The soil metagenome – a rich resource for the discovery of novel natural products Rolf Daniel Soil microorganisms have been the most valuable source of natural products, providing industrially important antibiotics and biocatalysts. But, of late, the discovery rate of novel biomolecules using traditional cultivation techniques has been extremely low, as most soil microorganisms cannot be cultured in this way. The development of novel cultivation-dependent and molecular cultivation-independent approaches has paved the way for a new era of product recovery from soil microorganisms. In particular, gene-mining based on the construction and screening of complex libraries derived from the soil metagenome provides opportunities to fully explore and exploit the enormous genetic and metabolic diversity of soil microorganisms. This strategy has already resulted in the isolation of novel biocatalysts and bioactive molecules. Addresses Institut fu ¨ r Mikrobiologie und Genetik der Georg-August-Universita ¨ t, Grisebachstrasse 8, 37077 Go ¨ ttingen, Germany e-mail: rdaniel@gwdg.de Current Opinion in Biotechnology 2004, 15:199–204 This review comes from a themed issue on Environmental biotechnology Edited by Michael Y Galperin and Alan JM Baker Available online 30th April 2004 0958-1669/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2004.04.005 Abbreviation BAC bacterial artificial chromosome Introduction Soil is a very complex habitat dominated by the soil solid phase. In contrast to water systems, soil is relatively recalcitrant to mixing, but soluble components of the solid soil matrix may dissolve in soil water and repreci- pitate at other sites. The soil microorganisms are localized in close association with soil particles, such as complexes of clay–organic matter [1]. Microorganisms can be found as single cells or microcolonies, which are often em- bedded in a polysaccharide matrix. Their metabolism and interactions with other organisms and with soil par- ticles is dependent on the conditions at the microhabitat level, which often differ between microhabitats even over very small distances. The microhabitats for soil micro- organisms include micropores and the surfaces of soil aggregates of various composition and sizes [2,3]. Thus, soil can be regarded as very heterogeneous with respect to conditions for microbial growth and for the distribution of microorganisms and matrix substances. This heterogene- ity results in a wide variety of microbial niches and a high diversity of soil microorganisms. The microbial diversity in soils exceeds that of other environments and is far greater than that of eukaryotic organisms: one gram of soil can contain up to 10 billion microorganisms of possibly thousands of different species [4]. The genetic complex- ity of microbial soil communities has been estimated by reassociation of community DNA. Not taking into account the genomes of rare and unrecovered microor- ganisms, such analyses have shown that the soil commu- nity size is equivalent to 6000–10 000 Escherichia coli genomes [5,6]. In another study, the analysis of the reassociation kinetics of the total bacterial DNA in a 30 g soil sample revealed that it contained more than 500 000 species [7]. These results illustrate that the entirety of the microbial genomes found in soil samples, termed the soil metagenome throughout this review, harbors more genetic information than is contained in the approximately 5000 to 6000 individual prokaryotic microorganisms available in the different culture collec- tions. Thus, the genetic diversity of the soil metagenome is a rich and widely unexplored resource for new indus- trial enzymes and bioactive compounds. In this review, the recent progress in recovering novel biocatalysts and other biomolecules from the soil meta- genome is summarized. An overview of novel cultivation- dependent and molecular cultivation-independent approaches to access and exploit the diversity of soil microbial communities with respect to natural product discovery is given. Cultivation-dependent approaches The diversity of soil microorganisms has been exploited for many years based on the cultivation and isolation of microbial species. Most natural products of economic value such as antibiotics or other pharmaceuticals are derived from cultured soil microorganisms, but the rate of discovery of novel natural products derived from iso- lated microorganisms has significantly decreased during the past couple of years [8]. This is mainly due to a high rediscovery frequency, as the same organisms are recov- ered repeatedly by using standard cultivation techniques. It has been estimated that 0.1–1% of the total soil popula- tion can be cultured by applying these techniques. In a study by Kellenberger [9], approximately 10 7 cells were counted in 1 g of soil, but only 0.1% of the cells were culturable. Thus, 99.9% of the genetic diversity present in this population was lost owing to difficulties in enrich- ing and isolating microorganisms. These unrecovered www.sciencedirect.com Current Opinion in Biotechnology 2004, 15:199–204