Rational design of thiolase substrate specificity for metabolic engineering applications

Abstract

Metabolic engineering efforts require enzymes that are both highly active and specific toward the synthesis of a desired output product to be commercially feasible. The 3-hydroxyacid (3HA) pathway, also known as the reverse β-oxidation or coenzyme-A-dependent chain-elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities. However, this pathway suffers from byproduct formation, which lowers the yields of the desired longer chain products, as well as increases downstream separation costs. The thiolase enzyme catalyzes the first reaction in this pathway, and its substrate specificity at each of its two catalytic steps sets the chain length and composition of the chemical scaffold upon which the other downstream enzymes act. However, there have been few attempts reported in the literature to rationally engineer thiolase substrate specificity. In this study, we present a model-guided, rational design study of ordered substrate binding applied to two biosynthetic thiolases, with the goal of increasing the ratio of C6/C4 products formed by the 3HA pathway, 3-hydroxy-hexanoic acid and 3-hydroxybutyric acid. We identify thiolase mutants that result in nearly 10-fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elongation, as well as expand our knowledge of sequence-structure-function relationship for this important class of enzymes.

Open Culture Ethanol-Based Chain Elongation to Form Medium Chain Branched Carboxylates and Alcohols

“…However, research has already shown that it is possible to increase the affinity of butyrate relative to acetate for an engineered thiolase (Bonk et al, 2018), as well as first efforts to modify thiolases to use branched carboxylates as primers (Clomburg et al, 2018). Further efforts to tailor the thiolase and other involved enzymes via metabolic engineering could offer perspectives where the k cat and K m values for branched carboxylates and their conversion intermediates are increased.…”

Machine learning modeling of family wide enzyme-substrate specificity screens

“…Instead, work to date to expand the substrate scope of an enzyme class of interest often relies upon time consuming and ad hoc rational engineering based upon structure [26,27], simple similarity searches between the native substrate and desired substrate of interest [28], or trial-and-error experimental sampling [29]. Once an enzyme with some activity for a substrate of interest is found practitioners can resume directed evolution strategies similar to those described above to increase efficiency [29][30][31][32].…”

Two possible candidate enzymes from Ulva lactuca-associated epiphytic bacteria obtained through PCR and functional evaluation

“…Although no data was recovered for the abg3 gene reported by Burke [42], we identified a 1.0 kb fragment corresponding to a "thiolase gene". Thiolases are a group of enzymes that have important uses on cellular engineering for industrial processes [70]. These enzymes appear to be functionally closely related to lipases and esterases since they are involved in the same lipid-degrading pathways and show genetic proximity to thioesterases [71].…”