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Sweet protein thaumatin, a promising alternative to table sugar, can overcome several drawbacks imposed by other sugar alternatives in the market today. Recombinant technology involving Komagataella pha#ii (previously known as Pichia pastoris) has the potential to facilitate a uniform and large-scale production of this protein. To ensure maximal yield from the bioprocess, this study performed a screening to identify the media composition that exhibits the highest secretion yield of recombinant thaumatin n and favourable growth of the host organism. To achieve this, the most recommended media formulations for K. pham namely, BSM, FM22, MGY, BMGY, MBSM and d'Anjou media were investigated. It was identified from this study that the highest yields of recombinant thaumatin II were achieved from BMGY, FM22 and BSM media, irrespective of not exhibiting the highest cell growth. Despite exhibiting a lower cell density than FM22, the BMGY medium resulted in 62.79 mg/L of recombinant thaumatin II, which is 1-2 times higher than the former. This observation facilitated an understanding that the optimal medium for cell growth is not necessarily optimal for recombinant protein production. Moreover, a comparison between baffled and non-baffled flasks demonstrated that the former leads to a higher secretion yield, thereby indicating the importance of sufficient oxygen transfer into the medium. Furthermore, casamino acids and vitamins were supplemented to the media, where the latter contributed to an increased yield of thaumatin. As such, the highest protein yield of 68.60 mg/L was achieved for BMGY supplemented with vitamins. [ABSTRACT FROM AUTHOR]

The manufacturing of recombinant proteins is increasing phenomenally, and yeasts serve as one of the most popular platforms for the large-scale production of these industrially relevant products. Protein analysis is a traditional step for the control and optimisation of such bioprocesses and often necessitates the combination of laborious sample treatments and expensive instruments. A hassle-free and cost-effective approach is always preferred for routine analyses and instigates a need for replacing these laborious tasks. In this study, a fast, simple, and reliable RP-HPLC method for the determination of the upcoming sweetener in the market, the sweet protein thaumatin was established. There is currently no reference method available for the quantification of this protein in fermentation samples. Due to an increasing industrial relevance foreseen for this sweetener, Pichia pastoris was used as a host for its bioproduction. The developed analytical method does not require a pre-treatment step of the biological sample and enables an easy quantification using a conventionally available HPLC system. The method facilitates the identfication and quantification of recombinant thaumatin n and was validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines(ICH, 2021). The developed technique enables the monitoring of thaumatin levels in fermentations, thereby facilitating bioprocess control and optimisation. [ABSTRACT FROM AUTHOR]