MPS 587 - Advanced Plant Biochemistry Course Fall Semester 2011 Lecture 11 Lipids III 9. Triacylglycerol synthesis 10. Engineering triacylglycerol fatty acid composition
Today s topics on the Arabidopsis node map 2
Tissue and function of triacylglycerol accumulation in plants 1. Seed, storage oil energy for germinating seedlings 1. Soybeans 2. Brassica napus (Canola) 2. Fruit, animal attractant? 1. Avocado mesocarp 2. Palm fruit 3. Leaf, intermediate in lipid breakdown during senescence 4. Anthers and pollen, may be used for energy in pollen tube growth 3
Lipids in oil bodies (oleosomes) Contain s. Surrounded by protein-rich membrane; size 1-2 um. ER contains biosynthetic enzymes; oil bodies are spun off from ER microdomains. Oleosins: 15-25 kda proteins; conserved hydrophobic core; amphipathic flanking sequences; present only in seed and pollen. 4
Plant triacylglycerol biosynthesis overview LIPID ASSEMBLY Pyruvate Acetyl-CoA ACCase Malonyl-CoA FAS Acyl-ACP (16:0;18:0) Desaturase Acyl-ACP (18:1) FA SYNTHESIS Thioesterase FA ENDOPLASMIC RETICULUM G3P GPAT LPA LPAT LACS PA Acyl-CoA PAP DGAT PDAT LPC ACYL EDITING PC PLASTID FA MODIFICATION Lipid linked desaturases Acyl-CoA elongases Oil body 5
Enzymes of triacylgycerol () synthesis Acyl-CoA : Diacylglycerol Acyltransferase (DGAT) 1,2- OH Acyl-CoA CoA Three classes of plant DGAT DGAT1 and DGAT2 are found in many eukaryotes and greatly differ in structure. DGAT3 is soluble and has only been characterized in peanut seeds. (Li-Beisson Y et. al. (2010) Acyl-Lipid Metabolism. The Arabidopsis Book 8:e0133. doi:10.1199/tab.0133) Phospholipid : Diacylglycerol Acyltransferase (PDAT) 1,2- OH Phospholipid P-Headgroup + + 2-lyso-phospholipid OH P-Headgroup Identified in plants and yeast, PC appears to be the preferred substrate in plants while PE is the preferred substrate in yeast Diacylglycerol Transacylase 1,2- OH 1,2-2-monoacylglycerol + OH + OH OH (Dahlqvist et al. (2000) Proc. Natl. Acad. Sci. USA 97: 6487 ) Enzymatic activity demonstrated in safflower seeds. The enzyme has yet to be isolated. (Stobart et al. (1997) Planta 203: 58) 6
DGAT1 and DGAT2 have different structures and may not have overlapping functions Schematic representations of murine DGAT1 and DGAT2 proteins. Major DGAT classes involved in synthesis in plants: Arabidopsis: DGAT1 Brassica napus: DGAT1 Castor bean: DGAT2 Tung Tree: DGAT2 DGAT1 and DGAT2 localize to separate subdomains of the ER (Yen et al. (2008) Journal of Lipid Research 49: 2283) 7
DGAT1 and PDAT1 are the only confirmed acyltransferases known in Arabidopsis synthesis LIPID ASSEMBLY Pyruvate Acetyl-CoA ACCase Malonyl-CoA FAS Acyl-ACP (16:0;18:0) Desaturase Acyl-ACP (18:1) FA SYNTHESIS Thioesterase FA ENDOPLASMIC RETICULUM G3P GPAT LPA LPAT LACS PA Acyl-CoA PAP DGAT PDAT LPC ACYL EDITING PC PLASTID FA MODIFICATION Lipid linked desaturases Acyl-CoA elongases Oil body 8
Determination of the enzymes responsible for triacylglycerol synthesis in Arabidopsis DGAT1 mutant has ~20% reduction in seed (Katavic et al. (1995) Plant Physiology 108: 399) DGAT2 cannot complement yeast DGAT knockouts as DGAT1 does http://arabidopsisacyllipids.plantbiology.msu.edu/enzymes/105 PDAT1 mutant has no effect on amounts of seed (Mhaske et al. (2005) Plant Physiology and Biochemistry 43: 413) DGAT1/PDAT1 double mutant is pollen lethal dgat1/dgat1 + PDAT1 RNAi or pdat1/pdat1 + DGAT1 RNAi has 70-80% reduction in seed oil content (Zhang et al. (2009) The Plant Cell 21: 3885) 9
The source of for synthesis may differ between plants and effect the fatty acid composition LIPID ASSEMBLY Pyruvate Acetyl-CoA ACCase Malonyl-CoA FAS Acyl-ACP (16:0;18:0) Desaturase Acyl-ACP (18:1) FA SYNTHESIS Thioesterase FA ENDOPLASMIC RETICULUM G3P GPAT LPA LPAT LACS PA Acyl-CoA PAP DGAT PDAT LPC ACYL EDITING PC PLASTID FA MODIFICATION Lipid linked desaturases Acyl-CoA elongases Oil body 10
source can affect composition desaturation, hydroxylation, acyl editing, etc. PC Cho P PC derived DGAT PDAT G3P LPA PA de novo P GPAT P LPAAT P PAP DGAT PDAT Acyl-CoA pool 11
Reduced oleate desaturation mutnat (rod1) encodes an enzyme that affects the flux of through PC into (Lu et al. (2009) Proceedings of the National Academy of Sciences in the USA 106: 18837) Phosphatidylcholine : Diacylglycerol CholinephosphoTransferase (PDCT) 1,2- PC Cho P PC Cho P + + 1,2-12
Determine relative flux through competing biosynthesis pathways with [ 14 C]glycerol labeling [ 14 C]glycerol PC Cho P PDCT rcpt PLC PLD/PAP PC derived DGAT PDAT CPT PDCT G3P LPA PA de novo P GPAT P LPAAT P PAP DGAT PDAT 13
DPM/ total lipid FAME Flux of glycerol through synthesis in WT Arabidopsis (Bates and Browse (2011) The Plant Journal, in press) [ 14 C]glycerol PC Cho P PDCT rcpt PLC PLD/PAP PC derived DGAT PDAT CPT PDCT G3P LPA PA de novo P GPAT P LPAAT P PAP DGAT PDAT 35 30 25 PC 20 15 10 5 14 0 0 5 10 15 20 25 30 35
DPM/ total lipid FAME Flux of glycerol through synthesis in WT Arabidopsis (Bates and Browse (2011) The Plant Journal, in press) [ 14 C]glycerol PC 14x CPT Cho P PDCT PDCT rcpt PLC PLD/PAP PC derived DGAT PDAT G3P LPA PA de novo P GPAT P LPAAT P PAP DGAT PDAT 1x 35 30 25 20 15 PC >90% of de novo fluxes through PC prior to synthesis 10 5 15 0 0 5 10 15 20 25 30 35
Flux of through PC for synthesis may depend on plant species Plant Use of PCderived Non-membrane lipid FA in Seed Oil content (weight %) Soybean yes no ~20% (Bates et al. (2009) Plant Physiology 150: 55) Arabidopsis yes ~20% ~37% (Bates & Browse (2011) The Plant Journal, in press) Castor bean No ~90% ~50% (Bafor et al. (1991) Biochemical Journal 280: 507) 16
Percent fatty acid Percent fatty acid Pcecent fatty acid Percent fatty acid World vegetable oil production: Four major crops (http://lipidlibrary.aocs.org/market/fourmain.htm) (http://www.scientificpsychic.com/fitness/fattyacids1.html) 17 70 60 50 40 30 20 10 0 70 60 50 40 30 20 10 0 70 60 50 40 30 20 10 0 70 60 50 40 30 20 10 0 Palm 16:0 18:0 18:1 18:2(n-6) 18:3(n-3) Soybean 16:0 18:0 18:1 18:2(n-6) 18:3(n-3) Canola 16:0 18:0 18:1 18:2(n-6) 18:3(n-3) Sunflower 16:0 18:0 18:1 18:2(n-6) 18:3(n-3)
Vegetable oil consumption (http://www.soystats.com/) World vegetable oil consumption 2010 146.9 Million Metric Tons U.S. Fats & Oils Edible Consumption 2010 9.64 Million Metric Tons U.S. Soybean Oil Consumption 2010 7.54 Million Metric Tons ~ US petroleum based Fuel consumption 2009 770 Million Metric Tons 18
Vegetable oil can replace petroleum for the chemical industry http://oilreset.com/blog/?p=159 19
Uses of vegetable oil and reasons for engineering fatty acid content Food oils, Healthy oils Engineering Strategies Low in saturated fatty acids High in ω-3 polyunsaturated fatty acids Fish type oils EPA and DHA Good cooking properties Low polyunsaturated fatty acids High in 18:1 Adjust proportions of endogenous fatty acids in crop plants Introduce new fatty acids into crop plants Industrial applications Medium-chain fatty acids (12:0): soaps, detergents, surfactants Long-chain (22:1): Lubricants, slip agents Epoxy fatty acids: Plasticizers, coatings, paints, Hydroxy fatty acids: Lubricants, polymers 20
Breeding changed rapeseed (Brassica napus) into Canola CANOLA = CANadian Oil Low Acid Natural isolates of a β ketoacyl-coa synthase used to remove Erucic acid (22:1) content LEAR Low Erucic Acid Rapeseed HEAR High Erucic Acid Rapeseed 21
New GM soybeans with improved cooking oil properties from Monsanto about to hit the market https://www.vistivegold.com/pages/about.aspx Higher oxidative stability Reduced polymerization during frying No need for hydrogenation Therefore no trans-fats Less saturated fat Vistive Gold soybeans have advanced to Phase IV (pre-launch), the final step within the R&D pipeline. Monsanto has recently completed submissions to the USDA and FDA in support of the trait. The U.S. Food and Drug Administration has issued a positive response letter to Monsanto s Generally Recognized as Safe notification. The FDA s letter supports the use of Vistive Gold under its intended uses. 22
Production of low saturate high oleic soybeans by Monsanto LIPID ASSEMBLY Pyruvate Acetyl-CoA ACCase Malonyl-CoA FAS Acyl-ACP (16:0;18:0) Desaturase FATb Acyl-ACP (18:1) FA SYNTHESIS Thioesterase FA Conventional breeding FAD3 mutant FAD3: desaturase 18:2 to 18:3 RNAi FAD2 FAD2: 18:1 to 18:2 Only wanted a semi KD therefore specifically targeted 2 of 4 FAD2 isoforms RNAi FATb FATb: 16:0-ACP thioesterase Reduces 16:0 and 18:0 fatty acid release from fatty acid synthesis, and promotes production of more 18:1 ENDOPLASMIC RETICULUM G3P GPAT LPA LPAT LACS PA Acyl-CoA PAP DGAT PDAT LPC ACYL EDITING PC 23 FA MODIFICATION Lipid linked desaturases 18:1-PC FAD2 18:2-PC FAD3 Oil body 18:3-PC
Industrially useful fatty acids are found throughout the plant kingdom but not always in suitable crop plants Ricinus communis (Castor bean) Vernonia galamensis (Iornweed) Fatty acid Ricinoleic acid Vernolic acid % unusual FA in oil ~90% Agronomic features ~50% oil by weight in seeds Highly toxic co-product Ricin Grows in the subtropics Uses Laxatives Chemotherapy carrier Lubricants Polymers Fuel additives ~80% ~40% oil by weight in seeds only grows near the equator Plastics Adhesives Paints Vernicia fordii (Tung tree) Eleostearic acid ~82% 24 Slow growing tree Only grows in the subtropics Susceptible to hurricanes and condominium plantations Drying oils Varnishes Paints
Engineering of industrial oils in Arabidopsis: Castor oil a case study Ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid) Phosphatidylcholine (PC) Cho P RcFAH12 Cho P ~ 90% ricinoleic acid RcFAH12 expressed in develoing Arabidopsis seeds, ~17% hydroxy fatty acids (HFA) mostly at sn-2 (Broun and Somerville (1997) Plant Physiology 113: 922) (Smith et al. (2003) Planta 217: 507) Lu et al. (2006) The Plant Journal 45: 847) 25
HFA specific biosynthetic increase accumulation of HFA in Arabidopsis and gave clues on HFA metabolism in Arabidopsis (Burgal et al. (2008) Plant Biotechnology Journal 6: 819) (van Erp et al. (2011) Plant Physiology 155: 683) LIPID ASSEMBLY Pyruvate Acetyl-CoA ACCase Malonyl-CoA FAS Acyl-ACP (16:0;18:0) Desaturase Acyl-ACP (18:1) FA SYNTHESIS Co-expression of RcFAH12 with Castor DGAT or PDAT increased seed HFA content from 17% to 25-28% Thioesterase Both RcDGAT and RcPDAT lower the amount of HFA that accumulate in PC FA GPAT G3P LPA LPAT LACS PA Acyl-CoA PAP RcDGAT RcPDAT LPC ACYL EDITING PC-HFA RcFAH12 Hydryoxylation of sn-2 PC Oil body HFA still mostly at sn-2 + sn-3 26
Flux analysis to determine bottlenecks in HFA synthesis (Bates and Browse (2011) The Plant Journal, in press) 1. RcFAH12 produces sn-2 HFA PC which can be converted to sn-2 HFA 2. HFA can also be utilized for de novo synthesis 1. HFA-de novo is turned over 3. In efficient utilization of HFA leads to inhibition of FA synthesis and reduced oil 4. HFA specific sn-3 acyltransferases allow HFA- accumulation RcFAH12 sn-2 hydroxylation PC Cho P PDCT rcpt PLC PLD/PAP PC derived HFA- DGAT PDAT [ 14 C]glycerol CPT PDCT G3P LPA PA de novo HFA- P GPAT P LPAAT P PAP HFA-CoA 27 F.A.S.
Future HFA engineering strategies 1. Inhibit enzymes that turnover HFA-containing de novo 2. Allow de novo to flux directly to RcFAH12 sn-2 hydroxylation PC Cho P PDCT rcpt PLC PLD/PAP PC derived HFA- DGAT PDAT CPT PDCT G3P LPA PA de novo HFA- P GPAT P LPAAT P PAP HFA-CoA 28