Biocatalytic conversion of vanillin to 3-carboxy muconate

The organic chemical industry permeates our daily lives, giving us products such as pharmaceuticals, plastics, adhesives and paints. 90 to 95% by mass of all organic chemicals produced are derived from petroleum oil or natural gas [1]. To move towards a more sustainable chemical industry, processes which transform renewable feed stocks need to be discovered and developed. Plant biomass is renewable, and typically contains 30% (w/w) lignin [2]. Lignin can be dismantled to yield a smaller and simpler compound, vanillin [3]. This project investigated the construction of a synthetic metabolic pathway to transform vanillin into the novel polymer building block 3-carboxy muconate (3CM). The metabolic pathway which performs this transformation consists of three enzymes; 4-hydroxy benzaldehyde dehydrogenase (HBD), vanillate monooxygenase (VMO) and protocatechuate 3,4-dioxygenase (P34O). These enzymes were cloned from Acinetobacter baylyi, expressed in Escherichia coli, and purified. Characteristics pertinent to the behaviour of these enzymes acting in a pathway, such as kinetics, had been reported for only P34O. This information for HBD and VMO was gained by characterising these enzymes for kinetic behaviour, substrate specificity and stability. The genes for all three enzymes were then combined within a single recombinant E. coli host. This whole cell biocatalyst transformed 1 mM vanillin into 1 mM 3CM. Evidence was gathered to show the rate limiting factor for the conversion was the expression level of VMO. In vitro characterisation had identified that this enzyme had markedly lower intrinsic activity than the other two enzymes, and required higher relative expression. The usefulness of 3CM as a polymer building block was explored. The butadiene system of 3CM was chemically isomerised during conversion to a trimethyl ester form. This trimethyl ester was found to copolymerise with styrene. The composition of the copolymer could be varied by varying the concentrations of monomers in the feed. This project increased the body of knowledge of the enzymology of the vanillin to 3CM pathway, demonstrated that 3CM can be made by biocatalytic transformation of the renewable compound vanillin, and that 3CM is a useful polymer building block.