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BIOTECHNOLOGICAL APPLICATION OF NOVEL ENZYMES FROM METAGENOME LIBRARIES

Final Activity Report Summary - BIOMELI (Biotechnological application of novel enzymes from metagenome libraries)

There is a pervasive perception that undiscovered microbial diversity is enormous and represents a treasure trove for new biotechnological applications. Therefore, one of the most exciting current research endeavours in the exploration of biological and functional diversity, especially the one existing in the extreme conditions which can occur at the limits of the biosphere, or in living organisms that inhabit environments characterised by harsh physicochemical conditions (extremophiles). This will not only lead to the discovery of unknown metabolic and physiological activities and molecular and cellular structures and reveal the range of potential life forms on Planet Earth, but shall also enable the definition of the mechanistic basis of life and enzymatic activities under the most hostile conditions at the extremities of the biosphere.

In this project we posed the question of whether new enzymatic diversity, exemplified by esterases, cellulases and amylase-like enzymes, was to be found in:

1. microbes present in deep sea hypersaline anoxic basins (DHABs), specifically in the brine, i.e. the seawater interface of the Urania west basin, one of five recently discovered DHABs in the southeastern Mediterranean Sea, with the others being the Bannock, Atalante, Discovery and Tyro basins,
2. the rumen content of a New Zealand dairy cow fed on a forage-based diet of ryegrass and clover that consisted of anaerobic and microaerophilic environments characterised by rapid degradation of plant polymers, high substrate fluxes and extreme pH, and
3. ferroplasma acidiphilum (DSM 12658T), a cell wall-lacking acidophilic archaeon with a growth optimum pH of 1.7 isolated from a pyrite-leaching bioreactor fed with pyrite ores from Bakyrtchik, Kazakhstan.

The results of this project, obtained via generation and screening of metagenome expression libraries created from deoxyribonucleic acid (DNA) isolated from the abovementioned environments and organisms, documented the discovery of:

1. five unique esterases from a deep sea Urania hypersaline anoxic basin of the eastern Mediterranean created between five and six million years ago. Interestingly, one enzyme showed rather surprising properties which were up to date unique; it had a unique sequence, contained three active centres and efficiently resolved pharmaceutical intermediates.
2. 22 novel hydrolases from bovine rumen microflora, among which which four enzymes exhibited no sequence similarity to enzymes from public databases and four did not match any putative catalytic residues involved in the catalytic function of esterase and cellulose-like enzymes.
3. three novel -glucosidases from the acidophilic archaeon ferroplasma acidophilum, one of which was a metalloenzyme that had no significant similarity with any known glycoside hydrolase classified in different families, possessed an unusual catalytic site and used a novel mechanism for sugar glycosylation or transglycosylation.

Summing up, the results revealed that the activity-based mining of metagenome libraries not only provided new enzymes for new biotechnological applications and the basis for new insights into protein structure and catalytic mechanisms, but also allowed for functional assignments of many proteins which were found in abundance in the databases and were currently designated as ‘hypothetical’ or ‘conserved hypothetical’ proteins.

Furthermore, the results of this project added new enzymes to the number of available biocatalysts and substantially expanded our knowledge regarding enzyme functions. In addition, the project exemplified Nature’s ability to evolve remarkable biocatalysts with no similarity to known enzymes that bear striking biochemical properties. The latter would be rather impossible to discover without implementing the metagenome approach.