8/12/2019 c3ob41885e.pdf
1/11
SI: Zi & Peters
S1
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
8/12/2019 c3ob41885e.pdf
2/11
SI: Zi & Peters
S2
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
tronic Supplementary Material (ESI) for Organic & Biomolecular Chemistryjournal is The Royal Society of Chemistry 2013
8/12/2019 c3ob41885e.pdf
3/11
SI: Zi & Peters
S3
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.
tronic Supplementary Material (ESI) for Organic & Biomolecular Chemistryjournal is The Royal Society of Chemistry 2013
8/12/2019 c3ob41885e.pdf
4/11
SI: Zi & Peters
S4
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 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 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
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
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
tronic Supplementary Material (ESI) for Organic & Biomolecular Chemistryjournal is The Royal Society of Chemistry 2013
8/12/2019 c3ob41885e.pdf
5/11
SI: Zi & Peters
S5
Figure S3.1H spectrum of Abietatriene (3)
Figure S4.13C spectrum of Abietatriene (3)
tronic Supplementary Material (ESI) for Organic & Biomolecular Chemistryjournal is The Royal Society of Chemistry 2013
8/12/2019 c3ob41885e.pdf
6/11
SI: Zi & Peters
S6
Figure S5. HSQC spectrum of Abietatriene (3)
Figure S6. HMBC spectrum of Abietatriene (3)
tronic Supplementary Material (ESI) for Organic & Biomolecular Chemistryjournal is The Royal Society of Chemistry 2013
8/12/2019 c3ob41885e.pdf
7/11
SI: Zi & Peters
S7
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
tronic Supplementary Material (ESI) for Organic & Biomolecular Chemistryjournal is The Royal Society of Chemistry 2013
8/12/2019 c3ob41885e.pdf
8/11
SI: Zi & Peters
S8
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
Retention Time (min.)
3
2
B)
Selected
IonCount 1
3
A)
TotalIonCount
1
3
A)
T
otalIonCount
19.519.018.518.017.517.016.5
Retention Time (min.)
1
3
B)
tronic Supplementary Material (ESI) for Organic & Biomolecular Chemistryjournal is The Royal Society of Chemistry 2013
8/12/2019 c3ob41885e.pdf
9/11
SI: Zi & Peters
S9
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
Retention Time (min.)
1
2
3
tronic Supplementary Material (ESI) for Organic & Biomolecular Chemistryjournal is The Royal Society of Chemistry 2013
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
tronic Supplementary Material (ESI) for Organic & Biomolecular Chemistryjournal is The Royal Society of Chemistry 2013
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
tronic Supplementary Material (ESI) for Organic & Biomolecular Chemistryjournal is The Royal Society of Chemistry 2013