Multi-Gene Transformation for Pathway Engineering of Secondary Metabolites

12Multi-Gene Transformation for Pathway Engineeringof Secondary MetabolitesHideyuki Suzuki, Eiji Takita, Kiyoshi Ohyama, Satoru Sawai, Hikaru Seki,Nozomu Sakurai, Toshiya Muranaka, Masao Ishimoto, Hiroshi Sudo,Kazuki Saito, and Daisuke Shibata

12.1Introduction

Triterpene saponins are widely distributed in higher plants [1,2] and belong to aclass of natural products that includes various bioactive compounds found inmedicinal plants [3]. The family Leguminosae, which includes plants such aslicorice (Glycyrrhiza uralensis) [4], soybean (Glycine max) [5] and alfalfa (Medicagosativa) [6], are well-known triterpene saponin-producing plants. The roots andstolons of licorice accumulate glycyrrhizin as a major bioactive triterpene saponin,while the hypocotyls of soybean accumulate soyasaponin I.Glycyrrhizin and soyasaponin I are synthesized from a common biosynthetic

intermediate, the triterpene aglycone b-amyrin, derived from the initial cyclizationof 2,3-oxidosqualene. Licorice b-amyrin 11-hydroxylase (CYP88D6) [7] and b-amyrin30-hydroxylase (CYP72A154) [8] catalyze the oxidation of b-amyrin at positions C-11(two-step oxidation) and C-30 (three-step oxidation), respectively, to produce glycyr-rhetinic acid, the triterpene aglycone of glycyrrhizin. In contrast, soybean b-amyrin22-hydroxylase [unpublished] and b-amyrin 24-hydroxylase (CYP93E1) [9] catalyzethe oxidation of b-amyrin at positions C-22 (one-step oxidation) and C-24 (one-stepoxidation), respectively, to produce soyasapogenol B (22-hydroxy, 24-hydroxy-b-amyrin), the triterpene aglycone of soyasaponin I (Figure 12.1).Construction of vector plasmids for multi-gene transformation is an essential

technique for pathway engineering of secondary metabolites. The construction ofcomplex plasmids for expressing multiple genes is considered to be a time-consuming process, but we were able to reduce the difficulty by connecting thegene cassettes in tandem at the restriction sites of homing endonucleases [10], whichrecognize 18 to 39 specific bases, and using a polymerase chain reaction (PCR)technique based on primer overlap extension [11].The terminator regulates the level of expression by controlling transcriptional

termination and 30-endprocessing of mRNA. Arabidopsis heat shock protein18.2

The Handbook of Plant Metabolomics, First Edition. Edited by Wolfram Weckwerth and Günter Kahl# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

j227

(HSP) terminator [12] increases mRNA levels of both transiently and stablyexpressed transgenes by approximately two fold when compared with nopalinesynthase terminator (NOS). To increase gene expression of introduced foreigngenes, we used dual terminators, nopaline synthase (NOS) and Arabidopsis heatshock protein 18.2 (HSP) terminators, to construct vector plasmids.In order to produce glycyrrhetinic acid in soybean, we attempted triterpene

pathway engineering using the multi-gene transformation technique, in whichthe genes involved in the biosynthesis of glycyrrhetinic acid were employed. Ouraim was to interrupt the endogenous soya saponin I biosynthetic pathway and to addthe glycyrrhizin biosynthetic pathway. We constructed a vector plasmid harboringthree cassettes, two were for over-expression of licorice CYP88D6 and CYP72A154

COOH11-Hydroxylase

(CYP88D6)

H

HOHO

HO

30 Hydroxylase

β-Amyrin 18-β-Glycyrrhetinic acid

in Glycyrrhiza uralensis

30-Hydroxylase(CYP72A154)

H

HO

OH

CH2OH

Soyasapogenol B

in Glycine max

24-Hydroxylase(CYP93E1)

22-Hydroxylase

Figure 12.1 Pathways of triterpene saponinglycone, glycyrrhetinic acid and soyasapogenolB biosynthetis in licorice and soybean.Glycyrrhizin and soyasaponin I arebiosynthesized from a common intermediate,glycone b-amyrin, derived from the initialcyclization of 2,3-oxidosqualene. Black arrows

indicate the reactions catalyzed by licoriceb-amyrin 11-hydroxylase (CYP88D6) [7] andb-amyrin 30-hydroxylase (CYP72A154) [8]. Grayarrows indicate reactions catalyzed by soybeanb-amyrin 22-hydroxylase [unpublished] andb-amyrin 24-hydroxylase (CYP93E1) [9].

228j 12 Multi-Gene Transformation for Pathway Engineering of Secondary Metabolites

genes, and the other was for down-regulation of the endogenous soybean CYP93E1gene, for soybean transformation by particle bombardment. Expression of eachcassette was controlled by a CaMV35S promoter and NOS-HSP dual terminators(Figures 12.2 and 12.3). Preliminary results on GC-MS analysis showed that oneof the six transgenic lines accumulated 30-hydroxy-11-oxo-b-amyrin and 11-oxo-b-amyrin, both of which are involved in glycyrrhetinic acid biosynthesis, in thehypocotyls (Figure 12.4). Further detailed study is necessary for evaluation ofthe three transgenes. Here, we provide the advanced protocols for plasmid con-struction using homing endonucleases and the primer overlap extension method,and those for the multi-gene transformation technique of soybean by particlebombardment, in addition to describing the results of GC-MS analysis of transgenicsoybeans.

P35S

CYP88 or CYP72

P35S

CYP93 sense

Intron1

CYP93 antisense

DT (Thsp-Tnos)

(a)

P35S-CYP88 or CYP72-DT

PI-Psp II-Sce I

DT (Thsp-Tnos)

Digestion with I-Sce I and PI-Psp I

Overlap Extension

P35S-CYP93E1sense-Intron1

Overlap Extension Overlap Extension

CYP93E1antisense-DT

pHSG299 CSPS


Ligation

pHSG299 CSPS 35S-CYP88-DTorpHSG299 CSPS 35S-CYP72-DT

PI-Psp I PI-Sce I

Digestion with PI-Psp I and PI-Sce I

P35S-CYP93(RNAi)-DT

Overlap Extension

pHSG299 CSPS

Ligation

pHSG299 CSPS 35S-CYP93(RNAi)-DT

Digestion with PI-Psp I and PI-Sce I

Figure 12.2 Schematic illustrations ofconstruction of plasmids harboring three-keyP450 genes involved in oxidative reactions intriterpene saponin biosynthesis. Procedure forconnecting gene cassettes by PCR based onprimer overlap extension at homingendonuclease restriction sites. DT indicatesNOS-HSP dual terminators. The restrictionends of I-SceI and PI-PspI are compatible with

one another. Abbreviations: P35S, CauliflowerMosaic Virus (CaMV) 35S promoter; CYP88,licorice CYP88D6 gene; CYP72, licoriceCYP72A154 gene, DT, NOS-HSP dualterminators; Thsp, Arabidopsis heat shock protein18.2 (HSP) terminator; Tnos, nopaline synthase(NOS) terminator; CYP93, soybean CYP93E1gene; HRT, hygromycin resistance gene;DsRed2, red fluorescent protein.

12.1 Introduction j229

I-C

euI

I-S

ceI

PI-

Psp

IP

I-S

ceI

(b)

P35S-CYP88-DT

PI-Psp II-Sce I


Ligation

pUHR KS CSPS Thsp

HRT DsRed2Thsp

I-Sce IPI-Psp I

PI S I

HRT DsRed2

I-Ceu I PI-Sce I

Thsp P35S-CYP88-DT

P35S-CYP72-DT

PI-Psp II-Sce IDigestion with PI-Psp I

Ligation

HRT DsRed2

I-Ceu II-Sce I

PI-Psp I

PI-Sce I

Thsp P35S-CYP88-DT P35S-CYP72-DT

PI-Sce IPI-Psp I

Digestion with PI-Psp I and PI-Sce IP35S-CYP93(RNAi)-DT

Digestion with PI Psp I and PI Sce I

Ligation

HRT DsRed2

I-Ceu II-Sce I

PI-Sce I

Thsp P35S-CYP88-DT P35S-CYP72-DT P35S-CYP93(RNAi)-DT

PI-Psp I

Figure 12.2 (Continued )

Figure 12.3 Diagram of cloning vectors(pHSG299CSPS, pUHR KS, pUHR KS CSPS,pUHR KS CSPS Thsp, pHSG299 CSPS 35S-CYP88-DT, pHSG299 CSPS 35S-CYP72-DT andpUHR KS CSPS 35S-CYP93(RNAi)-DT).Positions of recognition sites of restrictionendonucleases and homing endonucleases areindicated. Abbreviations: CSPS,CSPSrestrictionsites, which combine I-CeuI, I-SceI,PI-Psp1 and PI-SceI; Ori, replication origin inE. coli; LacZ, structural gene of the lac operon in

E. coli; Kmr, kanamycin resistance gene; Spmr,spectinomycin resistance gene; P35S,Cauliflower Mosaic Virus (CaMV) 35SPromoter; CYP88, licorice CYP88D6 gene;CYP72, licorice CYP72A154 gene, DT, NOS-HSPdual terminators; Thsp, Arabidopsis heat shockprotein 18.2 (HSP) terminator; Tnos, nopalinesynthase (NOS) terminator; CYP93, soybeanCYP93E1 gene; HRT, hygromycin resistancegene; DsRed2, red fluorescent protein.

——————————————————————————————————I


Ori lacZ

Kmr

Sph

IS

bfI

Asc

II-

Ceu

II-

Sce

IP

I-P

spI

PI-

Sce

IS

rfI

Fse

IN

otI

Eco

R I

pHSG299 CSPS(2.8 kb)

III

pUHR KS(6.5 kb)

HRT

Ampr

Asc

I

Pac

I

pUC19 (2.7kb)

Tnos P35S DsRed2P35S Tnos

Sac

I

Xba

I

Spe

I

Bam

H

Sm

aI

Eco

RI

Him

dI I

Kpn

I

Apa

I

Xho

I

Sal

I

dIII

uI

eI

spI

ceI

R I

pUHR KS CSPS(6.7 kb)

HRT

Ampr

Asc

I

Pac

I

pUC19 (2.7kb)

Tnos P35S DsRed2P35S TnosH

imd

Sph

IS

bfI

Asc

II-

Ceu

I-S

ceP

I-P

sP

I-S

cS

rfI

Fse

IN

otI

Eco

R

HRT

Ampr

Asc

I

Pac

I

pUC19 (2.7kb)

Tnos P35S DsRed2P35S Tnos

Sal

I I-C

euI

I-S

ceI

PI-

Psp

IP

I-S

ceI

pUHR KS CSPS Thsp(7.0 kb) Thsp

pHSG299 CSPS 35S-CYP88-DT(5.7 kb)

P35S

2.9 kb

PI-

Psp

I

pHSG299 CSPS

I-S

ceI

CYP88 TnosThsp

spI

eI

PI-

Sce

I

P35S

3.0 kb

PI-

Ps

pHSG299 CSPS

I-S

ce

CYP72 TnosThsp

pHSG299 CSPS 35S-CYP72-DT(5.8 kb)

PI-

Psp

I

CYP93CYP93 P

pUHR KS CSPS 35S-CYP93(RNAi)-DT(5.2 kb)

P35S

2.4 kb

P

pHSG299 CSPS

Intron1 TnosThsp

antisenseC 93sense

pUHR KS CSPS Thsp CYP88-CYP72-CYP93 (RNAi)(15.4 kb)

PI-

Sce

I

X.X kb

pUHR KS CSPS Thsp

DTP35S

PI-

Psp

I

I-S

ceI

CYP93(RNAi)DTP35SDTP35S 27PYC88PYCpshT

8.4 kb

I-C

euI

Figure 12.3 (Continued )

12.1 Introduction j231

Figure 12.4 GC-MS analysis of hypocotyls oftransgenic soybean obtained from particlebombardment using plasmids harboring threekey P450 genes involved in oxidative reactionsof triterpene saponin biosynthesis. A) and B)GC-MS analysis of hypocotyls of T1 seedsderived from self-pollinated T0 plants. A) Upperchromatogram shows the selected ionchromatogram (m/z 383) of 11-oxo-b-amyrinchemical standards. Middle chromatogramshows the selected ion chromatogram (m/z383) in plant materials transformed with pUHR(control vector). The lower chromatogramshows the selected ion chromatogram(m/z383)

in plant materials transformed with pUHR KSCSPS Thsp-CYP88-CYP72-CYP93 (RNAi).Arrows indicate mass spectra for 11-oxo-b-amyrin. B) Upper chromatogram shows theselected ion chromatogram (m/z 361) of 30-hydroxy-11-oxo-b-amyrin chemical standards.Middle chromatogram shows the selected ionchromatogram (m/z 361) in plant materialstransformed with pUHR (control vector). Lowerchromatogram shows the selected ionchromatogram (m/z 361) in plant materialstransformed with pUHR KS CSPS Thsp-CYP88-CYP72-CYP93 (RNAi). Arrows indicate massspectra for 30-hydroxy-11-oxo-b-amyrin.


12.2Methods and Protocols

12.2.1Chemicals

� Chemical standards of 30-hydroxy-11-oxo-b-amyrin and 11-oxo-b-amyrin weresynthesized from b-amyrin as described elsewhere [7].

12.2.2Plasmid Construction of Multi-Gene Transformation

� Figure 12.2 shows a schematic illustration for the construction of the plasmidharboring three key P450 genes involved in the oxidative reactions in triterpenesaponin biosynthesis. All cloning vectors are shown in Figure 12.3. All primersare summarized in Table 12.1. Primers for overlap extension were designed tohave the same Tm values in overlapping sequence domains, and the annealingtemperature for PCR was set at 65 �C.

� All restriction enzymes were purchased from New England BioLabs (Hitchin,Hertfordshire, UK).

� All amplification steps by PCR were followed by purification of PCR productsusing the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI,USA), mixing with phenol/chloroform/isoamyl alcohol (25 : 24 : 1) (Wako, Osaka,Japan), and ethanol precipitation.

� Ligation of DNA fragments was performed using DNA Ligation Kit Mighty Mix(TaKaRa Biomedicals, Osaka, Japan).

� Transformation of Escherichia coli was performed using E.coli DH5a CompetentCells (TaKaRa Biomedicals, Osaka, Japan).

� Before ligation into cloning vector plasmids double digested with the appropriaterestriction enzymes, plasmids were extracted from agarose gels using the WizardSV Gel and PCR Clean-Up System (Promega, Madison, WI, USA).

12.2.3Preparation of Dual Terminator (DT) Fragment by PCR-Based Overlap ExtensionMethod

� Arabidopsisheat shock protein(HSP) 18.2 terminator was amplified by PCRwith the plasmid vector GUS-HSP-pBI101 [11] as a template, and primers1 and 2.

� Nopaline synthase(NOS) terminator was amplified by PCR with the plasmid vectorpBI121 [13] as a template, and primers 3 and 4.

� After purification, PCR products were purified using the Wizard SV Gel and PCRClean-Up System, mixed with phenol/chloroform/isoamyl alcohol (25 : 24 : 1), andprecipitated with ethanol.

� After calculating the DNA concentration of the PCR products with an spectro-photometer, equal amounts (0.05 pmol) of the PCR products were combined and

12.2 Methods and Protocols j233

Table12.1

Prim

ersused

forplasmid

constructio

nin

multi-gene

tran

sformation.

Prim

erNo.

Sequ

ence

(50 --3

0 )Plasmid

Descriptio

n

1GGATCCGAGCTCATA

TGAAGATGAAGATGAAATA

TTT

GUS-HSP

-pBI101

Amplification

ofHSP

fragmen

t2

AATGTTTGAACGATCGGGGAAATTCGGTA

CCCTTA

TCTTTA

ATCATA

TTCCATA

GTC

GUS-HSP

-pBI101

Amplification

ofHSP

fragmen

t

3GAATTTCCCCGATCGTTCAAACATT

pBI121

Amplification

ofNOSfragmen

t4

GAATTCCCGATCTA

GTA

ACATA

GATGACAC

pBI121

Amplification

ofNOSfragmen

t5

aaggaaaaAAGCTTGCATGCCCTGCAGGCGCGCCTA

ACTA

pHSG

299CSP

SAmplification

ofCSP

Sfragmen

t,HindIIIsite

6aaggaaaaGAATTCGCGGCCGCGGCCGGCCCGGGCT

pHSG

299CSP

SAmplification

ofCSP

Sfragmen

t,EcoRIsite

7aaggaaaaGTCGACGAGCTCATA

TGAAGATGAAGATGAAATA

GUS-HSP

-pBI101

Amplification

ofHSP

fragmen

t,SalIsite

8aaggaaaaTTCGCTA

CCTTA

GGACCGTTA

TAGTTA

GAATTCCTTA

TCTTTA

ATCATA

TTCCATA

GGUS-HSP

-pBI101

Amplification

ofHPSfragmen

t,I-Ceu

Isite

9aaggaaaaATTA

CCCTGTTA

TCCCTA

AAGCTTGCATGCCTGCAGGTCCC

pBI121

Amplification

ofP35Sfragmen

t,I-SceI

site

10CGTGTTCTCTCCAAATGAAATGAACTTCCT

pBI121

Amplification

ofP35Sfragmen

t11

AGGAAGTTCATTTCATTTGGAGAGAACACGGGGGACTCTA

GACA

CCATGGAAGTA

CATTGGGTT

pCYP88

Amplification

ofCYP88

fragmen

t

12TTCATCTTCATCTTCATA

TGAGCTCGGATCCCTA

AGCACATGAAACCTTTA

TC

pCYP88

Amplification

ofCYP88

fragmen

t13

GATCCGAGCTCATA

TGAAGATGAAGATGAA

PCRProdu

ctsforDT

Amplification

ofDTfragmen

t14

aaggaaaaACCCATA

ATA

CCCATA

ATA

GCTGTTTGCCAGAATTCCCGATCTA

GTA

ACATA

GATGACACC

PCRProdu

ctsforDT

Amplification

ofDTfragmen

t,PI-PspIsite

15CGTGTTCTCTCCAAATGAAATGAACTTCCT

pBI121

Amplification

ofP35Sfragmen

t16

AGGAAGTTCATTTCATTTGGAGAGAACACGGGGGACTCTA

GACACCATGG

ATGCATCTTCCACA

pCYP72

Amplification

ofCYP72

fragmen

t


17TCGGATCCTTA

CAGTTTA

TGCAGAATGATG

pCYP72

Amplification

ofCYP72

fragmen

t18

CATCATTCTGCATA

AACTGTA

AGGATCCGAGCTCATA

TGAAGAT

PCRProdu

ctsforDT

Amplification

ofDTfragmen

t19

GTTGAGTA

GTTCTTCATCATA

GTCTTA

AA

SoybeancD

NA

Amplification

ofCYP93

fragmen

t20

GCTCTCGCGGATTCTCACGAAATG

SoybeancD

NA

Amplification

ofCYP93

fragmen

t21

aagg

aaaaTGGCAAACAGCTA

TTA

TGGGTA

TTA

TGGGTA

AGCTTGCATGCCTG

CAGGTCCC

pBI121

Amplification

ofP35

Sfragmen

t,PI-PspIsite

22CCGTGTTCTCTCCAAATGAAATGAA

pBI121

Amplification

ofP35

Sfragmen

t23

TTCATTTCATTTGGAGAGAACACGGGGGACTCTA

GAGTTGAGT

AGTTCTTCATCATA

GpC

YP93

Amplification

ofCYP93

sense

fragmen

t24

TTA

TAGATA

GCGAAAGAGATCGAGAGAGGAGAAGGAGAAGTA

AGATC

TCACCATCCCGACGCCACCACCACGTGCTTC

pCYP93

Amplification

ofCYP93

sense

fragmen

t25

TCCTCTCTCGATCTCTTTCGCTA

TCTA

TAA

Arabido

psisgenom

icDNA

Amplification

ofintron

fragmen

t

26CGTA

CACCTA

CATTA

CACAAACCAATGAAG

Arabido

psisgenom

icDNA

Amplification

ofintron

fragmen

t

27CTTCATTGGTTTGTGTA

ATGTA

GGTGTA

CGACGCCACCACCACGTGCTTC

pCYP93

Amplification

ofCYP93

antisense

fragmen

t28

TTCATCTTCATCTTCATA

TGAGCTCGGATCCGTTGAGTA

GTTCTTCA

TCATA

GTCTT

pCYP93

Amplification

ofCYP93

antisense

fragmen

t29

GATCCGAGCTCATA

TGAAGATGAAGATGAA

PCRProdu

ctsforDT

Amplification

ofDTfragmen

t30

aagg

aaaaATTA

CCTCTTTCTCCGCACCCGACATA

GATGAATTCCCGATCTA

GT

AACATA

GATGACACC

PCRProdu

ctsforDT

Amplification

ofDTfragmen

t,PI-SceI

site

Smalllettersin

sequ

encesindicate

addition

alsequ

encesforcuttingby

restrictionen

zymes.

Boldlettersin

sequ

ence

indicatesoverlapsequ

encesforPCR-based

overlapextension

method

.Underlineindicatesrestrictionen

zymesite.

Abb

reviations:DT,NOS-HSP

dualterm

inators;HSP

,Arabido

psisheatshockprotein18.2term

inator;N

OS,

nop

alinesynthaseterm

inator;P

35S,

Cau

liflow

erMosaicVirus

35S

Promoter;CYP88,CYP88D6gene;

CYP72,CYP72A154gene;

CYP93,CYP93E1gene.


amplified by PCR-based overlap extension with primers 1 and 4, into a finalvolume of 50 ml (final concentration of PCR products: 1 nM).

� After calculating the Tm value of the overlapping sequence domain, the annealingtemperature was set at 65 �C.

� After purification, the resulting PCR products were designated as DT fragments.

12.2.4Plasmid Construction of pUHR KS CSPS Thsp

� CSPS restriction sites, which are a combination of I-CeuI, I-SceI, PI-Psp1 andPI-SceI, were amplified by PCR with pHSG299 CSPS plasmid [14] as a template,and primers 5 and 6.

� After purification using the Wizard SV Gel and PCR Clean-Up System, PCRproducts were digested with HindIII and EcoRI.

� The resulting double-digested CSPS fragments were purified by mixing withphenol:chloroform:isoamyl alcohol (25: 24: 1) and ethanol precipitation.

� After calculating the DNA concentration of the CSPS fragments with a spectro-photometer, ligation into a pUHR KS plasmid vector [15] digested with HindIIIand EcoRI was performed to produce the pUHR KS CSPS plasmid vector.

� HSP fragments were amplified by PCR with the plasmid vector GUS-HSP-pBI101 as a template, and primers 7 and 8.

� After digestion of PCR products with SalI and I-CeuI, ligation into a pUHR KSCSPS plasmid vector digested with HindIII and EcoRI was performed to givepUHR KS CSPS Thsp plasmid vector (Figure 12.3).

� The new plasmid vector containing the CSPS restriction sites for homingendonuclases, the hsp terminator, and the DsRed2 and hpt expression cassetteswas designated as pUHR KS CSPS Thsp (Figure 12.3).

12.2.5Construction of pHSG299 CSPS 35S-CYP88-DT (Figure 12.2a)

� Connection of DNA fragments was carried out using a PCR-based overlapextension strategy (Figure 12.2a).

� The Cauliflower Mosaic Virus 35S Promoter (P35S) was amplified by PCR withthe plasmid vector pBI121 [14] as a template, and primers 9 and 10.

� The b-amyrin 11-hydroxylase (CYP88D6) gene was amplified by PCR with theplasmid vector pCYP88 [7] as a template, and primers 11 and 12.

� DT were amplified by PCR with the above DT fragments as templates, andprimers 13 and 14.

� After purification using the Wizard SV Gel and PCR Clean-Up System, PCRproducts were further purified by mixing with phenol/chloroform/isoamylalcohol (25 : 24 : 1) and ethanol precipitation.

� After calculating the DNA concentration of the PCR products with a spectro-photometer, equal amounts (0.05 pmol) of the PCR products were combined andamplified by PCR-based overlap extension with primers 9 and 14.


� The resulting PCR products were sequentially digested with I-SceI and PI-PspI,two homing endonuclases, as follows.

� After purification using the Wizard SV Gel and PCR Clean-Up System, PCRproducts were digested with I-SceI.

� After purification bymixture with phenol/chloroform/isoamyl alcohol (25 : 24 : 1)and ethanol precipitation, PCR products were further digested with PI-PspI.

� The double-digested pHSG299 CSPS plasmid vector was extracted from anagarose gel using the Wizard SV Gel and PCR Clean-Up System, and wasthen ligated with the double-digested PCR products using DNA Ligation KitMighty Mix.

The new plasmid vector containing the P35S, CYP88D6 gene and DT wasdesignated as pHSG299 CSPS 35S-CYP88-DT (Figure 12.4).

12.2.6Construction of pHSG299 CSPS 35S-CYP72-DT2 (Figure 12.2a)

� Construction of pHSG299 CSPS 35S-CYP72-DT2 was performed using the sameprocedure as above, with the b-amyrin 30-hydroxylase (CYP72A154) gene beingused instead of the b-amyrin 11-hydroxylase (CYP88D6) gene

� P35S fragments were amplified by PCR with the plasmid vector pBI121 as atemplate, and primers 9 and 15.

� The b-amyrin 30-hydroxylase (CYP72A154) gene was amplified by PCR with theplasmid vector pCYP72 [8] as a template, and primers 16 and 17.

� DT were amplified by PCR with the above DT fragments as templates, andprimers 18 and 14.



� The resulting PCR products were sequentially digested with the homing endo-nuclases I-SceI and PI-PspI as follows.


� After further purification bymixing with phenol/chloroform/isoamyl alcohol (25 :24 : 1) and ethanol precipitation, PCR products were digested with PI-PspI.

� The double-digested pHSG299 CSPS plasmid vector was extracted from anagarose gel using the Wizard SV Gel and PCR Clean-Up System, andligated with the double-digested PCR products using DNA Ligation Kit MightyMix.

The new plasmid vector containing the P35S, CYP72 gene andDTwas designatedas pHSG299 CSPS 35S-CYP72-DT (Figure 12.3).


12.2.7Construction of pHSG299-CYP93(RNAi)-DT (Figure 12.2a)

� Soybean cDNA was synthesized from Glycine max (L.) seeds of the cultivar Jack.� For the cloning of the b-amyrin 24-hydroxylase (CYP93E1) gene, which contains

54 bp of 50UTR and 456 bp of protein coding sequences, Soybean cDNA wasamplified by PCR with the primers 19 and 20.

� The resulting PCR products were ligated with the pTA2 cloning vector (ToyoboCo., Osaka, Japan) using DNA Ligation Kit Mighty Mix and used as a PCRtemplate, according to the following procedures, and were designated aspCYP93.

� P35S fragments were amplified by PCR with plasmid vector pBI121 as a template,and primers 21 and 22.

� Sense-orientated fragments of the b-amyrin 24-hydroxylase (CYP93E1) gene wereamplified by PCR with the plasmid vector pCYP93 as a template, and primers 23and 24.

� Arabidopsis intron fragments, which are the first introns of At1g54340 (NADP-specific isocitrate dehydrogenase) gene, were amplified by PCR with Arabidopsisthaliana (Wassilewskija) genomic DNA as a template, and primers 25 and 26.



� The resulting PCR products containing the P35S, CYP93 sense and Arabidopsisintron 1 were used as a PCR template, according to the following procedures, andwere designated as the P35S-CYP93 sense-intron.

� The antisense-orientated fragments of the b-amyrin 24-hydroxylase (CYP93E1)gene were amplified by PCR with the plasmid vector pCYP93 as a template, andprimers 27 and 28.

� DT were amplified by PCR with the above DT fragments as the template, andprimers 29 and 30.



� The resulting PCR products containing the CYP93 antisense and DTwere used asa PCR template, according to the following procedure, and were designated asCYP93 antisense-DT.

� Equal amounts (0.05 pmol) of P35S-CYP93 sense-intron and CYP93 antisense-DTwere combined and amplified by PCR-based overlap extension with primers 19and 28.


� After calculating the Tm value in overlapping sequence domains, the annealingtemperature was set at 65 �C.


� After further purification by mixing with phenol/chloroform/isoamyl alco-hol (25 : 24 : 1) and ethanol precipitation, PCR products were digested withPI-PspI.

� The double-digested pHSG299 CSPS plasmid vector was extracted from agarosegel using the Wizard SV Gel and PCR Clean-Up System, and was ligated with thedouble-digested PCR products using DNA Ligation Kit Mighty Mix.

The new plasmid vector containing P35S, CYP93 sense gene, Arabidopsis intron 1,CYP93 antisense gene and DT was designated as pHSG299 CSPS 35S-CYP93(RNAi)-DT (Figure 12.3).

12.2.8Construction of pUHR KS CSPS Thsp-CYP88-CYP72-CYP93 (RNAi)

� We constructed a final new plasmid for soybean transformation according to thefollowing three-step procedure (Figure 12.2b).

� After digesting P35S-CYP88-DT fragments from the pHSG299 CSPS 35S-CYP88-DT plasmid vector with I-SceI and PI-PspI, they were ligated into a pUHR KSplasmid vector [15] digested with I-SceI and PI-PspI to give pUHR KS CSPS P35S-CYP88-DT plasmid vector.

� After digesting P35S-CYP72-DT fragments from the pHSG299 CSPS 35S-CYP72-DT plasmid vector with I-SceI and PI-PspI, they were ligated into a pUHR KSCSPS P35S-CYP88-DT plasmid vector digested with PI-PspI to give pUHR KSCSPS P35S-CYP88-DT P35S-CYP72-DT plasmid vector.

� After digesting 35S-CYP93(RNAi)-DT fragments from the pHSG299 CSPS 35S-CYP93(RNAi)-DTplasmid vector with I-SceI and PI-PspI, they were ligated into apUHR KS CSPS P35S-CYP88-DT P35S-CYP72-DT plasmid vector digested withPI-PspI and PI-SceI to give the pUHRKSCSPS Thsp-CYP88-CYP72-CYP93(RNAi)plasmid vector.

� The final new plasmid vector containing 35S-CYP88-DT, 35S-CYP72-DT andpHSG299-CYP93(RNAi)-DT, as well as the hsp terminator, the DsRed2 and hptexpression cassettes, was designated as pUHR KS CSPS Thsp-CYP88-CYP72-CYP93(RNAi) (Figure 12.3).

12.2.9Transformation of Soybean by Particle Bombardment

12.2.9.1 Preparation of Embryogenic Suspension Tissue Culture

� Soybean [Glycine max (L.) Merrill] plants, cultivar Jack, were grown in soil undernatural light conditions and controlled temperature, 28 �C (day) and 22 �C (night),in a glasshouse


� Developing green pods were collected when immature cotyledons were 4 to 5mmin length.

� After sterilization of the pod surface with 70% ethanol for 2min followed by threewashes with sterile water, the immature seeds were extracted, the embryonic axiswas removed, and the cotyledons were placed with the adaxial side up on MSD40medium [MS salts [16], and B5 vitamins [17] supplemented with 3% sucrose and2,4-dichlorophenoxyacetic acid (2,4-D), 40mg/l, adjusted to pH 7.0, andsolidified with 0.2% Gelrite (Wako, Osaka, Japan) in 90-mm disposable plasticPetri dishes.

� Embryogenic tissue was initiated and allowed to proliferate by culture at 25 �Cunder cool white fluorescent light (23-h-light, 1-h-dark cycle, 5 to 10mol/m2s) in100-ml flasks containing 25ml of fresh FN Lite liquid medium [FN Lite macro-salts, MS micro-salts, B5 vitamin, asparagine (1 g/l), 2,4-D (5mg/l) and 1%sucrose, adjusted to pH 5.8] [18].

� Cultures were maintained on a rotary shaker at 100 rpm and sub-cultured once aweek into 25ml of fresh FN Lite liquid medium in a 100-ml flask.

� About 0.8 g of embryogenic suspension tissue was arranged to the center of a9-cm plastic plate containing MSD20 medium [MS salts, B5 vitamin, 3% sucrose,asparagine (1 g/l), 2,4-D (20mg/l), and 0.2% Gerlite (Wako), adjusted to pH 5.8].

12.2.9.2 Preparation of Plasmid DNA for Particle Bombardment

� Plasmid DNA was isolated from E. coli DH5 a harboring pUHR KS CSPS hspter-CYP88-CYP72-CYP93(RNAi) or pUHR KS (control plasmid) using a PlasmidMidi kit (Qiagen Valencia, CA, USA).

� Next, 50 ml of gold particle suspension (0.6 mm in diameter, 60mg/ml in 50%glycerol) was mixed with 50 ml of plasmid DNA (0.8 mg), 50 ml of 2.5M CaCl2, and20 ml of 0.1M spermidine in a 1.5-ml plastic microtube.

� The mixture was incubated for 3min at room temperature, followed by centrifu-gation to isolate gold particles, which were then washed with 70% ethanol and re-suspended in 300 ml of 99.5% ethanol

� Before use, the gold particle suspension was stored at 4 �C.

12.2.9.3 Conditions of Particle Bombardment

� Bombardment was performed twice at an acceleration pressure of 7.6MPa(1100 psi) and a distance of 6 cm with gold particles (0.6 mm in diameter andcoated with 0.8 mg of plasmid DNA) with the use of a Biolistic PDS-1000/HeParticle Delivery System (Bio-Rad, Richmond, CA).

12.2.9.4 Selection and Generation of Transgenic Soybean Plants

� Twenty-four hours after bombardment, expression of DsRed2 fluorescence wasmonitored with a fluorescence stereomicroscope (Leica, Wetzlar, Germany)equipped with a filter set for excitation at 530–560 nm and emission at 590–650 nm. Embyrogenic tissue was returned to fresh FN Lite liquid medium.


� After 1 week, the suspension cultures were incubated for 2 weeks in fresh FN Liteliquid medium supplemented with hygromycin B (15mg/l) (Roche Diagnostics,Mannheim, Germany), with a change of medium after 1 week. Clumps of somatictissue exhibiting bright green fluorescence were then transferred every otherweek into the fresh FN Lite medium containing hygromycin B at a concentrationof 30mg/l for 3 weeks. Hygromycin-tolerant somatic embryos were then sub-jected to selection in FN Litemedium containing hygromycin B at a concentrationof 45mg/l for 1 week.

� Somatic embryos tolerant to hygromycin B were selected and suspended inFNL0S3S3 liquid medium [FN Lite macro salts, MS micro salts, and B5 vitaminssupplemented with asparagine (1 g/l), 3% sucrose, and 3% sorbitol, and adjustedto pH 5.8].

� After 4–5 weeks, excess liquid was removed from the fully developed somaticembryos and they were desiccated in dry Petri dishes for 3–5 days before transferto MS0 medium (MS medium without hormones).

� Germinating plantlets were grown on 0.5� B5 medium.� After root and shoot elongation, plantlets were transferred to pots containing soil

and were maintained under high humidity.� Plantlets were gradually adapted to ambient humidity and placed under natural

light conditions at a controlled temperature of 28 �C (day) and 22 �C (night) in aglasshouse.

� T2 seeds were harvested from individual T1 plants derived from self-pollinated T0plants (original transgenic plants) in the glasshouse.

12.2.10GC-MS Analysis for Triterpene Aglycone

12.2.10.1 Extraction of Metabolite

� Hypocotyls were collected from soybean seeds divided with pliers.� Portions (30mg) of the hypocotyls of transgenic soybeans were ground with a

Multi-beads Shocker (Yasui Kikai, Osaka, Japan), for 20 sec each.� Powder was transferred to a glass tube.� Next, 2.0ml of MeOH/CHCl3 (1 : 1) solution was added to the tube and mixed.� Themixture was then incubated for 60min at 80 �C and centrifuged at 17,500� g

for 5min, and the supernatant was transferred to a 10-ml glass tube.� To the pellet, 2.0ml of MeOH/CHCl3 (1 : 1) solution was added.� This extraction was repeated three times.� The supernatant was then dried with an evaporator.

12.2.10.2 Acid Treatment of Extracted Metabolites

� To dried extracts, 2.0ml of MeOH/4M HCl (1 : 1) solution was added.� The mixture was incubated for 60min at 80 �C.� To this MeOH/4M HCl solution, 2.0ml of hexane/ethyl acetate (1 : 2) solution

was added, followed by mixing.


� The mixture was centrifuged at 17,500� g for 5min, and the upper layer wastransferred to a 6-ml glass tube.

� This extraction was repeated three times.� The collected organic solution was then dried.

12.2.10.3 Derivatization of Metabolites

� To the dried pellet 450 ml of CHCl3 was added.� Of this solution, 150 ml was transferred to a 1.5-ml glass tube followed by

evaporation of CHCl3.� To the dried pellet, 50 ml of N-methyl-N-trimethylsilyltrifluoroacetamide was

added.� The mixture was incubated for 30min at 80 �C.

12.2.11GC-MS Conditions

� GC 6890 (Agilent Technologies, Palo Alto, CA) was operated under electronicpressure control, and was equipped with a split/splitless capillary inlet.

� Samples were injected at 1 ml in splitless mode with an injection temperature of250 �C.

� Separation was achieved with a temperature program of 80 �C for 1min,increasing at 20 �C min�1 to 300 �C, which was held for 23min, on a 30-mDB-1 column (0.25mm ID� 30m, 0.25-mm film thickness; J&W Scientific) and aconstant flow of 1.0mlmin�1 of Helium gas.

� The transfer line to the mass spectrometer was set at 280 �C.� MS analysis was performed with a JEOL MS system (JMS-AM SUM200, JEOL)

with an electron impact ionization (EI) source set to 250 �C. Mass spectra wererecorded at 20 scans s�1 with an m/z 50–750 scanning range.

12.3Application of Technology

The homing endonuclease sites have the advantage of being extremely rare innatural sequences, and this allows unidirectional cloning. The use of a homingendonuclease in molecular cloning vectors can eliminate time-consuming stepswhen producing complex plasmids using multiple genes for secondary metabolitepathway engineering. We selected four homing endonucleases, sequentially com-bining I-CeuI, I-SceI, PI-Psp1 and PI-SceI, and designated this combination as CSPSrestriction sites, for the construction of cloning vectors. This cloning technology forexpressing multiple genes has been applied to increasing the amount and compo-sition of carotenoids in Brassica napus (canola) transgenic seeds by using seven keyenzymes involved in ketocarotenoid formation [14].Transient expression and stable transformation in soybean by particle bombard-

ment has been established by optimizing the bombardment conditions using green


fluorescent protein (GFP) [19] or red fluorescent protein (DsRed2) [15] as a visualreporter. As a preliminary result, GC-MS analysis detected two intermediates,30hydroxy, 11-oxo-b-amyrin and 11-oxo-b-amyrin, involved in glycyrrhetinic acidbiosynthesis in the hypocotyls of transgenic soybean (Figure 12.4). Genetic manip-ulation of triterpene saponins in soybean is a useful model for triterpene saponinpathway engineering.

12.4Perspectives

Multiple-gene plant-transformation vector systems should provide the user with aneasy route for cloning genes of interest. Several versatile vector systems for multiplegene expressionhave already been reported [20–22]. A binary vector forAgrobacterium-mediated plant transformation, which has a multiple cloning site consisting of 13hexa-nucleotide restriction sites, 6 octa-nucleotide restriction sites and 5 homingendonuclease sites has also been constructed [21]. In addition, a Gateway-based vectorsystem to enablemultiple transgenes to be directly linked or fused bymultiple roundsof LR recombination reactions has been reported [22]. Thus, the construction ofcomplex plasmids has become greatly simplified by such vector systems.The disadvantage of multiple gene plant-transformation vector systems is plasmid

instability in Agrobacterium strains due to repeated promoter and terminatordomains, and the induction of RNA silencing by strongly transcribed transgenesin plant cells. Single terminators were found to be leaky for terminating transcrip-tion of transgenes driven by the strong CaMV 35S promoter [23]. The use of the dualterminators substantially reduces mRNA 30-readthrough, resulting in transcriptsthat are properly terminated [23]. To avoid the use of repeated promoters andterminators, new promoters and terminators that are inducible and that have tissue/organ specificity are necessary for multiple-gene plant-transformation vectors.

Acknowledgments

This study was supported in part by a grant from the New Energy and IndustrialTechnologyDevelopment Organization (NEDO), Japan.

References

1 Vincken, J.-P., Heng, L., de Groot, A., andGruppen, H. (2007) Saponins,classification and occurrence in the plantkingdom. Phytochemistry, 68, 275–297.

2 Sparg, S.G., Light, M.E., and van Staden, J.(2004) Biological activities and distributionof plant saponins. J. Ethnopharmacol., 94,219–243.

3 Waller, G.-R. and Yamasaki, K. (eds) (1996)Saponins Used in Food and Agriculture:Advances in Experimental Medicine andBiology, vol. 405, Plenum Press, New York.

4 Gibson, M.R. (1978) Glycyrrhiza in old andnew perspectives. Lloydia, 41, 348–354.

5 Kitagawa, I., Yoshikawa, M., and Yosioka, I.(1976) Saponin and sapogenol. XIII.

References j243

structures of three soybean saponins:soyasaponin I, soyasaponin II, andsoyasaponin III. Chem. Pharm. Bull., 24,121–129.

6 Huhman, D.V. and Sumner, L.W. (2002)Metabolic profiling of saponins inMedicagosativa andMedicago truncatula using HPLCcoupled to an electrospray ion-trap massspectrometer. Phytochemistry, 59, 347–360.

7 Seki, H., Ohyama, K., Sawai, S., Mizutani,M., Ohnishi, T., Sudo, H., Akashi, T., Aoki,T., Saito, K., and Muranaka, T. (2008)Licorice b-amyrin 11-oxidase, a cytochromeP450 with a key role in the biosynthesis ofthe triterpene sweetener glycyrrhizin. Proc.Natl. Acad. Sci. USA, 105, 14204–14209.

8 Seki, H., Sawai, S., Ohyama, K., Mizutani,M., Ohnishi, T., Sudo, H., Fukushima, E.O., Akashi, T., Aoki, T., Saito, K., andMuranaka, T. (2011) Triterpene functionalgenomics in licorice for identification ofCYP72A154 involved in the biosynthesis ofglycyrrhizin. Plant Cell, 23 (11), 4112–4123.

9 Shibuya, M., Hoshino, M., Katsube, Y.,Hayashi, H., Kushiro, T., and Ebizuka, Y.(2006) Identification of b-amyrin andsophoradiol 24-hydroxylase by expressedsequence tag mining and functionalexpression assay. FEBS J., 273, 948–959.

10 Jasin, M. (1996) Genetic manipulation ofgenomes with rare-cutting endonucleases.Trends Genet., 12, 224–228.

11 Pogulis, R.J., Vallejo, A.N., and Pease, L.R.(1996) In vitro recombination andmutagenesis by overlap extension PCR.Methods Mol. Biol., 57, 167–176.

12 Nagaya, S., Kawamura, K., Shinmyo, A.,and Kato, K. (2010) TheHSP terminator ofArabidopsis thaliana increases geneexpression in plant cells. Plant Cell Physiol.,51, 328–332.

13 Jefferson, R.A., Kavanagh, T.A., and Bevan,M.W. (1987) GUS fusions: beta-glucuronidase as a sensitive and versatilegene fusion marker in higher plants.EMBO J., 6 (13), 3901–3907.

14 Fujisawa, M., Takita, E., Harada, H.,Sakurai, N., Suzuki, H., Ohyama, K.,Shibata, D., and Misawa, N. (2009)

Pathway engineering of Brassica napusseeds using multiple key enzyme genesinvolved in ketocarotenoid formation.J. Exp. Bot., 60, 1319–1332.

15 Nishizawa, K., Kita, Y., Kitayama, M., andIshimoto, M. (2006) A red fluorescentprotein, DsRed2, as a visual reporter fortransient expression and stabletransformation in soybean. Plant Cell Rep.,25, 1355–1361.

16 Murashige, T. and Skoog, F. (1962) Arevised medium for rapid growth andbioassays with tobacco tissue culture.Physiol. Plant., 15, 473–497.

17 Gamborg, O., Miller, R., and Ojima, K.(1968) Nutrient requirements ofsuspension cultures of soybean root cells.Exp. Cell Res., 50, 151–158.

18 Finer, J.J. and Nagasawa, A. (1988)Development of an embryogenicsuspension culture of soybean Glycine maxL.Merrill. Plant Cell Tiss Org. Cult., 15, 125–136.

19 El-Shemy, H.A., Teraishi, M., Khalafalla,M.M., Katsube-Tanaka, T., Utsumi, S., andIshimoto, M. (2004) Isolation of soybeanplants with stable transgene expression byvisual selection based on green fluorescentprotein.Mol. Breed, 14, 227–238.

20 Chungm, S.M. Frankman, E.L., and Tzfira,T. (2005) A versatile vector system formultiple gene expression in plants. TrendsPlant Sci., 10, 357–361.

21 Goderis, I.J., De Bolle, M.F., FranScois, I.E.,Wouters, P.F., Broekaert, W.F., andCammue, B.P. (2002) A set of modularplant transformation vectors allowingflexible insertion of up to six expressionunits. Plant Mol. Biol., 50, 17–27.

22 Chen, Q.J., Zhou, H.M., Chen, J., andWang, X.C. (2006) A Gateway-basedplatform for multigene planttransformation. Plant Mol. Biol., 62, 927–936.

23 Luo, Z. and Chen, Z. (2007) Improperlyterminated, unpolyadenylated mRNA ofsense transgenesis targeted by RDR6-mediated RNA silencing in Arabidopsis.The Plant Cell, 19, 943–958.


Multi-Gene Transformation for Pathway Engineering of Secondary Metabolites

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