Recombinant protein expression in Komagataella pastoris and the scheduling of a multistage, multiproduct facility producing alternative sweeteners from A-molasses
Thesis (MEng)--Stellenbosch University, 2025.Mamba, P. E. 2025. Recombinant protein expression in Komagataella pastoris and the scheduling of a multistage, multiproduct facility producing alternative sweeteners from A-molasses. Unpublished masters thesis. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/111d8af1-ea00-44b1-add4-d5ea3af09b44The South African Sugar Industry (SASI) is financially distressed due to challenges such as subsided sugar prices in the global markets, increasing energy costs, and decreasing local sugar demand. The latter leaves the SASI with surplus sugar that is then exported to distorted global markets at discounted prices, resulting in revenue losses. Diversifying the product range of the SASI by co-producing bioproducts such as low-calorie sweeteners and commodity chemicals such lactic acid has gained traction as viable options to help revitalise the SASI. The low-calorie content and the generally regarded as safe (GRAS) status of alternative sweeteners such as allulose, short-chain fructooligosaccharides (scFOS), isomaltulose, and thaumatin have made them potential replacements for sucrose in food and beverages. A-molasses, an intermediate stream in sugar manufacturing has been identified as a potential feedstock for co-producing these sweeteners. The aim of this study was to recombinantly express thaumatin and D-psicose 3-epimerase (DPEase) enzyme used in allulose production in Komagataella pastoris, and design a multistage, multiproduct facility producing thaumatin, allulose, scFOS, and isomaltulose from A-molasses. This was followed by the economic viability assessment of the designed facility. A β-fructofuranosidase (FFase) enzyme-producing strain of K. pastoris was cultivated in an A-molasses derived fermentation medium to empirically elucidate the potential use of A-molasses in such applications. Herein, the FFase enzyme was used as a reporter protein.
Biomass titres of 136.4 gdcw/L and 137.9 gdcw/L were achieved from strains transformed with the native and codon-optimised thaumatin gene, respectively. Despite the successful growth resulting in high biomass, there was no evidence of recombinant thaumatin in the culture supernatants of both strains. Therefore, the strains were concluded to be incompetent in expressing recombinant thaumatin. Biomass titres of 137.7 gDCW/L were obtained from the K. pastoris strain expressing recombinant DPE enzyme under the GAP promoter. The DPE enzyme was successfully expressed but it was inactive and incompetent in converting D-fructose to D-allulose. Biomass titres of 125.05 gDCW/L were achieved when the K. pastoris strain expressing FFase enzyme was cultured in hydrolysed A-molasses medium. The highest FFase enzyme activity of 1784 U/ml was obtained after 55 hrs of fermentation. The satisfactory biomass titre and enzyme activity indicated A-molasses' ability to replace synthetic medium such as BSM with minimal effects on biomass and protein titres. Moreover, A-molasses cultures showed lower oxygen demand compared to BSM cultures of the same strain.
The inability to express thaumatin and functional DPE enzyme created limitations and uncertainty for the scheduling model and techno-economic analysis. Experimental values of the protein titre and enzyme activity were intended for use as model input data but were replaced with literature values considering the experimental challenges.
Following a mixed product campaign (MPC), the equipment sizing, batching, and scheduling of a multistage, multiproduct facility producing four sweeteners from A-molasses were successful. The developed schedule executed all the required batches within the stipulated time horizon of 5000 hours. The determined equipment sizes were deemed acceptable and were within design limits, with the highest processing rate of 60.1 tons/hr obtained for the simulated moving bed chromatography. Deterministic techno-economic analysis using an internal discount rate of 20% yielded an internal rate of return (IRR) of 52% with a payback period of 3.08 years. The high IRR obtained indicated that the multiproduct facility is economically profitable.Master
