A Novel Acyl-CoA Beta-Transaminase Characterized from a Metagenome (original) (raw)
Identification of the Last Unknown Genes in the Fermentation Pathway of Lysine
Journal of Biological Chemistry, 2006
Although the proteins of the lysine fermentation pathway were biochemically characterized more than thirty years ago, the genes encoding the proteins that catalyze three steps of this pathway are still unknown. We combined gene context, similarity of enzymatic mechanisms, and molecular weight comparisons with known proteins to select candidate genes for these three orphan proteins. We used a wastewater metagenomic collection of sequences to find and characterize the missing genes of the lysine fermentation pathway. After recombinant protein production and purification following cloning in Escherichia coli, we demonstrated that these genes (named kdd, kce, and kal) encode a L-erythro-3,5-diaminohexanoate dehydrogenase, a 3-keto-5-aminohexanoate cleavage enzyme, and a 3-aminobutyryl-CoA ammonia lyase, respectively. Because all of the genes of the pathway are now identified, we used this breakthrough to detect lysine-fermenting bacteria in sequenced genomes. We identified twelve bacteria that possess these genes and thus are expected to ferment lysine, and their gene organization is discussed.
Journal of Biological Chemistry, 2004
We recently reported a new metabolic competency for Escherichia coli, the ability to degrade and utilize fatty acids of various chain lengths as sole carbon and energy sources (Campbell, J. W., Morgan-Kiss, R. M., and Cronan J. E. (2003) Mol. Microbiol. 47, 793-805). This -oxidation pathway is distinct from the previously described aerobic fatty acid degradation pathway and requires enzymes encoded by two operons, yfcYX and ydiQRSTD. The yfcYX operon (renamed fadIJ) encodes enzymes required for hydration, oxidation, and thiolytic cleavage of the acyl chain. The ydiQRSTD operon encodes a putative acyl-CoA synthetase, ydiD (renamed fadK), as well as putative electron transport chain components. We report that FadK is as an acyl-CoA synthetase that has a preference for short chain length fatty acid substrates (<10 C atoms).
Microbial metagenomes: moving forward industrial biotechnology
Journal of Chemical Technology & Biotechnology, 2007
Biotechnology, in terms of exploitation of catalytic activities for industrial applications, is increasingly recognized as one of the pillars of the knowledge-based economy that we are heading for. Comprehensive knowledge of enzymology should be of practical importance for effective intervention on whole cell processes and enzymatic networks. Over the last decade metagenome-based technologies have been developed to take us farther and deeper into the enzyme universe from uncultivable microbes. This sophisticated platform, which identifies new enzymes from vast genetic pools available, and assesses their potential for novel chemical applications, should be increasingly important in the discovery of advanced biotechnological resources.
Scientific Reports, 2016
DNA derived from environmental samples is a rich source of novel bioactive molecules. The choice of the habitat to be sampled predefines the properties of the biomolecules to be discovered due to the physiological adaptation of the microbial community to the prevailing environmental conditions. We have constructed a metagenomic library in Escherichia coli DH10b with environmental DNA (eDNA) isolated from the microbial community of a slaughterhouse drain biofilm consisting mainly of species from the family Flavobacteriaceae. By functional screening of this library we have identified several lipases, proteases and two clones (SA343 and SA354) with biosurfactant and hemolytic activities. Sequence analysis of the respective eDNA fragments and subsequent structure homology modelling identified genes encoding putative N-acyl amino acid synthases with a unique two-domain organisation. The produced biosurfactants were identified by NMR spectroscopy as N-acyltyrosines with N-myristoyltyrosine as the predominant species. Critical micelle concentration and reduction of surface tension were similar to those of chemically synthesised N-myristoyltyrosine. Furthermore, we showed that the newly isolated N-acyltyrosines exhibit antibiotic activity against various bacteria. This is the first report describing the successful application of functional high-throughput screening assays for the identification of biosurfactant producing clones within a metagenomic library. Metagenomics allow to access novel biocatalysts and metabolites from organisms that are not cultivable 1-3. In sequence-based approaches, genes are detected using DNA probes or degenerate oligonucleotides derived from known genes encoding the protein family of interest or by homology search of datasets obtained from eDNA deep sequencing 1,2,4. In contrast, phenotypic screening approaches apply activity-based assays enabling the discovery of so far unknown proteins belonging to completely novel families. Since the environmental conditions shape the microbial diversity, the choice of the respective habitat is essential for successful mining for novel biocatalysts as shown for cold, hot, and halophilic, habitats 5,6. Furthermore, nutrient availability largely determines the spectrum of enzymes to be identified 7,8. Regarding phenotypic screening approaches, functional expression of the cognate genes in standard laboratory host strains as well as the availability of efficient screening assays are necessary prerequisites 9,10. Many biotechnological relevant enzymes including hydrolases and oxidoreductases have already been discovered by metagenomic screenings 7,11,12. In addition, several secondary metabolites including patellamide D, violaceins, and polytheonamides were successfully isolated in metagenome studies 2,13,14. However, the discovery of secondary metabolites still remains challenging, probably due to the demand of proper precursor molecules, functionally interacting auxiliary proteins and the necessity for heterologous expression of large gene clusters 15,16. Another
AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization
Archaea, 2006
Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1.
Impact of Genomics and Genetics on the Elucidation of Bacterial Metabolism
Methods, 2000
In the last few years, the emergence of complete genome sequences has had profound effects on all fields of biology. While the existence of these genome sequences has served to facilitate experimental work, it has also highlighted the gaps in our knowledge of bacterial metabolism. Our current knowledge of metabolism is primarily the result of data accumulated from decades of study by biochemists and geneticists. In general these studies focused on discrete pathways and their regulation. The technical innovations of the last decade, culminating with the sequencing of complete genomes, provide us with the ability to address the next frontier in physiology, metabolic integration. Herein we describe current approaches that can be used to complement classic genetic approaches and further our understanding of both novel metabolic functions and metabolic integration in microorganisms.
Journal of Bacteriology, 1996
The enzymes beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase (BHBD), crotonase, and butyryl-CoA dehydrogenase (BCD) from Clostridium acetobutylicum are responsible for the formation of butyryl-CoA from acetoacetyl-CoA. These enzymes are essential to both acid formation and solvent formation by clostridia. Clustered genes encoding BHBD, crotonase, BCD, and putative electron transfer flavoprotein alpha and beta subunits have been cloned and sequenced. The nucleotide sequence of the crt gene indicates that it encodes crotonase, a protein with 261 amino acid residues and a calculated molecular mass of 28.2 kDa; the hbd gene encodes BHBD, with 282 residues and a molecular mass of 30.5 kDa. Three open reading frames (bcd, etfB, and etfA) are located between crt and hbd. The nucleotide sequence of bcd indicates that it encodes BCD, which consists of 379 amino acid residues and has high levels of homology with various acyl-CoA dehydrogenases. Open reading frames etfB and etfA, located dow...
Carbohydrate actives enzymes derived from metagenomes: from microbial ecology to enzymology
"In the last ten years, the intensive mining of various environmental metagenomes has led to the discovery of numerous new genes and corresponding putative enzymes. Some enzymes were isolated for their ability to hydrolyze carbohydrates, including starch, xylan, chitin and cellulose derivatives. The accurate characterization of these proteins highlights their variability and their biophysical adaptation in order to cope with specific environmental conditions. In this perspective, the sampling of extreme environments for metagenomic library construction resulted in the isolation of enzymes harbouring tailor made properties aimed at their implementation in various industrial processes. Although these new catalysts appear to be of particular interest for biotechnological applications, little is known about their physiological functions in their natural host. In the field of glycosides hydrolases, different functions have been suggested including both hydrolysis and synthesis of polymers. On the one hand, indeed in the environment microorganisms compete for ecological niches by producing enzymes active against vast ranges of substrates which allow them to thrive on various carbon sources. On the other hand, production of structural (cellulose) or reserve (glycogen) polymers by bacteria such as Gluconacetobacter sp. was reported. Polysaccharides can be associated with bacterial biofilm and feed stock, compounds that are required for bacteria to live in various environments. Interestingly, the synthesis of these polymers requires enzymes which act on carbohydrate including enzymes referred to as glycoside hydrolases acting as transglycosylases. In this chapter, a review of the representative glycoside hydrolases isolated by metagenomic and their possible physiological functions are presented."
Novel metabolic features in Acinetobacter baylyi ADP1 revealed by a multiomics approach
Metabolomics, 2014
Expansive knowledge of bacterial metabolism has been gained from genome sequencing output, but the high proportion of genes lacking a proper functional annotation in a given genome still impedes the accurate prediction of the metabolism of a cell. To access to a more global view of the functioning of the soil bacterium Acinetobacter baylyi ADP1, we adopted a multi 'omics' approach. Application of RNA-seq transcriptomics and LC/MS-based metabolomics, along with the systematic phenotyping of the complete collection of single-gene deletion mutants of A. baylyi ADP1 made possible to interrogate on the metabolic perturbations encountered by the bacterium upon a biotic change. Shifting the sole carbon source from succinate to quinate elicited in the cell not only a specific transcriptional response, necessary to catabolize the new carbon source, but also a major reorganization of the transcription pattern. Here, the expression of more than 12 % of the total number of genes was affected, most of them being of unknown function. These perturbations were ultimately reflected in the metabolome, in which the concentration of about 50 % of the LC/MSdetected metabolites was impacted. And the differential regulation of many genes of unknown function is probably related to the synthesis of the numerous unidentified compounds that were present exclusively in quinate-grown cells. Together, these data suggest that A. baylyi ADP1 metabolism involves unsuspected enzymatic reactions that await discovery.