Nylon-6 is a versatile polyamide with numerous applications and is industrially produced by ring-opening polymerization of caprolactam which is synthesized from petrochemical resources. The ongoing depletion of fossil resources and poor sustainability metrics of current synthetic routes necessitate exploration of bio-based solutions for the demands of modern societies. Alternatives for the production of the precursors caprolactam and 6-aminocaproic acid (6-ACA) are worth considering for this reason. Whereas a route for production from α-ketoglutarate over α-ketopimelate to 6-ACA in E. coli has been described, productivity is rather low. The modest production levels for this artificial biosynthetic pathway are due to the deflection of intermediates by competing endogenous enzymes into non-productive routes leading to side product formation. This thesis’ work is aimed at exploring solutions that remedy this undesired formation. One way to circumvent the side product formation could be the creation of an orthogonal artificial pathway by using a D-enantiomer as intermediate. In nature, the abundance of D-amino acids and enzymes working on D-stereo centers is rather low and, therefore, this pathway would not interfere with the metabolism of the host. Supported by computational redesign tools, we engineered two enzymes for this orthogonal pathway, a dehydrogenase and a decarboxylase. To our knowledge, it is the only fermentation pathway including a D-amino acid functionality as central intermediate. Besides that, we investigated a new-found enzyme for the hydrolysis of caprolactam (caprolactamase), of which we solved the structure and of which the catalytic mechanism has been proposed, which has not been described yet.
|Qualification||Doctor of Philosophy|
|Place of Publication||[Groningen]|
|Publication status||Published - 2021|