title: Towards the Industrial Production of Omega-3 Long Chain Polyunsaturated Fatty Acids from a Genetically Modified Diatom Phaeodactylum tricornutum.
abstract: The marine diatom Phaeodactylum tricornutum can accumulate up to 30% of the omega-3 long chain polyunsaturated fatty acid (LC-PUFA) eicosapentaenoic acid (EPA) and, as such, is considered a good source for the industrial production of EPA. However, P. tricornutum does not naturally accumulate significant levels of the more valuable omega-3 LC-PUFA docosahexaenoic acid (DHA). Previously, we have engineered P. tricornutum to accumulate elevated levels of DHA and docosapentaenoic acid (DPA) by overexpressing heterologous genes encoding enzyme activities of the LC-PUFA biosynthetic pathway. Here, the transgenic strain Pt_Elo5 has been investigated for the scalable production of EPA and DHA. Studies have been performed at the laboratory scale on the cultures growing in up to 1 L flasks a 3.5 L bubble column, a 550 L closed photobioreactor and a 1250 L raceway pond with artificial illumination. Detailed studies were carried out on the effect of different media, carbon sources and illumination on omega-3 LC-PUFAs production by transgenic strain Pt_Elo5 and wild type P. tricornutum grown in 3.5 L bubble columns. The highest content of DHA (7.5% of total fatty acids, TFA) in transgenic strain was achieved in cultures grown in seawater salts, Instant Ocean (IO), supplemented with F/2 nutrients (F2N) under continuous light. After identifying the optimal conditions for omega-3 LC-PUFA accumulation in the small-scale experiments we compared EPA and DHA levels of the transgenic strain grown in a larger fence-style tubular photobioreactor and a raceway pond. We observed a significant production of DHA over EPA, generating an EPA/DPA/DHA profile of 8.7%/4.5%/12.3% of TFA in cells grown in a photobioreactor, equivalent to 6.4 μg/mg dry weight DHA in a mid-exponentially growing algal culture. Omega-3 LC-PUFAs production in a raceway pond at ambient temperature but supplemented with artificial illumination (110 μmol photons m-2s-1) on a 16:8h light:dark cycle, in natural seawater and F/2 nutrients was 24.8% EPA and 10.3% DHA. Transgenic strain grown in RP produced the highest levels of EPA (12.8%) incorporated in neutral lipids. However, the highest partitioning of DHA in neutral lipids was observed in cultures grown in PBR (7.1%). Our results clearly demonstrate the potential for the development of the transgenic Pt_Elo5 as a platform for the commercial production of EPA and DHA.
authors.affiliation: Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, AL5 2JQ, United Kingdom.
authors.forename: Mary L
authors.initials: ML
authors.lastname: Hamilton
authors.affiliation: Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, PL1 3DH, United Kingdom.
authors.forename: Joanna
authors.initials: J
authors.lastname: Warwick
authors.affiliation: Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, PL1 3DH, United Kingdom.
authors.forename: Anya
authors.initials: A
authors.lastname: Terry
authors.affiliation: Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, PL1 3DH, United Kingdom.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Allen
authors.affiliation: Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, AL5 2JQ, United Kingdom.
authors.forename: Johnathan A
authors.initials: JA
authors.lastname: Napier
authors.affiliation: Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, AL5 2JQ, United Kingdom.
authors.forename: Olga
authors.initials: O
authors.lastname: Sayanova
mesh_terms: D019149:Bioreactors; D053898:Biosynthetic Pathways / Q000235:genetics*; D001709:Biotechnology / Q000379:methods; D017377:Diatoms / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D004281:Docosahexaenoic Acids / Q000096:biosynthesis* / Q000302:isolation & purification; D015118:Eicosapentaenoic Acid / Q000096:biosynthesis* / Q000302:isolation & purification; D006801:Humans; D008027:Light; D050356:Lipid Metabolism / Q000235:genetics; D060847:Metabolic Engineering; D030781:Organisms, Genetically Modified; D017440:Photoperiod; D012492:Salts / Q000378:metabolism / Q000494:pharmacology; D012623:Seawater / Q000737:chemistry; D013696:Temperature
chemical_list: D012492:Salts; D004281:Docosahexaenoic Acids; D015118:Eicosapentaenoic Acid