Metabolic engineering of the fungal D-galacturonate pathway:Dissertation

Industrial biotechnology is one of the enabling technologies for biorefineries. In addition to biofuels, several platform and fine chemicals can be produced from biomass taking advantage of metabolic pathways in the cell. However, genetic engineering is often needed to redirect the cellular metabolism towards a product of interest. In this thesis, one of these metabolic pathways catabolizing constituents of pectin - the catabolic D-galacturonate pathway in filamentous fungi- was engineered and redirected to desired end products. Biotechnological production of L-galactonic acid, a potential platform chemical, was demonstrated in this thesis for first time. The production was obtained in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei) by deleting the second gene, encoding a dehydratase, from the fungal D-galacturonate pathway. In addition to the production from pure D-galacturonic acid, a consolidated bioprocess from citrus processing waste, a pectin-rich biomass was investigated. L-galactonic acid can be lactonised and further oxidised to L-ascorbic acid (vitamin C) via chemical or biochemical routes. In this thesis, an A. niger strain was engineered for direct conversion of D-galacturonic acid to L-ascorbic acid by introducing two plant genes: L-galactono-1,4-lactone lactonase and dehydrogenase. The resulting strains were capable of L-ascorbic acid production from pure D-galacturonic acid or citrus processing waste. In addition to lactonization, two other biochemical reactions towards L-galactonic acid are known: dehydration, which is the second reaction in the fungal Dgalacturonate pathway, and oxidation to D-tagaturonic acid, which occurs in the catabolic L-galactonic acid pathway in bacteria. Both of these biochemical reactions and responsible enzymes from A. niger and Escherichia coli were investigated more detailed. As a result, the substrate specifities for four dehydratases from A. niger were determined and the bacterial L-galactonate-5-dehydrogenase was characterised and applied in a colorimetric assay for Lgalactonic and L-gulonic acids. Pectin-rich biomass has potential as a raw material for the production of renewable chemicals. This thesis presents new ways to utilise this residual biomass by using industrial biotechnology. In addition, the thesis broadens basic understanding of the fungal catabolic D-galacturonate pathway and how it can be engineered for production of useful chemicals