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

Abstract

The putative lipoxygenase (LOX) from the proteobacterium Shewanella hanedai was determined to be an 82kDa 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 (kcat/Km) 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, 0.2g/l cells, and 7mM ARA as substrate in a 500ml-Erlenmeyer baffled flask with 50ml reaction solution with agitation at 200rpm in the presence of 10mM cysteine as a reduction agent, respectively. Under these conditions, 6.4mM 9S-hydroxyeicosatetraenoic acid, 6.2mM 9S-hydroxyeicosapentaenoic acid, and 5.9mM 11S-hydroxydocosahexaenoic acid were produced in 30min, 40min, and 60min with specific productivities of 1067μmol/min/g, 775μmol/min/g, and 492μmol/min/g, volumetric productivities of 213μM/min, 155μM/min, and 98μM/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.