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Industrial Biotechnology, 2005      A biodiversity-based approach to development of performance enzymes
Eric J. Mathur, Gerardo Toledo, Brian D. Green, Mircea Podar, Toby H. Richardson, Michael Kulwiec, and Hwai W. Chang
Industrial Biotechnology, 2005

Many industrial processes catalyzed by chemical reactions could benefit from the use of enzymes; cost reductions, increased efficiency, improved recovery of products, and reduced use and disposal of toxic chemicals are just some of the advantages that enzymes can deliver. Industrial processes often take place under harsh reaction conditions of temperature and pH that also occur in natural environments. Through the targeted use of applied metagenomics and directed evolution, genes from uncultured microorganisms residing in these extreme environments can be recovered and optimized to produce enzymes with specificities and stabilities tuned to particular industrial reactor conditions.
It has become increasingly clear that we know very little about microorganisms that inhabit the natural environment. During the past hundred years of what might be considered modern microbiology, only about 10,000 strains have been well characterized. Yet, using cultivation-independent approaches to assess microbial diversity, single biotopes containing over 15,000 unique microbial genomes appear to be common in the biosphere. When attempting to isolate microorganisms from extreme environments (extremophiles), the situation is further exacerbated; very few genera of extremophiles are amenable to cultivation under laboratory conditions. As a result, the genes and enzymes produced from extremophilic microorganisms have been difficult to identify, recover, and commercialize.
At Diversa Corporation, we have developed technologies to rapidly obtain enzymes from uncultivatable microorganisms. For the past eleven years, Diversa has worked to advance the field of applied metagenomics by developing and optimizing a series of cultivationindependent recombinant techniques that enable the screening, cloning, and expression of genes and pathways derived from environmental microorganisms at previously unobserved rates. In addition, we have developed complementary directed evolution tools that can be used to improve and optimize the characteristics of the discovered enzymes to match industrial conditions. This Methods paper provides an overview of Diversa's technology platforms and describes how these are used to discover and evolve enzymes from nature. Two examples of commercial products that have been developed will be presented to illustrate this integrative approach for enzyme discovery.

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