Supplementary Data O 173.3 -H -O OH 120 173.2 Relative absorbance 100 80 295.3 60 313.3 40 20 0 100 150 200 250 300 m/z Supplementary Fig. 1 LC-MS/MS spectrum of 8-HPOME obtained from conversion of αlinolenic acid using diol synthase from A. nidulans double-site (H1004A-C1006S) variant. The inset shows the major fragments formed. 120 Relative activity (%) 100 80 60 40 20 0 25 30 35 40 45 50 55 60 o Temperature ( C) Supplementary Fig. 2 Effect of temperature on the production of 5,8-diHOME from oleic acid using whole recombinant cells expressing diol synthase from A. nidulans. The reactions were performed by varying the temperature from 30 to 55 °C for 10 min in 50 mM HEPES buffer (pH 7.5) containing 2 g cells l–1 and 0.14 g oleic acid l–1. Data represent the means of three separate experiments and error bars represent the standard deviation. 120 Relative activity (%) 100 80 60 40 20 0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 pH Supplementary Fig. 3 Effect of pH on the production of 5,8-diHOME from oleic acid using whole recombinant cells expressing diol synthase from A. nidulans. The reactions were performed by varying the pH from 6.0 to 8.0 at 40 °C for 10 min in 50 mM HEPES buffer (filled circle, pH 6.0−8.0) and 50 mM EPPS buffer (empty circle, pH 8.0−8.5) containing 2 g cells l−1 and 0.14 g oleic acid l−1. Data represent the means of three separate experiments and error bars represent the standard deviation. 120 Residual activity (%) 100 80 60 40 20 0 25 30 35 40 45 50 55 o Temperature ( C) Supplementary Fig. 4 Effect of enzyme stability on the production of 5,8-diHOME from oleic acid using whole recombinant cells expressing diol synthase from A. nidulans. The reactions were performed in 50 mM HEPES buffer (pH 7.5) containing 2 g cells l−1 and 0.14 g oleic acid l−1 after incubation for 1 h at temperatures from 25 to 50 °C. Initial activity was set at 100 %, and the residual activity after incubation is presented. Data represent the means of three separate experiments and error bars represent the standard deviation. 140 Relative activity (%) 120 100 80 60 40 20 0 ol ol ol te ol ne de ol r ne ol ntr an an eta an ta xi an the xa tan Co EthoMethyl ac Prop o-ocsulfo Hexhyl e He Bu Is yl h et Et im eth D m Di Supplementary Fig. 5 Effect of solvents on the production of 5,8-diHOME from oleic acid using whole recombinant cells expressing diol synthase from A. nidulans. The reactions were performed at 40 °C for 10 min in 50 mM HEPES buffer (pH 7.5) containing 2 g cells l−1, 0.14 g oleic acid l−1, and 5 % (filled bar) or 10 % (empty bar) (v/v) solvent. Data represent the means of three separate experiments and error bars represent the standard deviation. 140 Relative activity (%) 120 100 80 60 40 20 0 0 2 4 6 8 10 12 14 16 18 Dimethyl sulfoxide concentration (%, v/v) Supplementary Fig. 6 Effect of dimethyl sulfoxide concentration on the production of 5,8diHOME from oleic acid using whole recombinant cells expressing diol synthase from A. nidulans. The reactions were performed at 40 °C for 10 min in 50 mM HEPES buffer (pH 7.5) containing 2 g cells l−1, 0.14 g oleic acid l−1, and DMSO. Data represent the means of three separate experiments and error bars represent the standard deviation.