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Supporting Information for:

Characterization of CYP76AH4 clarifies phenolic diterpenoid

biosynthesis in the Lamiaceae

Jiachen Zi and Reuben J. Peters

Dept. Biochem., Biophys. & Mol. Biol., Iowa State University, Ames, IA 50011 USA

Materials and Methods

General

Unless otherwise noted, chemicals were purchased from Fisher Scientific and molecular biologyreagents from Invitrogen. Sequence analyses were carried out with the CLC Main Workbench

6.9 software package (CLCbio). HPLC separations were carried out with an Agilent 1100 seriesinstrument equipped with fraction collector and diode array detector. Gas chromatography with

mass spectrometric detection (GC-MS) analyses were performed using a Varian (Palo Alto, CA,USA) 3900 instrument with HP-5ms column and Saturn 2100 ion trap mass spectrometer in

electron ionization (70 eV) mode. Samples (1 !L) were injected in splitless mode at 50 C and,after a 3 min. hold, the temperature raised at 14 C/min. to 300 C, where it was held for 3 min.

MS data was collected from m/zfrom 90 to 600, starting 12 min. after the injection until the endof the run. GC with flame ionization detection was carried out using an Agilent (Santa Clara, CA,

USA) 6890 GC with HP-5ms column, and the same protocol utilized as for GC-MS analyses.

Discovery of CYP76AH4-7

BLAST searches of the rosemary transcriptome (http://www.medicinalplantgenomics.msu.edu)using CYP76AH1 (GenBank JX422213)

1 as the query sequence yielded a number of hits. At

least five appeared to be full-length and fall within the CYP76AH sub-family (i.e., the translated

amino acid sequences were > 55% identical to CYP76AH1). Although three of these wereannotated as a single locus (roa_locus_3663), they shared less than 97% aa sequence identity

(using isotigs 5, 8 & 9 as representative), suggesting that they might be distinct loci. The othertwo were more clearly distinct (roa_locus_4381 and roa_locus_31503). These were submitted to

Prof. David Nelson (Univ. Tenn. Health Sci. Center) for determination of CYP nomenclature,which was assigned as follows: CYP76AH4 (roa_locus_3663_iso_9), with the other two isotigs

from this locus differing essentially only in a 54 aa stretch in the middle these were suggested tobe isoforms/variants CYP76AH5v1 (roa_locus_3663_iso_8) and CYP76AH5v2

(roa_locus_3663_iso_5), along with the more distinct CYP76AH6 (roa_locus_4381), andCYP76AH7 (roa_locus_31503).

Synthesis and cloning of CYP76AH1 and 4-7 for recombinant expression

Completely recoded genes for CYP76AH1 and CYP76AH4-7, including both isoforms of

CYP76AH5, as well as the CYP reductase (CPR) from Danshen (DsCPR1, GenBankFR693803),

1were synthesized (Genscript) with optimized codon usage for expression in E. coli

and, in the case of the CYP, also modified N-termini2 (see sequences below). These were

tronic Supplementary Material (ESI) for Organic & Biomolecular Chemistryjournal is The Royal Society of Chemistry 2013

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amplified via PCR, inserted into pENTR/SD/D-TOPO vectors, and each construct verified byfull gene sequencing. DsCPR1 was sub-cloned into the multiple cloning site (MSC) 2 of

pCDFDuet (Novagen) using BglII and KpnI restriction sites, which were introduced at the 5' and3' ends (respectively) of the synthetic gene during sub-cloning. CYP76AH1 & 4-7 were sub-

cloned into the MCS1 of pETDuet using the EcoRI and HindIII restriction sites, which were

introduced at the 5' and 3' ends (respectively) of the synthetic genes during sub-cloning. Finally,to enable co-production of miltiradiene (1), the previously described DsKSL3 was sub-cloned

into the EcoRI and HindIII restriction sites of the MSC1 of a pCDFDuet vector carrying either

DsCPR1 or a previously cloned4 CPR from Arabidopsis thaliana (AtCPR15, 6), which wereinserted into MCS2 using NdeI and KpnI restriction sites.

Characterization of CYP76AH1 and 4-7 via metabolic engineering

The ability of CYP76AH1 & 4-7 to react with 1 (actually, as eventually realized, 3) wasinvestigated using a previously described modular metabolic engineering system.

7 Briefly, E.

coli strain OverExpress C41 (Lucigen) was transformed with pGGnC, harboring ageranylgeranyl diphosphate synthase and copalyl diphosphate synthase, the pCDFDuet/CPR/

DsKSL and pETDuet/CYP76AHx constructs described above, as well as a previously describedpIRS vector overexpressing key enzymes in the upstream isoprenoid precursor supply pathway.8

The resulting recombinant strains were grown at 37 C in 50 mL TB (terrific broth) with

appropriate antibiotics to an OD600of 0.4!0.6, when the temperature was lowered to 16 C, the

cultures were supplemented with !-aminolevulinic acid (75 mg/L) and riboflavin (3 mg/L), and

after one hour equilibration time induced with 1 mM IPTG (isopropyl "-D-thiogalacto-pyranoside). After fermentation for an additional 72 h at 16 C, the cultures were extracted withan equal volume of hexanes, which was dried under nitrogen gas (N2), and resuspended in 200

!L hexanes for analysis by GC-MS. From these studies, of CP76AH4-7, CYP76AH4 gave thebest yield of ferruginol (2), with co-expression of AtCPR1 or DsCPR1 making no difference (the

data shown is here from co-expression with AtCPR1).

Characterization of CYP76AH1 & 4 via in vitro reactions

CYP76AH1 or CYP76AH4 constructs were co-expressed with DsCPR in E. coli strain

OverExpress C43, and in vitro reactions were carried out with the resulting clarified lysates asdescribed previously.

9This enabled kinetic assays, which were carried out much as previously

described.4Serial concentrations of abietatriene (5 M, 10 M, 15 M, 20 M, 30 M, 40 M,

60 M, 80 M and 120 M) together with 0.4 mM NADPH were assays in 500 L enzymereactions. The resulting solutions were extracted twice with 1 mL hexanes, the extracts dried

under N2, with the residue resuspended in 150 L hexanes for quantification by GC analysis.

Isolation of miltiradiene, abietatriene and ferruginol

To obtain miltiradiene, 3 L of recombinant E. coliC41 (Table S1) transformed with pGGnC,pIRS and pDEST14/DsKSL was grown as described previously.

3These were extracted with an

equal volume of hexanes, which was dried using a rotovap, and the residue resuspended in 3 mL

of hexanes, which was subjected to flash chromatography over a 4 g-silica column, with elutionby hexanes. After being dried under N2, 15 mg miltiradiene (1) was obtained. Abietatriene (3)

was produced by putting an open glass vial containing 3 mg of 1upside down on an UV lightplate, with the sample repeatedly dissolved in hexanes and evaporated under N2every other hour.

After 6 h, 1was completely converted into 3(2.6 mg). Ferruginol (2) was obtained from Salvia


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miltiorrhizahairy root cultures, which we have described previously.1After induction by yeast

extract (100 g/mL) and silver nitrate (30 M), the cultures were grown for another week, and atotal of 62 g tissues were frozen in liquid N2, ground into powder that was resuspended in 200mL methanol, and extracted with 200 mL hexanes. The hexane phase was dried using a rotovap,

and the residue was resuspended in 2 mL of a 1:1 mixture of acetonitrile and water and separated

by reversed-phase HPLC over an Agilent Eclipse XDB-C8 column (5m, 9.4 "250 mm), withelution via a linear gradient of 67%-100% acetonitrile in water to afford ferruginol (2.3 mg). The

isolated ferruginol (2) and abietatriene (3) were confirmed by NMR analysis (Table S1 andFigure S2). Briefly, the purified compound was dried under a gentle stream of N2, and then

dissolved in 0.5 mL deuterated chloroform (CDCl3; Sigma-Aldrich), with this evaporation-resuspension process repeated once more to completely remove the protonated acetonitrile

solvent. This sample was placed in NMR microtubes (Shigemi, Allison Park, PA) for analyses,and chemical shifts were referenced using known chloroform-d (

13C 77.23,

1H 7.24 ppm) signals

offset from TMS. Structural analysis was performed using 1D1H, and 2D DQF-COSY, HSQC,

HMQC, HMBC, and NOESY experiment spectra acquired at 700 MHz, and 1D13

C and

DEPT135 spectra (174 MHz) using standard experiments from the Bruker TopSpin v1.4

software.

Phytochemical analyses

To verify the presence of 1 3, rosemary aerial tissue (5.3 g) or induced S. miltiorrhizahairyroots (3.8 g) were frozen in liquid nitrogen and grinded to a fine powder. This material was

extracted with 2 "50 mL hexanes and then 50 mL methanol, which was re-extracted with anadditional 50 mL of hexanes. The hexanes extracts were pooled and evaporated under N2, with

the resulting residues resuspended in 50 L hexanes for analysis by GC-MS.

References

1.J. Guo, Y. J. Zhou, M. L. Hillwig, Y. Shen, L. Yang, Y. Wang, X. Zhang, W. Liu, R. J. Peters,

X. Chen, Z. K. Zhao and L. Huang, Proc Natl Acad Sci U S A, 2013, 110, 12108-12113.2.E. E. Scott, M. Spatzenegger and J. R. Halpert, Arch. Biochem. Biophys., 2001, 395, 57-68.3.W. Gao, M. L. Hillwig, L. Huang, G. Cui, X. Wang, J. Kong, B. Yang and R. J. Peters, Org.

Lett., 2009, 11, 5170-5173.4.D. Morrone, X. Chen, R. M. Coates and R. J. Peters, Biochem. J., 2010, 431, 337-344.

5.P. Urban, C. Mignotte, M. Kazmaier, F. Delorme and D. Pompon, J. Biol. Chem., 1997, 272,19176-19186.

6.M. Mizutani and D. Ohta, Plant Physiol., 1998, 116, 357-367.7.A. Cyr, P. R. Wilderman, M. Determan and R. J. Peters, J. Am. Chem. Soc., 2007, 129, 6684-

6685.8.D. Morrone, L. Lowry, M. K. Determan, D. M. Hershey, M. Xu and R. J. Peters, Appl.

Microbiol. Biotechnol., 2010, 85, 1893-1906.9.Y. Wu, M. L. Hillwig, Q. Wang and R. J. Peters, FEBS Lett., 2011, 585, 3446-3451.


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Figure S1.Alignment of CYP76AH1 (Danshen) with CYP76AH4-7 (rosemary).

Figure S2.Numbering and selected1H-

1H COSY and HMBC correlations of abietatriene (3)

M D S - - F P L L A A L F F I AA T I T F L S F - - - - - - - R R R R N L P P G P F P Y P I V GN M L Q L GA N P H Q V F A K L S K R Y G P L M S I H L G S L Y T V I V S S P E M A K E I L H R H GQ V F S

M D S - - F P L L V A L F F I AV T - T F L S F - - - - - - - R R P R N L P P G P F P L P I V GN M L Q L GT Q P H E T F A K L S K K Y GP L M S I H L G S L Y T V I V S S P E MA K E I M H K Y GQ V F S



M E F - - S T L L I A L F S I - - T L T Y F I F - K S G S K R R SG A K L P P G P Y P L P I V G N I F Q L G T K P H Q S L A Q L A KT H G P L M S L R F G S V Y T V I V T S P EM A R E I F V R H DQ D F L

M D S L F F P L L V G L S L V G - - L T G F L L L R S G S R P R R VQ P L P P G P K P L P V V G N F L Q L G K K P Y E T L T Q L A K T H G P L M S I H L G S L Y T V I V S S P E MA K E L L Q H H GQ V F S

G R T I A Q A V H A C D H D K I SM G F L P V A S E W R DM R K I C K E QM F S N Q S M E A S Q G L R R Q K L Q Q L L D H V Q K C S D S G R A V D I R E A A F I T T L N L M S A T L F S S Q A T E F D S K A

G R T V A Q A V H A C G H D K I S M G F L P V G G E WR D M R K I C K E Q M F S H Q S M E D S Q WL R K Q K L Q Q L L E Y A Q K C S E R G R A I D I R E A A F I T T L N L M S A T L F S MQ A T E F D S K V



N R T V V E A V H A HD H D S I S MA F M D V GA E WR T L R R I C K E Q I F S V Q S L E A S Q G L R R E K L H Q L R E Y V L R C C D A GR V V D I R E A S F V T T L N L M S A S L F S I Q A T E F D S T A

G R T T A Q V MH A R D L N K L S M A F T P T G K E WR D K R K I C K E Q V F S E R S L D G S E A L R Q E K L Q Q L V E H V R R H C D RG D V V D I H D A M L V T N L N L M L T T L F S T R S P D F E S A I

T M E F K E I I E G V A T I V G V P N F A D Y F P I L R P F D P Q GV K R R A D V F F G K L L A K I E G Y L N E R L E S K R A N P N A P K KD D F L E I V V D I I - Q A N E F K L K T H H F T H L M L D L F

T M E F K E I I E G V A T I V G V P N F A D Y F P I L R P F D P Q GV K R R A D V Y F G R L L A I I E G F L N E R I E S R RT N P N A P K K D D F L E T L V D T L - Q T N D N K L K T D H L T H L M L D L F

T M E F K E I I E G V A T I V G V P N F A D Y F P I L R P F D P Q GV K R R A N AY F G K L L A K I E G Y L S E R I A S R K AN P N A P K K D D F L E T L V D I I - E A N E Y K L K T D H F T H L M L D L F

T M E F K E I I E G V A T I V G V P N F A D Y F P I L R P F D P Q GV K R R A D V Y F G R L L A I I E G F L N E R I E S R RT N P N A P K K D D F L E T L V D T L - Q T N D N K L K T D H L T H L M L D L F

T E E F R E I M E G V A S I V G D P N F A D Y F P I L K R F D P Q GV K R K A E L Y F G K M L V L V Q D L L E K R Q E E R R R A P N Y V K KN D L L E K L V D V L N E E S E Y K L T T K H I T H L L L D L F

T R E F K E I V E I I A V A V A V P N F A D Y F P I L K P F D P Q G I K S K A E L Y F G K L F Q K F E G F L H E R L E S R R N N P GA P R E H D L L E T L V E - I S E G ND Y N L T T E E I P Y L I F D L F

V G G S D T N T T S I E WA M S E L V M N P D K M A R L K A E L K S V A G D E K I V D E S A M P K L P Y L Q A V I K E V M R I H P P G P L L L P R K A E S D Q E V N G Y L I P K G T Q I L I N A Y A I G RD

V G G S E T S T T S I E WT M S E L V M N P E K M A K L K A E L K S V A G D E K I V D E S E I A K L P Y L Q A V I K E V M R I H P P G P L L L P R K A E S D Q E V N G Y L I P K G T Q V L I N A WA I GR D

V G G S E T S T T S I E W T M S E L V M N P E K M A K L K A E L K S V M G E E K V V D E S Q M P R L P Y L Q A V V K E S M R L H P P G P L L L P R K A E S D Q V V N G Y L I P K G A Q V L I N A W A M G R D

V G G S E T S T T S I E W T M S E L V M N P E K M A K L K A E L K S V M G E E K V V D E S Q M P R L P Y L Q A V V K E S M R L H P P G P L L L P R K A E S D Q V V N G Y L I P K G A Q V L I N A W A M G R D

V G G S E T T T T S V E W I M S E L L I N P E K L E K T K E E L R R V V G E K NQ V Q E S D I P R L P Y F E A V L K E V F R L H P P G P L L L P R K A E R E V Q I G E Y T I P K D T Q I L I N A WA I G RD

V G G S E T N S T S I E W I M T E L L F H P D K L H K L K E E L N R V V G E K E Q I R E S D I P R L P Y L Q A V I K E T L R Y H P P G P L L L P R R S E A D Q V V N G Y M I P K G A Q I L F N V WA M GR D

P S I WT D P E T F D P E R F L D N K I D F K G QD Y E L L P F G S G R R V C P GM P L A T R I L H MA T A T L V H N F D WK L E D D S T A A A D H A G E L F G V A V R R AV P L R I I P I V K S *

P S V WK N P D S F E P E R F L E Q K I D F K GQ D F E L L P F G S G R R V C P G L P L A S R I L H M T A A T L V HN F D WK L E D E A T A E A D H A G E L F G L A V R R A V P L R I I P I V K S *

P S I WK N P D S F E P E R F L D Q K I D F K GT D Y E L I P F G S G R R V C P GM P L A N R I L H T V T A T L V H N F H WE L E R P D A S H A E H QG V L F G F A V R R A V P L K I V P F - - K V

P S I WK N P D S F E P E R F L D Q K I D F K GQ D Y E L I P F G S G R R V C P GM P L A N R I L H T V T A T L V H N F H W E L E R P D A SH A E H Q GV L F G F A V R R A V P L K I V P F - - K V

P S I W P N P E A F E P E R F L A Q K K D Y K G Q D F E L I P F G S G R R M C P G L S F A N R M L P M T V A T L I H N F D WK L E V E A N A Q D V H K G E M F G I A V R R A V P L R A Y P I - - S H

P S I W PN P D S F E P E R F L D K K V D F K G QH F E L I P F G A GR R I C P GM P L A T R I L QM T V A V L V H N F E WK L E K E K D - H A D H K G E V L G V A L R R A V P L R A I P I - - K N

1H-1H COSY

HMBC

Abietatriene (3)Abietatriene (3)

1

3 56

7

9

10

11 13

14

15

16

17

18 19

20


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Figure S3.1H spectrum of Abietatriene (3)

Figure S4.13C spectrum of Abietatriene (3)


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Figure S5. HSQC spectrum of Abietatriene (3)

Figure S6. HMBC spectrum of Abietatriene (3)


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Figure S7. NOESY spectrum of Abietatriene (3)

Table S1.NMR Data (in CD3Cl)

Position Abietatriene (3) Ferruginol (2)

!H !C !H !C

1 a 2.18 (1H, br.d,J= 12.5 Hz) 39.4 2.15 (1H, br.d,J= 12.4 Hz) 39.1b 1.31 (1H, br.d,J= 12.5 Hz) 1.36(1H, br.d,J= 12.4, 3.4 Hz)

2 a 1.66 (1H, m ) 19.9 1.71 (1H, m) 19.5

b 1.51 (1H, m ) 1.57 (1H, m)

3 a 1.38 (1H, d,J= 13.2 Hz) 42.3 1.45 (1H, br.d,J= 13.1 Hz) 41.9

b 1.12 (1H, m) 1.18 (1H, m)

4 34.0 33.5

5 1.26 (1H, d,J= 12.3 Hz) 51.0 1.30 (1H, dd,J= 12.4, 1.8 Hz) 50.6

6 a 1.78 (1H, dd,J= 13.1, 7.6) 19.7 1.83 (1H, m) 19.4

b 1.62 (1H, m) 1.64 (1H, m)

7 a 2.83 (1H, dd,J= 17.2, 6.6 Hz) 31.1 2.84 (1H, dd,J= 16.5, 6.4 Hz) 30.0

b 2.78 (1H, m) 2.75 (1H, ddd,J= 16.5, 11.4, 7.6 Hz)

8 135.5 127.5

9 148.2 148.9

10 38.1 37.7

11 7.09 (1H, d,J= 8.2 Hz) 124.9 6.61 (1H, s) 111.212 6.90 (1H, br.d,J= 8.2 Hz) 124.4 150.9

13 146.0 131.5

14 6.80 (1H, br.s) 127.4 6.81 (1H, s) 126.8

15 2.74 (1H, m) 34.0 3.08 (1H, m) 27.0

16 1.14 (3H, d,J= 6.9 Hz) 24.57 1.22 (3H, d,J= 7.0 Hz) 22.8

17 1.14 (3H, d,J= 6.9 Hz) 24.59 1.20 (3H, d,J= 7.0 Hz) 22.9

18 0.86 (3H, s) 33.9 0.92 (3H, s) 33.5

19 0.84 (3H, s) 22.2 0.89 (3H, s) 21.8

20 1.09 (3H, s) 25.5 1.15 (3H, s) 25.0


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Figure S8. 1 is spontaneously converted to 3. GC-MS chromatogram of extract from E. coli

expressing only enzymes for the production of 1(i.e., no CYP or CPR).

Figure S9.CYP76AH1 also only hydroxylates 3. GC-MS chromatograms of in vitro reactions

with (A) 1(selected ions m/z= 272 + 286) or (B) 3(selected ions m/z= 255 + 286).

Figure S10. GC-MS analysis of 1 incubated in phosphate buffer, pH 7.5 for 3 days at roomtemperature. (A) In the presence of O2. (B) In the absence of O2 (removed by bubbling N2

through solution for 15 min.).

TotalIonCount

19.519.018.518.017.517.016.5

Retention Time (min.)

1

3

SelectedIonCount

19.519.018.518.017.517.016.5


3

2

B)

Selected

IonCount 1

3

A)

TotalIonCount

1

3

A)

T

otalIonCount

19.519.018.518.017.517.016.5


1

3

B)


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Figure S11.GC-MS analysis of S. miltiorrhizahairy root extract.

Synthetic gene sequences

CYP76AH1

ATGGCTAAGAAAACCTCGTCAAAGGGTAAACTGCCGCCGGGTCCGTTCCCGTATCCGATTGTGGGTAACATGCTGCAACTGGGTGC

TAACCCGCATCAGGTGTTTGCAAAACTGTCGAAGCGTTATGGCCCGCTGATGAGTATTCACCTGGGTTCCCTGTACACGGTCATCG

TGAGCTCTCCGGAAATGGCCAAAGAAATTCTGCATCGTCACGGCCAGGTGTTTAGCGGCCGTACCATTGCCCAAGCAGTCCATGCA

TGCGATCACGACAAAATCTCTATGGGCTTTCTGCCGGTTGCTAGTGAATGGCGTGATATGCGCAAAATCTGTAAGGAACAGATGTT

CAGCAATCAATCTATGGAAGCATCTCAGGGCCTGCGTCGCCAAAAGCTGCAGCAACTGCTGGATCATGTGCAGAAATGCTCAGATT

CGGGTCGTGCTGTTGACATTCGCGAAGCGGCCTTTATCACCACGCTGAACCTGATGTCGGCCACCCTGTTTAGTTCCCAAGCAACG

GAATTTGATAGCAAAGCTACGATGGAATTTAAGGAAATTATCGAAGGCGTGGCGACCATTGTTGGTGTCCCGAATTTTGCCGATTA

TTTCCCGATCCTGCGTCCGTTCGACCCGCAGGGTGTTAAACGTCGCGCAGATGTCTTTTTCGGCAAACTGCTGGCTAAGATTGAAG

GTTACCTGAACGAACGTCTGGAAAGTAAACGCGCTAACCCGAATGCGCCGAAAAAGGATGACTTTCTGGAAATCGTGGTTGATATT

ATCCAGGCGAATGAATTTAAACTGAAGACCCATCACTTCACGCATCTGATGCTGGACCTGTTTGTCGGCGGTTCAGACACCAACAC

CACGTCGATTGAATGGGCGATGAGCGAACTGGTTATGAATCCGGATAAAATGGCCCGCCTGAAAGCAGAACTGAAGTCCGTGGCCG

GCGATGAAAAAATCGTTGACGAATCAGCGATGCCGAAGCTGCCGTATCTGCAGGCCGTCATTAAAGAAGTGATGCGTATCCACCCG

CCGGGTCCGCTGCTGCTGCCGCGCAAGGCGGAATCTGACCAGGAAGTGAACGGCTATCTGATTCCGAAAGGCACCCAAATTCTGAT

CAATGCTTACGCGATTGGCCGTGATCCGAGTATCTGGACCGATCCGGAAACGTTTGACCCGGAACGCTTCCTGGATAACAAAATTG

ACTTTAAGGGCCAGGATTACGAACTGCTGCCGTTCGGCAGCGGTCGTCGCGTGTGTCCGGGTATGCCGCTGGCAACGCGTATCCTG

CACATGGCCACCGCAACGCTGGTTCACAATTTTGACTGGAAACTGGAAGATGACTCCACCGCAGCTGCGGATCATGCCGGCGAACT

GTTCGGTGTGGCTGTTCGTCGCGCGGTCCCGCTGCGTATTATCCCGATTGTGAAATCATAA

CYP76AH4

ATGGCTAAGAAAACCTCGTCCAAGGGTAAACTGCCGCCGGGTCCGTTCCCGCTGCCGATTGTGGGTAACATGCTGCAACTGGGTAC

GCAGCCGCATGAAACGTTCGCCAAACTGTCTAAAAAGTATGGCCCGCTGATGTCTATTCACCTGGGTAGTCTGTACACCGTGATCG

TTAGCTCTCCGGAAATGGCCAAAGAAATTATGCATAAGTATGGCCAGGTGTTTTCGGGTCGTACCGTTGCGCAAGCCGTCCATGCA

TGCGGCCACGATAAAATTAGCATGGGTTTCCTGCCGGTTGGCGGTGAATGGCGTGACATGCGCAAAATCTGTAAGGAACAGATGTT

TAGTCACCAAAGCATGGAAGATTCTCAGTGGCTGCGTAAACAAAAGCTGCAGCAACTGCTGGAATACGCTCAGAAATGCAGTGAAC

GTGGCCGCGCGATTGATATCCGCGAAGCGGCCTTTATCACCACGCTGAATCTGATGTCCGCTACCCTGTTCTCAATGCAAGCGACG

GAATTTGATAGCAAAGTCACGATGGAATTTAAGGAAATTATCGAAGGCGTGGCCACCATTGTCGGTGTGCCGAATTTTGCAGACTA

TTTCCCGATCCTGCGCCCGTTTGATCCGCAGGGTGTTAAACGTCGCGCTGACGTCTACTTCGGCCGTCTGCTGGCGATTATCGAAG

GTTTTCTGAACGAACGCATTGAATCCCGTCGCACGAACCCGAATGCGCCGAAAAAGGATGACTTCCTGGAAACCCTGGTGGATACC

CTGCAGACGAACGACAATAAACTGAAGACCGATCATCTGACGCACCTGATGCTGGACCTGTTTGTGGGCGGTTCAGAAACGTCGAC

CACGAGCATCGAATGGACCATGAGTGAACTGGTTATGAACCCGGAAAAAATGGCTAAACTGAAGGCGGAACTGAAATCTGTGGCAG

GCGATGAAAAGATTGTTGACGAAAGTGAAATCGCCAAACTGCCGTATCTGCAGGCAGTGATTAAGGAAGTTATGCGTATCCATCCG

CCGGGTCCGCTGCTGCTGCCGCGCAAAGCGGAAAGCGATCAGGAAGTTAACGGCTACCTGATTCCGAAAGGCACCCAAGTCCTGAT

TAATGCATGGGCTATCGGCCGTGACCCGTCGGTGTGGAAAAACCCGGATAGCTTTGAACCGGAACGCTTCCTGGAACAGAAAATTG

ATTTTAAGGGCCAAGACTTCGAACTGCTGCCGTTTGGCTCCGGTCGTCGCGTCTGTCCGGGTCTGCCGCTGGCTTCACGTATCCTG

CACATGACCGCAGCTACGCTGGTTCACAATTTCGATTGGAAACTGGAAGACGAAGCGACCGCCGAAGCAGATCACGCCGGCGAACT

GTTTGGTCTGGCAGTCCGTCGCGCAGTGCCGCTGCGTATTATCCCGATTGTTAAAAGCTAA

T

otalIonCount

19.519.018.518.017.517.016.5


1

2

3


8/12/2019 c3ob41885e.pdf

10/11

SI: Zi & Peters

S10

CYP76AH5v1

ATGGCTAAGAAAACCTCGTCAAAGGGTAAACTGCCGCCGGGTCCGTTCCCGCTGCCGATTGTGGGTAACATGCTGCAACTGGGTAC

GCAGCCGCATGAAACCTTTGCCAAACTGAGCAAAAAGTATGGCCCGCTGATGTCTATTCACCTGGGTAGTCTGTACACGGTGATCG

TTAGCTCTCCGGAAATGGCTAAGGAAATCATGCATAAGTACGGCCAGGTGTTTTCGGGTCGTACCGTTGCACAAGCTGTCCATGCC

TGCGGCCACGATAAAATTAGCATGGGTTTTCTGCCGGTTGGCGGTGAATGGCGTGACATGCGCAAAATCTGTAAGGAACAGATGTT

CAGTCACCAAAGCATGGAAGATTCCCAGTGGCTGCGTAAACAAAAGCTGCAGCAACTGCTGGAATACGCTCAGAAATGCTCAGAAC

GTGGCCGCGCGATTGATATCCGCGAAGCGGCCTTTATCACCACGCTGAATCTGATGTCCGCCACGCTGTTTTCAATGCAAGCAACC

GAATTTGACTCTAAGGTGACGATGGAATTTAAGGAAATCATCGAAGGCGTTGCCACCATTGTCGGTGTGCCGAATTTTGCAGATTA

TTTCCCGATCCTGCGTCCGTTTGACCCGCAGGGTGTGAAACGTCGCGCGAACGCCTATTTCGGCAAACTGCTGGCAAAGATTGAAG

GTTACCTGTCGGAACGTATCGCAAGCCGCAAAGCTAACCCGAATGCGCCGAAAAAGGATGACTTTCTGGAAACCCTGGTTGATATT

ATCGAAGCGAATGAATACAAACTGAAGACGGATCATTTTACCCACCTGATGCTGGACCTGTTCGTCGGCGGTTCAGAAACCTCGAC

CACGAGCATTGAATGGACGATGAGTGAACTGGTCATGAACCCGGAAAAAATGGCCAAACTGAAGGCAGAACTGAAATCCGTGATGG

GCGAAGAAAAGGTGGTTGATGAATCACAGATGCCGCGCCTGCCGTATCTGCAAGCGGTCGTGAAAGAATCTATGCGTCTGCATCCG

CCGGGTCCGCTGCTGCTGCCGCGCAAAGCCGAAAGTGACCAGGTTGTCAACGGCTACCTGATTCCGAAAGGTGCACAAGTTCTGAT

CAATGCATGGGCTATGGGCCGTGATCCGTCTATTTGGAAAAACCCGGACAGTTTTGAACCGGAACGCTTCCTGGATCAGAAAATTG

ACTTTAAGGGCACCGATTATGAACTGATCCCGTTCGGCTCGGGTCGTCGCGTGTGTCCGGGTATGCCGCTGGCTAATCGTATTCTG

CATACCGTTACGGCGACCCTGGTCCATAACTTTCACTGGGAACTGGAACGCCCGGATGCGTCACATGCCGAACACCAGGGCGTTCT

GTTTGGTTTCGCTGTGCGTCGCGCGGTTCCGCTGAAAATCGTCCCGTTCAAGGTGTAA

CYP76AH5v2

ATGGCTAAGAAAACCTCCTCCAAGGGTAAACTGCCGCCGGGTCCGTTCCCGCTGCCGATTGTGGGTAACATGCTGCAACTGGGTAC

GCAGCCGCATGAAACCTTTGCGAAACTGTCTAAAAAGTATGGCCCGCTGATGTCTATTCACCTGGGTAGTCTGTACACCGTGATCG

TTAGCTCTCCGGAAATGGCGAAAGAAATTATGCATAAGTATGGCCAGGTGTTTTCCGGTCGTACCGTTGCACAAGCTGTCCATGCC

TGCGGCCACGATAAAATTTCAATGGGTTTTCTGCCGGTTGGCGGTGAATGGCGTGACATGCGCAAAATCTGTAAGGAACAGATGTT

CAGTCACCAAAGCATGGAAGATTCTCAGTGGCTGCGTAAACAAAAGCTGCAGCAACTGCTGGAATACGCACAGAAATGCAGTGAAC

GTGGCCGCGCTATTGATATCCGCGAAGCGGCCTTTATCACCACGCTGAATCTGATGTCCGCGACGCTGTTTTCAATGCAGGCCACC

GAATTTGACAGCAAAGTCACGATGGAATTTAAGGAAATTATCGAAGGCGTGGCGACCATTGTCGGTGTGCCGAATTTTGCCGACTA

TTTCCCGATCCTGCGTCCGTTCGATCCGCAGGGTGTGAAACGTCGCGCAGACGTTTACTTTGGCCGTCTGCTGGCTATTATCGAAG

GTTTCCTGAACGAACGCATTGAAAGCCGTCGCACCAACCCGAATGCACCGAAAAAGGATGACTTTCTGGAAACCCTGGTGGATACG

CTGCAGACCAACGACAATAAACTGAAGACGGATCATCTGACCCACCTGATGCTGGACCTGTTTGTTGGCGGTTCAGAAACCTCGAC

CACGAGCATCGAATGGACGATGAGCGAACTGGTTATGAACCCGGAAAAAATGGCGAAACTGAAGGCCGAACTGAAATCTGTCATGG

GCGAAGAAAAGGTGGTTGATGAAAGTCAGATGCCGCGCCTGCCGTATCTGCAAGCCGTCGTGAAAGAATCGATGCGTCTGCATCCG

CCGGGTCCGCTGCTGCTGCCGCGCAAAGCAGAAAGCGACCAGGTTGTCAACGGCTACCTGATTCCGAAAGGTGCTCAAGTGCTGAT

CAATGCGTGGGCGATGGGCCGTGACCCGTCGATTTGGAAAAACCCGGATAGCTTTGAACCGGAACGCTTCCTGGATCAGAAAATTG

ACTTTAAGGGCCAAGATTATGAACTGATCCCGTTCGGCTCCGGTCGTCGCGTGTGTCCGGGTATGCCGCTGGCAAATCGTATTCTG

CATACCGTTACGGCCACCCTGGTCCATAACTTTCACTGGGAACTGGAACGCCCGGATGCAAGTCATGCTGAACACCAGGGCGTTCT

GTTTGGTTTCGCAGTGCGTCGCGCTGTTCCGCTGAAAATCGTCCCGTTCAAGGTGTAA

CYP76AH6

ATGGCTAAGAAGACGTCCTCAAAGGGTAAACTGCCGCCGGGTCCGTATCCGCTGCCGATTGTGGGTAACATTTTTCAACTGGGTAC

GAAACCGCACCAGTCCCTGGCACAACTGGCAAAGACCCACGGCCCGCTGATGAGCCTGCGCTTTGGTTCTGTTTATACCGTGATTG

TTACGTCACCGGAAATGGCCCGCGAAATCTTTGTTCGTCACGATCAGGACTTCCTGAACCGCACCGTGGTTGAAGCAGTCCATGCT

CACGATCATGACAGCATTTCTATGGCATTTATGGATGTGGGCGCTGAATGGCGTACGCTGCGTCGCATTTGCAAAGAACAGATCTT

CTCAGTGCAATCGCTGGAAGCATCGCAGGGTCTGCGTCGCGAAAAACTGCATCAACTGCGCGAATACGTTCTGCGTTGCTGTGATG

CCGGTCGCGTCGTGGACATTCGTGAAGCAAGCTTTGTCACCACGCTGAACCTGATGAGTGCGTCCCTGTTTTCGATCCAGGCCACC

GAATTTGATAGCACCGCAACGGAAGAATTTCGCGAAATTATGGAAGGCGTGGCTTCTATCGTTGGTGATCCGAATTTTGCGGACTA

TTTCCCGATTCTGAAACGCTTTGATCCGCAGGGCGTTAAGCGTAAGGCCGAACTGTACTTCGGTAAAATGCTGGTCCTGGTGCAGG

ACCTGCTGGAAAAGCGTCAAGAAGAACGTCGCCGTGCACCGAACTATGTGAAAAAGAATGATCTGCTGGAAAAACTGGTTGACGTC

CTGAACGAAGAAAGCGAATACAAACTGACCACGAAGCACATTACCCATCTGCTGCTGGACCTGTTTGTCGGCGGTAGTGAAACCAC

GACCACGTCCGTGGAATGGATTATGTCAGAACTGCTGATCAACCCGGAAAAACTGGAAAAAACCAAGGAAGAACTGCGCCGTGTTG

TCGGCGAAAAGAACCAGGTTCAAGAATCTGATATCCCGCGCCTGCCGTATTTTGAAGCTGTCCTGAAAGAAGTGTTCCGTCTGCAC

CCGCCGGGTCCGCTGCTGCTGCCGCGCAAAGCGGAACGTGAAGTGCAGATTGGTGAATACACCATCCCGAAGGATACGCAAATTCT

GATCAACGCGTGGGCCATTGGTCGCGACCCGAGTATCTGGCCGAATCCGGAAGCTTTTGAACCGGAACGTTTCCTGGCGCAGAAAA

AGGATTATAAAGGCCAAGACTTTGAACTGATTCCGTTCGGCTCTGGTCGCCGTATGTGTCCGGGTCTGAGTTTTGCCAATCGCATG

CTGCCGATGACCGTGGCAACGCTGATCCACAACTTCGATTGGAAACTGGAAGTGGAAGCAAATGCTCAGGACGTTCATAAGGGCGA

AATGTTTGGTATTGCAGTTCGCCGTGCCGTCCCGCTGCGTGCATACCCGATCTCCCATTAA


8/12/2019 c3ob41885e.pdf

11/11

SI: Zi & Peters

S11

CYP76AH7

ATGGCTAAGAAAACCTCGTCAAAGGGTAAACTGCCGCCGGGTCCGAAACCGCTGCCGGTCGTGGGTAACTTCCTGCAACTGGGTAA

AAAACCGTATGAAACCCTGACGCAGCTGGCTAAGACCCACGGCCCGCTGATGAGTATTCACCTGGGTTCCCTGTACACGGTGATCG

TTAGCTCTCCGGAAATGGCAAAAGAACTGCTGCAGCATCACGGCCAAGTGTTTAGCGGTCGTACCACGGCCCAGGTTATGCATGCA

CGCGATCTGAATAAACTGTCTATGGCCTTTACCCCGACGGGCAAGGAATGGCGTGATAAACGCAAGATTTGCAAAGAACAGGTGTT

CTCAGAACGTTCGCTGGACGGTAGCGAAGCGCTGCGCCAAGAAAAACTGCAGCAACTGGTCGAACATGTGCGTCGCCACTGTGATC

GTGGCGACGTGGTTGATATCCACGACGCCATGCTGGTGACCAACCTGAATCTGATGCTGACCACGCTGTTTAGCACCCGTTCTCCG

GATTTCGAAAGCGCAATTACGCGCGAATTTAAAGAAATCGTTGAAATTATCGCGGTCGCCGTGGCAGTTCCGAACTTCGCTGATTA

TTTTCCGATTCTGAAACCGTTTGACCCGCAGGGTATCAAATCTAAGGCGGAACTGTACTTCGGCAAACTGTTTCAAAAGTTCGAAG

GTTTTCTGCATGAACGTCTGGAAAGTCGTCGCAACAATCCGGGTGCACCGCGCGAACACGATCTGCTGGAAACCCTGGTCGAAATT

TCCGAGGGTAACGACTATAATCTGACCACGGAAGAAATTCCGTACCTGATCTTCGACCTGTTTGTGGGCGGTAGCGAAACCAATAG

TACGTCCATTGAATGGATCATGACCGAACTGCTGTTTCATCCGGATAAGCTGCACAAACTGAAGGAAGAACTGAACCGTGTCGTGG

GCGAAAAAGAACAGATTCGTGAATCAGACATCCCGCGCCTGCCGTATCTGCAAGCTGTGATTAAAGAAACCCTGCGCTACCATCCG

CCGGGTCCGCTGCTGCTGCCGCGTCGCTCGGAAGCGGATCAGGTTGTCAACGGCTATATGATTCCGAAAGGTGCTCAAATCCTGTT

TAATGTTTGGGCGATGGGCCGTGATCCGTCAATTTGGCCGAACCCGGACTCGTTCGAACCGGAACGCTTTCTGGATAAAAAGGTCG

ACTTCAAAGGCCAGCATTTTGAACTGATTCCGTTCGGTGCAGGTCGTCGCATCTGCCCGGGTATGCCGCTGGCAACCCGTATCCTG

CAGATGACGGTCGCAGTGCTGGTTCACAATTTCGAATGGAAGCTGGAAAAAGAAAAGGATCATGCCGACCACAAAGGCGAAGTTCT

GGGTGTCGCTCTGCGTCGCGCGGTTCCGCTGCGTGCAATTCCGATCAAAAACTAA


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