Biocatalyst Engineering: Metabolic Engineering, Kinetic modeling and metagenomics applied to industrial biotechnology

Sammanfattning: Industrial biotechnology has been defined as the use and application of biotechnology for the sustainable processing and production of chemicals, materials and fuels. It makes use of biocatalysts such as microbial communities, whole-cell microorganisms or purified enzymes. Although biocatalysts are considered advantageous, since they operate under mild conditions regarding temperature and pH and enable chemo-, regio-, and stereoselective reactions, their utilization on the industrial scale can be impeded by sub-optimal performance. The present study was aimed at the improvement of two biocatalytic processes: whole-cell bioreduction for the production of optically pure alcohols, and ethanol production from lignocellulosic feedstock. The reduction of the bicyclic diketone, bicyclo[2.2.2]octane-2,6-dione into 1R,4S,6S-6-hydroxy-bicyclo[2.2.2]octane-2-one (endo-alcohol) and 1S,4R,6S-6- hydroxy-bicyclo[2.2.2]octane-2-one (exo-alcohol) was used as a model bioreduction reaction. The identification and overexpression of an exo-reductase encoding gene in Candida tropicalis enabled the production of the uncommon exo-alcohol as major product. In parallel, the advantages and disadvantages of metabolically engineered Saccharomyces cerevisiae and Escherichia coli as host for whole-cell bioreduction were compared for the production of the endoalcohol. Both these microorganisms gave about the same product purity. While E. coli showed a three times higher reduction rate, higher cell viability was maintained during the bioreductions with recombinant S. cerevisiae, which resulted in higher final conversion (95%) and indicated that yeast could be recycled. Improvement of bioethanol production from xylose was achieved through the construction and use of a kinetic model as a simulation tool for metabolic engineering of recombinant S. cerevisiae strains. In parallel, novel xylose isomerases and reductases were isolated from soil metagenome libraries by sequence- and activity-based screening methods. In addition a novel indirect protocol for soil DNA extraction that enabled the isolation of environmental DNA at high yield and purity was developed. This study enabled the expansion of a biocatalyst toolbox by providing new catalysts, screening methods and generating new recombinant strains with improved properties, which can be utilized in the pharmaceutical and bioenergy sectors, and thus constitutes a step forward in the development of novel biobased processes.