Braun Galleani, SC; (2014) Exploring the potential for recombinant protein production in microalgae. Doctoral thesis , UCL (University College London).

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Abstract

Microalgae are considered as attractive platforms for the synthesis of high-value heterologous proteins due to their many beneficial attributes including ease of cultivation, lack of pathogenic agents, and low-cost downstream processing. However, recombinant protein levels are low compared to microbial platforms and commercial production is not a reality yet. Promising research using the model microalga Chlamydomonas reinhardtii has highlighted this potential, particularly for transgene expression in the chloroplast. The objective of this research was to study different strategies for expression in the C. reinhardtii chloroplast aimed at increasing growth rate and recombinant protein production, by means of cell engineering and bioprocess optimisation. Two main approaches were considered for this purpose. Firstly, the insertion of the light-harvesting protein proteorhodopsin (PR) into the cell membrane, that has been reported to increase growth rate and culture lifespan in bacterial systems. This has resulted in PR accumulation to low yet detectable levels providing a moderate increase (12 %) of specific cell growth. Noteworthy, this is the first demonstration of an integral membrane recombinant protein expressed in the chloroplast. The second approach was to express a gene for a novel fluorescent protein (VFP) under the control of different regulatory elements. Detectable VFP levels were produced, with increased protein levels when using the psaA promoter/5’UTR element, and with co-expression of a gene for the Spy chaperone. These strains were used to study the effect of temperature, media and light intensity on recombinant protein production and cell growth. Protein levels and fluorescence allowed determining improved cultivation conditions as 30 °C under mixotrophic mode, and these conditions were tested for the accumulation of a therapeutic protein (Cpl-1 endolysin). In conclusion, protein productivity was observed to be protein-specific and improved conditions that increase protein levels for one protein cannot necessarily be extrapolated to the accumulation of a different protein.

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