Production of C20 9S- and C22 11S-hydroxy fatty acids by cells expressing Shewanella hanedai arachidonate 9S-lipoxygenase.

Autor: Kim MJ; Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea., Lee J; Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea., Kim SE; Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea., Shin KC; Department of Integrative Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea., Oh DK; Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea. deokkun@konkuk.ac.kr.; Department of Integrative Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea. deokkun@konkuk.ac.kr.
Jazyk: angličtina
Zdroj: Applied microbiology and biotechnology [Appl Microbiol Biotechnol] 2023 Jan; Vol. 107 (1), pp. 247-260. Date of Electronic Publication: 2022 Nov 28.
DOI: 10.1007/s00253-022-12285-3
Abstrakt: The putative lipoxygenase (LOX) from the proteobacterium Shewanella hanedai was determined to be an 82 kDa monomeric enzyme by SDS-PAGE and gel filtration chromatography analysis. LOX was identified as a single-dioxygenating arachidonate (ARA) 9S-LOX by analyzing ARA-derived bioconversion products using high-performance liquid chromatography with reverse-, normal-, and chiral-phase columns and evaluating kinetic parameters for C20- and C22-polyunsaturated fatty acids (PUFAs). The catalytic efficiency (k cat /K m ) values of 9S-LOX from S. hanedai for ARA, eicosapentaenoic acid, and docosahexaenoic acid were 3.1-, 4.1-, and 2.5-fold higher, respectively, than those only reported 9S-LOX from Sphingopyxis macrogoltabida with double-dioxygenating activity. To promote the production of C20 9S- and C22 11S-hydroxy fatty acids (HFAs) using Escherichia coli expressing 9S-LOX from S. hanedai, bioconversion conditions, including temperature, pH, solvent type and its concentration, concentrations of cells, and substrate, were optimized to 25 °C, pH 8.5, 6% (v/v) dimethyl sulfoxide, 0.2 g/l cells, and 7 mM ARA as substrate in a 500 ml-Erlenmeyer baffled flask with 50 ml reaction solution with agitation at 200 rpm in the presence of 10 mM cysteine as a reduction agent, respectively. Under these conditions, 6.4 mM 9S-hydroxyeicosatetraenoic acid, 6.2 mM 9S-hydroxyeicosapentaenoic acid, and 5.9 mM 11S-hydroxydocosahexaenoic acid were produced in 30 min, 40 min, and 60 min with specific productivities of 1067 μmol/min/g, 775 μmol/min/g, and 492 μmol/min/g, volumetric productivities of 213 μM/min, 155 μM/min, and 98 μM/min, and conversion yields of 91.4%, 88.6%, and 84.3%, respectively. To date, these are the highest specific productivities reported for the bioconversion of C20- and C22-PUFAs into HFAs. KEY POINTS: • Lipoxygenase from Shewanella hanedai was identified as arachidonate 9S-lipoxygenase • Optimization led to increased production of C20 9S- and C22 11S-hydroxy fatty acids • We reported the highest specific productivities of C20- and C22-hydroxy fatty acids.
(© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
Databáze: MEDLINE
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