(164o) A Novel Thiolase with Enhanced Activity of ATP-Independent Triacetic Acid Lactone (TAL) Production | AIChE

(164o) A Novel Thiolase with Enhanced Activity of ATP-Independent Triacetic Acid Lactone (TAL) Production


Cheong, S. - Presenter, Rice University
Wang, Z., UC Berkeley
Pereira, J. H., Lawrence Berkeley National Laboratory
Degiovanni, A., Lawrence Berkeley National Laboratory
Araujo Barcelos, C., Lawrence Berkeley National Laboratory
Haushalter, R. W., Lawrence Berkeley National Lab
Adams, P. D., Joint BioEnergy Institute
Keasling, J., UC Berkeley
Thiolase is the enzyme that catalyzes the non-decarboxylative Claisen condensation reaction forming a carbon-carbon bond between the carbonyl carbon of an acyl-CoA serving as the primer and alpha carbon of another acyl-CoA serving as the extension unit. This type of carbon bond forming reaction does not require the beta-carboxylation of extension unit acyl-CoA and uses acetyl-CoA instead of malonyl-CoA as extension unit in most cases, hence allowing more flexibility in substrate structure and bypassing the ATP-consuming acetyl-CoA carboxylation step to supply malonyl-CoA. Therefore, many researchers are exploiting thiolase for carbon-chain elongation in bioproduction of chemicals. In one recent exemplary report (Tan et al. 2020. Nature Catalysis, 3, 593-603), researchers discovered that Cupriavidus necator thiolase BktB is able to condense between acetoacetyl-CoA primer and acetyl-CoA extension unit to generate triacetic acid lactone (TAL), a platform chemical for productions of valuable chemicals and a potential monomer of recyclable polymers, and Escherichia coli strains expressing BktB exhibited higher titer than strains expressing 2-pyrone synthase, a native and commonly used polyketide synthase for TAL production, due to higher ATP energy efficiency.

While C. necator BktB is already an energy-efficient TAL producing enzyme, through screening selected BktB homologs with different phylogenetic distances, this research discovered a new thiolase with ~30 times higher in vitro TAL production activity and ~10 times higher TAL titer from E. coli in in vivo small-scale plate fermentation compared to C. necator BktB. When growing in a larger scale fermenter, E. coli strain expressing this thiolase produced ~ 3 g/L of TAL, higher than the highest reported TAL titer by E. coli despite the lethality of TAL upon E. coli. Successful crystallization and subsequent substrate docking further helped explain the reaction mechanism and reveal the contributing residues for enhanced TAL synthesis activity of this enzyme.

This new discovered thiolase with much higher ATP-independent TAL production activity could benefit the biorenewable TAL production industry in microbial platforms like E. coli or other organisms with higher TAL tolerance, and its structural analysis results could aid the future rational designs for activity improvement or expansion of functional and structural range of substrates hence expanding the potential product diversity of biorefinery.