Microbial biotechnology meets environmental microbiology

Abstract

The isolation of commercially valuable bacteria from the environment has been a cornerstone of microbial biotechnology for many decades. The environment has yielded organisms capable of producing valuable fermentation products such as alcohols and amino acids, strains able to produce diverse pharmacologically active secondary metabolites, as well as microorganisms that can affect highly selective chemical transformations and convert recalcitrant pollutants into non‐toxic metabolites. As Microbial Biotechnology (sister journal to the now well established Environmental Microbiology) celebrates its first birthday, we would like to speculate upon the way in which the ongoing revolution in the characterization of uncultured environmental microorganisms may facilitate discovery of valuable microbial enzymes and pathways that are currently beyond reach. The great majority of microorganisms in natural environments have never been obtained in pure culture and represent an important source of microbial diversity that biotechnologists cannot ignore. Cloning of environmental DNA (metagenomics) has already emerged as a rich source of new biocatalysts for production of bulk and high‐value chemicals (reviewed in Steele et al., 2009). Metagenomics is reliant on the cloning of genes from complex samples that contain DNA from all manner of organisms, some relevant to the biotechnologist but most otherwise. Hence methodology for specifically increasing the abundance of functional genes (genes encoding key target enzymes) of interest would be of great value in increasing the proportion of the relevant biodiversity that could be accessed. In the sphere of environmental microbiology, stable isotope probing (SIP) techniques employ enrichment cultures containing a 13C‐labelled growth substrate, in which the DNA of organisms growing on the labelled substrate becomes enriched in the heavy isotope and can be separated from bulk environmental DNA by means of CsCl density gradient centrifugation. Originally developed to identify organisms actively metabolizing one‐carbon compounds via analysis of 16S rRNA and functional genes, SIP has since been applied to characterize microorganisms utilizing a wide range of microbiological growth substrates (reviewed in Dumont and Murrell, 2005; Friedrich, 2006). In principle, SIP is ideal for increasing the abundance of target genes for subsequent direct cloning or amplification by means of PCR. A report from Daniel and co‐workers (Schwarz et al., 2006) was the first that indicated the feasibility of such applications. Daniel and co‐workers focused on glycerol dehydratase, a key enzyme during biosynthesis of the valuable product propane‐1,3‐diol. SIP with glycerol‐13C3 led to an increase of up to 3.8‐fold in the frequency of recovery of glycerol dehydratase genes per megabase of cloned environmental DNA, compared with parallel metagenomics experiments where SIP enrichment was not used. While the increase in sensitivity that SIP yielded in this pilot study was modest, we predict that through careful manipulations of enrichment conditions, SIP and related techniques can be developed into a key tool in gene mining. DNA‐SIP would give access to valuable functional genes that are present at very low abundance in inhospitable extreme environments or at low levels in complex ecosystems and which are below the threshold of detection of current technology. This would generate a pool of potentially novel target genes that could then be screened in expression libraries or used in gene shuffling experiments in order to generate novel biocatalysts. In addition, heavy DNA in the SIP experiments will become enriched in the genomes of target organisms, thus allowing focussed or targeted metagenomics, and isolation of potentially novel catabolic (or biosynthetic) gene clusters of biotechnological relevance.