Transient heterologous protein expression in green microalgae

Stevens, Dylan

Transient heterologous protein expression in green microalgae

Thesis / Dissertation

2025

Publisher

Universiy of Cape Town

Department

Department of Chemical Engineering

Faculty

Faculty of Engineering and the Built Environment

Abstract

Introduction: Recombinant proteins are produced using recombinant DNA technology and are widely used in numerous industrial applications, as well as innovative therapeutics. As the requirements of industries and pharma grow more complex chemically, traditional chemical engineering is, at times, limited by sets of synthetic chemical reactions currently available. This drives the need for applying novel chemical biology. When recombinant proteins are produced in a host organism other than the gene's original source, it is referred to as heterologous protein expression. The baculovirus insect cell culture, yeast, and plants, such as Nicotiana benthamiana, are only a few of the commercial expression systems where this technology has been extensively used. These systems, however, often have disadvantages in terms of their cost, safety and scalability. The potential of green microalgae as an innovative heterologous expression system for the production of recombinant proteins is examined in this thesis. To enable this, more specifically, the preparation of algal biomass of various axenic isolates, and their incorporation into a packed-bed matrix mediated by diatomaceous earth was investigated as an approach to be exploited for the expression of various heterologous proteins, using transient gene insertion rather than stable, nuclear gene integration (GMO). Microalgae is ideally suited for this as they are some of the most diverse, complex cells found in nature. Advantages of Green Microalgae: The various benefits of green microalgae become apparent when compared with conventional expression systems. These photosynthetic organisms grow quickly and can be cultivated in large numbers using very straightforward methods as well as simple inexpensive media compositions. Furthermore, unlike all other eukaryotic plants of higher order such as angiosperms, bryophytes and pteridophytes, green microalgae are able to shift their metabolism from light-mediated photosynthetic reduction of carbon dioxide to oxidative respiration and reduction of complex carbon (this includes glucose, acetate and glycerol) to fulfil their energy requirements for growth, reproduction and metabolism. Known as heterotrophy, this results in volumetric feeding of biomass rather than surface area based growth profiles, as is the case with light-dependent photosynthesis; with a resultant increase in algal growth rates as well as exponential increases in biomass yield. Green microalgae have been used to make a variety of recombinant proteins in the past. Chlamydomonas reinhardtii, for example, has been thoroughly characterised and investigated as a host of heterologous protein expression in both the chloroplast and within the cell nucleus. By developing a more ubiquitous approach to establishing transient expression systems in green microalgae, their metabolic diversity can be harnessed. Plant Expression Systems: Due to their affordability, scalability, and safety compared to conventional expression systems, plants are being investigated more and more as a source of heterologous protein production. While the potential of transgenesis has been studied for decades, Rhizobium radiobacter-mediated transient plant expression (formerly known as Agrobacterium tumefaciens-mediated plant expression before taxonomy revision), which involves the introduction of a recombinant plasmid into plant cells, is nowadays one of the most frequently used plant expression systems. In the present study, harnessing the single-celled nature of eukaryotic microalgae, as well as their superior biodiversity to fill the gaps left by plant based systems, R. radiobacter-mediated transient protein expression in green microalgae was investigated. Development of a Microalgal Library: In the development of a microalgal library, which is described in this thesis, microalgae were grown axenically. The library was screened for native isolate resistance to various antibiotic agents, as well as for media preference for growth rate maximisation and their ability to be grown axenically and withstand cryogenesis at -80 °C. Finally, applicable species were identified using 18s RNA sequencing. The collection is a useful tool for finding and selecting strains with high performance for heterologous protein production, with intention to further up scale for implementation as a feasible industrial platform for the expression of various heterologous protein products. Cell-Pack Column System Construction: A column system was conceptualised and built to enable intentional concentration of microalgae, their contacting with chemical and biological agents including transfection agents, growth, expression and product and biomass recovery or separation or both. Following design, it was used to investigate enhancing the growth, yield, and stability of green microalgae, which used photosynthetically growing biomass, but also looked at how this biomass could withstand the column environment which is light impermeable. Initially, the system development process looked at algal biomass harvest efficiency, formation of the packed bed or matrix-mediated algal cell pack within the column, and material selection, with a final investigation into up-scale of column formation. Thereafter a methodology was developed for the separation of an algal ecology by size as well as preliminary findings of the cell pack's ability to induce cryptic, latent viral sequences, harboured within the algal genome, into a state of cell lysis. Microalgal biomass was able to grow and reproduce within the column, with additional demonstration of the matrix-mediated column's ability to separate microalgal ecologies by size for further library investigation and development of novel microalgal libraries. This offers a useful production platform for large-scale operations, as well as a novel tool for the generation of axenic algal libraries from ecological sources. Investigation of Heterologous Protein Expression: Several heterologous proteins were selected for expression in the novel system using R. radiobacter-mediated transient expression in green microalgae through contacting in the matrix-mediated algal cell pack column. These included nptII stable expression with the plasmid vector pTRAkc ERH::rfp, β-glucuronidase (GUS) with the vector pCAMBIA1301, green fluorescent protein (GFP) with the vector pTRAc::eGFP, horseradish peroxidase (HRP) with the vectors pTRAc::HRPDC and pRIC::HRPDC, as well as HPV16 hL1, transient expression and virus-like particle (VLP) formation with the vector pTRAkc CTP rbcs1:: HPV16 hL1. All were proven to be expressible within the Matrix-mediated Algal cell pack. Conclusion: The power of dove-tailing various green microalgal isolates with their ideal transient expression vector, in a high throughput system was demonstrated by this study. In order to not only identify microalgal isolates amenable to industrial application, but in the case of heterologous protein expression, the vectors that allow for maximal protein expression yields, must rely on high throughput approaches, such as this novel diatomaceous earth-based column approach. This firstly allows for cheap media removal and biomass concentration. Secondly, it allows for identification of ideal isolate- vector pairing for the expression of recombinant proteins. The creation of both a novel microalgal library and the development of a diatomaceous earth-based column expression system, as well as their application to the R. radiobacter-mediated transient expression of a range of heterologous proteins in microalgae, are significant advances within biotechnology, demonstrating the potential to harness the synthetic biology of novel microalgae and heterologous expression through novel process engineering. These advances will open the door for the use of green microalgae as a vehicle for commercial expression. The findings of this study serve to demonstrate the value of a novel column-based expression system, which allows for discovery and experimentation of novel microalgae for their application in a column system for biomass harvesting without centrifugation and growth. Furthermore, the findings demonstrate the value of an effective contacting system for microalgae and transfection agents or other chemicals. The results presented in this thesis regarding use of the algal cell pack column for expression of various heterologous proteins and centrifuge-free biomass harvesting and as well as the column system itself will prove invaluable for the expression-development and production of recombinant proteins in the future using planktonic organisms, as well as delivering a research tool that allows for greater study of novel microalgae from different aquatic ecologies.