Biobased Adipic Acid - Challenges in Establishing a Cell Factory

Sammanfattning: Growing concern regarding climate change calls for sustainable solutions to significantly reduce our dependency on non-renewable fossil-based raw materials. One potential solution is the development of biorefineries where biobased, renewable raw materials are converted into valuable products via enzymatic, chemical or microbial conversion. This work focuses on the microbial production of adipic acid, a precursor in the nylon industry, currently derived from fossil-based raw material. No known naturally occurring microorganism is able to produce adipic acid, and genetic engineering of a suitable host is therefore required. The aim of the work presented in this thesis was to engineer a microorganism for the production of adipic acid from glucose, more specifically, from glucose streams derived from lignocellulosic forest residues. Theoretical evaluation of metabolic pathways for adipic acid production revealed several obstacles, including redox imbalance and the discovery or engineering of enzymes to catalyze novel reactions. Mining of enzyme databases for alternative paths proved fruitful, and the number of biochemical reactions in the lysine pathway employing as yet unidentified enzymes was reduced from three to two, without affecting the thermo­dynamics of the pathway. A combined approach of in vitro and in silico analysis suggested potential enzyme engineering strategies for one of the reactions, for which there are as yet no identified enzymes, namely, the reduction of unsaturated α,β bonds of 6-aminohex-2-enoic acid and 2-hexenedioic acid. When defining a suitable host for microbial adipic acid production, tolerance to high concentrations of adipic acid (50-100 g L-1) is important to ensure an economically feasible process, preferably at low pH (below 5) to further reduce the overall process cost. Screening of bacteria, yeasts and a filamentous fungus grown in increasing concentrations of adipic acid (0-100 g L-1) and at different pH revealed Candida viswanathii to be a promising host to engineer for adipic acid production. A comparative study of C. viswanathii with Saccharomyces cerevisiae in controlled batch cultivations at increasing adipic acid concentrations (0-95 g L-1) and low pH (pH 4 and pH 5) revealed significant differences in their tolerance to adipic acid; C. viswanathii being able to grow, almost unaffected, under all the conditions investigated, whereas S. cerevisiae was unable to grow at 95 g L-1. Lipid analysis of their cell membranes revealed C. viswanathii to have a thicker and more compact cell membrane, which is probably less permeable to adipic acid.