As an alternative to petroleum and biomass derived starting materials, this dissertation proposes utilization of CH4 and CO2-derived acetylenecarboxylic acid (ACA) as feedstock for commodity chemical production. These commercial building block chemicals such as 3-hydroxypropionic acid are being targeted for production via biocatalytic methods over petroleum and biomass derived methods. We look to biotechnology as an alternative route to produce these valued chemicals through enzyme biocatalysis. Here we evaluate the tautomerase enzyme cis-CaaD, which almost exclusively converts ACA to intermediate malonic semialdehyde (MSA). MSA can then be further be enzymatically converted to high priority target chemicals. We also compare cis-CaaD to the previously evaluated tautomerase Cg10062, which largely decarboxylates to acetaldehyde and produces minimal MSA. In addition, although cis-CaaD produces predominantly MSA, Cg10062 is more catalytically efficient. In our efforts to construct a highly catalytic MSA-forming cis-CaaD enzyme we used kinetic studies and protein crystallography to understand the cis-CaaD mechanism and gain a deeper understanding for the different product profiles these two enzymes produce. Previous efforts have shown the in vitro production of 3-hydroxypropionic acid from ACA. Here we look at in vivo production. Earlier experimental trials have shown that E. coli as well as many other bacterial species are not viable with ACA as a sole carbon source. However, the use of a tautomerase modified fusion protein allows the conversion of ACA to MSA in media followed by the uptake of MSA by E. coli cells. This will allow ongoing in vivo biocatalytic conversion of MSA to high priority chemicals.
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