Compartmentalized metabolic engineering of plant for artemisinin

biosynthesis

Shashi Kumar Group Leader,

Metabolic Engineering International Centre for Genetic Engineering and Biotechnology,

U. N. Organization, New Delhi, India skrhode@icgeb.res.in

Artemisinin biosynthesis by sequential metabolic engineering. Six genes AACT, HMGS, HMGRt, MVK, PMK and PMD (in red font) encoding yeast MEV pathway were integrated into chloroplast genome to generate a high IPP pool. Homoplastomic plant’s nuclear genome was transformed with six genes (ADS, CYP71AV1, AACPR, DBR2, IDI and FPP) of artemisinin biosynthetic pathway. Subcellular targeting of DBR2, AACPR and CYP71AV1 were done by chloroplast transit peptide. Dihydroartemisinic acid (DHAA) converts itself to artemisinin via self photochemical-oxidation, a non-enzymatic reaction.

Compartmentalized MBE (chloroplast, mitochondria and nuclear organelles) for artemisinin biosynthesis

P. falciparum

IPP M

EV/M

eval

on

ate

pat

hw

ay

MEP

/DX

P p

ath

way

Bacteria, and malaria parasites have MEP while higher plants have both MEP (plastids) and MEV (cytosol)

Pathways produces two five-carbon building blocks called isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are used to make a diverse class of over 50,000 known biomolecules such as cholesterol, heme, vitamin K, coenzyme Q10, and all steroid hormones, metabolic drugs, latex, squalene etc.

MBE of yeast Mev pathway into chloroplasts to enhance IPP

RF MVKPMK PMDAACT HMGRt HMGSPLF aadA

Functionality of Yeast MEV (IPP) pathway in chloroplast

Fosmidomycin containing medium

doi:10.1016/j.ymben.2011.11.005

Metabolic Engineering

Volume 14, Issue 1, January 2012, Pages 19–28

Remodeling the isoprenoid pathway in tobacco by expressing

the cytoplasmic mevalonate pathway in chloroplasts

Shashi Kumara, b, 1, Frederick M. Hahna, Edward Baidooc, Talwinder S. Kahlona, Delilah F. Wooda, Colleen

M. McMahana, Katrina Cornishb, 2, Jay D. Keaslingc, Henry Danielld, Maureen C. Whalena, ,

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doi:10.1016/j.ymben.2011.11.005

Abstract

Metabolic engineering to enhance production of isoprenoid metabolites for industrial and

medical purposes is an important goal. The substrate for isoprenoid synthesis in plants is

produced by the mevalonate pathway (MEV) in the cytosol and by the 2-C-methyl-D-

erythritol 4-phosphate (MEP) pathway in plastids. A multi-gene approach was employed

to insert the entire cytosolic MEV pathway into the tobacco chloroplast genome.

Molecular analysis confirmed the site-specific insertion of seven transgenes and

homoplasmy. Functionality was demonstrated by unimpeded growth on fosmidomycin,

which specifically inhibits the MEP pathway. Transplastomic plants containing the MEV

pathway genes accumulated higher levels of mevalonate, carotenoids, squalene,

sterols, and triacyglycerols than control plants. This is the first time an entire eukaryotic

pathway with six enzymes has been transplastomically expressed in plants. Thus, we

have developed an important tool to redirect metabolic fluxes in the isoprenoid

biosynthesis pathway and a viable multigene strategy for engineering metabolism in

plants.

Highlights

▶ Cytosol isoprenoid substrate synthesis pathway genes were inserted into chloroplast

genome. ▶ An entire eukaryotic pathway with six enzymes has been expressed in

chloroplasts for the first time. ▶ Chloroplast metabolic engineering enhanced production

of isoprenoids and linked metabolites. ▶ This multigene strategy is a tool to redirect and

engineer isoprenoid metabolism in plants.

Abbreviations

MEV, mevalonate; MEP, 2-C-methyl-D-erythritol 4-phosphate; IPP, isopentenyl

diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP,

farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; HMGR, 3-hydroxy-

3methylglutaryl-coenzyme A reductase; FPPS, FPP synthase; PTS, patchoulol

synthase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-

phosphate reductoisomerase; PMK, phosphomevalonate kinase; MVK, mevalonate

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44

))

( 4

Shashi Kumar 1,…… Jay D Keasling2, Henry Daniell3, Katrina Cornish4, Colleen McMahan5, Maureen Whalen5

1International Centre for Genetic Engineering and Biotechnology, New Delhi, India 2Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, United States 3University of Pennsylvania, Philadelphia, PA, USA 4The Ohio State University, Wooster, OH, USA 5WRRC, USDA, Albany CA, USA

Squalene

Lipid

Carotenoid

Mev

alo

nat

e C

on

c. (

nm

ole

s/g

DW

)

WT EV MEV6

Artemisinin is produced by trichomes on Artemisia annua leaves

artemisinin... ...is produced by trichomes... ...found on Artemisia annua leaves...

Use of excess flux of IPP to produce artemisinic acid

(precursor to artemisinin)

Antimalarial and Anticancer drug Low artemisinin is

produced by trichomes

on Artemisia annua

leaves

Artemisinic acid biosynthesis in chloroplast

Negative Electrospray Ionization (ESI) Mass spectrometric (MS) m/z spectra of artemisinic acid

13C NMR Spectra of artemisinic acid (a) control

J Biosci. 2014 Mar;39(1):33-41 Metabolic engineering of chloroplasts for artemisinic acid biosynthesis and impact on plant growth Saxena B, Subramaniyan M, Malhotra K, Bhavesh NS, Kumar S

PMK

MVK MDD AACT HMGS HMGRt IPP FPP ADS CYP71AV1 aadA AACPR trnI trnA

P-rrn16S P-psbA-5’UTR 3’UTR 3’UTR

Mevalonate pathway Artemisinic acid pathway

P2 P1 T T

SphI

Probe

SphI Southern hybridization (~ 6.1 kb)

(0.84kb)

SalI

PCR (~ 2.2 kb) Sp

ec-m

f

23

S-R

(8.1 kb) (6.8 kb)

0 400800

1200

1600

2000

2400

28

00

32

00

36

00

4000

4400

4800

5200

5600

600064006800

7200

7600

8000

8400

8800

92

00

96

00

1000

010

400

10800

11200

11600

12000

1240012800

AACPR

=ADS

CY

P7

1A

V1

DB

R2

FPP

35

Sd

e

35Sde

IPP

CsVMV

CsV

MV

=

CsVMV=

rbcS

1

P-APX

Co

x4

Mlu

I

DraI (9879)

PacI (3320)

PvuII (3235)

AccI (3339)

CTP

DraI (10163)

35S-PolyA T

SphI (2533)

AccI (2258)

EcoRV (8726)

BclI (1823)

KDEL

st op

EcoRI (4843)

ocs-polyA

XmaI (4859)

SmaI (4861)

ApaLI (4 935)Nos-polyA

St op

KDEL

EcoRV (7885)

EcoRI (11893)

ocs-polyA

ApaLI (11985)

SspI (12091)

CTP

EcoRI (12202)

PvuII (5695)

ClaI (12270)

ApaI (7401)

PacI (5780)

EcoRV (619)

XbaI (613)

SpeI (12739)

DraI (6183)AccI (6 827)

35S-PolyA T

BclI (6651)

BclI (6492)

PvuII (6601)

XhoI (13098)

KpnI (2 3)

Pst I (17)

St uI (13119)

AccI (8 )

SalI (7)

BamHI (13123)

pCox-Nuc-Art e13128bp

Rationalized MBE for Artemisinin via dihydroartemisinic acid (DHAA) pathway

Mitochondria, Chloroplast

genome targeted via nuclear

transformation

T-MEV6 with additional IPP

+

•Dual transformation •Targeted enzymes to three different subcellular compartments

DHAA (in transgenics)

+O2

Advantage of expressing dihydroartemisinic acid (DHAA) biosynthesis pathway

(Spontaneous Conversion of DHAA to ART)

Artemisinin

• Conversion of DHAA to artemisinin is a spontaneous photo-oxidative process and does not require additional enzymes and energy

• The exposure of light after harvesting the plant favored this bioconversion (Farhi et al., 2011). The maximum bioconversion of DHAA to artemisinin was achieved when A. annua plants were sun-dried (Ferreira and Luthria, 2010).

• To minimize adverse effects and negative impact of artemisinin on growth of transgenic lines (Toxic)

Nuclear genome transformation of homoplastomic plant

Hygro

+Spec

WT DT

Identification of pathway-metabolites in DT

Isopentenyl diphosphate (LC-MS/MS)

Amorphadiene (GC-MS)

Dihydroartemisinic acid (LC-MS/MS)

Artemisinin (LC-MS/MS)

Artemisinin biosynthesis in alternative plant

Bioconversion of

DHAA to

artemisinin

spontaneously in

presence of light

and oxygen

Maximum in

DT4 transgenic

line

Maximum titer 0.8µg/g DW

(~0.08%)

DHAA analysis by LC-MS & Amorphadiene by GC-MS of DT plants

XIC of +MRM (2 pairs): 237.200/163.100 Da fro... Max. 4114.0 cps.

5 10 15 20Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

Inte

nsity, cps

11.10

XIC of +MRM (2 pairs): 237.200/163.100 Da fro... Max. 1049.6 cps.

5 10 15 20Time, min

0

200

400

600

800

1000

1184

Inte

nsity, cps

10.99

5.38

1.89

9.714.10 5.77 11.330.60 13.17 17.8515.108.43

XIC of +MRM (2 pairs): 237.200/163.100 Da from ... Max. 1143.0 cps.

5 10 15 20Time, min

0

200

400

600

800

1000

1143

Inte

ns

ity

, c

ps

10.85

5.24

1.71

9.593.91 5.58 11.292.82 13.988.70 17.79

DT

Std-

DHAA

A. Annua

DHAA

amorphadiene

DT

Mass spectra

amorphadiene

Std-amorphadiene

Enhanced accumulation of isoprenoid IPP

IPP/DMAPP are universal

isoprenoid precursors

IID gene introduction in

nuclear genome for

maintaining equilibrium ratio

between IPP and DMAPP

Nearly ~3-fold enhancement

in IPP

Sufficient IPP is essential for

high artemisinin biosynthesis

Functionality of Artemisinin

24 hours 48 hours

DT

4

D

T3

D

T2

D

T1

W

T

DM

SO

Extracts

from DT1-

DT4

inhibited

parasite

Growth in

24-48h

assay

Oral feeding

of intact

whole plant

DT4 in

Balb/C mice

reduced the

parasitemia

levels.

Mechanism of action of artemisinin

Fluorescent Artemisinin Nitrobenzodiazole

Figure - Confocal images of parasite

infected RBC incubated with Fluorescent

Artemisinin Nitrobenzodiazole conjugate

without iron chelator DFO before (1a) and

after wash (1b), and with DFO (100 µM)

before (1c) and after wash (1d)

In 1991, Meshnick and

collaborators showed that

artemisinin interacted with

intraparasitic heme, and

suggested that

intraparasitic heme or iron

might function

to activate artemisinin

inside the parasite into

toxic free radicals

(Meshnick et al., 1991). The

malaria parasite is rich in

heme-iron, derived from

the proteolysis of host

cell hemoglobin

(Rosenthal and Meshnick,

1996). This could explain

why artemisinin is

selectively toxic to parasites.

• ART is frontline treatment for rapid clearance of malarial parasitemia

• Lengthy biosynthesis period (18 months) and low yield of ART from native plant. Higher cost of chemical synthesis limits the ART supply for malarial treatment.

• Low supply from the natural sources, and the non-availability of an antimalarial vaccine, has necessitated producing this drug with an alternative method.

• Chloroplast and nuclear genome transformation has produced the complete artemisinin, stop the growth of Plasmodium falciparum

• Higher biomass and rapid growth of tobacco plant may provide sufficient supply of ART for endemic regions

Conclusions

Acknowledgments

Funding

MBE Group

Collaborators

DBT

Karan Irshad Nitin Amit Kunbhi Charli Prachi Randhir Amita

International Centre for Genetic Engineering and Biotechnology colleagues, friends and ICGEB Director Prof. Dinakar Salunke