Chemical biotechnology strategies for adipic acid synthesis
Item statusRestricted Access
Embargo end date25/01/2024
Suitor, Jack Timothy
Adipic acid is one of the most widely-used and valuable platform chemicals. However, industrial adipic acid synthesis is environmentally damaging, as it produces greenhouse gas and relies on non-renewable petrochemical feedstocks. Biotechnology promises new, sustainable ways to synthesise adipic acid enzymatically without producing greenhouse gas. This study aimed to develop such sustainable biotechnology strategies for adipic acid production from aromatic substrates. These substrates were selected as they can be derived from lignin, an abundant and renewable waste by-product of the lumber and agriculture industries and from post-consumer plastic. Therefore, producing adipic acid from these substrates would represent an overall transformation of unwanted wastes into a valuable product without resulting in the environmental harm traditionally associated adipic acid production. First, Escherichia coli was engineered to express the enzymes GcoAB, CatA, and BcER, which enables the bacterium to synthesise adipic acid from guaiacol, a molecule obtained from depolymerised lignin. It was found that optimising recombinant protein folding and expression was crucial to achieving adipic acid synthesis. After optimisation, the engineered E. coli cells quantitatively converted guaiacol to adipic acid, an efficient reaction that had never before been achieved. During this research, it was observed that GcoB, the redox partner for the catalytic GcoA enzyme, expressed poorly in E. coli. Given recent advances in photobiocatalysis, it was hypothesised that GcoA could be activated by an irradiated photoredox catalyst instead of GcoB. By incubating cells GcoA-expressing cells with irradiated riboflavin, the photobiocatalytic conversion of guaiacol to catechol was achieved. Co-expression of the CatA enzyme with GcoA then enabled the photobiocatalytic synthesis of muconic acid, another valuable platform chemical, from guaiacol. This reaction occurred without the requirement of any additional carbon sources like glucose, which are traditionally required for lignin valorisation. As a result, this strategy proposed an alternative way to transform lignin into valuable chemicals using a novel type .of chemical synthesis. Chemical Biotechnology Strategies for Adipic Acid Synthesis Finally, it was shown that engineering E. coli to express the TPADO, AroY, and KpdB enzymes in conjunction with BcER and CatA enabled the synthesis of adipic acid from terephthalate, a monomer derived from PET plastic waste. As a result, the use of terephthalate enables the valorisation of plastic waste that would otherwise accumulate in landfills or the environment. To achieve adipic acid synthesis, the sequences of the recombinant plasmids were optimised using a variety of synthetic biology strategies, and reactions were further improved by immobilising E. coli cells in alginate beads. This achieved improved reaction rates and adipic acid titres and enabled the synthesis of adipic acid directly from a post-consumer plastic bottle. Overall, this work highlighted the potential for chemical biotechnology to enable sustainable adipic acid synthesis from waste feedstocks.