Chemoenzymatic Cascade Conversion of Linoleic Acid into a Secondary Fatty Alcohol Using a Combination of 13S-Lipoxygenase, Chemical Reduction, and a Photo-Activated Decarboxylase

Abstract

Linoleic acid serves as a starting material in the production of various oleochemicals. Here, we have investigated the transformation of linoleic acid into 13S-hydroxy-(9Z,11E)-octadecadienoic acid (13-HOD) (17) and 6S-hydroxy-(7E,9Z)heptadecadiene (6-HHD) (18) by using 13S-lipoxygenase from Pseudomonas aeruginosa (Pa-LOX) and photo-activated decarboxylase from Chlorella variabilis NC64A (Cv-FAP). Remarkably, the recombinant Escherichia coli expressing Pa-LOX was able to produce 13S-hydroperoxy-(9Z,11E)-octadecadienoic acid ( 16) at a maximum rate of 850 μmol/g dry cells/min. This allowed the accumulation of 13-HOD (17) to 161 mM (48 g/L) concentration from 200 mM linoleic acid in the reaction medium within 3.5 h. We have also demonstrated that the fatty acids, including CC bonds in cis-and trans-forms [e.g., 13-HOD (17)], were subjected to photo-activated decarboxylation by Cv-FAP. Ultimately, the secondary fatty alcohol [i.e., 6-HHD ( 18)] was produced from linoleic acid through the chemo/enzymatic cascade transformation, consisting of dioxygenation of linoleic acid by intracellular Pa-LOX and reduction of the hydroperoxy fatty acid (16) by Tris(2-carboxyethyl) phosphine or cysteine. Moreover, the photoactivated decarboxylation of the hydroxy fatty acid (17) by intracellular Cv-FAP achieved a conversion of ca. 74% in a one-pot process. This study will contribute to the valorization of γ-linolenic and arachidonic acid, as well as linoleic acid, which are the substrates of Pa-LOX.

Valorization of Soy Lecithin by Enzyme Cascade Reactions Including a Phospholipase A2, a Fatty Acid Double-Bond Hydratase, and/or a Photoactivated Decarboxylase

“…The Cv-FAP expression was induced by the addition of each 0.5 mM IPTG into the cultivation medium to facilitate soluble expression in E. coli in 20 °C, 200 rpm. 35,36 Chemicals and Reagents. L-α-Phosphatidylcholine from soybean (Type II-S, 14−29% choline basis, P5638), antibiotics, sulfuric acid, Tris-EDTA buffer solution, IPTG, and pyridine were purchased from Sigma-Aldrich (St. Louis, MO, USA).…”

“…The Cv-FAP expression was induced by the addition of each 0.5 mM IPTG into the cultivation medium to facilitate soluble expression in E. coli in 20 °C, 200 rpm. , …”

Production of 8,11‐dihydroxy fatty acids from oleic and palmitoleic acids by Escherichia coli cells expressing variant 6,8‐linoleate diol synthases from Penicillium oxalicum

“…Hydroxy fatty acids (HFAs), which can be derived from unsaturated fatty acids by the action of fatty acid oxygenases, exhibit higher reactivity, hydrophilicity, viscosity, and miscibility in polar solvents than unsaturated fatty acids. [1][2][3][4][5] These properties have facilitated the use of HFAs as precursors of carboxylic acids and lactones 6,7 ; additives in paints, emulsifiers, and stabilizers; 8 as well as starting materials for biopolymers, polyurethanes, and plastics. 9 Dihydroxy fatty acids (DiHFAs) are considered to be better surfactants than monoHFAs 10 and have been applied in the manufacture of skincare products and rigid polyurethane foam.…”

A light-controlled biocatalytic system for precise regulation of enzymatic decarboxylation

“…Hollmann lab and Park lab successfully developed CvFAP-dependent enzymatic cascades to produce valuable chemicals. [4][5][6][7] Via protein engineering, the range of CvFAP substrates was extended from fatty acids to natural and unnatural hydroxy acids or amino acids in Wu's lab, Scrutton's lab, and our lab, where D,L-phosphinothricin (D,L-PPT) was investigated as a substrate for CvFAP. [8][9][10][11][12] Another interesting advancement in optogenetic fields includes a series of circuits for optogenetic regulation of the lac operon (OptoLAC) developed to control gene expression under blue light.…”

“…Hollmann lab and Park lab successfully developed Cv FAP-dependent enzymatic cascades to produce valuable chemicals. 4–7 Via protein engineering, the range of Cv FAP substrates was extended from fatty acids to natural and unnatural hydroxy acids or amino acids in Wu's lab, Scrutton's lab, and our lab, where d , l -phosphinothricin ( d , l -PPT) was investigated as a substrate for Cv FAP. 8–12…”