Metabolic engineering of natural products for plant, animal, and human health Richard Dixon Plant Biology Division Samuel Roberts Noble Foundation Ardmore, Oklahoma and Centre for Novel Agricultural Products, Department of Biology, University of York Molecular biology of plant natural products

We study…… We do…….

•Lignin • Phytochemistry – Forage digestibility • Enzymology • • Gene discovery through – Phytoalexins genomics – Phytoestrogens • Pathway engineering • Condensed tannins – Understanding pathways – Pasture bloat – Addressing function – Health benefits – Improving traits • Triterpene saponins – Palatability – Plant defense – Adjuvants Natural products from legumes 650 genera, >18,000 examined phytochemically • Lignins • Flavonoids • Isoflavonoids • Terpenoids (di-, sesqui- and tri-triterpenes) • Alkaloids (eg. quinolizidine, dipiperidine) • Coumarins • Anthraquinones • Non-protein amino acids • Cyanogenic glycosides IntegratedIntegrated functionalfunctional genomicsgenomics ofof thethe modelmodel legumelegume MedicagoMedicago truncatulatruncatula

AAAA AAAA

MDKLLV...

OH

HO CH2OH Agronomic traits in Medicago truncatula

• Nitrogen fixation • Mineral nutrition • Forage quality – Protein content – Digestibility – Palatability –Bloat – Silage properties • Disease resistance • Drought tolerance • Grazing tolerance • Source of health-promoting natural products Re-designing alfalfa as the perfect forage

Alfalfa has a high protein content and is an excellent quality forage, BUT….. Disease problems (cotton root rot) Digestibility decreases with increasing maturity Causes pasture bloat AA structuralstructural modelmodel ofof ligninlignin Lignin and forage digestibility USDFRC estimates that

15 a 10% increase in fiber digestibility would result in an annual $350 10 million increase in milk/beef production and = 2.8 million tons 5 decrease in manure

Klason lignin (% of DM) solids produced each year. 0 1/2345678 Internode number COMT and CCOMT: targets for lignin engineering

CCOMT

COMT EffectsEffects ofof COMTCOMT andand CCOMTCCOMT downdown-- regulationregulation onon ligninlignin compositioncomposition

Transgenic COMT CCOMT S G 5-OHG Lines pkat/mg pkat/mg Lignin Lignin Lignin

Wild type 5.98 22.35 158.8 305.4 0.0

COMT downregulated 1.24 22.26 8.6 246.1 8.8

CCOMT 14.39 0.78 159.1 150.2 0.0 downregulated COMT & CCOMT 0.78 5.59 54.0 223.7 0.0 downregulated

Guo et al. (2001) Plant Cell 13: 73-88 In-rumen digestibility of transgenic alfalfa forage in fistulated steers

0.9

0.88 ck 0.86 SC5 0.84 ACC305 D ig estib ility Guo et al. Transgenic 0.82 Research 10, 457- 0.8

0.78 464, 2001 020406080 Time in Rumen (h) Summary of field data (3 States)

59 58 •LSD .05 = 1.14 57 56 55 54 In vitro digestibility 53 IVD % 52 51 50 49 48 4 5 - 0 Null -3 -315 WL342 MT-310 MT COMT COMT-5 O OMT C 10.5 CC CCO

10.0

9.5

9.0 Acid detergent lignin ADL % ADL 8.5 (lower stem)

8.0 l ul N 342 -5 L 310 05 W MT -3 15 COMT-4 O C MT COMT- CCO CCOMT-3 Profiling of soluble phenolics by HPLC 16K M. truncatula oligo array vs alfalfa stem RNA

Absorbence (mAU) Control (leaf)

Control (stem)

COMT (stem)

CCOMT (stem)

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 Retention time (min)

E Factor1

Leaf F

Factor2

B

A

Factor3 Stem C

D

Principle component analysis Data of Fang Chen and Srinu Reddy

2-D NMR reveals novel structures in lignin from COMT down-regulated alfalfa

Marita et al. Phytochemistry, 2003 Re-designing alfalfa as the perfect forage

• Alfalfa has a high protein content and is an excellent quality forage, BUT….. – Disease problems (cotton root rot) – Digestibility decreases with increasing maturity – Causes pasture bloat Condensed tannins (proanthocyanidins) are oligomers or polymers of flavonoid (i.e.flavan-3-ol) linked R by carbon-carbon bonds. 2 OH 5' 4' may contain 2-50 or greater 1' HO O 2' flavonoid units. 3' 1 2 R1 3 differ in flavonoid units (R1, 4 R n OH 2 R2 =H or OH) and interflavan OH bond sites. OH 5'4' 1' B HO O 2' 8 1 3' R 7 2 1 produce anthocyanidin after A C 3 5 4 HCl-butanol hydrolysis OH produce blue coloration with OH dimethylaminocinnamaldehyde Activities of condensed tannins

• Bind to proteins, and reduce their rate of degradation (eg. tanning of leather, reduction of rumen fermentation). Help prevent pasture bloat. •In Medicago and white clover, CTs accumulate in the seed coat but not in the leaves • Toxic to fungi, bacteria and insects. • Antioxidant-cardiovascular benefits (polymers and monomers) • Flavor and astringency (tea, wine, fruit juices) • Urinary tract health (cranberry)

BiosynthesisBiosynthesis ofof condensedcondensed tanninstannins andand anthocyaninsanthocyanins..

ban WT M.truncatula homolog of Arabidopsis BAN gene was identified by EST database mining -developing seed library.

1 MASIKQIEIEKKKACVIGGTGFVASLLIKQLLEKGYAVNT 1 MDQT-LTHTGSKKACVIGGTGNLASILIKHLLQSGYKVNT

41 TVRDLDSANKTSHLIALQSLGELNLFKAELTIEEDFDAPI 40 TVRDPENEKKIAHLRQLQELGDLKIFKADLTDEDSFESSF

81 SGCELVFQLATPVNFASQDPENDMIKPAIKGVLNVLKACV 80 SGCEYIFHVATPINFKSEDPEKDMIKPAIQGVINVLKSCL

121 RAKEVKRVILTSSAAAVTINELEGTGHVMDETNWSDVEFL 120 KSKSVKRVIYTSSAAAVSINNLSGTGLVMNEENWTDIDFL

161 NTAKPPTWGYPVSKVLAEKAAWKFAEENNIDLITVIPTLT 160 TEEKPFNWGYPISKVLAEKKAWEFAEENKINLVTVIPALI

201 IGPSLTQDIPSSVAMGMSLLTGNDFLINALKGMQFLSGSI 200 AGNSLLSDPPSSLSLSMSFITGKEMHVTGLKEMQKLSGSI

241 SITHVEDICRAHIFVAEKESTSGRYICCAHNTSVPELAKF 240 SFVHVDDLARAHLFLAEKETASGRYICCAYNTSVPEIADF

281 LSKRYPQYKVPTEFDDFPSKAKLIISSGKLIKEGFSFKHS 280 LIQRYPKYNVLSEFEEGLSIPKLTLSSQKLINEGFRFEYG MtBAN 321 IAETFDQTVEYLKTQGI----K. Identity, 59% 320 INEMYDQMIEYFESKGLIKAKES AtABN R2 R2 OH OH 6' 5' 5' 4' 4' 1' 1' HO 7 O 2' LAR HO 7 O 2' 8 1 3' R 3' 2 1 8 1 2 R1 6 3 6 3 5 4 5 4 OH OH OH OH WT OH Flavan-3-ol Leucoanthocyanidin 35S-BAN R2 ANS R2 6' OH 5' OH 4' 6' 5' 1' 4' HO 7 O 2' 1' 8 + 3' R HO 7 O 2' 2 1 8 1 3' R 6 ANR 2 1 5 4 6 3 3 OH 5 4 OH OH Anthocyanidin ban OH R2 OH 5' 4' 1' HO O 2' 3' 1 2 R1 3 4 R n OH 2 OH OH 5'4' 1' HO O 2' 8 1 3' R 7 2 1 3 5 4 OH OH WT Anthocyanins Condensed tannins CT Xie et al. Science, 299, 396-399, 2003 Ectopic expression of MtBAN in tobacco flowers reduces anthocyanin content and results in accumulation of tannins

7 6 5 4 Comparison of 3 528nm) 2 anthocyanin content 1 0 Anthocyanidin (absorbance at -B -C -5 C-4 C -13 -19-A -19 B B B B-21-B 121-1-B 121-4-B Plant lines

70 60 50 40

FW) 30 Comparison of 20 10 CT content 0 CT (µg cyanidin equivalents/g 4 5 -B -A -C -B - - -B -B 3 9 9 1 C C -1 -4 -1 -1 -1 -2 1 1 2 2 B B B B 1 1 Plant lines R 1 OH Leucoanthocyanidin HO O Genetic support for R 1=R2=H, leucopelargonidin R 2 R 1=OH, R2=H, leucocyanidin OH R 1=R2=OH, leucodelphinidin new pathway to OH OH

ANS LAR R condensed R 1 1 OH OH

HO O HO O tannins via BAN + R R 2 2 OH OH BAN OH OH Anthocyanidin: (ANR) 2,3-trans-2R,3S-flavan-3-ol R =R =H, pelargonidin 1 2 R R =OH, R =H, cyanidin 1 1 2 OH R 1=R2=OH, delphinidin HO O 2 R 2 4 3 OH 3GT OH 2,3-cis-2R,3R-flavan-3-ol R 1=R2=H, (-)-epiafzelechin R 1=OH, R2=H, (-)-epicatechin R 1=R2=OH, (-)epigallocatechin Anthocyanins R1 Col tt18 ban OH HO O R 2 R n OH 1 OH OH HO O R 2 OH OH Condensed tannins Species and tissue ANR enzyme activity (pmol/min/mg protein) Lotus corniculatus Leaf 721 Desmodium uncinatum Leaf 563 Flower 342 Pod 122 Grape Skin 143 Barley Grain 33* Alfalfa Leaf No reaction

Data of Deyu Xie Moving tannins into alfalfa

Provision of anthocyanin substrate? Need for LAR? Need for transporters, etc? Anthocyanin flower pigments- natural or engineered? WT BAN PAPIxBAN PAP1

Anthocyanin Epicatechin Production of WT --epicatechin BAN --in transgenic tobacco PAP1 ++++++ - leaves PAP1xBAN ++ ++ Redesigning alfalfa – triterpene saponins

• Growth inhibitory to poultry • Anti-palatability and possibly toxic to monogastric animals • Anti-insect • Antifungal (avenacins in oats) • Hypocholesterolemic • Anticancer (desert Acacia spp.) Profiling saponins in Medicago spp

Intens. x10 6 (a) 22 [Sumner and Huhman, Alfalfa 1.5 21 (cv. Radius) Phytochemistry, 2002] 16 23 1.0 56 8 0.5 4 10 25 LC/MS profiling reveals 35 0.0 0 1020304050Time [min]

Intens. more than 30 triterpene x10 6 22 1.50 (b) 21 Alfalfa saponins in M. truncatula 1.25 16 (cv. Kleszczewska) 1.00 6 23 (more complex pattern 5 0.75 8

0.50 10 than alfalfa) 4 25 0.25 35

0.00 0 10 20 30 40 50 Time [min]

Intens. x10 6 21 (c) 22 Medicago truncatula CO2H 1.25 16 HO 27 (Jemalong a17) 1.00 15 23 25 26 0.75 30 4 10 24 31 HO 29 0.50 9 CO2H 1 5 0.25

MedicagenicAcid 0.00 0 1020304050Time [min] Saponin aglycones from Medicago truncatula

30 29 Medicagenic Acid Soyasapogenol B 20 mw = 502 19 21 mw = 458 12 18 22 11 13 17 OH 25 26 CO H 14 16 28 2 15 HO 1 9 2 10 8 E 3 7 27 4 5 6 C HO D COOR HO 24 CO H HO 2 CH OH 23 2 2 A B

R1O HOH2C Bayogenin mw = 488 O COOR2

R1O HO HOH2C CH2OH Hederagenin Soyasapogenol E mw = 472 mw = 456 Early steps in the biosynthesis of triterpenes and sterols in plants

PPO

Sesquiterpenes Mevalonic acid FPP

Squalene synthase (SS)

Squalene

Squalene epoxidase (SE)

O 2,3-Oxidosqualene

β-Amyrin synthase (AS)

30 29

19 20 21 12 13 18 22 11 25 26 17 O 1 9 14 28 2 16 10 8 15 3 5 4 27 HO 7 HO Cycloartenol 6 β-Amyrin 24 23 700 Time post-elicitation (hr) 600 500 01 2 4 8 12 24 48 400 SS 300 SS 200 100 SS 0 0 1020304050

700

600

500 400 SE2 300 SE2 SE2 200

) 100

0

%

( 0 1020304050

l 3000

e 2500

v

e 2000 l βAS

βAS 1500

A 1000 βAS

N

R 500

m 0 0 1020304050

e

v 180 i

PAL t

a 150 l PAL

e 120

R 90 PAL 60

30

CHS 0 0 1020304050 120

90

60 rRNA CHS CHS 30

0 Differential responsivenes of 0 1020304050 triterpene and phenylpropanoid Time post-elicitation (hr) biosynthesis to MeJA in M. Suzuki et al. Plant J. 32, 1033 (2002) truncatula suspension cultures Saponin biosynthesis in M. truncatula involves cytochrome P450s and glycosyltransferases

E

C D

AB β-Amyrin HO

P450s

OH O COOH COOH COOH HO HO

HO HO HO HO CH OH COOH CH OH HO CH2OH 2 2 CH2OH BayogeninHederagenin Medicagenic acidSoyasapogenol B Soyasapogenol E

Glycosyltransferases

OH

O CH2OH COOH O CO HO HO O O O CH OH O CH2OH 2 CH2OH CH2OH HO O O OH CO HO OH HO HO HO HO O O O O O CH2OH COOH CH OH O OH 2 O OH O HO OH HO HO OH HO HO OH OH OH 3-Glc, 28Glc-Medicagenic acid 3-Glc-Ara, 28-Glc-Hederagenin Soyasaponin I (3-Rha-Gal-GlcA-Soyasapogenol B) Filters for triterpene pathway gene selection • P450 or GT involved in natural product biosynthesis • Micro/macroarray analysis for up-regulation by methyl jasmonate • In silico expression analysis: expressed in libraries where β-AS is expressed; not expressed in libraries where β-AS is not expressed. • Induction kinetics cf β-AS (RNA gel blots) • Functional expression in yeast (P450s) or E. coli (GTs)

Collaboration with Seiichi Matsuda, Rice University Cluster all P450 TCs Cluster all GT TCs

Clustering P450- candidates Clustering GT- candidates Functional characterization

P450s- express in yeast harboring β- amyrin synthase and plant P450 reductase

GTs- express in E.coli, use trterpene aglycones as substrates 0 1 2 4 8 12 24 48

β-AS

rRNA

0 1 2 4 8 12 24 48

0 1 2 4 8 12 24 48 0 1 2 4 8 12 24 48

(3/8)P012G (5/8) GT068

Coordinated expression of candidate P450 and GT genes with β-amyrin synthase in MeJA-treated M. truncatula cell suspensions Biological activities of compounds • Antimicrobial phytoalexins • Allelochemicals • Anti-insect and piscicidal activity • Nodulation gene inducers (soybean/Bradyrhizobium) • Phytoestrogens (, ): anti-osteoporosis • Anti-cancer Structural similarities between phytoestrogens and estradiol Beneficial effects of genistein on human health ? Isoflavonoid biosynthesis in soybean

Phenylalanine 4-Coumaroyl-CoA 3 x Malonyl-CoA CHS (CHR) Chalcone OH HO O CHI H HO O

OH O OH H O Genistein (OH) Flavanone HO O OH 2-HYDROXY ISOFLAVANONE PHYTOESTROGENS SYNTHASE (“IFS”) H O (OH) OH PHYTOALEXIN 2-Hydroxyisoflavanone O O HO O OH

O H O OH Daidzein OH Steele et al. Arch Biochem Biophys 367: 146-150, 1999 2 = Glu-Rha-genistein OH A 40 Transgenic 3 = Rha-genistein HO o 30 2. 3. 4. OH 20 OH O IFS 10 0

HO O B 40 Control

OHOH O OH 30 20

10

0

10 15 20 25 30 35 40 45 50 55 60 Time (min)

Production of genistein conjugates in transgenic Arabidopsis

Successful engineering of in Arabidopsis requires blocking flux into formation of flavonols

Competition between endogenous and introduced pathways

Flavanone IFS F3H Genistein

Flavonol

Liu et al. PNAS 99, 14778, 2002 Accumulation of genistein in alfalfa leaves expressing 35S-IFS

140

120

100

80

60

40

20 Genistein (nmol/gFW)

0 C5 A2 A6 A8 A5 A4 C6 A9 B9 B8 B4 B2 B1 B3 B5 B6 C17 C23 A11 C21 A13 C11 A15 A17 C13 A20 C12 A12 C16 C22 A10 A14 A16 A18 A19 B13 B10 B20 B11 B12 B14 B16 Line number mAU G mAU

200 200 A 150

150 1 3 100 A 100 2 50 B 50 5 0 4 20 30 40 50 60 min

0 mAU 20 30 40 50 60 min

200 mAU

150 200 A 100 150

50 100

0 50 20 30 40 50 60 min

0 20 30 40 50 60 min Retention time Retention time data of Bettina Deavours Genes for engineering complex (iso)flavonoids. -importance of prenyltransferases

O O

OH O HO O Kraussianone 1

• Component of roots of Eriosema kraussianum • South African shrublet • Component of “uBangalala” • Used by Zulus to treat impotency and infertility -75% activity of Viagra- PhytoViagra? Interconversions among isoflavonoids- role of isoflavone 2’- and 3’-hydroxylases

HO O OH 8 O 7 o HO o IFS A H 1 Flavanone 2 Chickpea IOMT(DH) I3’H 3' OH 6 4 5 3 3' O B R OH O OH O OH OCH3 CYP81E9 OCH3 IOMT (DH) pratensin A HO 7 o H o B HO (I3’H) o o H 3' OH o O 4' ' 2 OCH3 CYP81E9 O OCH3 H O OCH3 CYP(X) CYP81E1/7 (I2’H) OCH3 HO o (rotenoid) HO o O HO o o O ? O O O Alfalfa? HO OCH3 O 2’-hydroxyformononetin CYP81E7 (I2’H) O medicagol IFR HO o (coumestan) O VR O HO O

HO o HO o CH3O o OH O 6a O O H OCH3 O O O O maackiain pisatin Alfalfa Chickpea Pea 0.02 0.02 81E8 81E7 0.01 81E9 0.01 0.01

0 0 0 Absorbance -0.01 -0.01 -0.01 390 410 430 450 470 490 390 410 430 450 470 490 390 410 430 450 470 490 Wavelength (nm) Wavelength (nm) Wavelength (nm)

Expression of M. truncatula CYP81E enzymes in yeast

Liu et al. Plant Journal 36: 471-484 A genomics approach for the discovery of (iso)flavonoid prenyltransferases

Data mining of EST libraries from: *Elicited soybean cell cultures *Lupin roots *Maclura pomifera fruit *Hop peltate trichomes (at U. of York)

Gglu

1400 2’-OH-Gglu 11.076 1200 19.198 8.923 1000 8.719 Prenylated isoflavones

800

600 6.242 400 10.283 33.928 13.913 10.013 Absorbance (mAU) 34.776 12.054 200 14.497 32.171

0 0 5 10 15 20 25 30 35 Time (min)

Production of prenylated isoflavonoids by lupin roots Conclusions • Legumes are a rich source of natural products that impart useful traits • Medicago truncatula is an excellent model system for legume genomics • Phenylpropanoid compounds can positively affect plant, animal and human health • Genomics approaches can now be applied for natural product pathway gene discovery – increased efforts in structural biology will facilitate progress • Commercial development of transgenics modified in natural product pathways (output traits) is in the pipeline • The possibility exists to produce “isometabolic” plant lines for testing effects of plant natural products on animal health Metabolic engineering of (iso)flavonoids for validating effects on human and animal health

PAP1

Flavonols

Anthocyanins ANR

Epicatechin

Tannins Complex isoflavonoids

Funding from:

••SamuelSamuel RobertsRoberts NobleNoble FoundationFoundation ••NationalNational ScienceScience FoundationFoundation PlantPlant GenomeGenome ProgramProgram ••DepartmentDepartment ofof EnergyEnergy BiosciencesBiosciences ProgramProgram ••OklahomaOklahoma CenterCenter forfor thethe AdvancementAdvancement ofof ScienceScience andand TechnologyTechnology HealthHealth ResearchResearch ProgramProgram ••ForageForage GeneticsGenetics InternationalInternational