Biotransformation of lauric acid into 1,12-dodecanedioic acid using CYP52A17 expressed in Saccharomyces cerevisiae and its application in refining coconut factory wastewater

1,12-Dodecanedioic acid (DDA), a primary compound and an intermediate precursor for various chemicalproducts, is normally produced by chemical synthesis, which presents potential disadvantages. Instead, thebiosynthesis of 1,12-DDA by recombinant (r) microorganisms may offer a viable production route. CytochromeP450 (CYP) can terminally oxidize fatty acids to ω-hydroxy-fatty acids and further to dicarboxylic acids (ω-oxidation). The wild type (r) CYP52A17LL and its engineered L261S/L490S form, in which the two leucineresidues were changed into serine (rCYP52A17SS), were expressed in Pichia pastoris and a strain of Saccharomycescerevisiae coexpressing the yeast NADPH cytochrome P450 reductase gene. Both organisms produced 12-hydroxydodecanoicacid (HDDA) and 1,12-DDA from lauric acid. In vitro, microsomes containing rCYP52A17SSextracted from the S. cerevisiae strain BY(2R)/pYeDP60-CYP52A17SS were able to convert lauric acid to 12-HDDA quite efficiently. Biotransformation of lauric acid using S. cerevisiae BY(2R)/pYeDP60-CYP52A17SS inculture gave the highest level of 12-HDDA (45.8 μM) at 24 h, which was oxidized to yield 20.8 μM of 1,12-DDA at72 h. The recombinant S. cerevisiae BY(2R)/pYeDP60-CYP52A17SS, which was initially cultured in YPGE, producedthe highest yield of 1,12-DDA from coconut milk wastewater at 24 h. Hence, our designed S. cerevisiaestrain BY(2R)/pYeDP60-CYP52A17SS can potentially produce 1,12-DDA for industrial applications.1. Introductionα,ω-Dicarboxylic acids (DCAs) are important versatile chemicalintermediates for the preparation of nylon and other polyamides,polyesters, perfumes, polymers, lubricants, cosmetic ingredients andcoatings. For example, dodecanedioic acid (DDA) is the precursor ofpolyamide 6,12 (nylon 6,12) (Funk et al., 2017; Huf et al., 2011;Waché, 2013). Currently, DCA production is mainly performed viachemical synthesis, but this has limitations and drawbacks, such as thesynthesis of unwanted by-products, the cost of removal of these alternatemolecules produce

Whole-Cell Biotransformation of 1,12-Dodecanedioic Acid from Coconut Milk Factory Wastewater by Recombinant CYP52A17 SS Expressing Saccharomyces cerevisiae

Biotransformation of fatty acids from renewable wastewater as feedstock to value-added chemicals is a fascinating commercial opportunity. α,ω-Dicarboxylic acids (DCAs) are building blocks in many industries, such as polymers, cosmetic intermediates, and pharmaceuticals, and can be obtained by chemical synthesis under extreme conditions. However, biological synthesis can replace the traditional chemical synthesis using cytochrome P450 enzymes to oxidize fatty acids to DCAs. Saccharomyces cerevisiae BY(2R)/pYeDP60-CYP52A17 SS (BCM), a transgenic strain expressing the galactose-inducible CYP52A17 SS cytochrome P450 enzyme, was able to grow in a coconut milk factory wastewater (CCW) medium and produced 12-hydroxydodecanoic acid (HDDA) and 1,12-dodecanedioic acid (DDA). The supplementation of CCW with 10 g/L yeast extract and 20 g/L peptone (YPCCW) markedly increased the yeast growth rate and the yields of 12-HDDA and 1,12-DDA, with the highest levels of approximately 60 and 38 µg/L, respectively, obtained at 30 • C and pH 5. The incubation temperature and medium pH strongly influenced the yeast growth and 1,12-DDA yield, with the highest 1,12-DDA formation at 30 • C and pH 5-5.5. Hence, the S. cerevisiae BCM strain can potentially be used for producing value-added products from CCW