ABSTRACT
Polyketides and nonribosomal peptides constitute two large families of microbial natural products. Over the past 20 years a broad range of microbial polyketide and nonribosomal peptide biosynthetic pathways have been characterized leading to a surfeit of genetic data on polyketide and nonribosomal peptide biosynthesis. We developed the ClusterMine360 database, which stores the antiSMASH-based annotation of gene clusters in the NCBI database, linking the structure of the natural product to the biosynthetic gene cluster. This database is searchable and enables the user to access multiple sequence files for phylogenetic analysis of polyketide and nonribosomal peptide biosynthetic genes. Herein we describe how to add compound families and gene clusters to the database and search it using key words or structures to identify specific gene clusters. We also describe how to download multiple sequence files for specific catalytic domains from polyketide and nonribosomal peptide biosynthesis.
Subject(s)
Bacteria/genetics , Biosynthetic Pathways , Genomics/methods , Multigene Family , Peptide Biosynthesis, Nucleic Acid-Independent , Polyketides/metabolism , Bacteria/metabolism , Biological Products/chemistry , Biological Products/metabolism , Databases, Genetic , Peptides/chemistry , Peptides/metabolism , Polyketides/chemistryABSTRACT
BACKGROUND: Heterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products. METHODOLOGY: We have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ(54) directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ(54) promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways. CONCLUSIONS: We have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ(54) may be a viable method for the production of additional polyketide products.
Subject(s)
Biosynthetic Pathways , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Polyketides/metabolism , RNA Polymerase Sigma 54/metabolism , Anti-Bacterial Agents/pharmacology , Base Sequence , Benzothiazoles , Diamines , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial/drug effects , Molecular Sequence Data , Organic Chemicals/metabolism , Oxytetracycline/biosynthesis , Oxytetracycline/chemistry , Oxytetracycline/pharmacology , Peptides/metabolism , Polyketides/chemistry , Promoter Regions, Genetic/genetics , Quinolines , RNA Polymerase Sigma 54/genetics , Transcription, Genetic/drug effectsABSTRACT
ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and non-ribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data consistency and quality. The database currently has >200 gene clusters from >185 compound families. It also features a unique sequence repository containing >10 000 polyketide synthase/non-ribosomal peptide synthetase domains. The sequences are filterable and downloadable as individual or multiple sequence FASTA files. We are confident that this database will be a useful resource for members of the polyketide synthases/non-ribosomal peptide synthetases research community, enabling them to keep up with the growing number of sequenced gene clusters and rapidly mine these clusters for functional information.
