Supplementary materials and methods

Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum

bicolor into tobacco chloroplasts for light-driven synthesis

Thiyagarajan Gnanasekaran, Daniel Karcher, Agnieszka Zygadlo Nielsen, Helle Juel Martens,

Stephanie Ruf, Xenia Kroop, Carl Erik Olsen, Mohammed Saddik Motawie, Mathias Pribil, Birger

Lindberg Møller, Ralph Bock and Poul Erik Jensen

Expression of the individual CYP79A1 and CYP71E1 enzymes and their

targeting to the thylakoids of genetically transformed N. benthamiana

Materials and methods

Plant material and growth conditions

Tobacco (N. benthamiana) plants were grown under greenhouse conditions (16 h light/8 h dark cycle;

25°C/20°C; light intensity of 200–600 µmol m-2

s-1

).

Cloning, vector construction and transformation of A. tumefaciens

Single constructs harboring the coding region of the CYP79A1 or CYP71E1 gene fused to the

ferredoxin transit peptide (Fd(TP)) were built (Nielsen et al., 2013) and incorporated into the pEAQ-

HT plasmid (Sainsbury et al., 2009). Electrocompetent A. tumefacians (LBA4404) cells were prepared

(Dulk-Ras and Hooykaas, 1995) and transformed with the vector pEAQ-HT harboring the Fd(TP)-

CYP79A1 and Fd(TP)-CYP71E1 constructs by electroporation (400 ohm resistance, 2.5 kV, 25 µF

capacitance; Gene Pulser, Biorad). The transformed cells were plated on YEP plates with 50 mg/l

kanamycin and 50 mg/l rifampicin for selection.

Nuclear transformation of N. benthamiana

The A.tumefacians transformants containing the vector pEAQ-HT carrying Fd (TP)-CYP79A1 and

Fd(TP)-CYP71E1 were grown for 48-72 h at 28°C in YEP media supplied with 25 mg/l rifampicin and

50 mg/l kanamycin. Nuclear transformation of N. benthamiana was carried out (Horsch RB, 1985).

Seed sterilization and regeneration of nuclear transformed lines

Seeds obtained from nuclear transformed N. benthamiana lines were surface sterilized (Fischer and

Hain, 1995), placed on plates with MS medium containing 50 mg/l kanamycin, and germination was

initiated by incubation in the dark for 48 h at 4°C followed by a 2 h incubation in the light (100-200

µmol m-2

s-1

) at room temperature. The plates were then incubated at room temperature in the dark for 2

d and kept in the growth chamber with a light intensity of 200 µmol m-2

s-1

at 25°C.

Genomic DNA purification and PCR

Genomic DNA was isolated from putative nuclear N. benthamiana transformants(Edwards et al.,

1991). To confirm successful transformation, 1 µL of the genomic DNA was used as a template for

PCR using the primer pairs 79-F and 79-R for Fd(TP)-CYP79A1 transgenic lines and 71-F and 71-R for

Fd(TP)-CYP71E1 transgenic lines (Supplementary Table S1).

Results

Each individual P450 of the dhurrin pathway was targeted to the chloroplast of N. benthamiana by

fusing the N-terminus of the protein to the first 52 amino acids of the transit peptide of the Arabidopsis

thaliana ferredoxin protein FedA (NCBI gene ID: 22136515; Fd(TP); Fig. S2A). Using the pEAQ-HT

expression vector, the two fusion constructs (Fd(TP)-CYP79A1 and Fd(TP)-CYP71E1) were

transformed into the nuclear genome of N. benthamiana. For each construct, 3-5 transformed lines

were obtained from two independent transformation events and analyzed. Genomic DNA was extracted

and successful integration of the individual genes in the nuclear genome of the plants was verified by

PCR. The size of the PCR product obtained for the CYP79A1 and CYP71E1 lines were 190 bp and 545

bp, respectively, which was in accordance with the expected size (Fig. S2B).

In vitro activity assays were performed with thylakoids prepared from the transgenic lines to

assess the functionality of the incorporated P450s. The in vitro activity assays were carried out using

radiolabeled substrates allowing a sensitive detection of enzymatic activity (Jensen et al., 2011; Nielsen

et al., 2013). The thylakoids were solubilized with n-dodecyl-β-D-maltoside (β-DM) and incubated

with exogenous ferredoxin (Fd) and the radiolabeled substrates [14

C]-tyrosine for CYP79A1 thylakoids

or [14

C]-p-hydroxyphenylacetaldoxime for CYP71E1 thylakoids. The reaction mixtures were incubated

in both darkness and light (Fig. S2C). The presence of de novo synthesized radioactively labelled

products in the light-incubated samples clearly demonstrates that both P450s are present and active in

the thylakoids of the transgenic plants. In addition, the products are only formed in the light thus

demonstrating the requirement for light-driven electron transport to the P450s for these to catalyze the

metabolic conversions, i.e. [14

C]-p-hydroxyphenylacetaldoxime formation by the CYP79A1 thylakoids,

and [14

C]-p-hydroxyphenylacetonitrile and [14

C]-p-hydroxybenzaldehyde formation by the CYP71E1

thylakoids. The CYP79A1 and CYP71E1-containing thylakoid preparations were probed with specific

antibodies for CYP79A1 and CYP71E1. The immunoblot analyses did not reveal detectable expression

of either CYP79A1 or CYP71E1 demonstrating that the P450s are present at very low levels in the

thylakoids of the stably transformed lines.

References

Dulk-Ras A, Hooykaas PJ. 1995. Electroporation of Agrobacterium tumefaciens. In: Nickoloff J, ed.

Plant Cell Electroporation and Electrofusion Protocols, Vol. 55: Springer New York, 63-72.

Edwards K, Johnstone C, Thompson C. 1991. A simple and rapid method for the preparation of

plant genomic DNA for PCR analysis. Nucleic Acids Research 19, 1349.

Fischer R, Hain R. 1995. Chapter 29 Tobacco protoplast transformation and use for functional

analysis of newly isolated genes and gene constructs. Methods Cell Biology 50, 401-410.

Horsch RB FJ, Hoffmann NL, Rogers SG and Fraley RT. 1985. A simple and general method for

transferring genes into plants. Science 227, 1229-1231.

Jensen K, Jensen PE, Moller BL. 2011. Light-driven cytochrome P450 hydroxylations.

ACS Chemical Biology 6, 533-539.

Nielsen AZ, Ziersen B, Jensen K, Lassen LM, Olsen CE, Møller BL, Jensen PE. 2013. Redirecting

photosynthetic reducing power toward bioactive natural product synthesis.

ACS Synthetic Biology 2, 308-315.

Sainsbury F, Thuenemann EC, Lomonossoff GP. 2009. pEAQ: versatile expression vectors for easy

and quick transient expression of heterologous proteins in plants. Plant Biotechnology Journal 7,

682-693.

Table S1: Details of primer pairs used for confirming the integration of Fd(TP)-CYP79A1 and

Fd(TP)-CYP71E1 gene constructs in the nuclear genome.

Forward primer Reverse primer Comments

79-F

CTCCGCGATCCTCAAACTGC

79-R

ATCTCCGGCAGGTTGCCGA

Primer pair used for confirming

Fd-CYP79A1 transgene

integration into the nucleus of

N. benthamiana

71-F

CGCACTTCGAGCTCATACCG

71-R

ATCGCAAGACCGGCAACAG

Primer pair used for confirming

Fd-CYP79A1 transgene

integration into the nucleus of

N. benthamiana

Wild type Hetroplastomic line Homoplastomic line

Fig. S1. Seed tests to assess homoplasmy of transplastomic lines. Seeds from a wild-type plant (left), a hetero-plastomic plant (center) and a homoplastomic plant (right) were germinated on synthetic medium containing 500 mg/L spectinomycin.

LB

RB

CaMV P35S

CpMV5’UTR

Fd(TP) CYP79A1CpMV3’UTR

T Nos Npt II

LB

RB

CaMV P35S

CpMV5’UTR

Fd(TP) CYP71E1CpMV3’UTR

T Nos Npt II

Fd(TP)-CYP79A1 Fd(TP)-CYP71E1

Fd(TP)-CYP79A1 Fd(TP)-CYP71E1

650 bp

1 2 3 4 5 6 WT C WT 1 2 3 4 5 C1 kb

100 bp

A

B

C1st Generation 2nd Generation

WT CYP79A1 WT CYP79A1 L D L D L D L D C

1st Generation 2nd Generation WT CYP71E1 WT CYP71E1

C L D L D L D L D [14C] -nitrile

[14C] -aldehyde

[14C] -oxime

6.3 kb6.2 kb

Fig.S2. Nuclear transformation of N. benthamiana with the genes encoding the two P450s involved in the dhur-rin pathway. (A) Gene constructs encoding the two P450s fused to the ferredoxin transit peptide at the N-terminus (Fd(TP)-CYP79A1 and Fd(TP)-CYP71E1) used for the nuclear integration in N. benthamiana. The genes are driven by the strong constitutive CaMV 35S promoter. (B) PCR analysis performed on genomic DNA extracted from the transformed lines: the CYP79A1 engineered line (left) and the CYP71E1 engineered line (right). The numbers 1-6 represent analysis of 6 independent transformed lines. “WT” indicates PCRs performed on genomic DNA extracted from the wild-type N. benthamiana and “C" indicates PCRs performed on plasmids harboring Fd(TP)-CYP79A1 or Fd(TP)-CYP71E1. The primer binding sites are marked in small black arrows. (C) In vitro light-driven assay carried out with thylakoids isolated from the lines transformed with CYP79A1 (left) and CYP71E1 (right) genes. To assay CYP79A1 [14C]-tyrosine was used as substrate and to assay CYP71E1 [14C]-p-hydroxyphenylacetaldoxime (oxime) was used as substrate. The assay was carried out in the light (L) or the dark (D), and analyzed by TLC. After incuba-tion, the assay mixtures were extracted with EtOAc and the organic phase was applied to the TLC.

3 1.5 0.75 0.38 0.19 0.1 0.05

5A 10A 29A 30A 0.4 0.2 0.1 0.05 0.02 0.01 0.005

5A 10A 29A 30A 0.8 0.4 0.2 0.1 0.05

WT 5A 10A 29A 30A

A

B

C

PsaD in picomoles

CYP79A1 in picomoles

CYP71E1 in picomoles

Fig.S3. The quantification of A) PsaD subunit B) CYP79A1 and C) CYP71E1 in the thylakoids of wildtype and the DhuOp lines. For quantification of PsaD subunit of PSI, thylakoid amount corresponding to 0.1 µg of total chlorophyll for the wildtype, 10A, 29A, 30 A lines and 0.03 µg of total chlorophyll for the line 5A was separated by SDS-PAGE, blotted and probed with PsaD primary antibody. For quantification of CYP79A1 and CYP71E1, thylakoids amount corresponding to 5µg and 2µg of total chlorophyll from the DhuOp lines, respectively. The proteins were separated by SDS-PAGE, blotted and probed with CYP79A1 and CYP71E1 primary antibodies. As standards, known concentrations of the purified PsaD fused to maltose binding protein, CYP79A1 and CYP71E1 were used. As the secondary antibody, polyclonal swine anti rabbit antibody was used.

163.0

309.1485.1

+MS, 2.8min

184.9

323.0

+MS2(485.1), 2.8min

127x10

Intens.

02466x10

200 400 600 m/z

334.1+MS, 5.7min

184.8307.0

+MS2(334.1), 5.7min

24

8x10Intens.

012

7x10

200 400 600 m/z

162.9 623.0

323.0+MS, 3.6min

184.9306.5

451.5576.7

+MS2(323.0), 3.6min

2

7x10Intens.

0

2

44x10

200 400 600 m/z

163.0

323.1+MS, 5.5min

184.8

+MS2(323.1), 5.5min

0.5

7x10Intens.

0.00.51.0

6x10

200 400 600 m/z

163.0 367.1 461.2

309.0+MS, 2.6min

184.8

290.8662.6

+MS2(309.0), 2.6min

1

27x10

Intens.

0246

4x10

200 400 600 m/z

595.1

309.0+MS, 5.3min

132.9

202.8

248.9 337.7

+MS2(309.0), 5.3min

0.5

8x10Intens.

0

12

5x10

200 400 600 m/z

Dhurrin p-glucosyloxy benzoic acid

p-hydroxy benzoyl glucose

p-glucosyloxy benzyl alcohol

p-hydroxybenzyl glucose

p-glucosyloxy benzyl glucose

Fig.S4. Mass fragmentation pattern for dhurrin and selected glucosides produced from p-hydroxybenzaldehyde detected using LC-MS method.

EIC (m/z)- 334.1; 329.1; 309.1; 323.1; 485.1

0.5

1.0

1.5

2.0

2.5

8x10Intens.

2

4

6

8x10Intens.

2

4

6

8x10Intens.

0

1

2

3

4

5

8x10Intens.

2.5 3.0 3.5 4.0 4.5 5.0 5.5 Time [min]

EIC (m/z) - 334.1; 329.1; 309.1; 323.1; 485.1

EIC (m/z) - 334.1; 329.1; 309.1; 323.1; 485.1

Dhurrin standard EIC (m/z)-334

Top leaf-DhuOp -line 5A

Middle leaf-DhuOp -line 5A

Bottom leaf-DhuOp -line 5A

p-hydroxybenzoyl glucose

p-glucosyloxybenzoic acid

p-glucosyloxybenzyl alcohol p-glucosyloxybenzoyl glucose

p-hydroxybenzyl glucose

Fig.S5. LC-MS analysis of dhurrin and glucosides on the extracts from leaves of different age from the DhuOp line 5A. The top leaf represents the newly sprouted leaf (third leaf from the top), middle leaf represent the matured leaf, and the bottom leaf represents senescing leaves.