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Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli.

ABSTRACT: Environmental DNA libraries prepared from three different soil samples were screened for genes conferring lipolytic activity on Escherichia coli clones. Screening on triolein agar revealed 1 positive clone out of 730,000 clones, and screening on tributyrin agar revealed 3 positive clones out of 286,000 E. coli clones. Substrate specificity analysis revealed that one recombinant strain harbored a lipase and the other three contained esterases. The genes responsible for the lipolytic activity were identified and characterized.

SUBMITTER: Henne A

PROVIDER: S-EPMC92121 | biostudies-literature | 2000 Jul

REPOSITORIES: biostudies-literature

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Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli.

Henne A A Schmitz R A RA Bömeke M M Gottschalk G G Daniel R R

Applied and environmental microbiology 20000701 7

Environmental DNA libraries prepared from three different soil samples were screened for genes conferring lipolytic activity on Escherichia coli clones. Screening on triolein agar revealed 1 positive clone out of 730,000 clones, and screening on tributyrin agar revealed 3 positive clones out of 286,000 E. coli clones. Substrate specificity analysis revealed that one recombinant strain harbored a lipase and the other three contained esterases. The genes responsible for the lipolytic activity were ...[more]

PMID: 10877816

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Project description:Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C(2) to C(4)) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any other known gene product.

Project description:Lipolytic enzymes are one of the most important enzyme types for application in various industrial processes. Despite the continuously increasing demand, only a small portion of the so far encountered lipolytic enzymes exhibit adequate stability and activities for biotechnological applications. To explore novel and/or extremophilic lipolytic enzymes, microbial consortia in two composts at thermophilic stage were analyzed using function-driven and sequence-based metagenomic approaches. Analysis of community composition by amplicon-based 16S rRNA genes and transcripts, and direct metagenome sequencing revealed that the communities of the compost samples were dominated by members of the phyla Actinobacteria, Proteobacteria, Firmicutes, Bacteroidetes, and Chloroflexi. Function-driven screening of the metagenomic libraries constructed from the two samples yielded 115 unique lipolytic enzymes. The family assignment of these enzymes was conducted by analyzing the phylogenetic relationship and generation of a protein sequence similarity network according to an integrated classification system. The sequence-based screening was performed by using a newly developed database, containing a set of profile Hidden Markov models, highly sensitive and specific for detection of lipolytic enzymes. By comparing the lipolytic enzymes identified through both approaches, we demonstrated that the activity-directed complements sequence-based detection, and vice versa. The sequence-based comparative analysis of lipolytic genes regarding diversity, function and taxonomic origin derived from 175 metagenomes indicated significant differences between habitats. Analysis of the prevalent and distinct microbial groups providing the lipolytic genes revealed characteristic patterns and groups driven by ecological factors. The here presented data suggests that the diversity and distribution of lipolytic genes in metagenomes of various habitats are largely constrained by ecological factors.

Dataset Information

Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli.

Publications

Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli.

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