deFUME: Dynamic exploration of functional metagenomic sequencing data

BACKGROUND: Functional metagenomic selections represent a powerful technique that is widely applied for identification of novel genes from complex metagenomic sources. However, whereas hundreds to thousands of clones can be easily generated and sequenced over a few days of experiments, analyzing the data is time consuming and constitutes a major bottleneck for experimental researchers in the field. FINDINGS: Here we present the deFUME web server, an easy-to-use web-based interface for processing, annotation and visualization of functional metagenomics sequencing data, tailored to meet the requirements of non-bioinformaticians. The web-server integrates multiple analysis steps into one single workflow: read assembly, open reading frame prediction, and annotation with BLAST, InterPro and GO classifiers. Analysis results are visualized in an online dynamic web-interface. CONCLUSION: The deFUME webserver provides a fast track from raw sequence to a comprehensive visual data overview that facilitates effortless inspection of gene function, clustering and distribution. The webserver is available at cbs.dtu.dk/services/deFUME/and the source code is distributed at github.com/EvdH0/deFUME. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13104-015-1281-y) contains supplementary material, which is available to authorized users

Identification and Engineering of Transporters for Efficient Melatonin Production in Escherichia coli

Transporter discovery and engineering play an important role in cell factory development. Decreasing the intracellular concentration of the product reduces product inhibition and/or toxicity. Lowering intracellular concentrations is especially beneficial for achieving a robust strain at high titers. However, the identification of transporters for xenobiotic chemicals in the host strain is challenging. Here we present a high-throughput workflow to discover Escherichia coli transporters responsible for the efflux of the inhibitory xenobiotic compound melatonin. We took advantage of the Keio collection and screened about 400 transporter knockouts in the presence of a high concentration of melatonin. We found five transporters that when knocked out showed decreased tolerance to melatonin, indicating they are exporters of melatonin. We overexpressed these five genes individually in the production strain and found that one of them, yhjV, encoding a transporter with unknown substrates, resulted in a 27% titer increase in cultivation mimicking fed-batch fermentation. This study demonstrates how microbial cell factories can be improved through transporter identification and engineering. Further, these results lay the foundation for the scale-up of melatonin production in E. coli

A Novel Efficient L-Lysine Exporter Identified by Functional Metagenomics

Lack of active export system often limits the industrial bio-based production processes accumulating the intracellular product and hence complexing the purification steps. L-lysine, an essential amino acid, is produced biologically in quantities exceeding two million tons per year; yet, L-lysine production is challenged by efficient export system at high titers during fermentation. To address this issue, new exporter candidates for efficient efflux of L-lysine are needed. Using metagenomic functional selection, we identified 58 genes encoded on 28 unique metagenomic fragments from cow gut microbiome library that improved L-lysine tolerance. These genes include a novel L-lysine transporter, belonging to a previously uncharacterized EamA superfamily, which is further in vivo characterized as L-lysine exporter using Xenopus oocyte expression system as well as Escherichia coli host. This novel exporter improved L-lysine tolerance in E. coli by 40% and enhanced yield, titer, and the specific production of L-lysine in an industrial Corynebacterium glutamicum strain by 7.8%, 9.5%, and 12%, respectively. Our approach allows the sequence-independent discovery of novel exporters and can be deployed to increase titers and productivity of toxicity-limited bioprocesses