Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis

Abstract

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. 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.

Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production

“…3b; Additional file 4) during fermentations of the evolved hybrids, focusing on ACSH, YPDX (a rich medium containing glucose and xylose that matches their concentrations in ACSH), YPDX plus the HTs found in ACSH, and YPDX plus feruloyl amide (FA), a product of AFEX pretreatment previously shown to have inhibitory effects in E. coli [9, 12]. Despite lack of growth improvement in ACSH (Additional file 3), extracellular xylose concentrations in microaerobic ACSH fermentations decreased significantly faster in cultures of evolved hybrids than ancestral hybrids ( t test, p value <0.0368) (Figs.…”

“…Second, these hydrolysates contain potent fermentation inhibitors that are mainly derived from the deconstruction of biomass during the chemical pretreatments used to improve the accessibility of cellulose and hemicellulose to hydrolysis [9]. For example, after enzymatic treatment and the application of the ammonia fiber expansion (AFEX) method used to deconstruct corn stover [10], phenolic amides, phenolic acids, furans, and other small inhibitory molecules are generated [11]; these molecules are collectively termed “hydrolysate toxins (HTs).” Proposed mechanisms for their toxicity include the inhibition of key enzymatic steps, such as glutamine PRPP amidotransferase (PurF), which is important for de novo purine biosynthesis but inhibited by feruloyl amide in Escherichia coli [12]; decreased energy availability due to costly efflux pumps [13]; and redox imbalances caused by the detoxification of acids and aldehydes [9]. …”

Metabolic, Epigenetic, and Transgenerational Effects of Gut Bacterial Choline Consumption

“…Serum and hepatic metabolomes were measured using a C18 method previously published (Pisithkul et al, 2015). Samples were analyzed using an HPLC-tandem MS (HPLC-MS/MS) system consisting of a Thermo Scientific/Dionex UHPLC coupled by electrospray ionization (ESI) (negative mode) to a hybrid quadrupole– high-resolution mass spectrometer (Q Exactive Orbitrap; Thermo Scientific) operated in full scan mode for detection of targeted compounds based on their accurate masses.…”

PKA and HOG signaling contribute separable roles to anaerobic xylose fermentation in yeast engineered for biofuel production

“…There are many types of lignotoxins present in hydrolysates (51), and while their effects are somewhat understood, many of their specific targets remain to be elucidated (33). One possibility is that toxins are directly inhibiting enzymes required for anaerobic xylose fermentation, as shown in Escherichia coli where feruloyl and coumaroyl amides were discovered to be allosteric inhibitors of de novo nucleotide biosynthetic enzymes (78). Jayakody et al (2018) found that glycoaldehyde and methyglyoxal present in hydrolysate are key inhibitors of xylose fermentation (79).…”