Discovery and Engineering of Pathways for Production of α-Branched Organic Acids

Abstract

Cell-based synthesis offers many opportunities for preparing small molecules from simple renewable carbon sources by telescoping multiple reactions into a single fermentation step. One challenge in this area is the development of enzymatic carbon-carbon bond forming cycles that enable a modular disconnection of a target structure into cellular building blocks. In this regard, synthetic pathways based on thiolase enzymes to catalyze the initial carbon-carbon bond forming step between acyl coenzyme A (CoA) substrates offer a versatile route for biological synthesis, but the substrate diversity of such pathways is currently limited. In this report, we describe the identification and biochemical characterization of a thiolase-ketoreductase pair involved in production of branched acids in the roundworm, Ascaris suum, that demonstrates selectivity for forming products with an α-methyl branch using a propionyl-CoA extender unit. Engineering synthetic pathways for production of α-methyl acids in Escherichia coli using these enzymes allows the construction of microbial strains that produce either chiral 2-methyl-3-hydroxy acids (1.1 ± 0.2 g L) or branched enoic acids (1.12 ± 0.06 g L) in the presence of a dehydratase at 44% and 87% yield of fed propionate, respectively. In vitro characterization along with in vivo analysis indicates that the ketoreductase is the key driver for selectivity, forming predominantly α-branched products even when paired with a thiolase that highly prefers unbranched linear products. Our results expand the utility of thiolase-based pathways and provide biosynthetic access to α-branched compounds as precursors for polymers and other chemicals.

Structural and Biochemical Studies of Substrate Selectivity in Ascaris suum Thiolases

“…Acat2 (UniProt F1KYX0) and Acat5 (UniProt F1L3N8) used for X-ray crystallography were expressed and purified as described previously. 12 The Acat2-C91S mutant was purified using the same protocol.…”

“…12 These thiolases exhibited marked differences in selectivity toward the formation and degradation of linear compared to branched products despite their sequence similarity. 12 In this work, we report the free (wild type) and substrate-bound (C91S mutant) crystal structures of Acat2, an α-branch permissive thiolase, along with the structure of Acat5, a linear-selective thiolase. Comparison of these structures highlighted the potential role of a "covering" loop at the back of the active site in α-branch permissiveness.…”

Reverse β-oxidation pathways for efficient chemical production

“…In a separate study, ketoreductase was identified to be the key driver for selectivity, forming predominantly α-branched products even when paired with a thiolase that highly prefers unbranched products. Leveraging the specificity of this ketoreductase from Ascaris suum allowed production of chiral 2-methyl-3-hydroxy acids (1.1 ± 0.2 g/l) or branched enoic acids (1.12 ± 0.06 g/l) at 44% and 87% yields of fed propionate, respectively (Blaisse et al, 2017 ). Synthesis of a ω-1-branched product is also possible by using a branched acyl-CoA primer such as isobutyryl-CoA.…”

“…Moreover, in vitro prototyping allows for high-throughput screening of a large number of enzyme variants like thioesterases to identify special functional group and chain length specificity (McMahon & Prather, 2014 ). Alternatively, substrate specificity of core rBOX enzymes like ketoreductases could influence specificity of upstream thiolase, leading to improved selectivity (Blaisse et al, 2017 ). Furthermore, optimization of relative expression levels is critical for debottlenecking the pathway flux, which could be facilitated by rapid in vitro prototyping (Karim et al, 2020 ) and independent gene expression control using orthogonal inducible promoters (Meyer et al, 2019 ) for in vivo implementation.…”

Rational design of thiolase substrate specificity for metabolic engineering applications

“…The 3-hydroxyacid (3HA) pathway (Figure 1A), also referred to as the reverse β-oxidation (r-BOX) or CoA-dependent chain elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities, including acids, alcohols, alkanes and aldehydes, with applications in the pharmaceutical, polymer and flavor and fragrance industries (Clomburg et al, 2015; Kim et al, 2015; Sheppard et al, 2014; Tseng & Prather, 2012; Blaisse et al, 2017). This is due to the promiscuous activities of pathway enzymes, which on the one hand makes the biological synthesis of these compounds possible, but on the other, always results in a mixture of products at the end of the fermentation (Cheong et al 2016; Clomburg et al 2012).…”