Browsing by Author "Conacher, Cleo Gertrud"

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    Constitutive expression of enzymes in Pichia pastoris for use in lignin valorisation
    (Stellenbosch : Stellenbosch University, 2018-03) Conacher, Cleo Gertrud; Gorgens, Johann F.; Garcia-Aparicio, Maria P.; Stellenbosch University. Faculty of Engineering. Dept. of Process Engineering.
    ENGLISH SUMMARY: Lignin valorisation is a key breakthrough in developing efficient integrated biorefineries. Enzymatic modification of lignin wastes offers an environmentally benign, energy efficient avenue for lignin upgrading. However, for enzymatic lignin valorisation to be a viable option, large quantities of the selected enzymes are required at minimal cost. Heterologous enzyme production using recombinant P. pastoris is a well-studied method for the successful large-scale production of recombinant eukaryotic proteins. Considering the disadvantages associated with methanol-induction, the use of the constitutive PGAP promoter for heterologous protein production in P. pastoris is the preferred expression strategy. Three enzymes of interest for lignin degradation and modification were selected in this study for recombinant enzyme production: glucuronoyl esterase (GE), cellobiose dehydrogenase (CDH) and laccase (LCC). These enzymes were selected based on novelty and their broad range of functions in the lignocellulose biodegradation process. These enzymes are representative of carbohydrate degradation (GE, CDH: Fenton reaction) and lignin degradation and/or modification (LCC). The aim was to create, and provide fundamental data of, the capabilities of a constitutive P. pastoris expression system to produce three enzymes (CDH, LCC, and GE), that are associated with lignin valorisation. This study is rooted in aspects of molecular biology as well as bioprocess engineering. The DNA 2.0 (USA) pJexpress expression system, which is free of any proprietary restrictions, was used to create the expression constructs. The created recombinant P. pastoris strains were screened at shake-flask level, and based on these results, one strain for each enzyme was selected for further studies. The enzyme production process was scaled up to a 14 L bioreactor. A two-stage fermentation strategy, consisting of a batch phase, followed by a constant glycerol fed-batch stage was implemented. The fermentation culture was harvested and concentrated through a two-stage tangential filtration process, after which freezing was evaluated as a possible storage strategy. Glycerol was evaluated as a possible cryoprotectant. The constant glycerol feed strategy was shown to be effective, returning high biomass and protein yields. Results of the bioreactor fermentations showed similar biomass growth kinetics (μmax = 0.15 – 0.17 h-1) and biomass yields (119.54-136.47 gdcw/L) throughout the fermentation process for the three recombinant enzymes. High titres of recombinant protein were obtained, with the highest being glucuronoyl esterase, at 2778.01 mg/L, followed by cellobiose dehydrogenase, at 1489.3 mg/L, and lastly laccase, at 778.54 mg/L. The first incidence of constitutive expression of H. jecorina glucuronoyl esterase and N. crassa cellobiose dehydrogenase is reported here. In addition, the highest yield of constitutively expressed T. versicolor laccase lcc2 is also reported. Although the laccase fermentation returned the lowest productivity, preliminary experiments showed that lowering the fermentation temperature may improve this value by aiding in secretion and/or preventing temperature-related laccase degradation at 30 °C. No glycerol was accumulated during the glycerol fed-batch stage. A decline in growth rate was observed during the fed-batch stage due to constant feeding rate during biomass growth. However, since constitutive expression is largely growth associated, it may be beneficial to increase the glycerol feed rate in order to maintain the growth rate at values nearer to the maximum growth rate of the recombinant strains (0.15 – 0.17 h-1). Tangential flow filtration was successfully used to concentrate the cell-free enzyme extracts, where 5.20 – 6.01-fold increases in volumetric activity were obtained, with final volumetric activity yields of 49.57-60.04 %. It was found that glucuronoyl esterase was the most sensitive to volumetric activity loss after refrigeration and freezing, with a decrease of 10.71 % in volumetric activity after freezing at -20 °C. Cellobiose dehydrogenase showed no sensitivity to the freezing process, and laccase activity was successfully preserved with the use of 10 % glycerol as a cryoprotectant. This study has successfully reported the baseline capabilities of a constitutive P. pastoris expression system to produce three enzymes, namely cellobiose dehydrogenase, laccase, and glucuronoyl esterase, associated with lignin valorisation, using a bioreactor fermentation bioprocess. Even though an identical molecular and bioprocess strategy was used, there were significant differences in protein secretion of each enzyme, emphasising the effect of the selected gene on secretion levels in P. pastoris. The yields obtained in the current study may be improved at the microbiological level by improving secretion with the use of alternative secretion factors or gene coding sequences. The bioprocess may be improved by optimisation of the glycerol feed, to maintain the growth rate at a value nearer to the maximum growth rate of each strain. Further, preliminary data suggests that lowering the fermentation temperature may be beneficial for laccase production.
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    Exploring multispecies interactions between wine-associated yeasts
    (Stellenbosch : Stellenbosch University, 2021-12) Conacher, Cleo Gertrud; Bauer, Florian; Rossouw, Debra; Blassoples-Naidoo, Rene; Stellenbosch University. Faculty of AgriSciences. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: The fermentation of grape must to wine is catalysed by a diverse microbial community. Yeast are primary drivers of the associated alcoholic fermentation process and have therefore garnered considerable research interest. The diversity of yeast species present during wine fermentation influences the chemical composition and related sensory properties of wine as a result of the metabolic functioning of particular yeast species in response to abiotic and biotic factors. The latter is a relatively new research field, given that microbiological science has a significant monoculture bias, and as such, there is much still to be understood about the role and mechanisms of biotic stress in wine yeast ecosystems. Moreover, while the wine yeast ecosystem was the model used in this study, there are several other yeast ecosystems of biotechnological importance, including in biofuels production, bioremediation and other food and beverage industries, that would benefit from insight into these biotic stress mechanisms. The current basis of our understanding of the molecular mechanisms of yeast interactions in the wine ecosystem is based on two-species pairings, which keeps the system interaction network uncomplicated. However, there are many more role-players in natural ecosystems, and they do not interact in a linear fashion. At the micro- and macroscopic level, the importance of these often overlooked higher-order interactions has been highlighted in other ecosystems. There is very little information on higher-order interactions in the yeast ecology field, and this must be remedied for predictive understanding of these systems. Here, we sought to address the current status quo in multispecies yeast research, by aiming to develop new tools to investigate the mechanistic basis of interaction in systems comprised of more than two species. Furthermore, the study aimed to generate a greater depth of understanding of these systems, by investigating transcriptional responses of Saccharomyces cerevisiae to co-culture in mixed-species cultures of increasing complexity. Firstly, these aims were achieved by developing a fluorescence-based multi-colour flow cytometric method for tracking of a consortium consisting of wine-associated yeast species. This involved optimizing the genetic modification of the selected environmentally isolated yeast species, followed by extensive validation to confirm the representativeness of the system as well as development of the flow cytometric protocol. This was followed by addressing the pertinent issue of reproducibility in multispecies cultures, and showing the role of the physiological state of pre-cultures in determining their growth performance in three-species and four-species consortia. Finally, to contribute to our understanding of the molecular mechanisms of interaction in non-linear yeast systems, we showed that Saccharomyces cerevisiae expresses a combination of known pair-wise as well as unique genes when grown in a three-species system. By using interactive network visualizations of the generated transcriptomic data, we were able to functionally characterize the cellular responses in more detail than has been done before in similar studies.