Phenolic amides are potent inhibitors of De Novo nucleotide biosynthesis

Journal Article · Fri Jun 12 04:00:00 UTC 2015 · Applied and Environmental Microbiology

DOI: https://doi.org/10.1128/aem.01324-15 · OSTI ID:1345181

Pisithkul, Tippapha ^[1]; Jacobson, Tyler B. ^[2]; O'Brien, Thomas J. ^[2]; Stevenson, David M. ^[2]; Amador-Noguez, Daniel ^[2]

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1. Univ. of Wisconsin, Madison, WI (United States); DOE Office of Scientific and Technical Information (OSTI)
2. Univ. of Wisconsin, Madison, WI (United States)

An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using ¹³C-labeled sugars and [¹⁵N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. Furthermore, the results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals.

• Research Organization: Univ. of Wisconsin, Madison, WI (United States). DOE Great Lakes Bioenergy Research Center
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
• Grant/Contract Number: FC02-07ER64494
• OSTI ID: 1345181
• Journal Information: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 17 Vol. 81; ISSN 0099-2240
• Publisher: American Society for MicrobiologyCopyright Statement
• Country of Publication: United States
• Language: English