Abstract
Medium-chain esters such as isobutyl acetate (IBAc) and isoamyl acetate (IAAc) are high-volume solvents, flavors, and fragrances. Compared to long-chain esters, these short-chain esters are more volatile and are flavor components of many fruits. For example, IAAc has banana flavor and is widely used as food or beverage additives. Currently, they are mainly produced from petroleum feedstocks. Alternatively, metabolic engineering enables the total biosynthesis of IBAc and IAAc directly from glucose in Escherichia coli. The pathways harnessed the power of natural amino acid biosynthesis. In particular, the native valine and leucine pathways in E. coli were utilized to supply precursors. The key enzyme alcohol O-acyltransferases (AAT) will then catalyze esterification reactions to produce IBAc and IAAc. In vitro biochemical characterization of AAT can provide rational guidance for future enzyme engineering or identify new enzymes for other target substrates. The below protocol provides the detailed description of expression and purification of AAT, in vitro enzymatic assays, direct biosyntheses of IBAc or IAAc in E. coli from glucose, and scale-up production of these valuable products in a benchtop bioreactor.
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Tai, YS., Zhang, K. (2015). Designing Bacteria to Produce Esters. In: McGenity, T., Timmis, K., Nogales Fernández, B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_143
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DOI: https://doi.org/10.1007/8623_2015_143
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