Prediction and identification of sequences coding for orphan enzymes using genomic and metagenomic neighbours

Many characterized metabolic enzymes currently lack associated gene and protein sequences. Here, pathway and genomic neighbour data are used to assign genes to these ‘orphan enzymes,' and the predictions are validated with experimental assays and genome-scale metabolic modelling

Clostridium sticklandii, a specialist in amino acid degradation:revisiting its metabolism through its genome sequence.

International audienceBACKGROUND: Clostridium sticklandii belongs to a cluster of non-pathogenic proteolytic clostridia which utilize amino acids as carbon and energy sources. Isolated by T.C. Stadtman in 1954, it has been generally regarded as a "gold mine" for novel biochemical reactions and is used as a model organism for studying metabolic aspects such as the Stickland reaction, coenzyme-B12- and selenium-dependent reactions of amino acids. With the goal of revisiting its carbon, nitrogen, and energy metabolism, and comparing studies with other clostridia, its genome has been sequenced and analyzed. RESULTS: C. sticklandii is one of the best biochemically studied proteolytic clostridial species. Useful additional information has been obtained from the sequencing and annotation of its genome, which is presented in this paper. Besides, experimental procedures reveal that C. sticklandii degrades amino acids in a preferential and sequential way. The organism prefers threonine, arginine, serine, cysteine, proline, and glycine, whereas glutamate, aspartate and alanine are excreted. Energy conservation is primarily obtained by substrate-level phosphorylation in fermentative pathways. The reactions catalyzed by different ferredoxin oxidoreductases and the exergonic NADH-dependent reduction of crotonyl-CoA point to a possible chemiosmotic energy conservation via the Rnf complex. C. sticklandii possesses both the F-type and V-type ATPases. The discovery of an as yet unrecognized selenoprotein in the D-proline reductase operon suggests a more detailed mechanism for NADH-dependent D-proline reduction. A rather unusual metabolic feature is the presence of genes for all the enzymes involved in two different CO2-fixation pathways: C. sticklandii harbours both the glycine synthase/glycine reductase and the Wood-Ljungdahl pathways. This unusual pathway combination has retrospectively been observed in only four other sequenced microorganisms. CONCLUSIONS: Analysis of the C. sticklandii genome and additional experimental procedures have improved our understanding of anaerobic amino acid degradation. Several specific metabolic features have been detected, some of which are very unusual for anaerobic fermenting bacteria. Comparative genomics has provided the opportunity to study the lifestyle of pathogenic and non-pathogenic clostridial species as well as to elucidate the difference in metabolic features between clostridia and other anaerobes

A Novel Acyl-CoA Beta-Transaminase Characterized from a Metagenome

BACKGROUND: Bacteria are key components in all ecosystems. However, our knowledge of bacterial metabolism is based solely on the study of cultivated organisms which represent just a tiny fraction of microbial diversity. To access new enzymatic reactions and new or alternative pathways, we investigated bacterial metabolism through analyses of uncultivated bacterial consortia. METHODOLOGY/PRINCIPAL FINDINGS: We applied the gene context approach to assembled sequences of the metagenome of the anaerobic digester of a municipal wastewater treatment plant, and identified a new gene which may participate in an alternative pathway of lysine fermentation. CONCLUSIONS: We characterized a novel, unique aminotransferase that acts exclusively on Coenzyme A (CoA) esters, and proposed a variant route for lysine fermentation. Results suggest that most of the lysine fermenting organisms use this new pathway in the digester. Its presence in organisms representative of two distinct bacterial divisions indicate that it may also be present in other organisms

Characterization of L-Carnitine Metabolism in Sinorhizobium meliloti

International audienceL-Carnitine is a trimethylammonium compound mostly known for its contribution to fatty acid transport into mitochondria. In bacteria, it is synthesized from ␥-butyrobetaine (GBB) and can be used as a carbon source. L-Carnitine can be formed directly by GBB hydroxylation or synthesized via a biosynthetic route analogous to fatty acid degradation. However, this multistep pathway has not been experimentally characterized. In this work, we identified by gene context analysis a cluster of L-carnitine anabolic genes next to those involved in its catabolism and proceeded to the complete in vitro characterization of L-carnitine biosynthesis and degradation in Sinorhizobium meliloti. The five enzymes catalyzing the seven steps that convert GBB to glycine betaine are described. Metabolomic analysis confirmed the multistage synthesis of L-carnitine in GBB-grown cells but also revealed that GBB is synthesized by S. meliloti. To our knowledge, this is the first report of aerobic GBB synthesis in bacteria. The conservation of L-carnitine metabolism genes in different bacterial taxonomic classes underscores the role of L-carnitine as a ubiquitous nutrient. IMPORTANCE The experimental characterization of novel metabolic pathways is essential for realizing the value of genome sequences and improving our knowledge of the enzymatic capabilities of the bacterial world. However, 30% to 40% of genes of a typical genome remain unannotated or associated with a putative function. We used enzyme kinetics, liquid chromatography-mass spectroscopy (LC-MS)-based metabolomics, and mutant phenotyping for the characterization of the metabolism of L-carnitine in Sinorhizobium meliloti to provide an accurate annotation of the corresponding genes. The occurrence of conserved gene clusters for carnitine metabolism in soil, plant-associated, and marine bacteria underlines the environmental abundance of carnitine and suggests this molecule might make a significant contribution to ecosystem nitrogen and carbon cycling

Characterization of a novel β-alanine biosynthetic pathway consisting of promiscuous metabolic enzymes

International audienceBacteria adapt to utilize the nutrients available in their environment through a sophisticated metabolic system composed of highly specialized enzymes. Although these en-zymes can metabolize molecules other than those for which they evolved, their efficiency toward promiscuous substrates is considered too low to be of physiological relevance. Herein, we investigated the possibility that these promiscuous enzymes are actually efficient enough at metabolizing secondary substrates to modify the phenotype of the cell. For example, in the bacterium Acinetobacter baylyi ADP1 (ADP1), panD (coding for L- aspartate decarboxylase) encodes the only protein known to catalyze the synthesis of beta-alanine, an obligate intermediate in CoA synthesis. However, we show that the ADP1 delta panD mutant could also form this molecule through an unknown metabolic pathway arising from promiscuous enzymes and grow as efficiently as the wildtype strain. Using metabolomic analyses, we identified 1,3-diaminopropane and 3-aminopropanal as intermediates in this novel pathway. We also conducted activity screening and enzyme kinetics to elucidate candidate enzymes involved in this pathway, including 2,4-diaminobutyrate aminotransferase (Dat) and 2,4-diaminobutyrate decarboxylase (Ddc) and validated this pathway in vivo by analyzing the phenotype of mutant bacterial strains. Finally, we experimentally demonstrate that this novel metabolic route is not restricted to ADP1. We propose that the occurrence of conserved genes in hundreds of genomes across many phyla suggests that this previously undescribed pathway is widespread in prokaryotes

Microbial urate catabolism: characterization of HpyO, a non-homologous isofunctional isoform of the flavoprotein urate hydroxylase HpxO

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A Conserved Gene Cluster Rules Anaerobic Oxidative Degradation of l-Ornithine▿

For the ornithine fermentation pathway, described more than 70 years ago, genetic and biochemical information are still incomplete. We present here the experimental identification of the last four missing genes of this metabolic pathway. They encode l-ornithine racemase, (2R,4S)-2,4-diaminopentanoate dehydrogenase, and the two subunits of 2-amino-4-ketopentanoate thiolase. While described only for the Clostridiaceae to date, this pathway is shown to be more widespread

Implementation of a synthetic formate assimilation pathway in Escherichia coli by adaptive evolution

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Genetic and biocatalytic basis of formate dependent growth of Escherichia coli strains evolved in continuous culture

International audienceThe reductive glycine pathway was described as the most energetically favorable synthetic route of aerobic formate assimilation. Here we report the successful implementation of formatotrophy in Escherichia coli by means of a stepwise adaptive evolution strategy. Medium swap and turbidostat regimes of continuous culture were applied to force the channeling of carbon flux through the synthetic pathway to pyruvate establishing growth on formate and CO2 as sole carbon sources. Labeling with 13C-formate proved the assimilation of the C1 substrate via the pathway metabolites. Genetic analysis of intermediate isolates revealed a mutational path followed throughout the adaptation process. Mutations were detected affecting the copy number (gene ftfL) or the coding sequence (genes folD and lpd) of genes which specify enzymes implicated in the three steps forming glycine from formate and CO2, the central metabolite of the synthetic pathway. The mutation R191S present in methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) abolishes the inhibition of cyclohydrolase activity by the substrate formyl-tetrahydrofolate. The mutation R273H in lipoamide dehydrogenase (Lpd) alters substrate affinities as well as kinetics at physiological substrate concentrations likely favoring a reactional shift towards lipoamide reduction. In addition, genetic reconstructions proved the necessity of all three mutations for formate assimilation by the adapted cells. The largely unpredictable nature of these changes demonstrates the usefulness of the evolutionary approach enabling the selection of adaptive mutations crucial for pathway engineering of biotechnological model organisms