Stapelberg, Julanie (Rogers); (2024) Exploring thaumatin production in the algal chloroplast: towards a sweet and sustainable cellular factory. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Super-sweet proteins offer a range of potential health, economic and social benefits to society. The best characterised sweet protein is thaumatin extracted from the West African plant, Thaumatococcus daniellii. One hundred thousand times sweeter than sucrose it represents an attractive sweetener that would address dietary issues such as obesity, diabetes and dental caries. Commercial plant extraction of the native protein is not economically feasible and production of recombinant thaumatin in conventional expression systems is challenging since it requires the formation and folding of eight disulphide bonds. The chloroplast of the green microalga, Chlamydomonas reinhardtii, holds significant promise as a cellular factory for producing recombinant proteins. Furthermore, C. reinhardtii biomass is nutritional, has a high protein content and is Generally Recognised as Safe (GRAS). This means the whole cell may be used as a food ingredient, mitigating downstream costs. Employing synthetic biology tools together with a DNA library of interchangeable parts, a design strategy for thaumatin production was established. To identify factors optimising each stage of transgene expression, reporters were used to investigate the effect of gene copy number, plastome insertion site, and choice of cis-regulatory elements (promoters and 5’UTRs). The relative merits of targeting a disulphide-rich reporter protein either to the stroma or to the thylakoid lumen were also explored. Further features were engineered into the thaumatin strain including codon reassignment within the thaumatin gene for biocontainment of the transgene, and the incorporation of a phosphite oxidoreductase gene into the plastome for crop protection. The organoleptic properties were improved by employing a 'yellow-in-the dark' mutant strain, resulting in biomass free from chlorophyll when grown heterotrophically. The production of thaumatin under mixotrophic as well as heterotrophic conditions was compared. Finally, the bioprocessing of this "sweet Chlamy" strain was considered from a commercial perspective in order to produce a novel functional food ingredient.

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