Supporting Information Material and methods 5 Fatty acid substrates Fatty acid standards and substrates, including palmitoleic acid (C16:1∆9Z), elaidic acid (C18:1∆9E), oleic acid (C18:1∆9Z), 11-cis-octadecanoic acid(C18:1∆11Z),11-trans-octadecanoic acid(C18:1∆11E),12-trans-octadecanoic acid(C18:1∆12E), linoleic acid (C18:2∆9Z,12Z), conjugated linoleic acids (C18:2∆9Z,11E, C18:2∆9E,11E, and C18:2∆10E,12Z), α-linolenic acid (C18:3∆9Z,12Z,15Z), γ- 10 linolenic acid (C18:3∆6Z,9Z,12Z), pinolenic acid (C18:3∆5Z,9Z,12Z), stearidonic acid (C18:4∆6Z,9Z,12Z,15Z) were purchased from Nu-Check Prep (Elysian, MN) and Sigma Aldrich (St. Louis, MO). Strains and plasmids Strains and plasmids used in this study are listed in Table S1. For cloning, expression, and 15 biotransformation experiments, E. coli ER2566 was transformed with two constructed plasmids following standard procedures (Sambrook and Russell 2001). Table S1: The strains and plasmids used in this study Name Strains L. acidophilus E. coli ER2566 ER2566_ lht13 ER2566_lht10 Plasmids pET-15b pET-15b/LHT-10 pET-15b/LHT-13 Description Reference or source LMG 11470 (Synonym of ATCC 4796) F– λ– fhuA2 [lon] ompT lacZ::T7 gal endA1 [dcm] ER2566 harboring lht-13 in pET-15b ER2566 harboring lht-10 in pET-15b LMG NEB In this study In this study T7 promoter, T7 terminator, bacterial pBR322 origin (ori) and Ampr pET-15b carries cloned lht1 from L. acidophilus pET-15b carries cloned lht2 from L. acidophilus Novagen In this study In this study 20 1 Primers Primers for amplification of LHTs in this study were listed in Table S2. Amplification of LHT10 and LHT-13 using each forward and reverse primer set, L. acidophilus genomic DNA, and a phusion high-fidelity DNA polymerase (New England Biolabs, Herfordshire, UK) kit following 5 recommendation of suppliers and standard procedures . Table S2: Primers used for amplification of LHTs in this study. Target DNA name Sequence (5’ to 3’)* lht-1(HMPREF0492_0906) LHT-13 F 5′-TTCATATGTATTATTCCAATGGTAATTACGA-3′ LHT-13 R 5′-GCCTCGAGTTAGACTAAATTTGCTTCTTTAAGTA-3′ lht-2(HMPREF0492_0997) LHT-10 F 5′-ATCATATGCATTATAGTAGTGGTAATTATG-3′ LHT-10 R 5′-CGCTCGAGCTAAACCAACTTATACTTCTTAAGC-3′ * Restriction enzyme (NdeI and XhoI) site were underlined 10 Molecular mass determination The subunit molecular mass of the putative fatty acid hydratases, LHT-13 and LHT-10, from L. acidophilus were examined by SDS-PAGE. The molecular mass of the native enzyme was determined by gel filtration chromatography using a Sephacryl S-300 HR 16/60 preparative15 grade column (Amersham Biosciences, Uppsala, Sweden). The purified enzyme solution was applied to the column and eluted with 50 mM citrate-phosphate buffer (pH 5.0) containing 150 mM NaCl at a flow rate of 1 ml/min. The column was calibrated with ferritin (400 kDa), catalase (206 kDa), albumin (75 kDa), and carbonic anhydrase (29 kDa) as reference proteins (Amersham Biosciences). 20 Effects of reaction conditions on enzyme activity of 10- and 13-HOD conversion Unless otherwise stated, the reaction was performed in 50 mM citrate-phosphate buffer (pH 5.0) 2 containing 5 mM linoleic acid, 0.1 mg/ml (84 U/ml) linoleate 13-hydratase (LHT-13) or 0.5 mg/ml (39 U/ml) linoleate 10-hydratase (LHT-10), and 4% (v/v) ethanol at 35°C in a 2 ml-tube for 10 min. To examine the effects of pH and temperature on the activity of LHT-13 from L. acidophilus, the pH was varied from 4.0 to 6.0 using 50 mM citrate-phosphate buffer and the 5 temperature was varied from 25 to 45°C. The thermal stability of the enzyme was determined by incubating the enzyme for 90 min at temperatures ranging from 25 to 60°C. After incubation, the remaining activity was determined under the standard reaction conditions. The effect of solvent on the activity of the 13-LHT was investigated using ethanol, methanol, butanol, 2propanol, hexane, DMSO, and acetone. The effect of ethanol concentration on enzyme activity 10 was examined by varying its concentration from 0 to 8% (v/v). The determination of reaction conditions for LHT-10 was also performed as same manner. Preparation of the hydroxy fatty acid standards The enzymatic reaction products, including 10-HOD, 10-HODE (cis-12,15), 10-HSA, 13-HOD, 15 13-HODE (cis-5,9, -6.9, and cis-12,15) and 10,13-diHOD, were isolated through twice time extraction using two volumes of ethyl acetate. After the organic layers were separated from the aqueous phase, the organic extract containing 10-HOD, 10-HSA, and 13-HOD were dried with anhydrous Na2SO4 or MgSO4 and concentrated by evaporation in vacuo. The concentrate was purified using a silicic acid column chromatography (Risser and Perkins 1966) with methanol 20 gradient (0−5%, v/v) in chloroform by step wise manner. 10,13-diHOD was also obtained with the same method except for 1−10% methanol gradient in chloroform. 10-HODE (cis-12,15) and 13-HODE (cis-5,9, -6.9, and cis-12,15) were prepared by semi-preparative HPLC using HPLC system (Agilent 1260, Palo Alto, CA, USA) coupled to a UV detector at the absorbance at 202 nm with a reverse phase Nucleosil C18 column (250 × 10 mm, 5 μm particle size; Phenomenex, 25 Torrance, CA, USA). The products were eluted with a gradient of solvent A (acetonitrile/water/acetic acid; 50:50:0.1, v/v/v) and solvent B (acetonitrile/acetic acid; 100:0.1, 3 v/v) at 3.0 ml/min. The hydroxy fatty acids were obtained with high purity (>97%) and higher purity (>99%) were used for structure determination and standards for quantification, respectively. 5 Quantification methods details of GC-FID Prior to quantify the products, 0.5 mM of C18:0 as an internal standard was added to monitor the extraction recovery. The supernatant (200-500 μl) obtained from the centrifugation and filtration of biotransformation reactant was extracted twice times with twice volumes of ethyl acetate and dried with anhydrous MgSO4 or Na2SO4. Since the presence of water inhibited 10 sylilation, the supernatant was completely dried. - Calibration: Calibration curves were established with 13-HOD, 13-HODE, 10-HOD, and 10HSA that were purified from enzymatic conversion products by repetitive semi-preparative HPLC. The purity of each product was checked by GC-MS as over 99%. Each standard solution was prepared in triplicate by serial dilution in MeOH with 20 mM stock solution. The range of 15 standard for 13-HOD and 10-HSA were six points from 0.3 to 20 mM and 10-HOD and 13HODE were five points from 0.3 to 10 mM. The linearity of peak area versus concentration for each hydroxyl fatty acid product was determined within the concentration range, and the aliquots were injected and run. The calibration curve constructed was evaluated by its correlation coefficient (Supplementary Table 1). The calibration equation from minimum three 20 replicate experiments demonstrated the linearity of the method. The standard deviation of the slope for the calibration curve was as relative standard deviation (RSD). - Precision and accuracy: The precision of the analytic method was determined by repeatability. Five or six different concentration samples (0.3−10 mM or 0.3−20 mM) were analyzed three times. The RSD value for precision was below 3%. 4 - Recovery: To determine the recovery and to study the interference of formulation additives, the recovery experiments were performed by adding the known amount of HFA with three different concentrations. The percent analytical recovery values were calculated by comparing the concentration obtained from the known samples with additional concentrations. 5 A B C D 10 Figure S1. Calibration graphs of standard for quantification of the biotransformed hydroxy fatty acids in this study. (A) 13-HOD (B) 13-HODE (cis-6,9) (C) 10-HOD (D) 10-HSA. 5 Table S3. Summary of calibration and linearity of standards. * Correlation %RSD** Hydroxy fatty acids Range (points) Regression equation 13-HOD 0.3 – 20 mM (6) Y = 8.602x + 0.2414 0.9996 0.1254 13-HODE (cis-6,9) 0.3 – 10 mM (5) Y = 8.552x + 0.2338 0.9988 0.0853 10-HOD 0.3 – 20 mM (6) Y = 8.4617x + 0.245 0.9989 0.0813 10-HSA 0.3 – 10 mM (5) Y = 8.611x + 0.2227 0.9997 0.1755 * coefficient Y=ax+b, ** relative standard deviation O-acetyl-mandelic derivatization of HFAs for stereo analysis 5 A 10 mg sample of purified HFA was treated with 2 ml of a 12% borontrifluoride in dichloromethane and the mixture was heated on a water bath (at 90°C) for 10 min. The reaction mixture was evaporated to dryness under a N2 stream after extraction with 2 ml of n-hexane. The 1uL of sample was subjected to gas chromatography to identify the methyl ester. O-Acetyl-mandelic derivatization from HFA methyl ester was performed as followed by the 10 published methods (el-Sharkawy et al. 1992; Yang et al. 1993). Simply, the 10 mg (0.032 mmol) of methyl-13-hydroxy-9-octadecenoate and methyl-10-hydroxystearate was dissolved in 10 mg (0.47mmol) of dicyclohexylcarbodiimide in 2 ml of CH2C12 and then this mixture added to a 2 ml of CH2Cl2 solution of O-acetylmandelic acid (0.047 mmol) and 4-(N,N-dimethylamino)pyridine mixture. The mixture was stirred over an ice bath for 5 h and then filtered. The reactant 15 was dried under nitrogen to give a crude residue containing mainly (S)-(+)-O-acetylmandelate ester of HFA. The 5mg of methyl-10-hydroxy-12-octadecenotate and methyl-13-hydroxy-6,9octadecadienoate was used for synthesis of mandeleate derivatives by same procedures. The obtained products were dissolved in DMSO-d6 for proton-NMR analysis. Peak integration was performed using ACD/NMR processor. 20 6 Results Identification of two hyratases in L. acidophilus genome and sequence comparison Supplementary Figure 2 shows putative two hydratase genes in L. acidophilus ATCC 4356 5 genome. The genes encoding LHT-13 and LHT-10 was comprised 590 amino acids encoded by 1,783 base pairs (HMPREF0492_0906 gene) and EEJ75841 protein was comprised 591 amino acids encoded by 1,786 base pairs (HMPREF0492_0997 gene). EEJ76183 protein showed 58% amino acid sequence identity with EEJ75841 protein. EEJ76183 protein showed 94−100% and more than 90 % sequence identities with hydratases of L. acidophilus (Supplementary Table 3) 10 and its related strains including L. amylovorus, L. helveticus, and L. crispatus, respectively (Supplementary Table 4). EEJ76183 protein was also homologous with MCRA proteins from the genus Pediococcus (78% identity) and Streptococcus (70%). This protein showed 42−60% amino acid sequence identity with the characterized linoleate 10-hydratases and oleate hydratases (Table S5). EEJ75841 protein was higher identity with MCRA proteins from L. 15 acidophilus and related strains (more than 80% identity) but showed lower identity with two hydratases of L. mucosae (77% and 58% identity), L. buchneri (59% and 30%), Pediococcus claussenii (56% and 34%), respectively (Supplementary Table 4). EEJ75841 protein exhibited 45−68% amino acid sequence identity with the characterized linoleate 10-hydratases and oleate hydratases (Supplementary Table 5). Both proteins were homologous with 67 kDa Streptococcal 20 MCRA family proteins and oleate hydratases. 7 Figure S2. Genetic loci of the two hydratases in L. acidophilus ATCC 4356. Shade box and percentage means relative identity between LHT-13 and LHT-10. 5 8 8 Table S4: Comparison of two hydratase protein sequences in L. acidophilus strains containing 9 LHT-13 and LHT-10. Name Protein accession (amino acid residues) Gene name Identity (%) For LHT-13 L. acidophilus ATCC 4796 HMPREF0492_0906 EEJ76183 (590) 100 L. acidophilus CIP 76.31 LACIP7613_01498 CDF67336 100 L. acidophilus CIRM-BIA 442 LACIRM442_00103 CDF69017 (590) 100 L. acidophilus CIRM-BIA 445 LACIRM445_00351 CDF70787 (590) 100 L. acidophilus DSM 9126 LADSM9126_01212 CDF72610 (590) 100 L. acidophilus DSM 20242 LADSM20242_00691 CDF74590 (590) 100 L. acidophilus La-14 LA14_0588 YP_007937442 (590) 100 L. acidophilus NCFM LBA0555 YP_193467 (590) 100 L. acidophilus 30SC LAC30SC_02835 YP_004286876 (590) 94 L. acidophilus ATCC 4796 HMPREF0492_0997 EEJ75841 (591) 100 L. acidophilus CIP 76.31 LACIP7613_00033 CDF67433 99 L. acidophilus CIRM-BIA 442 LACIRM442_00386 CDF69112 (591) 99 L. acidophilus CIRM-BIA 445 LACIRM445_00448 CDF70883 (591) 99 L. acidophilus DSM 9126 LADSM9126_01305 CDF72702 (591) 99 L. acidophilus DSM 20242 LADSM20242_01398 CDF74685 (591) 99 L. acidophilus La-14 LA14_0679 YP_007937532 (591) 99 L. acidophilus NCFM LBA0649 YP_193559 (591) 99 L. acidophilus 30SC LAC30SC_03260 YP_004291763 (591) 99 (590) For LHT-10 10 11 -9- (591) 12 Table S5: Sequence identities of (A) LHT-13 and (B) LHT-10 from L. acidophilus ATCC 4356 13 with orthologous proteins in Lactobacillales. Strains Protein accession Amino acids in length Identity (%) For LHT-13 Lactobacillus acidophilus ATCC 4796 Lactobacillus amylovorus GRL 1112 Lactobacillus helveticus R0052 Lactobacillus crispatus ST1 Lactobacillus kefiranofaciens ZW3 Lactobacillus sp. ASF360 Lactobacillus mucosae LM1 Lactobacillus gasseri ATCC 33323 Pediococcus claussenii ATCC BAA-344 Streptococcus mutans UA159 Lactobacillus otakiensis JCM 15040 Lactobacillus buchneri NRRL B-30929 Pediococcus pentosaceus IE-3 For LHT-10 EEJ76183 YP_004031359 YP_006656360 YP_003601030 YP_004562912 EMZ19002 EHT17106 YP_814320 YP_005005459 NP_721921 GAD16444 YP_004397927 CCG90649 590 590 590 590 590 590 590 590 590 589 589 589 589 100 94 92 90 90 87 82 80 72 72 69 68 65 Lactobacillus acidophilus ATCC 4796 Lactobacillus ultunensis DSM 16047 Lactobacillus amylovorus GRL 1112 Lactobacillus crispatus ST1 Lactobacillus helveticus R0052 Lactobacillus kefiranofaciens ZW3 Lactobacillus sp. ASF360 Lactobacillus gasseri ATCC 33323 Lactobacillus mucosae LM1 EEJ75841 EEJ72450 YP_004031446 YP_003601030 YP_006656262 YP_004562795 EMZ18971 YP_815151 EHT17106 EHT15500 YP_005005459 YP_005004553 NP_721921 NP_720953 YP_004397927 YP_004397517 CCG90649 CCG90677 591 591 591 591 591 591 591 591 590 591 590 558 589 591 589 564 589 557 100 95 93 92 93 93 86 84 77 58 58 35 58 56 59 30 56 34 Pediococcus claussenii ATCC BAA-344 Streptococcus mutans UA159 Lactobacillus buchneri NRRL B-30929 Pediococcus pentosaceus IE-3 14 15 - 10 - 16 Table S6: Sequence identities of LHT-13 and LHT-10 from L. acidophilus ATCC 4356 with other characterized hydratases. Protein (Gene name) Microorganism Name of enzymes Accession number Identity (%) Reference L. acidophilus ATCC 4356 Macrococcus caseolyticus KCTC 3582 Lysinibacillus fusiformis KCTC 3454 L. acidophilus NCFM Streptococcus pyogenes NZ131 Stenotrophomonas maltophilia KCTC 1773 Bifidobacterium breve NCIMB 702258 Elizabethkingia meningoseptica 3266 Linoleate 13-hydratase Linoleate 10-hydratase Oleate hydratase Oleate hydratase Linoleate 10-hydratase Linoleate 10-hydratase Oleate hydratase Oleate hydratase Oleate hydratase EEJ76183 EEJ75841 B9E972 EFI68504 YP_193559 B5XK69.2 CAQ45596 ADY18551 ACT54545 100 58 60 60 58 56 52 50 42 This study This study (Joo et al. 2012a) (Kim et al. 2012) (Volkov et al. 2013) (Volkov et al. 2010) (Joo et al. 2012b) (Rosberg-Cody et al. 2011) (Bevers et al. 2009) L. acidophilus ATCC 4356 Linoleate 10-hydratase EEJ75841 100 This study L. acidophilus NCFM Streptococcus pyogenes NZ131 Lysinibacillus fusiformis KCTC 3454 Macrococcus caseolyticus KCTC 3582 Stenotrophomonas maltophilia KCTC 1773 Bifidobacterium breve NCIMB 702258 Elizabethkingia meningoseptica 3266 Linoleate 10-hydratase Linoleate 10-hydratase Oleate hydratase Oleate hydratase Oleate hydratase Oleate hydratase Oleate hydratase YP_193559 B5XK69.2 EFI68504 B9E972 CAQ45596 ADY18551 ACT54545 100 68 63 62 54 51 45 (Volkov et al. 2013) (Volkov et al. 2010) (Kim et al. 2012) (Joo et al. 2012a) (Joo et al. 2012b) (Rosberg-Cody et al. 2011) (Bevers et al. 2009) For LHT-13 EEJ76183 (HMPREF0492_0906) For LHT-10 EEJ75841 (HMPREF0492_0997) 17 - 11 - Expression, purification, and determination of molecular mass The both expressed and purified protein showed a single band in SDS-PAGE with a molecular mass of approximately 68 kDa, which is consistent with the calculated value of 68.1kDa based on the 590 and 591 amino acids and six histidine tag (Supplementary Fig. 3A and 3B). The molecular mass of the 5 native protein was estimated using gel filtration chromatography to be 68 kDa, indicating that it is a monomer (Supplementary Fig. 3C). The molecular masses of oleate hydratases of Elizabethkingia menungoseptica (Bevers et al. 2009), Bifidobacterium breve (Rosberg-Cody et al. 2011), and Macrococcus caseolyticus (Joo et al. 2012a), and linoleate hydratase of S. pyogenes (Volkov et al. 2010) were 65−70 kDa as a monomer. 10 Substrate specificity of purified recombinant LHT-13 and identification reaction products of LHT-13 and LHT-10. Supplementary Table 7 shows relative activity (as for LA is 100%) and ssubstrate specificity of purified recombinant LHT-13. The biotransformed 13-HODE(13-OH, 6Z,9Z) from GLA and 1315 HODE(13-OH, 9Z,15Z) from ALA were showed in Figure S4. No activity was detected for palmitoleic acid (C16:1∆9Z),elaidic acid (C18:1∆9E), oleic acid (C18:1∆9Z), 11-cis-octadecanoic acid(C18:1∆11Z),11-trans-octadecanoic acid(C18:1∆11E),12-trans-octadecanoic acid(C18:1∆12E),cis-9conjugated linoleic acid(C18:2∆9Z,11E), all trans-conjugated linoleic acid (C18:2∆9E,11E), eicosenoic acids (C20:1∆7Z, C20:1∆9Z, C20:1∆11Z), arachidonic acid (C20:4∆5Z,8Z,11Z,14Z), and erucic acid (C22:1∆13Z). 20 Identification of detailed structure determination by 1H and 13 C-NMR spectroscopy was shown in Figure S5 and S6. The biotransformed 10-HSA from OA and 10-HOD (10-OH, 12Z) from LA were showed in Figure S7. Optimized reaction conditions for the production of 13-HFAs by LHT-13. 25 To obtain maximal productivity of each 13-HFAs using LHT-13, optimum pH, temperature, and solvent concentrations were investigated. Maximal enzyme activity was observed at pH 5.0 (Supplementary Fig. 11A) and at 35°C (Supplementary Fig. 11B). The production of 13-HOD from 12 linoleic acid was highest using ethanol among the solvents tested (Supplementary Fig. 12A), and its optimal concentration was 4% (v/w) (Supplementary Fig. 12B). 13 A B 5 C 14 Figure S3. SDS-PAGE analysis of the purified hydratase for recombinant (A) EEJ76183 and (B) EEJ75841 from L. acidophilus in E. coli. Lane 1, molecular mass marker proteins (130, 100, 75, 55, 40, 35, and 25 kDa); lane 2, crude extract; lane 3, purified putative fatty acid hydratase. The protein extract was prepared from culture of recombinant E. coli cells grown in the Riesenberg medium and 20 h 5 after induction of the gene expression with 0.1mM IPTG. The expressed (LHT-13 and LHT-10) band was indicated by an arrow. (C) Determination of molecular mass of the purified native enzyme using gel filtration chromatography. The reference proteins were ferritine (400 kDa), catalase (206 kDa), conalbumin (75 kDa), and carbonic anhydrase (29 kDa). The putative fatty acid hydratase eluted at a position corresponding to 68 kDa. 10 15 Table S7: Substrate specificity of the putative fatty acid hydratase of L. acidophilus. Asterisk means hydroxylation position. Substrate LA(18:2 n-6, 9Z,12Z) Product Relative activity (%)a 13S-HOD (13-OH, 9Z) 100 GLA (18:3 n-6, 6Z,9Z,12Z) 13S-HODE(13-OH, 6Z,9Z) 13 PLA (18:3 n-6, 5Z,9Z,12Z) 13-HODE (13-OH, 5Z, 9Z) 7 10-HOD(18:1, 10-OH, 12Z) 10,13-diHOD 6 ALA (18:3 n-3, 9Z,12Z,15Z) 13S-HODE(13-OH, 9Z,15Z) Traceb CLA (18:2, 10E, 12Z) 13-HOD(13-OH, 10E) Trace 5 a The relative activity of 100% in the enzyme reactions was 814 U/mg. b Trace: Each product clearly formed peak at GC compared with control reaction but their conversion yield was no more than 3% or less. 16 A B 5 Figure S4. GC-MS of the product formed from (A) α- and (B) γ-linolenic acid by recombinant purified LHT-13 enzyme reaction. 10 17 H-NMR spectra (400 MHZ, CDCl3): δ ppm [integration, multiplicity, coupling constant J (HZ)], 5.35(1H, m, 10.8), 5.35(1H, m, 10.5), 3.60(1H, m), 2.32(2H, t, 7.4), 2.13(2H, m), 2.0(2H, q), 1.61(2H, m), 1.52(2H, m), 1.48(2H, m), 1.43(2H, m), 1.26 (2H, m), 1.26 (2H, m), 1.26 (2H, m), 1.26 (2H, m), 1.26 (2H, m), 1.26(2H, m), 1.26(2H, m), 0.89(3H, t, 6.8). 1 5 Figure S5. 1H-NMR spectra of 13-hydroxy-9(Z)-octadecenoic acid 10 18 C-NMR spectrum (100 MHZ, CDCl3): δ ppm (multiplicity), 178.9(s), 130.7(d), 129.4(d), 72.1(d), 13 37.6(t), 37.4(t), 34.0(t), 31.1(t), 29.6(t), 29.1(t), 29.0(t), 29.0(t), 27.2(t), 25.5(t), 24.8(t), 23.8(t), 22.8(t), 5 14.2(q). Figure S6. 13C-NMR spectra of 13-hydroxy-9(Z)-octadecenoic acid. 10 19 Proton-NMR spectral data of the minor products - 13S-hydroxy-6Z,9Z-octadecadienoic acid (500 MHz, CD3Cl): δ ppm [integration, multiplicity, coupling constant J (HZ)], 5.39 (m, 4H, 6Z, 10.7), 5.36 (m, 4H, H9, 10.6), 3.63 (m, 1H, H-13), 2.80 (dd, 2H), 2.35 (t, 2H), 2.22-2.06 (m, 6H), 1.69-1.25 (m, 12H), 0.89 (t, 3H, H-18). 5 - 13-hydroxy-5Z,9Z-octadecadienoic acid (500 MHz, CD3Cl): δ ppm [integration, multiplicity, coupling constant J (HZ)], 5.29 (m, 4H, H-5, 10.6), 5.35 (m. 4H, H-9, 10.8), 3.65 (m, 1H, H-13), 2.80 (dd, 2H), 2.79-2.0 (m, 8H), 1.49-1.29 (m, 12H), 0.86 (t, 3H, H-18). - 13S-hydroxy-9Z,15Z-otadecenoic acid (500 MHz, CD3Cl): δ ppm [integration, multiplicity, coupling constant J (HZ)], 5.44 (m, 2H, H9, 10.7), 5.33 (m, 2H, H10), 5.30 (m, H16), 5.24 (m, H15, 10.6), 10 3.51 (m, H-13), 2.72 (m, 2H), 2.25 (t, 2H), 2.20 (m, 2H), 2.22 (dd, 2H), 1.96 – 1.21 (m, 12H), 0.89 (t, 3H, H-18). - 13-hydroxy-10E-otadecenoic acid (500 MHz, CD3Cl) (partial): δ ppm [integration, multiplicity, coupling constant J (HZ)], 5.60 (m, 2H, H-9, 15.0), 5.44 (m, 1H, H-10), 3.82 (m, 1H, H-13), 2.32 (m, 2H), 2.04 (m, 2H), 1.93 (m, 2H), 1.82 (m, 2H), 1.60 (m, 2H), 1.40 – 1.18 (m, 12H), 0.86 (t, 15 3H, 7.5, H-18). - 10S-hydroxy-12Z-octadecenoic acid (500 MHz, CD3Cl) (partial): δ ppm [integration, multiplicity, coupling constant J (HZ)], 5.48 (m, 2H, H-12, 10.6), 3.54 (m, 1H, H-13), 2.33 (m, 2H), 2.22 (dd, 2H), 2.0 (m, 2H), 1.80 (m, 2H), 1.32 – 1.24 (m, 14H), 0.89 (t, 3H, 7.5, H-18). - 10-hydroxy-12Z,15Z-octadecadienoic acid (500 MHz, CD3Cl) (partial): δ ppm [integration, 20 multiplicity, coupling constant J (HZ)], 5.49 (m, 1H), 5.40 (m, 1H, 10.7, H-12), 5.36 (m, 1H), 5.30 (m, H15, 10.6), 3.64 (m, H-10), 2.79 (m, 2H), 2.35 (m, 2H), 2.26 (dd, 2H), 2.08 (m, 2H, H17), 1.60 (m, 2H, H-3), 1.45 (m, 2H, H-9) , 1.28 – 1.21 (m, 12H), 0 (t, 3H, 7.5, H-18). 20 Figure S7. 1H-NMR spectra of 13-hydroxy-6(Z),9(Z)-octadecenoic acid 5 Proton- (A) and 13 C- (B) NMR spectra of 13-hydroxyoctadec-6,9-dienoic acid. NMR data of the isolated product: H NMR (500 MHz, CDCl3) δ 5.39 (m, 2H, 10.7), 5.36 (m, 2H, 10.6), 3.63 (m, 1H, H-13), 2.80 (dd, 1 2H), 2.35 (t, 2H), 2.22-2.06 (m, 6H), 1.69-1.25 (m, 12H), 0.89 (t, 3H, H-18). 13C NMR (125 MHz, CDCl3) δ 178.8 (C-1), 129.5 (C-6), 129.3 (C-7), 128.4 (C-9, C-10), 71.8 (C-13), 37.3, 37.0, 33.7, 31.8, 28.9, 26.7, 25.6, 25.2, 24.3, 23.5, 22.6, 14.0 (C-18). 10 21 A B 5 Figure S8. GC-MS of the product formed from (A) oleic acid and (B) α-linolenic acid by LHT-10 10 enzyme reaction. 22 A B 5 Figure S9. Proton-NMR spectroscopy of (S)-(+)-O-acetylmandelic ester of 13S-hydroxy-9(Z)octadecenoic acid methyl ester for determination of stereochemistry. (A) Proton NMR spectra (600MHz, DMSO-d6), the peaks derived from mandelic acid was underlined: δ ppm [integration, 10 multiplicity, coupling constant J (HZ)], 7.45 (2H, dd), 7.36 - 7.39 (3H, m), 5.86 (1H, s), 5.7 - 5.3 (1H, m, H-9, 10.6), 4.79 (1H, m, H-10), 3.67 (1H, s, H-13), 3.33 (m, 2H), 3.19 (3H, s, OCH3), 2.28 (3H, t, CH3CO), 2.12 - 1.92 (4H, m), 1.72 - 1.23 (7H, m), 1.15 - 0.96 [18H, m], 0.88 (2H, m), 0.72 (t, 3H, H18). (B) Chiral (R/S) distribution of (S)-(+)-O-acetylmandelic ester of 13S-hydroxy-9(Z)-octadecenoic acid. Examined S-form was 98% and R-form was 2% and thus obtained enantiomeric excess (% ee) 15 was 96%. 23 5 A B C D Figure S10. Chiral analysis of the minor product of 13-LHT and 10-LHT using 1H-NMR 10 spectroscopy. (A) (S)-(+)-O-acetylmandelic ester of 13-hydroxy-9(Z),15(Z)-octadecadienoic acid methyl ester (B) (S)-(+)-O-acetylmandelic ester of 13-hydroxy-6(Z),9(Z)-octadecadienoic acid (C) (S)-(+)-O-acetylmandelic ester of 10-hydroxy-9(Z)-octadecenoic acid (D) (S)-(+)-O-acetylmandelic ester of 10-hydroxy-9(Z),15(Z)-octadecadienoic acid. Examined % ee was 98, 98, 97, and 82 %, respectively. 15 24 A B 5 Figure S11. Effects of pH and temperature on the activity of LHT-13 from L. acidophilus. (A) pH effect. The reactions were performed in 50 mM citrate-phosphate buffer (pH 4.0−6.0) containing 5 m M linoleic acid, 0.1 mg/ml enzyme, and 4% (v/v) ethanol at 35°C for 10 min. (B) Temperature effect. The reactions were performed by varying the temperature from 25 to 60°C in 50 mM citrate10 phosphate buffer (pH 6.5) containing 5 mM linoleic acid, 0.1 mg/ml enzyme, and 4% (v/v) ethanol for 10 min. 25 A B 5 Figure S12. Effect of solvent on the activity of LHT-13 from L. acidophilus. (A) Effect of solvent type. The reactions were performed in 50 mM citrate-phosphate buffer (pH 5.0) containing 5 mM linoleic acid, 0.1 mg/ml enzyme, and 2% (■) or 4% (□) (v/v) solvent at 35°C for 10 min. (B) Effect of solvent concentration. The reactions were performed by varying the ethanol concentration from 0 to 10 8%in 50 mM citrate-phosphate buffer (pH 5.0) containing 5 mM linoleic acid and 0.1 mg/ml enzyme at 35°C for 10 min. Data represent the means of three experiments and error bars represent the standard deviation. 26 A B 5 C Figure S13. Characteristics of cofactor effect on the 13-LHT activity. (A) Multiple alignment of FADbinding regions (1-98 of PDB 4IA6) from 13-LHT (EEJ76183 from L. acidophilus) with the 10 characterized oleate hydratases (ADY18551 from B. breve, CAQ45596 from S. maltophilia, ZP_07049769 from L. fusiformis, B9E972 from M. caseolyticus, ACT54545 from E. meningoseptica) and linoleate hydratases (EEJ75841 from L. acidophilus ATCC 4796, YP_193559 from L. acidophilus NCFM, B5XK69.2 from S. pyogenes) (see also Table S6). Conserved residues were indicated asterisk. Critical residues of FAD-binding region for enzyme activity were shown as black box, whereas 15 variable residues were shown as gray box. (B) UV-visible spectrum of holoenzyme (blue), apoenzyme (pink), and FAD (red). The concentrations of the holoenzyme and apoenzyme were approximately 10 mg/ml in 50 mM citrate-phosphate buffer (pH 5.0) and the concentration of FAD is 40 μM. (C) Effect 27 of FAD concentration on the activity of cis-12 linoleate hydratase from L. acidophilus. The effect on FAD concentration was evaluated.The reactions were performed by varying the concentration from 0 to 4 mMin 50 mM citrate-phosphate buffer (pH 5.0) containing 5 mM linoleic acid, 0.1 mg/ml enzyme, and 4% (v/v) ethanol at 35°C for 10 min. 5 28 H-NMR spectra (400 MHZ, CDCl3): δ ppm [integration, multiplicity, coupling constant J (HZ)], 3.87 1 (1H, m), 3.66 (1H, m), 2.34 (2H, t, 7.4), 2.21 (2H, m), 1.95 (2H, m), 1.63 (2H, m), 1.51 (2H, m), 1.48 5 (2H, m), 1.31 (2H, m), 1.31 (2H, m), 1.31 (2H, m), 1.31 (2H, m) 1.31 (2H, m) 1.31 (2H, m) 1.31 (2H, m) 1.31 (2H, m), 0.89 (3H, t, 6.8). Figure S14. 1H-NMR spectra of 10,13-diHOD 10 29 C-NMR spectrum (100 MHZ, CDCl3): δ ppm (multiplicity), 178.5(s), 73.3(d), 73.2(d), 42.8(t), 13 38.2(t), 33.9(t), 31.8(t), 29.4(t), 29.2(t), 29.1(t), 29.0(t), 28.9(t), 28.9(t), 25.2(t), 25.2(t), 24.6(t), 22.6(t), 5 14.0(q). Figure S15. 13C-NMR spectra of 10,13-diHOD 10 30 References 5 10 15 20 25 Bevers LE, Pinkse MWH, Verhaert PDEM, Hagen WR. 2009. 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