Optimisation of Pichia pastoris bioprocess for recombinant thaumatin II production

Pichia pastoris, a methylotrophic yeast, is one of the most popular hosts used currently for the bioproduction of industrially relevant ingredients. An ingredient emerging in the market today is the sweet protein thaumatin, a natural sugar replacer. Being the only sweet protein approved within the European Union, this protein is predicted to have a potential market in the coming years. Conventionally, this protein is extracted from a tropical plant grown in Western Africa. To ensure a continuous supply and sustainable production of this protein, precision fermentation can be considered. However, due to the novelty of this protein as well as limited studies associated with its production, an optimised bioprocess utilising P. pastoris has not been developed yet. Therefore, the overall objective of this research is to identify the optimal conditions including media composition, supplement addition, pH, temperature, etc. to maximise the bioproduction of recombinant thaumatin from P. pastoris. To achieve this, the following sub-objectives were defined; (i) develop analytical methodologies for the determination of metabolites and substrates to facilitate P. pastoris bioprocess optimisation; (ii) identify optimal environmental conditions for the growth of the host organism and maximisation of the protein yield; (iii) understand the cellular metabolism of the host system on a macroscopic level by investigating its growth and substrate consumption behaviour; (iv) upscaling of P. pastoris fermentation from shake flasks to bioreactor scale to achieve maximal production yield of recombinant thaumatin. The upscaled fed-batch fermentation was further optimised and resulted in about 250 mg/L thaumatin, which was 6 times higher than the titre attained in shake flasks and 2.5 times higher than the highest titre reported in literature.