Deep-sea anoxic brine pools of the Red Sea are considered one of the most remote and extreme environments on Earth while remaining one of the least studied. High salt concentrations (4.3 M), elevated temperatures (up to 68°C) and high metal content make them promising sources for novel enzymes with structures distinguished by evolutionary adaption. Reliable functional annotation of genomic data is the key-step in the discovery of novel enzymes. Therefore, we developed a Profile & Pattern Matching algorithm to minimize false positive annotations of far distant related genes [1]. Based on scientific and industrial interest 13 genes were selected for expression in halophilic hosts. To optimize expression, the Haloferax volcanii was genetically modified, creating the first halophilic expression system, which is applicable in a bioreactor [2]. This system achieves a 28-fold increase in total protein yield in comparison to shaking flasks. In this presentation, we will detail the expression of two genes isolated from Red Sea brine pool SAGs: an alcohol dehydrogenase and a carbonic anhydrase. Both enzymes are extremely tolerant to high temperatures (up to 80 °C), a broad range of salinity (from millimolar to saturation) and high solvent concentrations. Structural insights based on crystallographic data show principles of salt tolerance, adoption to elevated temperatures as well as promising amino acids for computationally guided evolution of this biocatalyst. We will discuss the development and verification of a complete pipeline to discover extremozymes from genetic data to structure. [1] Grötzinger, S. W.; et al., Frontiers in microbiology 2014, 5, 134 [2] Grötzinger, S. W.; et al., Appl. Microbiol. Biotechnol. 2016, 100 (3), 1183-1195

Discovery, characterization and applications of a novel extremozymes from the deep Red-Sea brine pools

253rd National Meeting (2017)

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