Recombinant protein production potential of South African microalgae by Julanie Stapelberg Submitted in partial fulfilment of the requirements for the degree of MAGISTER SCIENTIAE: PLANT BIOTECHNOLOGY In the Faculty of Natural and Agricultural Sciences University of Pretoria Pretoria May 2019 Supervisor: Dr Bridget Crampton Co-supervisors: Dr Robyn Roth and Dr Michael Crampton DECLARATION I, Julanie Stapelberg declare that the thesis, which I hereby submit for the degree of Magister Scientiae at the University of Pretoria, is my own work and has not previously been submitted by me for a degree at this or any other tertiary institution. SIGNATURE: DATE: Recombinant protein production potential of South African microalgae by Julanie Stapelberg Supervisor: Dr Bridget Crampton Co-supervisor: Dr Robyn Roth Co-supervisor: Dr Michael Crampton Department: Plant and Soil Sciences Degree: Magister Scientiae SUMMARY Therapeutic recombinant proteins, including complex glycoproteins, antibodies and other small molecules, have many current and future applications in biopharmaceuticals, biomaterials, nutraceuticals, agriculture, animal health and cosmeceutical industries (Rasala & Mayfield, 2015). Existing expression systems used to produce these proteins include mammalian, plant, insect and microbial culture systems, with each portraying their own strengths and weaknesses (Andersen & Krummen, 2002). For mammalian cell cultures, proteins, including those for human and animal function, can be manipulated and produced in their active form but the system is costly. Microbial systems are less expensive but may not have the necessary transcription, translation and post- translational tools to produce viable proteins without further alterations (Rasala & Mayfield, 2015). Therefore, the current expression systems available to produce recombinant proteins lack either in their ability to produce viable proteins, have low protein yields, and are not easily manipulated. Some of these systems also incur high a cost of processing. The benefit of a novel expression system, through algal culture, could overcome many challenges by offering low production costs, fast growth rates, easy culture, simple transgenic manipulation, and modified abilities of transcription and translation (Feng et al., 2014). The most advanced genetic work that has been done on green algae is that of Chlamydomonas reinhardtii. The nuclear, chloroplast and mitochondrial genomes of C. reinhardtii have been sequenced with optimised transformation vectors and protocols created for each (Merchant et al., 2007). The nuclear genome has already been engineered to successfully express an assortment of soluble proteins, enzymes or immunotoxins of therapeutic or industrial value. While C. reinhardtii is the model microalgae currently used in research, it may not be the best strain for large scale production of heterologous proteins (Rasala & Mayfield, 2015). Other microalgae strains might have faster growth rates and tolerate commercial culturing conditions better (Taunt et al., 2018). Microalgae represent a rich collection of species that offer numerous advantages for both biotechnology and biomedical industries (Doron et al., 2016). Many South African microalgae species have not been identified, let alone characterised for their biotechnology potential. The aim of this project was to establish and optimise the nuclear transformation protocols of C. reinhardtii within our current South African laboratory conditions. A previously successful vector system ii harbouring a C. reinhardtii (Cr) codon optimised green fluorescent protein (CrGFP) was used along with endogenous algal regulatory elements for an optimised molecular toolkit. South African microalgae isolated across the country were then cultured, screened and identified by genetic sequencing. From a database of over 500 indigenous microalgae isolates, nine isolates indicated potential as promising protein expression platforms. This project lays down the foundation for initiating a heterologous microalgae protein expression system within South Africa. iii PREFACE This Thesis is divided in four chapters as set out below: Chapter 1: Introduction to microalgae and their application as an expression system for recombinant protein production. This Chapter provides detail on green microalgae, on their structure, reproduction, habitats, environment and growth requirements. The role microalgae may play in the economy and the benefit of microalgal expression systems over current recombinant protein production platforms is discussed. Further details are then provided on the transformation methods, including nuclear and chloroplast transformation comparisons, and the molecular toolkit that has been explored for microalgal protein expression. Chapter 2: Transformation of two strains of the model green microalgae C. reinhardtii is described in this Chapter. A gene construct containing the C. reinhardtii codon optimised Crgfp transgene driven by the Hsp70-rbSc2 promoter with she ble antibiotic resistance gene as a selectable marker was electroporated into the nuclear genome. The CrGFP production was then quantified in live microalgae cells by fluorescence. Chapter 3: In this Chapter selected South African microalgae isolates, obtained from the microalgal culture collection of South Africa (MiCCSA), were subjected to growth curve analysis in comparison to C. reinhardtii. Growth conditions were on both mixotrophic and freshwater media with a variety of light intensities. The microalgae culturing conditions were also optimised. The South African microalgae with the fastest growth rates were identified based on compound microscopy and 18S rRNA Sanger sequencing. Chapter 4: This Chapter outlines the general conclusion of this study, proposes recommendations for future work, as well as exploring the broad potential of microalgae for South Africa’s bio-economy. Aspects of this study were presented at the following conferences: The 8th International Conference on Algal Biomass, Biofuels and Bioproducts. Seattle, USA. Stapelberg J, Roth RL, Crampton MC, Crampton BG. June 2018. Development of a heterologous expression platform in indigenous South African microalgae. (Poster Presentation) The 31st Congress of the Phycological Society of Southern Africa. Hartbeespoort, South Africa. Stapelberg J, Roth RL, Crampton MC, Crampton BG. July 2018. The recombinant protein production potential of South African microalgae. (Poster Presentation) South African Society for Bioinformatics Conference, Golden Gate, South Africa. Stapelberg J, Roth RL, Crampton MC, Crampton BG. October 2018. Why we should focus on microalgal biotechnology in South Africa. (Oral Presentation) South African Genetics Society Conference, Golden Gate, South Africa. Stapelberg J, Roth RL, Crampton MC, Crampton BG. October 2018. The identification, growth analysis and biotechnological potential of South African microalgae. (Poster Presentation) Chapter 3 has been written up as a journal article and will be submitted to a peer review journal for publication. iv ACKNOWLEDGEMENTS There are many people whom I would like to thank; this MSc was not simply a project for a degree but has taught me so many fundamental skills and has developed my passion for microalgae research. I truly believe that microalgae are invaluable to making our planet sustainable, with endless potential in several industries. (There is currently an entire building powered by algae in Germany with plans for an entire sustainable algae city in Sweden). I am therefore most grateful to everyone who allowed me the opportunity to pursue this passion. Thank you to my supervisor and role model, Dr Bridget Crampton, who knew I was interested in a microalgae protein expression and who opened the door to this MSc project. To Dr Michael Crampton as the initiator at the CSIR and to Dr Robyn Roth who was a constant support and who understood that I work in the laboratory at night and therefore granted me the freedom to experiment and learn from all my failures. To Monique Smit for tolerating the constant presence of my microalgae cultures in the Phycology room and for informing me when the laboratory moved locations. Thank you to the kind staff at CSIR who allowed me to use their equipment, and to all my colleagues, lab mates and my friends for the mutual motivation. Thank you to my funding bodies, the University of Pretoria and National Research Foundation for providing the resources and the CSIR for supporting project costs. A hearty thanks also to all the members, staff and friends at the Forestry and Agricultural Biotechnology Institute (FABI for allowing me to work there so that I may support my master’s studies and for allowing me to present my work- despite it being a somewhat off-topic from foci- Eucalyptus trees, avocadoes or fungi. Thank you especially to Dr Bernard Slippers for always challenging my love for microalgae and urging me to actively research its pros compared to fungi. A sincere thank you to the SANPCC community who allowed me the opportunity to attend their conference and get further inspired by the wonderful work South Africa is doing on algae. And finally, I owe a huge debt of gratitude to my family for their love and encouragement and continued support of my research passions. You mean more to me than you would ever know. Lastly to John Rogers, for his innate understanding, patience and reassurance. I am grateful for everyone on this journey, it has been an absolute privilege! v Table of Contents CHAPTER 1 1.1 General