A Novel Acyl-CoA Beta-Transaminase Characterized from a Metagenome (original) (raw)

Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase

Journal of Biological Chemistry

In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of B-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2kilobase NcoI-CZaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M, = 62,000 using SDS-

Metagenomic analysis of uncultured microorganisms and their enzymatic attributes

Journal of Microbiological Methods, 2018

Although second generation biofuel technology is a sustainable route for bioethanol production it is not currently a robust technology because of certain hindrances viz., unavailability of potential enzyme resources, low efficiency of enzymes and restricted availability of potent enzymes that work under harsh conditions in industrial processes. Therefore, bioprospecting of extremophilic microorganisms using metagenomics is a promising alternative to discover novel microbes and enzymes with efficient tolerance to unfavourable conditions and thus could revolutionize the energy sector. Metagenomics a recent field in "omics" technology enables the genomic study of uncultured microorganisms with the goal of better understanding microbial dynamics. Metagenomics in conjunction with NextGen Sequencing technology facilitates the sequencing of microbial DNA directly from environmental samples and has expanded, and transformed our knowledge of the microbial world. However, filtering the meaningful information from the millions of genomic sequences offers a serious challenge to bioinformaticians. The current review holds the opinion tool 'knowhow' to unravel the secrets of nature while expediting the bio-industrial world. We also discuss the novel biocatalytic agents discovered through metagenomics and how bioengineering plays a pivotal role to enhance their efficiency.

Identification of syntrophic acetate-oxidizing bacteria in anaerobic digesters by combined protein-based stable isotope probing and metagenomics

The ISME Journal, 2016

Inhibition of anaerobic digestion through accumulation of volatile fatty acids occasionally occurs as the result of unbalanced growth between acidogenic bacteria and methanogens. A fast recovery is a prerequisite for establishing an economical production of biogas. However, very little is known about the microorganisms facilitating this recovery. In this study, we investigated the organisms involved by a novel approach of mapping protein-stable isotope probing (protein-SIP) onto a binned metagenome. Under simulation of acetate accumulation conditions, formations of 13 C-labeled CO 2 and CH 4 were detected immediately following incubation with [U-13 C]acetate, indicating high turnover rate of acetate. The identified 13 C-labeled peptides were mapped onto a binned metagenome for improved identification of the organisms involved. The results revealed that Methanosarcina and Methanoculleus were actively involved in acetate turnover, as were five subspecies of Clostridia. The acetateconsuming organisms affiliating with Clostridia all contained the FTFHS gene for formyltetrahydrofolate synthetase, a key enzyme for reductive acetogenesis, indicating that these organisms are possible syntrophic acetate-oxidizing (SAO) bacteria that can facilitate acetate consumption via SAO, coupled with hydrogenotrophic methanogenesis (SAO-HM). This study represents the first study applying protein-SIP for analysis of complex biogas samples, a promising method for identifying key microorganisms utilizing specific pathways.

Comparative genomics and metabolic profiling of the genus Lysobacter

BMC Genomics, 2015

Background: Lysobacter species are Gram-negative bacteria widely distributed in soil, plant and freshwater habitats. Lysobacter owes its name to the lytic effects on other microorganisms. To better understand their ecology and interactions with other (micro)organisms, five Lysobacter strains representing the four species L. enzymogenes, L. capsici, L. gummosus and L. antibioticus were subjected to genomics and metabolomics analyses. Results: Comparative genomics revealed a diverse genome content among the Lysobacter species with a core genome of 2,891 and a pangenome of 10,028 coding sequences. Genes encoding type I, II, III, IV, V secretion systems and type IV pili were highly conserved in all five genomes, whereas type VI secretion systems were only found in L. enzymogenes and L. gummosus. Genes encoding components of the flagellar apparatus were absent in the two sequenced L. antibioticus strains. The genomes contained a large number of genes encoding extracellular enzymes including chitinases, glucanases and peptidases. Various nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) gene clusters encoding putative bioactive metabolites were identified but only few of these clusters were shared between the different species. Metabolic profiling by imaging mass spectrometry complemented, in part, the in silico genome analyses and allowed visualisation of the spatial distribution patterns of several secondary metabolites produced by or induced in Lysobacter species during interactions with the soil-borne fungus Rhizoctonia solani. Conclusions: Our work shows that mining the genomes of Lysobacter species in combination with metabolic profiling provides novel insights into the genomic and metabolic potential of this widely distributed but understudied and versatile bacterial genus.

Transcriptional and Functional Analysis of Oxalyl-Coenzyme A (CoA) Decarboxylase and Formyl-CoA Transferase Genes from Lactobacillus acidophilus

Applied and Environmental Microbiology, 2006

Oxalic acid is found in dietary sources (such as coffee, tea, and chocolate) or is produced by the intestinal microflora from metabolic precursors, like ascorbic acid. In the human intestine, oxalate may combine with calcium, sodium, magnesium, or potassium to form less soluble salts, which can cause pathological disorders such as hyperoxaluria, urolithiasis, and renal failure in humans. In this study, an operon containing genes homologous to a formyl coenzyme A transferase gene ( frc ) and an oxalyl coenzyme A decarboxylase gene ( oxc ) was identified in the genome of the probiotic bacterium Lactobacillus acidophilus . Physiological analysis of a mutant harboring a deleted version of the frc gene confirmed that frc expression specifically improves survival in the presence of oxalic acid at pH 3.5 compared with the survival of the wild-type strain. Moreover, the frc mutant was unable to degrade oxalate. These genes, which have not previously been described in lactobacilli, appear to...

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.

The Escherichia coli fadK (ydiD) Gene Encodes an Anerobically Regulated Short Chain Acyl-CoA Synthetase

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.

Metagenomic discovery of novel enzymes and biosurfactants in a slaughterhouse biofilm microbial community

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