The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins)

Applied Microbiology and Biotechnology
Aaron M Marcella, Adam W Barb

Abstract

The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and β-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min-1 mg-1. FabD R117A maintained K M values for holo-ACP (~ 40 μM) and displayed small changes in K M f...Continue Reading

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Citations

Sep 16, 2020·Proceedings of the National Academy of Sciences of the United States of America·Laetitia E MissonMichael D Burkart
Jun 3, 2018·Applied Microbiology and Biotechnology·Aaron M Marcella, Adam W Barb
Apr 11, 2021·Applied and Environmental Microbiology·Marco N Allemann, Eric E Allen

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Methods Mentioned

BETA
transacylation
X-ray
PCR
gel filtration
fluorescence assay

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