Browsing by Subject "Saccharomyces cerevisiae"

Aspergillus niger is a filamentous fungus that is known for its ability to degrade plant biomass polysaccharides. A total of 86 sugar transporters have been identified in A. niger, but only 10 of them have been thoroughly characterized. Sugar transporter proteins are crucial for fungi as they enable efficient utilization of sugars in their metabolism and therefore breakdown of plant biomass. Additionally, sugar transporters can be used in various biotechnological applications. L-arabinose is a pentose sugar present in plant biomass and A. niger can utilize it through the pentose catabolic pathway (PCP). Recently, a sugar transporter LatA was identified from A. niger, capable of transporting the PCP intermediate product L-arabitol into fungal cells. L-arabitol is a polyol similar to xylitol and can be used as a low-calorie sweetener in food and beverage industries. Although A. niger LatA has previously been shown to be specific to L-arabitol in vivo, its in vitro functional activity has not yet been described. This study aimed to in vitro characterize two potential L-arabitol transporters from A. niger, LatA and unpublished 9364, using the yeast Saccharomyces cerevisiae. As a platform strain, we used S. cerevisiae IMK1010 that is devoid of all hexose and disaccharide transporters, as well as disaccharide hydrolases. In addition, we used a disaccharide-polyol and a pentose metabolic strain which were generated from the IMK1010 strain. The metabolic strains carried pathways for maltose, saccharose, sorbitol and mannitol, and xylose and arabinose, respectively. This provided a controlled research environment for studying A. niger LatA and 9364 transporters. The sugar specificity of the transporters was tested through two different growth experiments on solid media with all the strains and in liquid media with IMK1010 strains. The tested sugars included D-glucose, D-fructose and D-mannose hexoses, D-xylose and L-arabinose pentoses, maltose and sucrose disaccharides, and D-mannitol and D-sorbitol polyols. In addition, LatA was examined through a disappearance assay, measuring the loss of sugar from the liquid growth medium. Altogether four different combination gene constructs, green fluorescent protein (GFP) gene fusions and plain sugar transporter gene constructs were successfully engineered and 21 different transformant yeast strains produced for this study. GFP gene fusions, were in addition to growth experiments, used to study the localization of the sugar transporters to the cell membrane. In strains containing combination gene constructs encoding sugar transporters and GFP, the sugar transporters were successfully localized to the cell membrane, showing already that the transporters potentially have transport activity in the heterologous expression system. Based on the results, A. niger 9364 transported the tested hexoses and maltose in the growth experiments but did not transport tested pentoses, disaccharides or D-mannitol and D-sorbitol polyols. As expected, A. niger LatA did not transport any of the tested sugars, confirming its specificity to L-arabitol polyol. However, in the disappearance assay LatA unexpectedly did not transport L-arabitol. This might be due to the possible toxicity of the polyols in high concentrations to yeast cells and many of them also serve as regulators of osmotic pressure in cells, which may lower the transport capacity of the sugar transporters. In the future the function of the transporters can be tested in different sugar concentrations and pH in disappearance assay. Alternatively, a L-arabitol metabolic strain could be constructed to investigate sugar specificity using the growth experiment instead of the disappearance assay. The study provided new information of A. niger 9364 and further insights into the sugar specificity of A. niger LatA. These sugar transporters could be used in various biotechnological applications in the future.

New alternative feedstocks are needed for biofuel production to fulfil the growing demand in the coming years. The industry is moving away from second-generation biofuels, produced from food and feed crops, to using waste streams from industrial processes. An abundant, cheap and attractive waste stream for processing in Europe is the pectin-rich pulp from sugar beet processing and fruit juice industry. Sugar beet pulp is particularly rich in D-galacturonic acid and arabinose, but neither are naturally used by the yeast Saccharomyces cerevisiae, which would be an interesting candidate for the microbial fermentation of the biomass. S. cerevisiae is one of the most used organisms in the industrial biotechnology, and methods for the genetic engineering of the organism are highly developed. To overcome the natural limitations of the yeast for D-galacturonic acid fermentation, the metabolic pathways present in other organisms could be integrated in the yeast genome. Two bacterial and one fungal pathway are known to convert D-galacturonic acid into metabolites of the yeast glycolytic and ethanol fermentation pathways, and are thus considered promising for engineering in yeast. A major engineering challenge in integrating the fungal pathway in yeast is the redox imbalance caused by the two NADPH-specific reducing enzymes. The aim of this thesis was to review the potential of different D-galacturonic acid pathways for yeast fermentation. S. cerevisiae is a well-characterised organism for heterologous protein expression, but at times functional expression of foreign proteins is not achieved. One approach to study the pathways was to clone and express enzymes of the bacterial isomerase and dehydratase pathways in S. cerevisiae, and to test their activity in culture lysates. In addition, to overcome the redox imbalance in the eukaryotic pathway, two approaches were used to obtain an NADH-spesific D-galacturonic acid reductase. First, a mutant library of the Trichoderma reesei gar1 reductase was designed with the structure-guided cofactor specificity reversal tool CSR-SALAD. An automated high-throughput screening method for expression in Escherichia coli was developed, and the library was screened for enzymatic activity. The second approach was to try to identify the sequence for the characterised NADH-utilising reductase from the single-cell algae Euglena gracilis. A cDNA library of the algae was made and screened with PCR and in vivo methods. The reductase uxaB of the isomerase pathway and dehydrogenase kduD of the dehydratase pathway were functionally expressed in S. cerevisiae, with specific activities of 1.1 µmol min-1 mg-1 and 0.22 µmol min-1 mg-1 , respectively. The enzymes dehydratase uxaA and isomerase kduI did not exhibit activity in activity assays. The galurD of the dehydratase pathway was expressed in E. coli, and the purified enzyme was successfully used to convert D-galacturonate to 5-keto-4-deoxy galacturonate. The approaches to change the cofactor specificity of the NADPH-specific reductase of the eukaryotic pathway did not lead to a discovery of a NADH-specific enzyme. More research is needed for engineering active enzymes for S. cerevisiae expression and constructing a fully functional D-galacturonic acid pathway for feasible D-galacturonic acid fermentation.