Catabolism of biomass-derived sugars in fungi and metabolic engineering as a tool for organic acid production: Dissertation

The use of metabolic engineering as a tool for production of biochemicals and biofuels requires profound understanding of cell metabolism. The pathways for the most abundant and most important hexoses have already been studied quite extensively but it is also important to get a more complete picture of sugar catabolism. In this thesis, catabolic pathways of L-rhamnose and D-galactose were studied in fungi. Both of these hexoses are present in plant biomass, such as in hemicellulose and pectin. Galactoglucomannan, a type of hemicellulose that is especially rich in softwood, is an abundant source of D-galactose. As biotechnology is moving from the usage of edible and easily metabolisable carbon sources towards the increased use of lignocellulosic biomass, it is important to understand how the different sugars can be efficiently turned into valuable biobased products. Identification of the first fungal L-rhamnose 1-dehydrogenase gene, which codes for the first enzyme of the fungal catabolic L-rhamnose pathway, showed that the protein belongs to a protein family of short-chain alcohol dehydrogenases. Sugar dehydrogenases oxidising a sugar to a sugar acid are not very common in fungi and thus the identification of the L-rhamnose dehydrogenase gene provides more understanding of oxidative sugar catabolism in eukaryotic microbes. Further studies characterising the L-rhamnose cluster in the yeast Scheffersomyces stipitis including the expression of the L-rhamnonate dehydratase in Saccharomyces cerevisiae finalised the biochemical characterisation of the enzymes acting on the pathway. In addition, more understanding of the regulation and evolution of the pathway was gained. D-Galactose catabolism was studied in the filamentous fungus Aspergillus niger. Two genes coding for the enzymes of the oxido-reductive pathway were identified. Galactitol dehydrogenase is the second enzyme of the pathway converting galactitol to L-xylo-3-hexulose. The galactitol dehydrogenase encoding gene ladB was identified and the deletion of the gene resulted in growth arrest on galactitol indicating that the enzyme is an essential part of the oxido-reductive galactose pathway in fungi. The last step of this pathway converts D-sorbitol to D-fructose by sorbitol dehydrogenase encoded by sdhA gene. Sorbitol dehydrogenase was found to be a medium chain dehydrogenase and transcription analysis suggested that the enzyme is involved in D-galactose and D-sorbitol catabolism. The thesis also demonstrates how the understanding of cell metabolism can be used to engineer yeast to produce glycolic acid. Glycolic acid is a chemical, which can be used for example in the cosmetic industry and as a precursor for biopolymers. Currently, glycolic acid is produced by chemical synthesis in a process requiring toxic formaldehyde and fossil fuels. Thus, a biochemical production route would be preferable from a sustainability point of view. Yeasts do not produce glycolic acid under normal conditions but it is a desired production host for acid production because of its natural tolerance to low pH conditions. As a proof of concept, pure model substrates, e.g. D-xylose and ethanol, were used as starting materials for glycolic acid production but the knowledge can be further applied to an expanded substrate range such as biomass derived sugars. Already the introduction of a heterologous glyoxylate reductase gene resulted in glycolic acid production in the yeasts S. cerevisiae and Kluyveromyces lactis. Further modifications of the glyoxylate cycle increased the production of glycolic acid and it was successfully produced in bioreactor cultivation. The challenge of biotechnology is to produce high value products from cheap raw materials in an economically feasible way. This thesis gives more basic understanding to the topic in the form of new information regarding L-rhamnose and D-galactose metabolism in eukaryotic microbes as well as provides an example on how cell metabolism can be engineered in order to turn the cell into a cell factory that is able to produce a useful chemical.