Leveraging synthetic biology and enzyme engineering to implement microbial production of new-to-nature molecules

September 18, 2026

The transition towards a sustainable industry requires the development of alternatives to the many molecules that are still being used as platform chemicals by industries and that stem from oil refining. Levulinic and methacrylic acids are two examples of such molecules, although their production in a bio-context is constrained by the absence of biochemical pathways that would turn their production easy to implement via microbial fermentation. This thesis is focused on the implementation of such pathways, exploring approaches based on synthetic biology that connect non-native links between enzymes and their non-native substrates. In case of levulinic acid (LA), a proposed biosynthetic pathway based on the promiscuous activities of L-lysine–8-amino-7-oxononanoate transaminase (BioK) and diaminopropionate ammonia-lyase (YgeX) enzymes was re-evaluated. Molecular docking was used to assess enzyme interactions with synthetic substrates and identify mutations which improve their recognition. In silico modifications targeting BioK residues A223 and A116 enabled the design of triple-point mutants with improved substrate affinity, while the YgeX D120R mutation improved substrate recognition. Enzymatic assays led to the detection of LA for the first time when BioK and YgeX enriched extracts were combined. For methacrylic acid (MAA) production, a similar in silico strategy evaluated the cis-aconitate decarboxylases use for the conversion of mesaconic acid into MAA. In the enzyme from Aspergillus terreus, H111G and H111D mutations showed the highest improvements in affinity for cis-aconitic acid (natural substrate) and mesaconic acid, respectively. Overall, these results highlight molecular docking’s potential to provide a sustainable LA and MAA production.

Authors in the community:

• 

  P

  Pedro Afonso Dias Paisana

  ist1102799

Supervisors of this institution:

• 

  N

  Nuno Gonçalo Pereira Mira

  ist148703

industrial-biotechnology - Industrial Biotechnology

eng - English

Instituto Superior Técnico