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.

A huge amount of phospholipids or lecithin is produced as a byproduct in the vegetable oil industry. However, most are just used as a feed additive. This study has focused on enzymatic valorization of lecithin. This was exploited by enzymatic transformation of soy lecithin into lysolecithin liposomes, including functional free fatty acids, hydroxy fatty acids, hydrocarbons, or secondary fatty alcohols. One of the representative examples was the preparation of lysolecithin liposomes containing secondary fatty alcohols [e.g., 9-Hydroxyheptadec-11-ene (9) and 9-heptadecanol (10)] by using a phospholipase A2 from Streptomyces violaceoruber, a fatty acid double-bond hydratase from Stenotrophomonas maltophilia, and a photoactivated decarboxylase from Chlorella variabilis NC64A. The engineered liposomes turned out to range ca. 144 nm in diameter by dynamic light scattering analysis. Thereby, this study will contribute to application of functional fatty acids and their derivatives as well as valorization of lecithin for the food and cosmetic industries.

Secretory phospholipase A2 (sPLA2), which hydrolyzes the sn-2 acyl bond of lecithin in a Ca2+-dependent manner, is an important enzyme in the oil and oleochemical industries. However, most sPLA2s are not stable under process conditions. Therefore, a thermostable sPLA2 was investigated in this study. A marine bacterial sPLA2 isolated from Sciscionella marina (Sm-sPLA2) was catalytically active even after 5 h of incubation at high temperatures of up to 50°C, which is outstanding compared with a representative bacterial sPLA2 (i.e. sPLA2 from Streptomyces violaceoruber; Sv-sPLA2). Consistent with this, the melting temperature of Sm-sPLA2 was measured to be 7.7°C higher than that of Sv-sPLA2. Furthermore, Sm-sPLA2 exhibited an improved biotransformation performance compared with Sv-sPLA2 in the hydrolysis of soy lecithin to lysolecithin and free fatty acids at 50°C. Structural and mutagenesis studies revealed that the Trp41-mediated anchoring of a Ca2+-binding loop into the rest of the protein body is directly linked to the thermal stability of Sm-sPLA2. This finding provides a novel structural insight into the thermostability of sPLA2 and could be applied to create mutant proteins with enhanced industrial potential.