Metabolic engineering of the fungal D- galacturonate pathway

Industrial biotechnology is one of the enabling technologies for biorefineries, where biomass is converted into value-added products. In addition to biofuels, several platform and fine chemicals can be produced from biomass using biotechnological routes taking advantage of metabolic pathways in the cell. Some of these metabolic pathways exist naturally in the cells that are used as production hosts. However, many of the desired chemical products are not naturally produced by the cellular metabolism. Consequently, genetic engineering is needed to redirect the cellular metabolism towards a product of interest. In this thesis, one of these metabolic pathways – the catabolic D-galacturonate pathway in filamentous fungi – was engineered and redirected to desired end products. D-Galacturonic acid is the main monomer of pectin, which is a common heteropolysaccharide in certain biomasses. Two examples of pectin-rich biomasses are citrus processing waste and sugar beet pulp from agro-industry. These residual biomasses are often poorly utilised. 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) strains by deleting the second gene, encoding a dehydratase, from the fungal D-galacturonate pathway. Overexpression of the first gene, encoding a D-galacturonate reductase, in the pathway improved the initial production rate in A. niger. In addition, production at low pH resulted in higher productivity and titres in cultivations with the engineered A. niger strains. Final titres between 7 and 9 g L-galactonic acid l and product yields close to 100% were observed from pure D-galacturonic acid with both of the production hosts. In addition to L-galactonic acid production from pure D-galacturonic acid, a consolidated bioprocess from citrus processing waste, a pectin-rich biomass, to Lgalactonic acid was investigated using the engineered strains of A. niger. Two different bioprocess types, submerged and solid state fermentation, were compared. As a result, similar final titres and product yields were observed to those obtained in the process from pure D-galacturonic acid. The highest product yield, approaching 90% of the theoretical maximum, was achieved in the solid state fermentation. The second reaction in the fungal D-galacturonate pathway is dehydration of Lgalactonic acid by the action of an L-galactonate dehydratase. Deletion of the gene gaaB encoding this enzyme in A. niger is crucial for L-galactonic acid production. Despite the deletion of gaaB, product yields (L-galactonic acid per consumed D-galacturonic acid) have remained below the theoretical maximum. In addition, catabolisation of mucic acid, an industrially potential dicarboxylic acid