Metabolic engineering plant seeds

Transcription

1 Metabolic engineering plant seeds with ihfish oil like lik levels l of DHA James Petrie CSIRO Food Futures Flagship 20 TH INTERNATIONAL SYMPOSIUM ON PLANT LIPIDS SEVILLE, JULY 2012

2 A partnership between CSIRO, Nuseed and GRDC. 20 TH INTERNATIONAL SYMPOSIUM ON PLANT LIPIDS SEVILLE, JULY 2012

3 Why DHA? The importance of omega 3 long chain chainpolyunsaturated fatty acids Omega 3 long chain ( C 20 ) polyunsaturated fatty acids have positive health effects. EPA (20:5ω3), DPA (22:5ω3) and DHA (22:6ω3) have important physiological roles. DHA is especially important for infant brain and retinal development and for aspects of cardiovascular health. Conversion of short chain to long chain omega 3 fatty acids, especially DHA, is relatively poor in humans. Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 3

4 Why DHA? Mechanisms are being uncovered. GPR120 is an omega 3 fatty acid receptor mediating potent anti inflammatory and insulinsensitizing effects. Da Young Oh et al. Cell 142, (2010). 0) Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 4

5 Why DHA? DHA Biosynthesis Seed DHA Levels Construct Design Summary Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 5

6 DHA Biosynthesis Pathways Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 6

7 DHA Biosynthesis Pathways Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 7

8 DHA Biosynthesis Isolation of an efficient DHA synthesis pathway Isolated numerous DHA synthesis genes Australian National Alga ae Culture Co ollection Tested multiple l pathways and pathway combinations: Δ9 elongase pathways Δ6 desaturase pathways Entire P. salina synthesis pathway Found an efficient combination of microalgal genes Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 8

9 DHA Biosynthesis Isolation of an efficient DHA synthesis pathway Australian National Algae Culture Collection Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 9

10 DHA Biosynthesis, ISPL 2010 An efficient microalgal Δ6 desaturase pathway with ω3 preference Demonstrated din Nicotiana i benthamiana leaf assay: Benth leaf TAG The challenge: achieve this profile in seed Required Δ12 and Δ15 desaturase activity to increase ALA Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 10

11 DHA Biosynthesis, ISPL 2010 An efficient microalgal Δ6 desaturase pathway with ω3 preference Demonstrated din Nicotiana i benthamiana leaf assay: Benth leaf TAG The challenge: achieve this profile in seed Required Δ12 and Δ15 desaturase activity to increase ALA Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 11

12 DHA Biosynthesis Isolation of an efficient DHA synthesis pathway Australian National Algae Culture Collection Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 12

13 DHA Biosynthesis Isolation of an efficient DHA synthesis pathway Australian National Algae Culture Collection Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 13

14 Fish oil like levels of DHA in seed and leaf Benth leaf TAG Arabidopsis seed Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 14

15 Why DHA? DHA Biosynthesis Seed DHA Levels Construct Design Summary Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 15

16 DHA Arabidopsis Seed Oil Events Arabidopsis: Dip T 0 plant Harvest T 1 seed T 1 plants T 2 seed T 2 plants T 3 seed Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 16

17 DHA Arabidopsis Seed Oil Events Arabidopsis: Dip T 0 plant Harvest T 1 seed T 1 plants T 2 seed T 2 plants T 3 seed Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 17

18 DHA Arabidopsis Seed Oil Events Arabidopsis: Dip T 0 plant Harvest T 1 seed T 1 plants T 2 seed T 2 plants T 3 seed Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 18

19 High/low DHA profiles are similar Presentation title Presenter name Page 19

20 DHA Seed Oil: T 4 Profile Ome ga 6 Om mega 3 Columbia T 4 Col_22.2 (weight % of TFA) OA 12.5% 3.3% LA 29.1% 4.3% ALA 18.3% 29.5% GLA 0.4% DGLA ARA 22:4ω6 DPA6 SDA 55% 5.5% ETA 0.8% EPA 1.8% DPA 1.5% DHA 15.1% Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 20

21 DHA Seed Oil: Pathway Conversions Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 21

22 DHA Seed Oil: Pathway Conversions Strong ALA production High 3: 6 = 11:1 including LA and ALA = 60:1 novel LC PUFA only Good interaction between desaturases and elongases Tri DHA detected by LC MS Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 22

23 DHA Seed Oil: Pathway Conversions Strong ALA production High 3: 6 = 11:1 including LA and ALA = 60:1 novel LC PUFA only Good interaction between desaturases and elongases Tri DHA detected by LC MS Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 23

24 DHA Seed Oil: Pathway Conversions Strong ALA production High 3: 6 = 11:1 including LA and ALA = 60:1 novel LC PUFA only Good interaction between desaturases and elongases Tri DHA detected by LC MS Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 24

25 DHA Seed Oil: Pathway Conversions Strong ALA production High 3: 6 = 11:1 including LA and ALA = 60:1 novel LC PUFA only Good interaction between desaturases and elongases Tri DHA detected by LC MS Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 25

26 DHA Seed Oil Characteristics DHA is enriched at the sn 1/3 positions of TAG: Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 26

27 Why DHA? DHA Biosynthesis Seed DHA Levels Construct Design Summary Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 27

28 Construct Design Lessons Learnt What didn t work well for us: Plant Methods Mthd 6:8 68(2010) Uni directional, multi promoter designs can be effective. Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 28

29 Construct Design Lessons Learnt What did work for us: However, a dual construct approach is undesirable. Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 29

30 Construct Design Extending the three gene theme to a single construct: FP1: B. napus truncated napin promoter FAE1: A. thaliana Fatty Acid Elongase 1 promoter Cnl1, Cnl2: L. usitatissimum conlinin1 and 2 promoters TMV transcriptional leader as 5 UTRs Still studying MAR effect it at least acts as a spacer. Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 30

31 DHA Seed Oil: Pathway Conversions Perfect? Of course not Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 31

32 DHA Seed Oil: Pathway Conversions Low 6 desaturation Perfect? Of course not Low 6 elongation Gene dosage effect Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 32

33 Construct Design: Improvements Several modified versions of the vector to increase DHA production. Expression levels differ: * ** ** ** *** ** ** Most modifications address/capitalise on expression differences Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 33

34 Why DHA? DHA Biosynthesis Seed DHA Levels Construct Design Summary Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 34

35 Summary Ahi Achieved dfish oil il like levels l of DHA in seed High 3: 6 = 11:1 with LA and ALA = 60:1 novel LC PUFA Good interaction between desaturases and elongases Learning how to build multi gene constructs, stable to at least T 5 DHA enriched at sn 1/3 Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 35

36 Summary Ahi Achieved dfish oil il like levels l of DHA in seed High 3: 6 = 11:1 with LA and ALA = 60:1 novel LC PUFA Good interaction between desaturases and elongases Learning how to build multi gene constructs, stable to at least T 5 DHA enriched at sn 1/3 Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 36

37 Summary Ahi Achieved dfish oil il like levels l of DHA in seed High 3: 6 = 11:1 with LA and ALA = 60:1 novel LC PUFA Good interaction between desaturases and elongases Learning how to build multi gene constructs, stable to at least T 5 DHA enriched at sn 1/3 Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 37

38 Summary Ahi Achieved dfish oil il like levels l of DHA in seed High 3: 6 = 11:1 with LA and ALA = 60:1 novel LC PUFA Good interaction between desaturases and elongases Learning how to build multi gene constructs, stable to at least T 5 DHA enriched at sn 1/3 Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 38

39 Summary Ahi Achieved dfish oil il like levels l of DHA in seed High 3: 6 = 11:1 with LA and ALA = 60:1 novel LC PUFA Good interaction between desaturases and elongases Learning how to build multi gene constructs, stable to at least T 5 DHA enriched at sn 1/3 Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 39

40 Summary Ahi Achieved dfish oil il like levels l of DHA in seed High 3: 6 = 11:1 with LA and ALA = 60:1 novel LC PUFA 1 Ha of canola at 12% will yield DHA from 10, fish Good interaction between desaturases and elongases Learning how to build multi gene constructs, stable to at least T 5 DHA enriched at sn 1/3 Metabolic engineering plant seeds with fish oil like levels of DHA James Petrie Slide 40

41 Surinder Singh Srinivas Belide Allan Green Dawar Hussain Yoko Kennedy Geraldine Lester Qing Liu Lina Ma Anne Mackenzie Peter Mansour Peter Nichols Nathalie Niesner James Petrie Pushkar Shrestha Lijun Tian Adam White Xue Rong Zhou Thank you CSIRO Food Futures Flagship James Petrie t e james.petrie@csiro.au 20 TH INTERNATIONAL SYMPOSIUM ON PLANT LIPIDS SEVILLE, JULY 2012