1

Seed Oil Biosynthesis in Brassica napus

Randall J. Weselake

(randall.weselake@ualberta.ca)

Professor and Canada Research Chair in Agricultural Lipid Biotechnology

Scientific Director of the Alberta Innovates Phytola Centre

Department of Agricultural, Food and Nutritional Science University of Alberta

Edmonton, Alberta, Canada

14th International Rapeseed Congress Saskatoon, Saskatchewan, Canada

July 5-9, 2015

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Presentation Outline • Triacylgycerol (TAG) biosynthesis in oleaginous developing seeds • Metabolic targets for increasing seed TAG content • Brassica napus diacylglycerol acyltransferase 1 (DGAT1) • Over-expression of B. napus DGAT1 during seed development in B. napus and metabolic control analysis of storage lipid biosynthesis • Substrate specificity properties of recombinant B. napus DGAT1 • Directed evolution of B. napus DGAT1 to increase enzyme activity • Purification and properties of recombinant B. napus DGAT1

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Seed Oil is Mainly Composed of Triacylglycerol (TAG)

Nelson DL, Cox MM (2005) Lehninger. Principles of Biochemistry, Fourth Edition, Freeman, New York

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Canola (Brassica napus) Production in Canada • Generates > $19 billion in economic activity for Canada

• 17 million acres • Exceeded non-durum wheat acreage in 2011 • Important in food, feed and industrial applications • A one percent increase in seed oil content could potentially result in an additional $100 million per year for the oilseed crushing and processing industry

Canola Council of Canada

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Oil Formation Occurs During Seed Development

Canola Council of Canada

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Photosynthesis

Sucrose

Carbon flow

Fatty acid biosynthesis & production of MUFA (18:1)

Cytosol

PLASTID

Acyl-CoA pool

Endoplasmic reticulum (ER)

- Fatty acid elongation - Acyl-exchange with ER acyl chains

TAG

PUFA formation

TAG assembly

CoA

Bicarbonate Acetyl-CoA ATP

TAG

Seed TAG Biosynthesis

Weselake RJ (2011) In: Canola: Description, Variety Development, Agronomy, Composition, and Utilization; JK Daun, D Hickling, NAM Eskin (editors); AOCS Press; Urbana, IL; pp 57-91

MUFA, monounsaturated fatty acid PUFA, polyunsaturated fatty acid

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Endoplasmic Reticulum

Cytosol

OH

P O

DHAP G3P P

OH HO

LPA PA

DAG

PC

FA-CoA pool

FA FA

PC

• PUFA formation on PC

• Acyl-exchange at sn-2 position of PC catalyzed by

TAG

FA FA

FA

Pi

P

FA FA

FA HO

PC FA

LPC

FA HO

P

• Further elongation of FA in FA-CoA pool can occur in the ER

OH

FA FA

CoA

CoA

CoA

ATP

Malonyl-CoA

Acetyl-CoA

FA synthesis

ATP HCO3

-

CO2

• Monounsaturated FA produced in this organelle

Plastid

FA

FA synthase complex

ACCase

ACS

NADH + H+ +

NAD+

PAP

PDCT

PLA2

PDAT

DGAT

LPCAT

FA-ACP (18:1,16:0)

=

CPT

Adapted from Weselake RJ et al. (2009) Biotechnol Adv 27: 866-876; Lu C et al. (2009) PNAS USA 106: 18837-18842

FAD2/FAD3

* LPAAT

GPAT

*

*

* G3PDH *

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Diacylglycerol Acyltransferase (DGAT) FA

FA OH

+ FA FA

FA FA

+ CoA

• Drives the acyl-CoA dependent acylation of diacylglycerol (DAG) to form triacylglycerol (TAG) (a final step in the formation of oils and fats)

• Typically assayed using [1-14C]acyl-CoA as an acyl donor

• Membrane bound DGATs: DGAT1, DGAT2 (Dga1 in yeast), bifunctional wax synthase/DGAT

• Soluble DGATs: DGAT3, defective cuticle ridge (DCR)

• In humans: reduce DGAT activity to combat obesity

• In plants and yeast: increase DGAT activity to increase oil accumulation (“pull effect”)

http://lipidlibrary.aocs.org/plantbio/tag_biosynth/index.htm

Liu Q et al. (2012) Prog Lipid Res 51: 350-377 Li Q et al. (2008) Microbiol Biotechnol 80: 749-756

DAG TAG

-Coenzyme A (CoA)

FA, fatty acyl

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10 20 30 40 50 60

0

1

2

3

4

5

0.0

0.5

1.0

1.5

2.0

2.5

0

4

8

12

16

0

10

20

30

40

(pm

oles

TA

G/m

in/s

eed)

DG

AT

spec

ific

activ

ity

DW

(mg/

seed

)

Lipi

d (m

g/se

ed)

(pm

oles

TA

G/m

in/m

g pr

otei

n)

Days after flowering

DG

AT

activ

ity

DGAT Activity, Lipid Content and Dry Weight of Maturing Seeds of Canola

Weselake RJ et al. (1993) Plant Physiol 102: 565-571

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Homology alignment of BnaDGAT1 polypeptides

B. napus DGAT1(BnaDGAT1) Sequence Homology

Coding sequence homology

BnaA.DGAT1.a and BnaA.DGAT1.b are from the B. rapa genome (A) BnaC.DGAT1.a and BnaC.DGAT1.b are from the B. oleracea genome (C) Named according to the nomenclature of Østergaard L, King GJ (2008) Plant Methods 4:10

BnaA.DGAT1.a

BnaC.DGAT1.b

BnaA.DGAT1.b

BnaC.DGAT1.a

Greer MS et al. (2014) Appl Microbiol Biotechnol 99: 2243-2253

Michael Greer

11 11

$

Before

After

Widening the Bottleneck in the Flow of Carbon into

Seed Oil

DGAT

More DGAT activity Weselake RJ et al. (2008) J Exp Bot 59: 3523-3549

Weselake RJ et al. (2009) Biotech Adv 27: 866-878

Taylor DC et al. (2009) Botany 87: 533-543

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Over-production of DGAT Increases Seed Oil Content

Weselake RJ et al. (2008) J Exp Bot 59: 3543–3549

BnaA.DGAT1.b in canola UrDGAT2A in soybean

Lardizabal KD et al. (2008) Plant Physiol 148: 89-96

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Untransformed Block B = 70% control B. napus L. cv Westar Transformed with BnaA.DGAT1.b Block B = 51% control

Weselake RJ et al. (2008) J Exp Bot 59: 3543-3549

Fatty Acid Production

Block A Acyl - CoA

Triacylglycerol Assembly

Block B

Increasing BnaDGAT1 Activity Reduces the Level of Control of Oil Formation by the TAG Assembly Block (B)

References on control analysis: Ramli US et al. (2002) Biochem J 364: 385-391 & 393-401 Harwood JL et al. (2013) Eur J Lipid Sci Technol 115: 12391246

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In vivo DGAT Activity in Saccharomyces cerevisiae Strain H12461 Cultures Producing BnaDGAT1 Isoforms

Determined Using Nile Red

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0.00

0.20

0.40

0.60

0.80

1.00

BnaC.DGAT1.a BnaA.DGAT1.a BnaA.DGAT1.b BnaC.DGAT1.b

∆F/O

D 600

cDNA cloned by Nykiforuk CL et al.2

1Sandagar L et al. (2002) J Biol Chem 277:6478-6482 2Nykiforuk CL et al. (1999) Plant Physiol 121:1057

Greer MS et al. (2014) Appl Microbiol Biotechnol 99: 2243-2253

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Acyl-CoA Substrate Specificity Properties of Recombinant BnaC.DGAT1 Isoforms in

S. cerevisiae H1246 Microsomes

Left to right: BnaA.DGAT1.a, BnaC.DGAT1.a, BnaA.DGAT1.b, BnaC.DGAT1.b | | Clade I Clade II

Clade II BnaDGAT1 isoforms exhibit enhanced specificity for linoleoyl (18:2)-CoA relative to clade I BnaDGAT1 isoforms

Greer MS et al. (unpublished data)

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Acyl-CoA Substrate Specificity and Selectivity of BnaC.DGAT1.a Using Two Molecular Species of Acyl-CoA

Greer MS et al. (2014) Lipids 49: 831-838

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Directed Evolution of BnaC.DGAT1.a to Increase Enzyme Activity

Kristian Caldo Gavin Chen Rodrigo Siloto Martin Truksa Sarena Xu

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Error Prone Polymerase Chain Reaction used to Introduce Mutations into BnaC.DGAT1.a

mutagenesis

cloning

transformation of microorganism

isolation of single events screening

selection

next round of mutagenesis

recombination

library

plasmid library

culture library

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Conventional DGAT Activity Assay

Scintillation Counter

DPM ~ Specific Activity

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Selection of Active Variants of BnaC.DGAT1.a

S. cerevisiae strain H1246 is a key component

Siloto RMP et al. (2009) Plant Physiol Biochem 47: 456-461 Siloto RMP et al. (2009) Lipids 44: 963-973

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Properties of Nile Red

Nile Red / Phospholipid

Nile Red / Triacylglycerol

Excitation Emission

Excitation Emission

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Nile Red Assay

Fluorescent detection of TAG in H1246

Normalization by cell density

Correlation with TAG in yeast culture

Siloto RMP et al. (2009) Lipids 44: 963-973

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Overview of DGAT High Throughput Screening

ISBAB - 2012 Siloto RMP, Weselake RJ (2010) Int J High Throughput Screening 1: 29-38

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High Throughput Screening Procedure

•Plasmid isolation

•Sequencing and alignment

•Re-transformation of

H1246 and wild type cells

Primary screening

Secondary screening Tertiary screening

ISBAB - 2012

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Screening of a BnaC.DGAT1.a Library

Primary screening of 1596 clones

50 SEQ

Secondary screening of 288 clones

ISBAB - 2012

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Tertiary Screening Re-transformation and analysis of yeast

Siloto RMP et al. (2009) Plant Physiol Biochem 47: 456-461 Siloto RMP et al. (2009) Lipids 44: 963-973 Chen G et al. (unpublished data)

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Locations of Amino Acid Substitutions

ISBAB - 2012

28 28

Alignment of the C-terminus Portion of Plant DGAT1 Sequences

**** * **** ****** * Oryza sativa (436) KFNNTMVGNMIFWFFFSILGQPMCVLLYYHDVMNRQQAQTNR Arabidopsis thaliana (482) RFG-STVGNMIFWFIFCIFGQPMCVLLYYHDLMNRKGSMS Perilla frutescens (495) KFKNSMVGNMMFWCFFCIFGQPMCVLLYYHDLMNRKASAR Tropaeolum majus (481) KFSNSMVGNMIFWFIFCILGQPMCVLLYYHDLINLKEK Olea europaea (493) KFQNSMVGNMIFWCFFSILGQPMCLLLYYHDLMNRKASAK Glycine max (459) KFRNSMVGNMIFWFIFSILGQPMCVLLYYHDLMNRKGKLD Euonymus alatus (468) KFRSSMVGNMMFWFSFCIFGQPMCLLLYYHDLMNRNGKME Lotus corniculatus (470) KFRNSMVGNMIFWFIFSILGQPMAVLLYYHDLMNRKSKLDQS Brassica juncea (465) RFG-SMVGNMIFWFSFCIFGQPMCVLLYYHDLMNRKGSMS Nicotiana tabacum (493) KFQSSMVGNMMFWCFFCILGQPMCVLLYYHDVMNRKSSAR Brassica napus (465) RFG-SMVGNMIFGSASCIFGQPMCGLLYYHDLMNRKGSMS Ricinus communis (484) KFRSSMVGNMIFWFFFCILGQPMCVLLYYHDLMNRDGN RcDGAT C1 (484) KFRSSMVGNMIFWFFFCILGQPMCVLLY BnDGAT1 mut 31 (465) RFG-SMVGNMIFGSASCIFGQPMCGLLYYHD

RcDGAT1 C1 inactive mutant

BnaC.DGAT1.a mut 31 active mutant

Siloto RMP et al. (2009) Lipids 44: 963-973

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0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

D9 D7 E7 D8 C8 G8 C3 G2 E8 E1 H12 A2 H9 A10 C7 B10 F8 B8 H5 B1 G12 C10 C4 A11 G1 WT

TAG

Con

tent

(%)

Variants

TAG content (%)

0%

20%

40%

60%

80%

100%

120%

Fatt

y Ac

id C

ompo

sito

n (%

)

Variants

C18:1

C16:1

C18:0

C16:0

Influence of BnaC.DGAT1.a Variants on Yeast TAG Content and Fatty Acid Composition of TAG

Chen G et al. (unpublished data)

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Purification and Properties of

Recombinant BnaC.DGAT1.a

Caldo KMP et al. (2015) FEBS Lett 589:773-778

Kristian Caldo Joanne Lemieux

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• Without a DGAT purified in active form, structure-function studies have been mainly accomplished through: a) bioinformatic analyses of published sequences b) mutational analysis of potentially important residues or

domains c) heterologous expression and analysis of the enzyme in

microsomes d) analysis of a purified recombinant BnaA.DGAT1.b

N-terminal fragment

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Insights into DGAT Action

Liu Q et al. (2012) Prog Lipid Res 51: 350-377 Weselake RJ et al. (2006) BMC Biochem 7: 24

32 32 32

microsomal pellet

-dissolved in various

detergents

1 2 3 4

105,000 x g 1 2 3 4

supernatant

1 2 3 4

pellet

Western blotting

or

Activity assay

P-pellet S-supernatant

P S P S P S P S

CHAPS DDM MEGA8 TX-100

BnaC.DGAT1.a 41% 68% 40% 55%

Western blot profile of pellet and supernatant following centrifugation of detergent-solubilized microsome at 105,000 x g

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Solubilization of Recombinant BnaC.DGAT1.a

CHAPS, 3-[(3-cholamidoproplyl)dimethylammonio]-1-propanesulfonate DDM, n-dodecyl-b-D-maltopyranoside MEGA8, n-octanoyl-N-methylglucamide TX-100, Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether)

[Detergent]=1%

33 33

33

Cobalt Ion Chelate Affinity Chromatography and Tag Removal

BnaC.DGAT1.a purification using immobilized cobalt ion affinity chromatography

Wash Elution

m SF FT W1 W2 E1 E2 E3 E4

50 60

40

kDa

m -TEV +TEV

BnaC.DGAT1.a before and after incubation with tobacco etch virus protease (TEV)

BnaC.DGAT1.a

BnaC.DGAT1.a-tag 50

60

40

kDa

33 m, molecular mass SF, solubilized fraction FT, flow through

[DDM]=0.1%

34 34

Sequence Modifications Charge MH+ [Da] SDSSnGLLPDSVTVSDADVR N5(Deamidated) 2 2034.94621

GDLLYGVER 2 1021.52904

ANPEVSYYVSLK 2 1369.69988

ANLAGENEIR 2 1086.55461

ESGGEAGGNVDVR 2 1246.56731

LIIENLmK M7(Oxidation) 2 989.56737

ESPLSSDAIFK 2 1193.60478

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In Gel Trypsin Digestion and LC MS/MS Sequencing

35

m Vo I II III V

SDS-PAGE of SEC fractions

Purified Active Recombinant BnaC.DGAT1.a Self-associates to Form Dimers and Tetramers

35

mAU

Elution volume (mL) -50

0

50

100

150

200

250

300

350

400

0.00 5.00 10.00 15.00 20.00 25.00

Vo

I III IV

V VI

Superdex 200 size-exclusion chromatography (SEC) profile of BnaC.DGAT1.a

II (11.87)

(10.46) BnaC.DGAT1.a 50 60

40

kDa

[DDM]=0.05%; m, molecular mass; Vo, void volume

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Fraction Volume (ml)

Total activity (nmol

TAG/min)

Total protein

(mg)

Recovery (%) Specific activity (nmol

TAG/min/mg protein)

Purification (fold)

Microsome

60.0 787.46 231.24 100 3.41 1.0

Solubilized fraction

58.0 33.64

162.89 4.3 (100)* 0.21 0.06 (1.0)*

ICAC

4.0 45.97 2.26 5.8 (137)* 20.39 6.0 (98)*

Peak II of SEC 1.0 1.68 0.065 0.2 (5)* 26.00 7.6 (126)*

*Recovery and purification fold relative to the solubilized fraction. ICAC, immobilized cobalt ion affinity chromatography SEC, size-exclusion chromatography TAG, triacylglycerol

Purification of BnaC.DGAT1.a from a Three Liter Yeast Culture

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Acyl-CoA Specificity of Purified BnaC.DGAT1.a

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

16:0 18:0 18:1 18:2 18:3

Spec

ific

activ

ity (n

mol

TA

G/m

g/m

in)

Acyl-CoA substrate

38 38

Closing Comments

• Seed-specific over-expression of BnaA.DGAT1.b in B. napus resulted in a significant

increase in seed oil content • Four closely related BnaDGAT1 isoforms use a range of acyl-CoAs which represent

the main fatty acids found in the seed oil • Clade II BnaDGAT1 isoforms exhibited enhanced specificity for 18:2-CoA relative to

clade I isoforms • Directed evolution was used to generate numerous variants of BnaC.DGAT1.a which

resulted in increased TAG content when produced in S. cerevisiae H1246 • The C-terminus is critical for maintaining plant DGAT1 activity

• DDM-solubilized BnaC.DGAT1.a oligomerized into apparent dimeric, tetrameric and

higher molecular mass states • Solubilized BnaC.DGAT1.a was purified 126-fold in active form. This achievement sets

the foundation for determining structure/function

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• Alberta Agricultural Research Institute

• Alberta Canola Producers Commission

• Alberta Crop Industry Development Fund

• Alberta Enterprise and Advanced Education

• Alberta Innovates Bio Solutions

• Alberta Innovates Technology Futures

• Agragen

• AVAC Ltd.

• Biotechnology and Biological Sciences Research Council (UK)

• Canada Foundation for Innovation

• Canada Research Chairs Program

• Cargill

• Genome Alberta, Genome Prairie & Genome Canada

• National Research Council of Canada

• Natural Sciences and Engineering Research Council of Canada

• United States Department of Agriculture

• University of Alberta

• Advisors to the Bioactive Oils Program & the Alberta Innovates Phytola Centre

Acknowledgements

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