Developing an enzymatic synthesis for acetoacetyl-CoA using acetyl-CoA acetyltransferase (AtoB) From Escherichia coli

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Brittney R. Ballentine (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Jason Reddick

Abstract: Polyketides are natural products that can be found in clinical drugs that are used as antibiotics, parasiticides, immunomodulators, and many others.1 Polyketide biosynthesis involves a complex pathway catalyzed by polyketide synthases. The Reddick laboratory at UNC-Greensboro has been studying the biosynthesis of the ß-branch from bacilliene and difficidin, both from Bacillus species. All the steps for the biosynthesis of the ß-branch of difficidin have been reconstituted except for the key C-C bond forming step catalyzed by the enzyme DfnL, which is a homolog of hydroxymethylglutaryl (HMG)-coenzyme A (CoA) synthase. Homologs of this enzyme adds an acetyl group to the nascent ß-ketoacyl intermediate to form an HMG-like intermediate which is dehydrated and decarboxylated to produce the ß-branch of difficidin. Since the true ß-ketoacyl substrate used during difficidin biosynthesis is a long and complex structure, the Reddick lab has used acetoacetyl-CoA as a shortened model of this ß-ketoacyl intermediate. For a while, many well-known science and technology companies, such as Sigma-Aldrich and Fisher, stopped producing acetoacetyl-CoA. Without access to acetoacetyl-CoA, we could not continue with experiments designed to complete the reconstitution of the biosynthetic pathway of the ß-branch for difficidin. To solve this ongoing issue of supply availability, we are attempting to develop our own method for producing acetoacetyl-CoA using enzymatic synthesis rather than organic synthesis; due to slow reaction rate and the difficulty with breaking carbon-carbon bonds in organic synthesis. In doing so we will be able to continue our research in completing the biosynthesis of the ß-branch of bacilliene and difficidin. The purpose of this project is to develop an enzymatic procedure preparing acetoacetyl-CoA using the enzyme encoded by the atoB gene from Escherichia coli (E. coli). The central hypothesis is that AtoB synthesizes acetoacetyl-CoA from acetyl-CoA. The rationale that underlies the research proposed here is that we will have access to acetoacetyl-CoA without the need to depend on chemical companies for a commercial supply. This will also be a benefit to any biochemistry project needing acetoacetyl-CoA. We tested this hypothesis by first cloning atoB into a vector for expression and purification of the enzyme, purified AtoB by Nickel-Affinity Column Chromatography, and then measured the activity using UV-Vis Spectrophotometry, while also attempting Liquid Chromatography-Mass Spectrometry (LC-MS). Acetoacetyl-CoA formation and activity has been detected by a UV-based assay. [This abstract may have been edited to remove characters that will not display in this system. Please see the PDF for the full abstract.]

Additional Information

Language: English
Date: 2022
Aceotoacetyl-CoA, Acetyl-CoA, AtoB, Polyketide
Polyketides $x Synthesis
Enzymes $x Synthesis
Escherichia coli

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