In efforts to reduce harmful greenhouse gasses such as methane (CH4) and carbon dioxide (CO2), we look to acetylenecarboxylic acid (ACA). ACA may be derived from CH4 and CO2 and converted into high priority target chemicals, such as 3-hydroxypropionic acid which is being targeted to be produced through biocatalytic methods over petroleum-derived, which are non-renewable, produces environmental pollution, and non-sustainable. Here we evaluate the tautomerase enzyme cis-CaaD and its almost exclusive conversion of ACA to intermediate product malonate semialdehyde (MSA) which can further be enzymatically converted to high priority target chemicals. We also compare cis-CaaD to the previously evaluated tautomerase Cg10062 which has 54% similarity yet in its native form does not afford primarily MSA (15% product formation) but rather follows a decarboxylation pathway to produce acetaldehyde (ACH, 85% product formation). Although cis-CaaD produces predominantly MSA, Cg10062 is more catalytically efficient. Through trapping of intermediates in atomic resolution crystal structures we are able to gain a better understanding of the covalent mechanism of cis- CaaD. Structural evaluation of cis-CaaD and Cg10062 variants gives rationale for different product profiles. By evaluating atomic interaction between active site residues, subtle positional changes of residue 114 in Cg10062 versus cis-CaaD lead to either hydration or hydration and decarboxylation. Additionally, structural evaluations of cis-CaaD and Cg10062 chimeras illustrate the C- terminal's necessity in activity by coordination of Arg70 into the active site. This further understanding of cis-CaaD and Cg10062 will enable better bioconversion of ACA to high value chemicals.

The β-hydroxycarboxylic acid functionality of 3-hydroxypropionic acid (3-HP) makes it a versatile building block chemical that can be converted to industrially relevant chemicals such as acrylic acid. Optimization of existing routes commonly utilizing glucose or glycerol as feedstocks is the most commonly targeted area of 3-HP research. In this study, 3-HP biosynthesis is demonstrated using a unique and novel carbon feedstock, acetylenecarboxylic acid (ACA), that can be C1-derived. ACA may be derived from methane and carbon dioxide by methane dehydrodimerization and subsequent monocarboxylation. A novel variant of tautomerase enzyme Cg10062 is used for transformation of ACA to malonate semialdehyde (MSA), a 3-HP precursor. Cg10062 activity is coupled with NADPH-dependent dehydrogenase YdfG to afford successful synthesis of 3-HP with cofactor regeneration. The two-step three-enzyme synthesis was demonstrated using as low as 0.001 equivalents of NADPH relative to ACA. A similar system was also demonstrated using soluble NADH-dependent MmsB.

Lactic acid (LA), a high-value chemical with extensive applications, is currently produced either from petrochemical sources, which are economically unviable or through fermentation of sugars with laborious pretreatment steps. We present acetylenedicarboxylate (ADCA), obtained from methane and carbon dioxide through commercial processes, as an alternative feedstock for LA production. In this study, we developed both in vitro and in vivo enzymatic routes for converting ADCA to LA employing FumA, MaeB, and LDH. We achieved LA yields of 0.96 mol/mol and 0.61 mol/mol for in vitro and in vivo approaches, respectively. We observed the complete conversion of 25 mM ADCA to lactate within 30 minutes, with 0.1 units of each enzyme. The in vivo approach involved using ALE isolates, strains harboring deletions in dcuC, and a fumA knockout mutant as a plasmid addiction strategy. These findings demonstrate the feasibility of using ADCA as a sustainable feedstock for LA production, offering a promising alternative for the production of commodity chemicals.