A Computational Screen for Type I Polyketide Synthases in Metagenomics Shotgun Data Konrad U. Foerstner, Tobias Doerks, Christopher J. Creevey, Anja Doerks, Peer Bork* European Molecular Biology Laboratory, Heidelberg, Germany Abstract Background: Polyketides are a diverse group of biotechnologically important secondary metabolites that are produced by multi domain enzymes called polyketide synthases (PKS). Methodology/Principal Findings: We have estimated frequencies of type I PKS (PKS I) – a PKS subgroup – in natural environments by using Hidden-Markov-Models of eight domains to screen predicted proteins from six metagenomic shotgun data sets. As the complex PKS I have similarities to other multi-domain enzymes (like those for the fatty acid biosynthesis) we increased the reliability and resolution of the dataset by maximum-likelihood trees. The combined information of these trees was then used to discriminate true PKS I domains from evolutionary related but functionally different ones. We were able to identify numerous novel PKS I proteins, the highest density of which was found in Minnesota farm soil with 136 proteins out of 183,536 predicted genes. We also applied the protocol to UniRef database to improve the annotation of proteins with so far unknown function and identified some new instances of horizontal gene transfer. Conclusions/Significance: The screening approach proved powerful in identifying PKS I sequences in large sequence data sets and is applicable to many other protein families. Citation: Foerstner KU, Doerks T, Creevey CJ, Doerks A, Bork P (2008) A Computational Screen for Type I Polyketide Synthases in Metagenomics Shotgun Data. PLoS ONE 3(10): e3515. doi:10.1371/journal.pone.0003515 Editor: Dawn Field, NERC Centre for Ecology and Hydrology, United Kingdom Received July 7, 2008; Accepted September 22, 2008; Published October 27, 2008 Copyright: 2008 Foerstner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: MetaHit (HEALTH-F4-2007-201052) Competing Interests: The authors have declared that no competing interests exist. * E-mail: bork@embl.de Introduction The majority of the microorganisms on earth cannot be cultured under standard laboratory conditions [1]. Therefore, uncultured organisms from environmental samples are promising sources of new enzymes and chemical compounds with biotechnological and pharmaceutical applications. Currently, three screening techniques are commonly applied for exploring protein functions in environ- mental samples: the function-based, the sequence-based and the substrate-induced gene-expression screening (SIGEX) [2]. Here we present a framework for sequence-based computational screens in environmental shotgun sequences, i.e. metagenomics data. It involves both homology-based and phylogenetic classification. While there has been some success in identifying important subfamilies in metagenomics data [3–6], there are also immense challenges ahead as tools and computational infrastructure often do not scale with the increase in metagenomics data and as many protein families have complicated evolutionary histories. In order to explore a difficult and also important protein family in the context of diverse metagenomics data sets, we have chosen type I polyketide synthases (PKS I) as target proteins for screening. They synthesize a highly diverse group of secondary metabolites that covers many biological functions and have considerable medical relevance. Polyketides in general can act among other functions as antibiotics, immunosuppressants, pigments but also as toxins or carcinogens [7] via different mechanisms. Antibiotics like Erythromycin, Rifamycin and Oleandomycin are only a few examples with medical relevance. Polyketides are usually large chemical compounds that are synthesized in a series of repetitive steps. Similar to the synthesis of fatty acids short acyl-units are added to the growing molecule and are modified. All of these steps are catalyzed by a combination of domains, namely a acyltrans- ferase domain (AT – transfers the acyl unit to the acyl carrier protein), a ketoacyl synthase domain (KS - performs the decarboxylative condensation), and an acyl carrier protein (PP - contains the phosphopantetheinyl arm) domain. Additionally the ketoreductase (KR), the dehydratase (DH), the enoyl reductase (ER) and the methyltransferase (MT) domain can modify the acyl unit after the condensation. The thioesterase domain (TE) releases the finished polyketide. PKS members have been found in bacteria, fungi, plants, slime mold [8], Alveolata [9] and animals [10,11]. Like the fatty acid synthases (FAS), PKS are classified according to the arrangement of their domains: type I with multiple domains per protein and type II in which each single domain represents an independent protein. Bacterial type I PKS are usually modular where each module is responsible for a single fusion step [12] while fungal type I PKS proteins usually occur as ‘‘iteratively’’ acting enzymes in which the domain combinations catalyze several steps. In plants a third class - PKS type III (chalcone synthases) – was discovered and later also described in bacteria [13]. It is common to classify the PKS into these three types although many exceptions of this classification are known [14,15] as the evolution of PKS is rather complex [10,12,16–18]. PLoS ONE | www.plosone.org 1 October 2008 | Volume 3 | Issue 10 | e3515