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Establishing sustainable approaches for human space exploration is key to achieve independency from terrestrial resources, as well as for ethical considerations. Here the authors highlight microbial biotechnologies that will support sustainable processes for space-based in situ resource utilization and loop-closure, and may be translatable to Earth applications.

Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (indel) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time.

The biomining microbes which extract metals from ores that have been applied in mining processes worldwide hold potential for harnessing space resources. Their cell growth and ability to extract metals from extraterrestrial minerals under microgravity environments, however, remains largely unknown. The present study used the model biomining bacterium Acidithiobacillus ferrooxidans to extract metals from lunar and Martian regolith simulants cultivated in a rotating clinostat with matched controls grown under the influence of terrestrial gravity. Analyses included assessments of final cell count, size, morphology, and soluble metal concentrations. Under Earth gravity, with the addition of Fe3+ and H2/CO2, A. ferrooxidans grew in the presence of regolith simulants to a final cell density comparable to controls without regoliths. The simulated microgravity appeared to enable cells to grow to a higher cell density in the presence of lunar regolith simulants. Clinostat cultures of A. ferrooxidans solubilised higher amounts of Si, Mn and Mg from lunar and Martian regolith simulants than abiotic controls. Electron microscopy observations revealed that microgravity stimulated the biosynthesis of intracellular nanoparticles (most likely magnetite) in anaerobically grown A. ferrooxidans cells. These results suggested that A. ferrooxidans has the potential for metal bioleaching and the production of useful nanoparticles in space.

A central goal in molecular evolution is to understand the ways in which genes and proteins evolve in response to changing environments. In the absence of intact DNA from fossils, ancestral sequence reconstruction (ASR) can be used to infer the evolutionary precursors of extant proteins. To date, ancestral proteins belonging to eubacteria, archaea, yeast and vertebrates have been inferred that have been hypothesized to date from between several million to over 3 billion years ago. ASR has yielded insights into the early history of life on Earth and the evolution of proteins and macromolecular complexes. Recently, however, ASR has developed from a tool for testing hypotheses about protein evolution to a useful means for designing novel proteins. The strength of this approach lies in the ability to infer ancestral sequences encoding proteins that have desirable properties compared with contemporary forms, particularly thermostability and broad substrate range, making them good starting points for laboratory evolution. Developments in technologies for DNA sequencing and synthesis and computational phylogenetic analysis have led to an escalation in the number of ancient proteins resurrected in the last decade and greatly facilitated the use of ASR in the burgeoning field of synthetic biology. However, the primary challenge of ASR remains in accurately inferring ancestral states, despite the uncertainty arising from evolutionary models, incomplete sequences and limited phylogenetic trees. This review will focus, firstly, on the use of ASR to uncover links between sequence and phenotype and, secondly, on the practical application of ASR in protein engineering. [ABSTRACT FROM AUTHOR]