Talon and Ni-NTA resins showed that Ni-NTA with its higher binding capacity was more suitable for the immobilization of Oxd. Immobilized Oxds were prepared from purified enzymes (OxdFv from Fusarium vanettenii and OxdBr1 from Bradyrhizobium sp.) or the corresponding cell-free extracts. The immobilization of cell-free extracts reduced time and cost of the catalyst production. The immobilized OxdBr1 was superior in terms of recyclability (22 cycles) in the synthesis of PAN from 15 mM E / Z -phenylacetaldoxime at pH 7.0 and 30 °C (100% conversion, 61% isolated yield after product purification). The volumetric and catalyst productivity was 10.5 g/L/h and 48.3 g/g of immobilized protein, respectively. [Display omitted] • Aldoxime dehydratases were immobilized on metal affinity resins, preferably Ni-NTA. • Immobilization of purified enzymes and cell-free extracts gave active catalysts. • Phenylacetonitrile and E -cinnamonitrile were obtained from the respective oximes. • Immobilized OxdBr1 from Bradyrhizobium was recycled over 20 times. • The catalyst productivity was 48 mg phenylacetonitrile/mg protein. [ABSTRACT FROM AUTHOR]

Mycobacterium marinum CAR (Mm CAR) is one of the most widely used CARs as the key enzyme for the synthesis of aldehydes, alcohols and further products from the respective carboxylic acids. Herein, we describe the first functionally secreted 131 kDa CAR and its isolated A-domain using Komagataella phaffii and a methanol-free constitutive expression strategy. Precipitated and lyophilized Mm CAR (500 µg) was isolated from the culture supernatant and showed no decrease in activity for piperonylic acid (80% conversion), even when stored for up to 3 weeks at 4°C. Lyophilized Mm CAR precipitate gave 48% yield of E/Z -nonanal-4-nitrobenzoyloxime from the reduction of nonanoic acid and in-situ derivatization with O -4-nitrobenzoyl-hydroxylamine. Furthermore, K. phaffii could successfully secrete the Mm CAR adenylation domain. Its activity was confirmed by the amidation of benzoic acid with n -hexylamine. Neither enzyme variant was glycosylated by the yeast. In summary, functional CAR can be secreted by K. phaffii and used for cell free conversion of carboxylic acids to various products. [Display omitted] • Active full-length Mm CAR and Mm CAR A-domain were secreted by Komagataella phaffii for the first time. • Mm CAR A-domain was active for amidation of benzoic acid with n-hexylamine. • Precipitated, lyophilized full-length Mm CAR can be stored without activity loss at 4°C for at least 3 weeks. [ABSTRACT FROM AUTHOR]

Background: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. Results: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB− 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. Conclusion: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry. [ABSTRACT FROM AUTHOR]

Background: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. Results: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB− 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. Conclusion: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry. [ABSTRACT FROM AUTHOR]