Cell-free enzymatic biosystem for the conversion of glucose to malate

Abstract

Platform chemicals are essential to industrial processes. Used as starting materials for the manufacture of diverse products, their cheap availability and efficient sourcing are an industrial requirement. Increasing concerns about the depletion of natural resources and growing environmental consciousness have led to a focus on the economics and ecological viability of biobased platform chemical production. Biobased strategies such as fermentation still have limitations that restrict their large scale industrial application. Current microbial biomanufacturing is hindered by the limitations caused by cells, as synthetic pathways and their optimizations are restricted by the physiological limits of the cellular production system. Cell-free metabolic engineering is pushing the boundaries of traditional bioengineering models by focusing on the use of in vitro combinations of catalytic enzymes prepared from purified proteins or crude lysates from cells, assembled in artificial cascades, for production of target commodities.This thesis describes a cofactor balanced, cell-free, 5 enzyme biosystem for the sustainable production of malic acid from glucose, whereby 5 thermophilic proteins (glucose dehydrogenase, dihydroxy-acid dehydratase, 2-keto-3-deoxygluconate aldolase, glyceraldehyde dehydrogenase and malate dehydrogenase) were successfully expressed, purified and demonstrated to display enzyme activity. Enzyme immobilization has been used by many researchers to overcome instability problems and facilitate the repetitive use of enzymes. Recombinant glucose dehydrogenase from S. solfataricus was successfully purified and immobilized onto novel supports and demonstrated great potential for gluconic acid production from glucose, as well as bread waste hydrolysate in a sustainable production approach. Coupling of cofactor recycling enzymes (glucose dehydrogenase and malate dehydrogenase) was also explored and successfully demonstrated the simultaneous production of chemicals and cofactor recycling capabilities. The experimental work also identified potential bottlenecks affecting the feasibility of the cell-free biosystem and paves the way for optimized characterization of enzymes in the cascade, in the free-state, individually immobilized and co-immobilized.