Seed Oil Biosynthesis in Brassica napus

Transcription

1 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 14 th International Rapeseed Congress Saskatoon, Saskatchewan, Canada July 5-9,

2 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 2

3 Seed Oil is Mainly Composed of Triacylglycerol (TAG) 3 Nelson DL, Cox MM (2005) Lehninger. Principles of Biochemistry, Fourth Edition, Freeman, New York

4 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 4

5 Oil Formation Occurs During Seed Development Canola Council of Canada 5

6 Seed TAG Biosynthesis Bicarbonate Acetyl-CoA ATP Acyl-CoA pool Fatty acid biosynthesis & production of MUFA (18:1) PLASTID CoA TAG assembly Endoplasmic reticulum (ER) - Fatty acid elongation - Acyl-exchange with ER acyl chains Carbon flow PUFA formation TAG Sucrose Cytosol TAG Photosynthesis MUFA, monounsaturated fatty acid PUFA, polyunsaturated fatty acid 6 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

7 NADH + H + + FA synthesis CO 2 HCO 3 - P = O OH DHAP Plastid Monounsaturated FA produced in this organelle Malonyl-CoA Acetyl-CoA ATP FA FA synthase complex ACCase NAD + HO FA-ACP (18:1,16:0) * * G3PDH P G3P ATP ACS OH Cytosol CoA * GPAT FA-CoA pool Further elongation of FA in FA-CoA pool can occur in the ER HO FA HO LPA TAG CoA FA FA LPC FA P FA PC Endoplasmic Reticulum LPAAT DGAT * * CoA FA FA P PA FA FA OH DAG CPT PDCT FA PDAT FA PC PLA 2 PC PAP PUFA formation on PC FAD2/FAD3 FA Acyl-exchange at sn-2 position of PC catalyzed by LPCAT P i 7 Adapted from Weselake RJ et al. (2009) Biotechnol Adv 27: ; Lu C et al. (2009) PNAS USA 106:

8 Diacylglycerol Acyltransferase (DGAT) FA FA OH + FA -Coenzyme A (CoA) FA FA FA + CoA DAG TAG 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 [1-14 C]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 ) 8 FA, fatty acyl Liu Q et al. (2012) Prog Lipid Res 51: Li Q et al. (2008) Microbiol Biotechnol 80:

9 DGAT Activity, Lipid Content and Dry Weight of Maturing Seeds of Canola DW (mg/seed) Lipid (mg/seed) DGAT specific activity (pmoles TAG/min/mg protein) DGAT activity (pmoles TAG/min/seed) Days after flowering Weselake RJ et al. (1993) Plant Physiol 102:

10 B. napus DGAT1(BnaDGAT1) Sequence Homology Coding sequence homology BnaA.DGAT1.a BnaC.DGAT1.a BnaA.DGAT1.b BnaC.DGAT1.b Michael Greer Homology alignment of BnaDGAT1 polypeptides 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 10 Greer MS et al. (2014) Appl Microbiol Biotechnol 99:

11 Before DGAT Widening the Bottleneck in the Flow of Carbon into Seed Oil After More DGAT activity Weselake RJ et al. (2008) J Exp Bot 59: Weselake RJ et al. (2009) Biotech Adv 27: Taylor DC et al. (2009) Botany 87: $ 11

12 Over-production of DGAT Increases Seed Oil Content BnaA.DGAT1.b in canola UrDGAT2A in soybean 12 Weselake RJ et al. (2008) J Exp Bot 59: Lardizabal KD et al. (2008) Plant Physiol 148: 89-96

13 Increasing BnaDGAT1 Activity Reduces the Level of Control of Oil Formation by the TAG Assembly Block (B) Fatty Acid Production Block A Acyl - CoA Triacylglycerol Assembly Block B Untransformed B. napus L. cv Westar Block B = 70% control Transformed with BnaA.DGAT1.b Block B = 51% control Weselake RJ et al. (2008) J Exp Bot 59: References on control analysis: Ramli US et al. (2002) Biochem J 364: & Harwood JL et al. (2013) Eur J Lipid Sci Technol 115:

14 1.00 In vivo DGAT Activity in Saccharomyces cerevisiae Strain H Cultures Producing BnaDGAT1 Isoforms Determined Using Nile Red 1 Sandagar L et al. (2002) J Biol Chem 277: Nykiforuk CL et al. (1999) Plant Physiol 121: F/OD cdna cloned by Nykiforuk CL et al BnaC.DGAT1.a BnaA.DGAT1.a BnaA.DGAT1.b BnaC.DGAT1.b 14 Greer MS et al. (2014) Appl Microbiol Biotechnol 99:

15 Acyl-CoA Substrate Specificity Properties of Recombinant BnaC.DGAT1 Isoforms in S. cerevisiae H1246 Microsomes Clade II BnaDGAT1 isoforms exhibit enhanced specificity for linoleoyl (18:2)-CoA relative to clade I BnaDGAT1 isoforms Left to right: BnaA.DGAT1.a, BnaC.DGAT1.a, BnaA.DGAT1.b, BnaC.DGAT1.b Clade I Clade II 15 Greer MS et al. (unpublished data)

16 Acyl-CoA Substrate Specificity and Selectivity of BnaC.DGAT1.a Using Two Molecular Species of Acyl-CoA Greer MS et al. (2014) Lipids 49:

17 Directed Evolution of BnaC.DGAT1.a to Increase Enzyme Activity Kristian Caldo Gavin Chen Rodrigo Siloto Martin Truksa Sarena Xu 17

18 Error Prone Polymerase Chain Reaction used to Introduce Mutations into BnaC.DGAT1.a mutagenesis library next round of mutagenesis recombination cloning plasmid library selection transformation of microorganism screening isolation of single events culture library 18

19 Conventional DGAT Activity Assay Scintillation Counter DPM ~ Specific Activity 19

20 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: Siloto RMP et al. (2009) Lipids 44:

21 Properties of Nile Red Nile Red / Phospholipid Nile Red / Triacylglycerol Excitation Emission Excitation Emission 21

22 Nile Red Assay Fluorescent detection of TAG in H1246 Normalization by cell density Correlation with TAG in yeast culture 22 Siloto RMP et al. (2009) Lipids 44:

23 Overview of DGAT High Throughput Screening 23 Siloto RMP, Weselake RJ (2010) Int J High Throughput Screening 1: ISBAB

24 High Throughput Screening Procedure Primary screening Secondary screening Plasmid isolation Sequencing and alignment Re-transformation of H1246 and wild type cells Tertiary screening 24 ISBAB

25 Screening of a BnaC.DGAT1.a Library Primary screening of 1596 clones Secondary screening of 288 clones 50 SEQ 25 ISBAB

26 Tertiary Screening Re-transformation and analysis of yeast Siloto RMP et al. (2009) Plant Physiol Biochem 47: Siloto RMP et al. (2009) Lipids 44: Chen G et al. (unpublished data)

27 Locations of Amino Acid Substitutions 27 ISBAB

28 Alignment of the C-terminus Portion of Plant DGAT1 Sequences Oryza sativa Arabidopsis thaliana Perilla frutescens Tropaeolum majus Olea europaea Glycine max Euonymus alatus Lotus corniculatus Brassica juncea Nicotiana tabacum Brassica napus Ricinus communis RcDGAT C1 BnDGAT1 mut 31 **** * **** ****** * (436) KFNNTMVGNMIFWFFFSILGQPMCVLLYYHDVMNRQQAQTNR (482) RFG-STVGNMIFWFIFCIFGQPMCVLLYYHDLMNRKGSMS (495) KFKNSMVGNMMFWCFFCIFGQPMCVLLYYHDLMNRKASAR (481) KFSNSMVGNMIFWFIFCILGQPMCVLLYYHDLINLKEK (493) KFQNSMVGNMIFWCFFSILGQPMCLLLYYHDLMNRKASAK (459) KFRNSMVGNMIFWFIFSILGQPMCVLLYYHDLMNRKGKLD (468) KFRSSMVGNMMFWFSFCIFGQPMCLLLYYHDLMNRNGKME (470) KFRNSMVGNMIFWFIFSILGQPMAVLLYYHDLMNRKSKLDQS (465) RFG-SMVGNMIFWFSFCIFGQPMCVLLYYHDLMNRKGSMS (493) KFQSSMVGNMMFWCFFCILGQPMCVLLYYHDVMNRKSSAR (465) RFG-SMVGNMIFGSASCIFGQPMCGLLYYHDLMNRKGSMS (484) KFRSSMVGNMIFWFFFCILGQPMCVLLYYHDLMNRDGN (484) KFRSSMVGNMIFWFFFCILGQPMCVLLY (465) RFG-SMVGNMIFGSASCIFGQPMCGLLYYHD RcDGAT1 C1 inactive mutant BnaC.DGAT1.a mut 31 active mutant Siloto RMP et al. (2009) Lipids 44:

29 TAG content (%) Influence of BnaC.DGAT1.a Variants on Yeast TAG Content and Fatty Acid Composition of TAG 2.50 TAG Content (%) 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 120% Variants 100% Fatty Acid Compositon (%) 80% 60% 40% 20% C18:1 C16:1 C18:0 C16:0 0% 29 Variants Chen G et al. (unpublished data)

30 Purification and Properties of Recombinant BnaC.DGAT1.a Kristian Caldo Joanne Lemieux Caldo KMP et al. (2015) FEBS Lett 589:

31 Insights into DGAT Action Without a DGAT purified in active form, structurefunction 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 Liu Q et al. (2012) Prog Lipid Res 51: Weselake RJ et al. (2006) BMC Biochem 7: 24 31

32 Solubilization of Recombinant BnaC.DGAT1.a -dissolved in various detergents ,000 x g supernatant Western blotting or microsomal pellet BnaC.DGAT1.a pellet CHAPS DDM MEGA8 TX-100 P S P S P S P S 41% 68% 40% 55% Activity assay [Detergent]=1% P-pellet S-supernatant Western blot profile of pellet and supernatant following centrifugation of detergent-solubilized microsome at 105,000 x g 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) 32 32

33 Cobalt Ion Chelate Affinity Chromatography and Tag Removal Wash Elution m SF FT W1 W2 E1 E2 E3 E4 m -TEV +TEV kda kda BnaC.DGAT1.a-tag BnaC.DGAT1.a BnaC.DGAT1.a purification using immobilized cobalt ion affinity chromatography m, molecular mass SF, solubilized fraction FT, flow through [DDM]=0.1% BnaC.DGAT1.a before and after incubation with tobacco etch virus protease (TEV) 33

34 In Gel Trypsin Digestion and LC MS/MS Sequencing Sequence Modifications Charge MH+ [Da] SDSSnGLLPDSVTVSDADVR N5(Deamidated) GDLLYGVER ANPEVSYYVSLK ANLAGENEIR ESGGEAGGNVDVR LIIENLmK M7(Oxidation) ESPLSSDAIFK

35 mau Purified Active Recombinant BnaC.DGAT1.a Self-associates to Form Dimers and Tetramers 50 V o I II (11.87) Elution volume (ml) Superdex 200 size-exclusion chromatography (SEC) profile of BnaC.DGAT1.a m V o I II III V (10.46) BnaC.DGAT1.a III IV V VI kda SDS-PAGE of SEC fractions [DDM]=0.05%; m, molecular mass; V o, void volume 35 35

36 Purification of BnaC.DGAT1.a from a Three Liter Yeast Culture Fraction Volume (ml) Total activity (nmol TAG/min) Total protein (mg) Recovery (%) Specific activity (nmol TAG/min/mg protein) Purification (fold) Microsome Solubilized fraction (100)* (1.0)* ICAC (137)* (98)* Peak II of SEC (5)* (126)* *Recovery and purification fold relative to the solubilized fraction. 36 ICAC, immobilized cobalt ion affinity chromatography SEC, size-exclusion chromatography TAG, triacylglycerol

37 Acyl-CoA Specificity of Purified BnaC.DGAT1.a Specific activity (nmol TAG/mg/min) :0 18:0 18:1 18:2 18:3 Acyl-CoA substrate 37

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 38

39 Acknowledgements 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 39

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