Supplementary Figure 4: Products synthesized through the use of glycolyl-CoA as ω-hydroxylated primer (a, c) or α-hydroxylated extender unit (b).
(a) Total ion GC-MS chromatogram showing peak of synthesized 4-hydroxybutyric acid. (b) Enlarged region of inset in (a) showing 2,3-dihydroxybutyric acid peak. 4-hydroxybutyric acid was produced through the platform utilizing glycolyl-CoA as the primer and acetyl-CoA as the extender unit, while 2,3-dihydroxybutyric acid was produced through the platform with same enzymatic components but utilizing acetyl-CoA as the primer and glycolyl-CoA as the extender unit with termination at β-hydroxyacyl-CoA node. The following enzymes provided the individual components of the pathway: BktB (thiolase) and PhaB1 (HACDH) from Ralstonia eutropha2,3, Aeromonas caviae PhaJ (ECH)4, Treponema denticola TdTer (ECR)5 with native enzymes catalyzing the acid-forming termination and Megasphaera elsdenii transferase Pct activating glycolic acid to glycolyl-CoA. MG1655 (DE3) ΔglcD served as the host strain. (c) Production of β-hydroxy-γ-butyrolactone through the engineered platform with enzymes Pct, BktB and PhaB1 utilizing the primer glycolyl-CoA and the extender unit acetyl-CoA with termination at β-hydroxyacyl-CoA node. β-hydroxy-γ-butyrolactone is the lactone of 3,4-dihydroxybutyric acid and is generated through spontaneous or endogenous enzyme-catalyzed lactonization of 3,4-dihydroxybutyric acid or β-hydroxy intermediate 3,4-dihydroxybutyryl-CoA. glcD encodes a subunit of glycolate oxidase, an enzyme involved in the degradation of glycolic acid. Functional groups from primer and extender unit are marked in red and blue, respectively. Strains were grown as described in supplementary methods.
