ABSTRACT
At the fundamental level the cellular metabolism is mediated through the enzymes that catalyze biotransformation reactions. e specic reactions transforming the particular substrate in an experimental assay (or in an in vivo system) may be or may not be of a practical interest. However, in case the research focuses on biotransformations-conversions and their biotechnological applications, in most cases a knowledge of a whole genetic circuit, particular pathway, and individual enzymes comprising the pathway are needed. Traditionally, the screening for a specic biocatalytic reaction was performed through the growth of a particular microorganism and the disappearance of the starting compound (reaction substrate) and detection of its disappearance, or/and occurrence and accumulation of
corresponding metabolites [1,2]. e structures of metabolites and the stoichiometry of their formation (determined both, experimentally or in silico) give some important hints to the specicity of an enzyme and its probable usefulness for the application [3]. Whatever the case, the reactions of an apparent new pathway are routinely elucidated via molecular biological methods, typically through molecular cloning and expression of the protein in a surrogate host, such as Escherichia coli, and consequent characterization of its enzymatic activity (Table 5.1). To obtain in a desired biocatalyst a known pathway or reaction
might be taken. e goal is then to obtain an ideal enzyme which is highly active with a given substrate or under specic set of conditions [4]. In this case, the screening method is important, as it might be necessary to look at hundreds or thousands of enzymes in the rst rounds of screening. e description of a wide range of screening methods can be seen in numerous publications, the most relevant being those by the groups of Omura and Cutler [5,6]. Moreover, fast, ecient, and high-throughput screening methods that could test vast numbers of enzymes for specic applications have been discovered in the last decades, bringing made to order enzymes a step closer [7]. Recently, a screening system utilizing the “uorescence activated cell sorting,” or FACS, a technology that enables the identication of biological activity within a single cell, has been developed by Diversa Co. (CA). is system incorporates a laser with multiple wavelength capabilities and the ability to screen up to 50,000 clones per second, or over 1 billion clones per day. Another approach is to test existing isolates of microorganisms to determine if they have an enzyme which show a high activity with the substrate of interest. e pure
cultures of microorganisms have passed the test of time well: those have successfully been used for over 100 years. However, will the advent of the genome-wide high-throughput techniques of enzymatic screening and in silico predictions [8], make the cultivation-based enzyme discovery excessive? Based on genome sequencing data it is currently possible to make a metabolic reconstruction in which all possible known biochemical conversions could be identied. us, a comprehensive metabolic reconstruction on aspergilli revealed up to 14,400 ORFs, 335 reactions, and 284 metabolites distributed over the intra-and extracellular compartments [9]. Moreover, the genomes of extremophilic bacteria and archaea could be even more sophisticated: those from ermotoga, Sulfolobus, Picrophilus spp., containing the ORF numbers in the range of 1,500-4,000, may comprise more complex biochemistries in terms of unique proteins, metabolites, and enzymes under extreme physical-chemical conditions existing in their natural environment [10-12]. Even though the valuable genomic information of individual organisms represents a good starting point, the cultivation of microbes oen represents a bottleneck. e biological diversity on our planet is mostly comprised of microorganisms, in terms of both absolute numbers of cells and in terms of the biomass and the numbers of species [13]. Recently it has been reported that up to 25,000 dierent genotypes in a millilitre of the seawater sample in some marine ecosystems have been found [14]. However, the majority of microbes in nature will never be cultured, and to access this great inexhaustable genetic and metabolic diversity the new “metagenomic” approach is used [15-21]. is approach uses harvesting DNA from an environmental sample (or from an enrichment), its archiving in the metagenomic libraries in appropriate hosts, and screening these libraries for a gene of interest or expressing the DNA and screening for the enzymatic activities of interest [17]. Alternatively, these libraries are subjected to a high throughput shotgun sequencing and automated annotation, which oen produces thousands of new genes whose functions oen remain unknown. is has been shown recently by the group of Venter [22], whose shotgun sequencing of environmental DNA from Sargasso Sea yielded 1,045 billion base pairs derived from 1,800 genomic species and deduced more than 1.2 million of previously unknown genes. However, the work reects diculties on genome assembly and other challenges in interpretation of large data sets. Metagenomics may result in the development of a number of new products and to provide the tools for the resolution of rare conversions which are not amenable to the existing biocatalysts. Below we focus on the issues, what are current challenges for enzymatic conversions and how the natural biodiversity may help to deal with these challenges.
