Preparation of 20-HETE using multifunctional enzyme type 2-negative Starmerella bombicola

Abstract

(19-HETE) and 20-HETE ( 2 ). 20-HETE has been shown to have an array of largely detrimental effects, inducing hypertension, endothelial dysfunction, infl ammation, cardiovascular diseases, angiogenesis, and tumor growth ( 3-8 ). Our recent research has shown that long-term administration of COX inhibitor rofecoxib caused a greater than 100-fold increase of circulating 20-HETE in a murine model, which suggests that the 20-HETE pathway contributes to the cardiovascular risks of COX inhibitors ( 9 ). More studies are needed to investigate the biological activities and mechanisms of 20-HETE.Development of a scalable method to prepare 20-HETE would greatly facilitate the evaluation of its biological activities and potential therapeutic applications, particularly in animal experiments that require relatively large amount of materials. 20-HETE is commercially available but prohibitively expensive ($4,000 per mg from Caymen Chemicals, Ann Arbor, MI). Chemical synthesis of 20-HETE has been reported ( 10 ); however, the multistep synthesis was challenging to carry out. Enzymatic conversion of ARA to 20-HETE using CYP enzyme(s) is attractive; however, the reaction is low-yield, is diffi cult to scale up, and requires the expensive cofactor NADPH or enzyme systems that generate NADPH. Biotransformation utilizing the nonpathogenic yeast Starmerella bombicola , which expresses CYP / -1-hydroxylases such as CYP52M1, is a promising alternative strategy ( 11 ). This yeast is known for its ability to produce sophorolipids, whose biosynthesis involves hydroxylation of fatty acids at the terminal or subterminal positions by the action of CYP / -1-hydroxylases in order to govern glycosidical coupling to the di-glucoside sophorose. The obtained sophorolipids can undergo further The lipid mediators derived from the arachidonic acid (ARA) cascade are important because these signaling lipids regulate many critical biological processes from infl ammation to tumorigenesis ( 1 ). Therefore, they are important therapeutic targets for many human diseases, and a significant proportion of drugs on the market targets lipid signaling of the ARA cascade. These lipid mediators are best known as prostaglandins and leukotrienes derived from the cyclooxygenase (COX) and lipoxygenase (LOX) pathways, respectively ( 1 ). Besides the intensively studied COX and LOX pathways, ARA is also a substrate of cytochrome P450 (CYP) / -1-hydroxylases (mainly CYP4A and CYP4F), which metabolize it to 19-hydroxyeicosatetraenoic acid This work was supported by National Institutes of Health Grants U24 DK097154, R01 ES-02710 (to B.D.H.), and P42 ES04699 (to B.D.H.

A hybrid strategy using global analysis of oxidized fatty acids and bioconversion by Bacillus circulans

“…To identify 21‐HDoHE and 22‐HDoHE clearly, their standards were synthesized by bioconversion, which is suitable for relatively rapid synthesis of a variety of metabolites. Omega and ω‐1 hydroxy fatty acids such as 20‐HETE and 19‐HETE are synthesized by cytochrome P450 (CYP), and 21‐HDoHE and 22‐HDoHE are also synthesized by CYP . Thus, we screened a microbial library for the synthesis of 21‐HDoHE and 22‐HDoHE.…”

“…If any authentic standards are not available, they need to be synthesized. Lipid standard synthesis is carried out in three major ways: chemical synthesis, enzymatic assay, and bioconversion using bacteria . Each approach has advantages and disadvantages.…”

“…Lipid standard synthesis is carried out in three major ways: chemical synthesis, [26][27][28] enzymatic assay, [29][30][31] and bioconversion using bacteria. [32][33][34][35] Each approach has advantages and disadvantages. In chemical synthesis, the molecule of interest can be synthesized specifically and stereoisomers can be discerned.…”