CSIRO Land & Water, Black Mountain Science and Innovation Park, Acton, ACT 2601, Australia

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1 Isolation of the (+)-pinoresinol-mineralizing Pseudomonas sp. SG-MS2 and elucidation of its catabolic pathway Madhura Shettigar a,b, Sahil Balotra a, David Cahill b, Andrew C. Warden a, Michael J. Lacey c, Hans-Peter E. Kohler d, Daniel Rentsch e, John G. Oakeshott a and Gunjan Pandey a,# a CSIRO Land & Water, Black Mountain Science and Innovation Park, Acton, ACT 2601, Australia b Deakin University, Geelong Waurn Ponds Campus, Geelong, VIC 3216, Australia c CSIRO National Collections & Marine Infrastructure, Black Mountain Science and Innovation Park Acton, ACT 2601, Australia d Swiss Federal Institute of Aquatic Science and Technology (EAWAG), Dübendorf 8600, Switzerland e Swiss Federal Laboratories for Materials Science and Technology (Empa), Dübendorf 8600, Switzerland Address correspondence to Gunjan Pandey, gunjan.pandey@csiro.au 1

2 Figure S1. Pinoresinol-dependent growth of Pseudomonas strain SG-MS2. 2

3 Figure S2. HPLC chromatogram depicting appearance of metabolite 1 in the culture supernatant of SG-MS2 during growth on pinoresinol as a sole source of carbon and energy. 3

4 Figure S3. LC-TOF/MS spectra of pinoresinol (A) and its metabolites (B-G). This data belongs to the degradation scheme shown in figure 7 of the main text. 4

5 S4. NMR analysis 1 H and 13 C NMR spectra were recorded at and MHz on a Bruker Avance III 400 NMR spectrometer (Bruker Biospin AG, Fällanden, Switzerland). The 1D 1 H NMR spectra, as well as the 2D 1 H- 1 H DQF-COSY, 1 H- 13 C HSQC, 1 H- 13 C HSQC-TOCS and 1 H- 13 C HMBC correlated NMR experiments were performed at 298 K using the Bruker standard pulse programs and parameter sets on a 5 mm CryoProbe Prodigy probe equipped with z-gradient applying 90 pulse lengths of 11.4 s ( 1 H) and 10.0 s ( 13 C). Since only a restricted amount of material was available ( 1 mg of a mixture of at least 5 metabolites) we used a susceptibility-matched Shigemi tube with about 190 L of the CDCl 3 solution. The 1 H and 13 C NMR chemical shifts (δ) in ppm are calibrated to the resonances of chloroform at δ = 7.26 / 77.0 ppm 1 H / 13 C, respectively. Coupling constants J are reported in Hz and for 1 H NMR data coupling patterns are described as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. For 13 C NMR data s = quaternary carbon, d = CH, t = CH 2, q = CH 3 and w = weak HMBC or DQF-COSY correlations. NMR analysis of non-derivatized, enriched fractions verified the presence of a hemiketal and ketoalcohol as metabolites 1A and 1B, respectively, and of the -lactone as metabolite 2 (chemical structures with numbering used for resonance assignment are shown in Fig. S4-A). Since only a small amount of material was available and, moreover, the major components of the mixture consisted of at least five very similar chemical species, the NMR chemical shift assignment to the individual species was not trivial. In a first step, the 1 H and 13 C NMR spin systems of aliphatic spin systems located between the aromatic rings of the pinoresinol moiety were identified by a 1 H- 13 C HSQC-TOCSY NMR experiment (expanded region with highlighted spin system assigned to metabolite 1B as an example are shown in Fig. S4-B). Once the five major spin systems were identified, in a 2 nd step the 1 H NMR connectivities were assigned for well-resolved peaks over the 1 H- 1 H DQF-COSY spectra (shown in Fig. S4- C) and the directly attached carbons over correlations observed in the 1 H- 13 C HSQC NMR experiment. In Fig. S4-D, expanded plots of the HSQC NMR chemical shift regions assigned to all methylene groups (positions 10 and 11, chemical structures with 5

6 numbering of positions shown in Fig. S4-A), for all -methine carbons (position 8) attached to the aromatic rings and all remaining correlations of methine carbons (positions 9 and 12) are shown together with the assignments to five mentioned individual metabolites. All HSQC correlations observable in the shown chemical shift regions are assigned to one of the five mentioned individual metabolites. It must be mentioned that on lower contour levels of the 2D NMR spectra some additional correlation peaks are present but, due to the much lower relative signal intensities, the respective chemical species could not be identified. In contrast to the aliphatic part of the 1 H and 13 C chemical shift regions, the aromatic parts of the spectra consisted of severely overlapping resonances and/or cross peaks and, a priori, no chemical shift assignment of these regions was possible. With the aid of long range correlations observed in the 1 H- 13 C HMBC NMR experiment (Fig. S4-E) the carbons at position 13 of candidates for metabolite 1B/1B (two different chemical species of a ketoalcohol, with 13 C = & ppm) and metabolite 1A/1A (two different species of a hemiketal with 13 C and ppm) were identified. Note that the 1 H and 13 C NMR chemical shifts assigned to species 1A and 1B apparently belong to a single chemical structure (see below). Additionally, a possible candidate for metabolite 2 was identified (-lactone with 13 C ppm). Subsequently, over long range correlations to carbons 8 or 13, at least parts of the 1 H and 13 C chemical shifts of the aromatic spin systems were assignable. In Fig. S4-E, expanded 1 H- 13 C HMBC spectra used for the chemical shift assignments of metabolites 1A, 1B and 2 are shown (for 1 H and 13 C chemical shifts, see section S4-F). For all spin systems, we observed correlations of H-2 and H-6 at the aromatic ring to the methine carbons at position 8, as well as correlations from H-15 and H-19 (if present) to C- 13. As mentioned above, for the ketoalcohol and the hemiketal, evidence for two species each was observed. It is plausible that there are two stereoisomeric forms of metabolite 1A (initial attack of OH at pinoresinol is, in principle, possible from either side of the molecule), however, this is not possible for the ketoalcohol species 1B. In the HMBC NMR spectrum, the protons at 3.35 and 3.00 ppm assigned to the CH 2-O group at position 10 in the ketoalcohol species 1B (Fig. S4-A) clearly show correlations to a carbon at ppm (Fig. S4-E). 6

7 This carbon, however, is part of the spin system assigned to the hemiketal species 1A. From the further observed HMBC correlations, it is clear that part 1B must contain two aromatic systems, whereas for part 1A we did not observe any correlations of aromatic protons to the carbon at position 13. Therefore, the postulated dimeric species consisting of a hemiketal and a ketoalcohol part could have been formed by attack of the - CH 2OH group of metabolite 1B at the lactone of 2. Although the chemical structure of the possible dimeric compound shown in Fig. S3 is rather unusual, so far we did not find any other explanation for all observed longrange NMR correlations. In the 1 H NMR spectrum (Fig. S4-G) distinct regions were identified where only resonances of one chemical species contributed to the respective signal intensity. Notably, for the spin systems assigned to species 1A and 1B, the same signal intensities were observed and, moreover, the two series of resonances belonging to the postulated dimeric species (Fig. S4-A) are the major components observed in the mixture. It remains unclear why we did not observe any indications in the NMR data for a second hemiketal species with the same mass as metabolite 1A. The fact that both species (1A and 1B) eluted as a single symmetrical peak in HPLC suggests the interconversion between the two is spontaneous. Furthermore, there is evidence in the literature for the analogous hemiketal-to-ketoalcohol conversion of α-hydroxylated (±)- syringaresinol by fungal peroxidases (1-3). 7

8 S4-A: Chemical structures with numbering of positions used for the NMR chemical shift assignment of metabolites 1A, 1B, 1A /1B and 2. 8

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11 S4-D: Expanded regions of 1 H- 13 C HSQC NMR spectra with signal assignments to metabolites 1A, 1B, 1A /1B and 2. Note that each cross peak in the aliphatic region of the spectra could be assigned to one of the chemical species. 11

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13 S4-F. Assigned 1 H and 13 C NMR data, HMBC and DQF-COSY correlations of metabolites 1A, 1B, 1A, 1B and 2. 1 H NMR (CDCl 3, MHz) /ppm: Metabolite 1A, 7.10 (d, J = 1.9, 1H, H-15); 7.08 (dd, J = , 1H, H-19); 6.91 (d, J = 8.1, 1H, H-18); 6.88 (m, 1H, H-2); 6.86 (m, 1H, H-5); 6.82 (m, 1H, H-6); 4.47 (d, J = 6.5, 1H, H-8); 4.36 (m, 1H, H-10a); 4.07 (dd, J = 8.9/1.6, 1H, H-10b); 3.92 (s, 3H, H-20); 3.89 (s, 3H, H-7); 3.85 (dd, J = 9.8/9.8, 1H, H-11a); 3.33 (s, 1H, H-12); 3.07 (dd, J = 9.8/8.1, 1H, H-11b); 3.06 (m, 1H, H-9). Metabolite 1B, 7.56 (d, J = 1.9, 1H, H-15); 7.54 (dd, J = 8.4/1.9, 1H, H-19); 6.94 (d, J = 8.4, 1H, H-18); 6.94 (d, J = 2.0, 1H, H-2); 6.90 (m, 1H, H-6); 6.89 (m, 1H, H-5); 4.99 (d, J = 6.6, 1H, H-8); 4.36 (dd, J = 9.7/2.1, 1H, H-11a); 4.29 (m, 1H, H-12); 4.27 (m, 1H, H-11a); 3.96 (s, 3H, H-20); 3.91 (s, 3H, H-7); 3.66 (m, 2H, H- 10); 2.63 (m, 1H, H-9). Metabolite 1A, 6.86 (d, J = 8.1, 1H, H-5); 6.74 (d, J = 1.8, 1H, H-2); 6.64 (dd, J = 8.1/1.8, 1H, H-6); 4.29 (m, 1H, H-8); 3.89 (s, 3H, H-7); 3.81 (dd, J = 9.5/2.0, 1H, H-10a); 3.71 (dd, J = 9.5/7.2, 1H, H-10b); 3.59 (dd, J = 9.5/8.4, 1H, H-11a); 2.88 (dd, J = 9.5/8.3, 1H, H-11b); 2.74 (q, J = 8.4, 1H, H-12); 2.23 (m, 1H, H-9). Metabolite 1B, 7.54 (dd, J = , 1H, H-19); 7.47 (d, J = 1.8, 1H, H-15); 6.93 (d, J = 8.4, 1H, H-18); 6.83 (d, J = 8.0, 1H, H-5); 6.72 (d, J = 1.8, 1H, H-2); 6.71 (dd, J = 8.0/1.8, 1H, H-6); 4.56 (d, J = 7.0, 1H, H-8); 4.29 (m, 1H, H-11a); 4.20 (m, 1H, H-12); 4.17 (ddd, J = 9.1/3.9/0.9, 1H, H-11b); 3.81 (s, 3H, H-20); 3.74 (s, 3H, H-7); 3.35 (dd, J = 9.7/9.7, 1H, H-10a); 3.00 (dd, J = 9.7/5.0, 1H, H-10b); 2.58 (m, 1H, H- 9). Metabolite 2, 6.90 (d, J = 8.2, 1H, H-5); 6.88 (d, J = 1.9, 1H, H-2); 6.8 (dd, J = , 1H, H-6); 4.61 (d, J = 6.9, 1H, H-8); 4.49 (dd, J = , 1H, H-10a); 4.36 (m, 1H, H-11a); 4.33 (dd, J = , 1H, H-10b); 4.18 (m, 1H, H-11b); 3.77 (s, 3H, H-7); 3.44 (ddd, J = 9.0/9.0/3.8, 1H, H-12); 3.12 (m, 1H, H-9). 13 C NMR (CDCl 3, MHz) /ppm: Metabolite 1A, (s, C-3); (s, C-17); (s, C-16); (s, C-4); (s, C-14); (s, C-1); (d, C-6); (d, C-19); (d, C-18); (d, C-5); (d, C-2); (d, C-15); (s, C-13); 87.7 (d, C-8); 70.7 (t, C-11); 69.6 (t, C-10); 56.1 (d, C-12); 56.0 (q, C-20); 55.9 (q, C-7); 53.0 (d, C-9). Metabolite 1B, (s, C-13); (s, C-17); (s, C-16); (s, C-3); (s, C-4); (s, C-1); (s, C-14); (d, C-19); (d, C-6); (d, C-5); (d, C-18); (d, C-15); (d, C-2); 83.1 (d, C-8); 70.9 (t, C-11); 60.7 (t, C-10); 56.0 (q, C-7); 56.0 (q, C- 13

14 20); 54.1 (d, C-9); 47.7 (d, C-12). Metabolite 1A, (s, C-3); (s, C-4); (s, C-1); (d, C- 6); (d, C-5); (s, C-13); (d, C-2); 87.9 (d, C-8); 70.1 (t, C-11); 70.0 (t, C-10); 56.7 (d, C-12); 56.0 (q, C-7); 52.7 (d, C-9). Metabolite 1B, (s, C-13); (s, C-17); (s, C-3); (s, C-16); (s, C-4); (s, C-1); (s, C-14); (d, C-19); (d, C-6); (d, C-5); (d, C-18); (d, C-15); (d, C-2); 83.4 (d, C-8); 70.6 (t, C-11); 58.9 (t, C-10); 55.9 (q, C-7); 55.8 (q, C-20); 52.4 (d, C-9); 47.3 (d, C-12). Metabolite 2, (s, C-13); (s, C-3); (s, C-4); (s, C-1); (d, C-6); (d, C-5); (d, C-2); 86.1 (d, C-8); 69.9 (t, C-11); 69.8 (t, C-10); 55.8 (q, C-7); 48.2 (d, C-9); 46 (d, C-12). HMBC correlations: Metabolite 1A, H-2 C-(8); H-5 C-(1, 3, 4); H-6 C-(2, 4, 8); H-7 C-(3); H-8 C-(1, 2, 6, 9w, 10, 11w, 12w); H-9 C-(1w, 8w, 11w, 12w, 13); H-10a C-(8); H-10b C-(8, 9, 12, 13); H-11a C-(8, 9, 13w ); H-11b C-(8w, 12w, 13w); H-12 C-(10w, 13w); H-15 C-(13, 14, 16, 17, 19); H-19 C-(13, 15, 16, 18); H-20 C-(16). Metabolite 1B, H-2 C-(4, 6, 8); H-6 C-(3, 8); H-7 C-(3); H-8 C-(1, 2, 6, 9, 10, 11w, 12); H-9 C-(13w); H-10 C-(8, 12); H-11a C-(8, 9, 13); H-11a C-(9, 13); H-15 C-(13, 14, 16, 17, 19); H-18 C-(14, 16, 17); H-19 C-(13, 15, 17); H-20 C-(16). Metabolite 1A, H-2 C-(3w, 4, 6, 8); H-5 C-(1, 2w, 3, 4); H-6 C-(2, 4, 5w, 8); H-7 C-(3); H-8 C-(1, 2, 6, 9, 10 or 11); H-9 C-(1, 11, 12, 13w); H-10a C-(8, 12, 13); H-10b C-(8); H-11a C-(13w); H-11b C-(8, 9, 12, 13); H-12 C-(8, 9, 10 or 11, 13). Metabolite 1B, H-2 C-(1, 3w, 4, 6, 8); H-5 C- (1, 3, 4w); H-6 C-(2, 4, 8); H-7 C-(3); H-8 C-(1, 2, 6, 9, 10, 11w, 12); H-9 C-(1w, 10, 11w, 12w, 13w); H-10a C-(8, 9, 12, 13 of 1A'); H-10b C-(8, 9, 12, 13 of 1A'); H-11a C-(8, 9, 12, 13); H-11b C-(8w, 9, 12, 13); H-12 C-(8w, 9, 10, 11, 13 ); H-15 C-(13, 14, 16, 17, 19); H-18 C-(13w, 14, 16, 17); H-19 C-(13, 15, 16w, 17); H-20 C-(16). Metabolite 2, H-2 C-(6, 8); H-5 C-(1, 3); H-6 C-(2, 4, 8); H-7 C-(3); H-8 C-(1, 2, 6, 10, 12); H-9 C-(1, 12w, 13); H-10a C-(8, 9w, 13w); H-10b C-(9w, 13); H-11a C-(8, 13); H-11b C-(8w, 9w, 13); H-12 C-(8, 9, 10, 13). DQF-COSY correlations: Metabolite 1A, H-8 H-(9); H-9 H-(8, 10a, 10b, 12); H-10a H-(9, 10b); H- 10b H-(9, 10a); H-11a H-(11b, 12); H-11b H-(11a, 12); H-12 H-(9, 11a, 11b); H-18 H-(19); H- 19 H-(18). Metabolite 1B, H-8 H-(9); H-9 H-(8, 10, 11aw, 11b, 12); H-10 H-(9); H-11a H- 14

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16 Figure S5. GC-MS spectra of pinoresinol (A), its metabolites (B-I) and authentic standards (where available) after double derivatization with methoxyamine hydrochloride and N-trimethylsilyl-N-methyl trifluoroacetamide. This GC-MS data belongs to the degradation scheme shown in figure 7 of the main text. 16

17 Figure S6. Transformation of pinoresinol by pinoresinol-induced cell free extract as measured by HPLC. 17

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19 References 1. Kamaya Y, Nakatsubo F, Higuchi T, Iwahara S Degradation of d,l-syringaresinol, a β-β linked lignin model compound, by Fusarium-Solani M Arch Microbiol 129: Iwahara S, Higuchi T Enzymic oxidation of d,l-syringaresinol. Agric Biol Chem 46: Kamaya Y, Higuchi T Degradation of d,l-syringaresinol and its derivatives, β-β' linked lignin substructure models, by Phanerochaete chrysosporium. Mokuzai Gakkaishi 29: