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Alkylation of β-Ketoester Enolates: Acetoacetic Ester Synthesis

Acetoacetic ester synthesis is a method to obtain ketones from alkyl halides and β-keto esters. The reaction occurs in the presence of an alkoxide base that abstracts the acidic proton of the β-keto esters. The step results in an enolate ion which is doubly stabilized. The enolate then reacts with an alkyl halide via the SN2 process to produce an alkylated ester intermediate with a new C–C bond. The hydrolysis of the intermediate, followed by acidification, results in an alkylated β-keto acid.
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Updated: May 14, 2026

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli
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Engineering an Acyl-CoA Ligase With Enhanced Activity Toward Synthetic CoA Alternatives.

Jared R Cossin1, Sarah A Taboada1,2, Gavin J Williams1,3

  • 1North Carolina State University, Raleigh, North Carolina, USA.

Chembiochem : a European Journal of Chemical Biology
|May 13, 2026
PubMed
Summary

Researchers engineered the acylCoA ligase AcsA to use synthetic N-acetylcysteamine (SNAC) instead of costly coenzyme A (CoA). This advance enables more efficient production of valuable compounds like polyketides.

Keywords:
acyl‐CoA ligasesbiocatalysisbiosynthesisdirected evolutionpolyketides

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Area of Science:

  • Biochemistry
  • Metabolic Engineering
  • Synthetic Biology

Background:

  • Fatty acyl-coenzyme A (CoA) thioesters are vital metabolic intermediates.
  • High CoA cost limits large-scale biosynthesis of fuels, chemicals, and pharmaceuticals.
  • Alternative strategies are needed for efficient precursor generation.

Purpose of the Study:

  • Engineer acylCoA ligase AcsAPc to utilize synthetic CoA surrogates.
  • Focus on N-acetylcysteamine (SNAC) as an inexpensive, membrane-permeable alternative.
  • Alleviate CoA-dependent biosynthetic bottlenecks.

Main Methods:

  • Structure-guided saturation and random mutagenesis of AcsAPc.
  • High-throughput colorimetric screening to select for altered thiol specificity.
  • Kinetic analyses to characterize enzyme variants.
  • Reconstitution of a polyketide system to assess functional relevance.

Main Results:

  • Developed a double mutant (F430W/D449E) with a 26-fold improvement in SNAC utilization.
  • Reduced wildtype CoA activity in the engineered enzyme.
  • Demonstrated enhanced pyrone formation (∼8-fold increase) using AcsA-generated acyl-SNACs.
  • Established AcsA as a tunable platform for orthogonal precursor generation.

Conclusions:

  • Engineered AcsAPc efficiently utilizes SNAC, overcoming CoA limitations.
  • Acyl-SNACs can effectively serve as precursors in biosynthetic pathways.
  • This work provides a general framework for improving precursor generation in biotechnology.