Cell-free reduction of carboxylic acids with secreted carboxylic acid reductase

0584677 - MBÚ 2025 RIV NL eng J - Journal Article
Goj, D. - Ebner, S. - Horvat, M. - Arhar, S. - Martínková, Ludmila - Winkler, M.
Cell-free reduction of carboxylic acids with secreted carboxylic acid reductase.
Journal of Biotechnology. Roč. 382, 20 February (2024), s. 44-50. ISSN 0168-1656. E-ISSN 1873-4863
R&D Projects: GA ČR(CZ) GF20-23532L
Institutional support: RVO:61388971
Keywords : expression * Carboxylic acid reductase * Komagataella phaffii * Biocatalysis * Natural nonanal * Bioconversion * Protein secretion
OECD category: Biochemistry and molecular biology
Impact factor: 4.1, year: 2022
Method of publishing: Open access
https://www.sciencedirect.com/science/article/pii/S0168165624000166?via%3Dihub

Mycobacterium marinum CAR (MmCAR) 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 MmCAR (500 mu g) 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 degrees C. Lyophilized MmCAR 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 MmCAR 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.
Permanent Link: https://hdl.handle.net/11104/0352585