Towards engineering the microalga Chlorella sorokiniana for the production of tailored high-value oils Xenia Spencer-Milnes UCL (University College London) A thesis submitted for the degree of Doctor of Philosophy (PhD) September 2018 1 DECLARATION DECLARATION I, Xenia Spencer-Milnes confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. …………………………………………………………………………………….. 2 ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS Firstly, I would like to thank my supervisor, Professor Saul Purton, for his continued support throughout these last four years and the opportunity to be part of such an interesting project. Also, many thanks go to my thesis committee and secondary supervisors: Dr Olga Sayanova for providing such expertise in the area of lipid metabolism and the opportunity to conduct some research at Rothamsted Research, Professor Kaila Srai for such constructive feedback, and Dr Vitor Pinheiro for providing invaluable support and advice through some difficult times. I would also like to thank all members of the Algal Oils by Design sLoLa group for continued stimulating discussion and inspiration at meetings. I would especially like to thank Dr Mary Hamilton and Dr Richard Smith from Rothamstead Research for their patience and support in teaching me new techniques and putting up with a myriad of questions. I also must thank former lab members Noreen Hiegle for showing me the ropes using Agrobacterium and Dr Sofie Vonlanthen who established much Chlorella work in the lab and was very quick to respond to my flurry of email questions in the beginning. Thanks is also due to the great bunch of people in the Purton lab who I have spent time with over the years and enjoyed getting up to mischief with at conferences and lab outings! Dr Rosie Young has been a great level head and provider of encouragement and advice during this process. Dr Laura Stoffels, Dr Priscilla Rajakumar, Dr Umaima Al Hoquani and Dr Alice Lui, Dr Max Blanshard and Dr Janet Waterhouse and Dr Fiona Li, thank you for all your help in the lab from knowing where things are to giving tips on protocols and solving little mis-haps along the way! To Saowalak Changko, Jing Cui and Lydia Mapstone, you’ve been great company and I wish you all the best with your continuing work. An enormous thanks is due to our lab manager Thushi Sivagnanam who keeps things running so smoothly and has so much patience for us. Dr Henry Taunt has been a valuable source of knowledge over the years and an excellent provider of suitable beverages. I would especially like to say a massive 3 ACKNOWLEDGEMENTS thank you to him for his incredible patience and encouragement during the final stages of the PhD. And to Juliana – we did it, woo! Thanks for being such a great partner in the lab and getting through the end together! The most important thanks must go to my parents Sally and John, and brother Zed, whose love and support I know is unconditional. Without them I would be at a loss. Finally, I would like to thank Andy for being a rock through this process, and my housemates Uther, Sophie, Lauren and Alex for being such wonderful company, especially after a hard day. 4 ABSTRACT ABSTRACT This project explores the production of LC-PUFAs, such as the nutritionally relevant omega-3 fatty acids EPA and DHA, in the freshwater microalga Chlorella sorokiniana (UTEX 1230). C. sorokiniana is of interest due to its rapid growth rate, tolerance to high light and temperature, and ability to accumulate a large proportion of cell weight as lipids. Since wild-type C. sorokiniana terminates fatty acid synthesis at ALA (C18:3n3), a genetic engineering approach is required to produce LC-PUFAs. Changes in FAME and lipid content in different growth conditions including carbon source, nitrogen source, and trophic state were assessed in this microalga. These investigations confirmed the common finding that nitrogen stress is a robust way to induce neutral lipid accumulation, but also highlighted the potential of pH change as an alternate stressor for TAG production. To facilitate the heterologous gene expression needed to increase the range of fatty-acids produced by C. sorokiniana, this work develops and begins to characterise a toolbox of genetic parts for nuclear transformation. This combinatorial parts library, compatible with a standard cloning syntax to enable sharing between groups, comprises five coding sequences, four promoter/5’UTRs and four 3’UTR/terminators. The coding sequences include two antibiotic reporters, two fluorescent reporters and one lipid gene. Among the regulatory sequences, two were novel putative promoters from chlorovirus, as identified from detailed bioinformatics analysis of published transcriptomic and genomic data. An attempt to improve an existing Agrobacterium-mediated transformation strategy was utilised to validate some of the parts within the library. Preliminary evidence suggests the putative chlorovirus promoters may be active within both C. sorokiniana and Chlamydomonas reinhardtii. Challenges with appropriate selectable markers and transformation efficiency highlight the difficulty in genetic engineering of microalgal strains, especially where these tools are not well established, but also showcase the pressing need for such foundational research to be conducted. 5 IMPACT STATEMENT IMPACT STATEMENT Microalgae are promising biotechnological platforms: lipid-rich biomass can be used for biofuels, and many species accumulate high-value products such as pigments and omega-3 oils, including EPA (20:5n-3) and DHA (22:6n-3), which are essential for human nutrition. However, there is often a trade-off in the production of these lipids, in that oil accumulation in the microalgae is stimulated when under stressful growth conditions such as nitrogen deprivation. The resulting decrease in growth rate and productivity can be a barrier to economic viability of industrial production. Chlorella sorokiniana UTEX 1230 is a small single-celled freshwater green alga of industrial relevance with regard to its high growth rate at warm temperatures and ability to accumulate lipids under optimised growth conditions. The wider aim of this project is to explore the potential for production of omega-3 fatty acids in C. sorokiniana. By working towards providing an alternative source of the nutritionally essential omega-3 oils EPA and DHA, the output of this project may contribute to the reduction of the unsustainable pressure on the fishing industries. Additionally, western diets are typically associated with consumption of too much omega-6 compared to omega-3, therefore additional sources of these oils may help increase the health of the population. The main outputs of this work relate to factors affecting lipid accumulation in C. sorokiniana, the development of a transformation methodology for this alga, and the creation of a library of shareable DNA parts for use in microalgae genetic engineering, including new putative regulatory sequences from chlorovirus genes. The study of lipid accumulation is important because it is relevant to improving process dynamics across several areas of the nascent algal biotech industry, including feed, food, and biofuels. Research conducted on the transformation methodology of C. sorokiniana informs and complements future efforts into this area. Finally, the parts library will allow for more rapid construction of algal expression cassettes in the Purton lab, and the use of a common syntax will encourage the sharing of parts across academic and industrial labs to facilitate future technological development. These 6 IMPACT STATEMENT findings will be useful for both microalgal biotechnology industries and further academic research into basic physiology of microalgae. 7 ABBREVIATIONS ABBREVIATIONS ACCase acetyl-CoA carboxylase ACP acyl carrier protein ALA Alpha-Linoleic-Acid (18:3n3) ARA Arachidonic acid (20:4n6) ATP adenosine 5’-triphosphate bp basepair CCAP Culture Collection of Algae and Protozoa cDNA complementary deoxyribonucleic acid CDS Coding Sequence CoA coenzyme A DAG diacylglycerol DGDG digalactosyl diacylglycerol DGTS Diacylglyceryltrimethylhomoserine DHA docosahexaenoic acid (22:6n3) DIG digoxigenin-dUTP DMSO diemthyl sulfoxide DNA deoxyribonucleic acid DNase deoxyribonuclease dNTP 2’deoxynucleoside 5’-triphosphate EDTA ethylenediaminetetraacetic acid (disodium salt) EPA eicosapentaenoic acid (20:5n3) FA fatty acid FAME fatty acid methyl ester FAS Fatty acid synthase GC gas chromatography GC-FID gas-chromatography-flame ionisation detection GC-MS gas chromatography-mass spectrometry GOI gene of interest GPAT glycerol-3-phosphate acyltransferase HPLC high performance liquid chromatography LA linoleic acid (18:2n6) LC-PUFA long-chain poly-unsaturated fatty-acid MGDG monogalactosyl diacylglycerol mRNA messenger ribonucleic acid MS mass spectrometry NAD(P)H Nicotinamide adenine dinucleotide phosphate nm nanometer NR nile red OD optical density PA phosphatidic acid PAT poly-A tail 8 ABBREVIATIONS PC phosphatidyl choline PCR polymerase chain reaction PDAT phospholipid diacylglycerol acyltransferase PE phosphatidylethanolamine PEG polyethylene glycol PG Phosphatidylglycerol PI phosphatidylinositol PSI, PSII photosystem I, photosystem II PUFA polyunsaturated fatty acid RACE Rapid amplification of cDNA ends RBCS ribulose-1,5-biphosphate carboxylase/oxygenase rDNA ribosomal deoxyribonucleic acid RNA ribonucleic acid RNase ribonuclease rRNA ribosomal ribonucleic acid