Expanding the known limits of life through adaptive laboratory evolution, functional metagenomics, and synthetic biology
Authors: Roberts Kingman Garrett , Rothschild Lynn 
Published: March 2022

&lt;p&gt;The intersection of environmental conditions with the conditions permissive for life defines habitability. Consequently, our understanding of habitability is fundamentally limited by our understanding of the multidimensional niche space for life, which up to now, is based on our one known data point: life on Earth. Terrestrial life has evolved to tolerate environmental conditions found on Earth, and as most physiological studies are limited to extant organisms, it is likely that life has potential for a far broader niche space than observed today.&lt;/p&gt;&lt;p&gt;Potentially habitable extraterrestrial environments present challenges not only in single environmental dimensions (temperature, pH, radiation, etc.), but also in combination. For example, Martian brines feature both low temperature and high concentrations of perchlorate, while Venusian clouds feature both desiccating conditions and extreme acidity. We do not know whether the inability of known life to reproduce under analogous conditions reflects a fundamental boundary condition or simply a lack of terrestrial selection pressure. A mixture of environmental challenges may be similarly common among exoplanets and other potentially habitable environments within our solar system.&lt;/p&gt;&lt;p&gt;We are addressing this key gap in our understanding of habitability by using adaptive laboratory evolution, functional metagenomics, and synthetic biology to expand the known environmental limits of life. First, we are determining and pushing the limits of pH (acidic and basic), salt (both chloride and perchlorate), and UV tolerance individually and in combination with temperature for &lt;em&gt;B. subtilis&lt;/em&gt;, &lt;em&gt;E. coli&lt;/em&gt;, and &lt;em&gt;D. radiodurans &lt;/em&gt;through adaptive laboratory evolution. This will define a multidimensional niche-space for these organisms and assess how firm these boundaries are. Second, we are taking advantage of the rich genetic diversity present on Earth to identify genetic elements providing transferable survival benefits under extreme environmental conditions. One of the most powerful resources available to us for this endeavor to expand the boundary conditions of life is the extensive biodiversity present on Earth, particularly those capable of surviving in extreme environments. Prior work demonstrates that extremophile genes can expand an organism&amp;#8217;s niche space, including increased resistance to desiccation, salinity, radiation, and low temperatures. However, despite all we have learned from them, at present it remains difficult and laborious to characterize their genetic mechanisms of adaptation and test their ability to facilitate an enlarged environmental niche. Through a combination of cDNA- and DNA-based libraries, we aim to establish a high throughput method of assaying novel organisms for additional mechanisms of expanding the niche-space of life. Third, we will use codon-optimized cassettes containing genes either identified in our screen or from published research to verify the synthetic acquisition of functional capabilities and to test if the same genetic constructs can expand the niche-space of multiple species.&lt;/p&gt;&lt;p&gt;Through these approaches, we will provide both selection pressure and genetic resources to challenge life to evolve beyond environmental conditions found naturally on Earth. Such work will improve our understanding of what environmental conditions are compatible with life as we know it and allow firm reclassification of some extraterrestrial environments from &amp;#8220;probably habitable&amp;#8221; to &amp;#8220;definitely habitable.&amp;#8221;&lt;/p&gt;