seed oil biosynthesis in brassica napus - ag-west · seed oil biosynthesis in brassica napus . ......
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Seed Oil Biosynthesis in Brassica napus
Randall J. Weselake
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
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$
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
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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
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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%
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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%
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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
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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
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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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