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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]

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]