Genetic modification of microalgae for the sustainable production of high value products

Transcription

1 Rothamsted Research where knowledge grows Genetic modification of microalgae for the sustainable production of high value products Olga Sayanova 9 May 2016 Cambridge

2 Metabolic Engineering of Microalgae for Sustainable Production of omega-3 LC-PUFAs The problem There is a growing demand for fishmeal and fish oil Alternative lipid and protein sources are required Potential solution Microalgae as a replacement for fishmeal and fish oils in aquafeeds Sustainable solution to omega-3 LC-PUFAs and protein supply Challenges To find an algal strain with a high lipid content and a fast growth rate that is easy to harvest; A cost-effective cultivation system Engineering of lipid pathways Growth optimization Harvest A novel aquaculture feed EPA/DHA Proteins Pigments

3 P. tricornutum as a new platform for high-value lipids and proteins A unicellular diatom Accumulates 20 to 30% TAG Synthesises up to 30% EPA and traces of DHA Completed genome sequence Established transformation system Easy to cultivate

4 Targeting specific steps of lipid metabolism in P. tricornutum Schematic representation of fatty acid metabolism in P. tricornutum FAS 9-des PtFAD2 PTD6 18:0 18:1 18:2 LA 18:3 GLA Acyl-CoA pool C18:0 D9 C18:1 Galactolipids PLASTID C16:0 D9 C16:1 C16:3 D6 PtFAD6 C16:2 ω3/ 15 6-elo 20:5 5-elo 4-des SDA 20:4 1 2 X PTD5 22:5 22:6 DHA EPA 3 X 5 Endoplasmic Reticulum LysoPC LPCAT PE PS PI PG PC PHOSPHOLIPIDS 4 PDAT G3P LPA PA DAG TAG GPAT LPAAT PAP DAGAT K e n n e d y /OILS p a t h w a y 1,2 and 4: developing strategies to channel EPA into TAGs; 3: Generation of high levels of DHA; 5: Targeting DHA into TAGs

5 Generation of P.tricornutum strain with elevated levels of omega-3 DHA pptos2 fcpap OtElo5 fcpat MCS3 ble fcpap OtD6 fcpat ble 13.5% 10.2% 9.2%

6 Profiling of TAG molecular species produced in WT and transgenic Elo5 algae using ESI-MS/MS TAGs containing DHA Cells were grown at 20 o C and sampled during stationary phases In transgenic strain DHA was efficiently incorporated into TAGs

7 From 50 ml to >1500 L : A scale-up story 10L bubble columns 1500L Raceway Pond 50ml flasks 500L PBR Buttercup PML Plymouth Marine Laboratory

8 Transgenic algae: from small to large scale 21% ω-3 LC 28% ω-3 LC 26% ω-3 LC 36% ω-3 LC Hamilton et al., 2016 PloS,

9 Open system Photoautotrophic systems Closed system Heterotrophic Production of Omega-3 LC-PUFAs The requirement for light to increase cell densities and production is the main objective and a limiting factor of the cultivation Heterotrophic Light limitation can be overcome by the cultivation of microalgae under heterotrophic conditions, meaning supplementation of growth media with organic carbon to meet cellular energy requirements

10 Trophic Conversion of P. tricornutum by transformation with a gene encoding a glucose transporter Light EcoRV OtElo5 ppha-otelo5 +Pp_glu-1 -Light + Glu fcpbt fcpat SacI +Light + Glu -Light + Glu 15% 9% 5% Cells were grown, at 20 C, 100 µmol photons/m 2 /s Hamilton et al., 2016, Marine Drugs Alternative technologies for the production of ω-3 LC-PUFAs have been developed

11 Growing diatoms as feedstock for sustainable production of omega-3 LC-PUFAs We have engineered the diatom Phaeodactylum tricornutum to accumulate elevated levels of the high value omega-3 LC-PUFA DHA in photo- and heterotrophically grown strains. DHA was shown to accumulate in TAG, with levels remaining stable at different growth stages up to 1,500 l culture volumes. These studies show the opportunity to establish P. tricornutum as a synthetic biology chassis for enhancing LC-PUFAs production in microalgae

12 Future directions Total Lipid Extract Flexible Analytical MS Platform Acyl-CoA Betaine lipids Cutins Diacylglycerol Free Fatty Acids Galactolipids Triacylglycerol Phytosterols Phospholipids Data Processing Using the RRes lipidomic platform we will continue our study of the biosynthesis of valuable lipids in microalgae to provide further knowledge for manipulating pathways for industrial applications We will screen available biodiversity in order to identify novel algal strains for the production of ingredients and proteins for food and feed, and of chemicals for pharmaceutical, cosmetics and nutraceutical applications.

13 Rothamsted Research where knowledge grows PML Plymouth Marine Laboratory Mike Allen Jo Warwick-Dugdale Thank you! Algal oils: Mary Hamilton Richard Smith Richard Haslam Johnathan Napier PALM-UK Chloe Economou Alex Pudney