Ancestral sequence reconstruction as an accessible tool for the engineering of biocatalyst stability

Synthetic biology is the engineering of life to imbue non-natural functionality. As such, synthetic biology has considerable commercial potential, where synthetic metabolic pathways are utilised to convert low value substrates into high value products. High temperature biocatalysis offers several system-level benefits to synthetic biology, including increased dilution of substrate, increased reaction rates and decreased contamination risk. However, the current gamut of tools available for the engineering of thermostable proteins are either expensive, unreliable, or poorly understood, meaning their adoption into synthetic biology workflows is treacherous. This thesis focuses on the development of an accessible tool for the engineering of protein thermostability, based on the evolutionary biology tool ancestral sequence reconstruction (ASR). ASR allows researchers to walk back in time along the branches of a phylogeny and predict the most likely representation of a protein family's ancestral state. It also has simple input requirements, and its output proteins are often observed to be thermostable, making ASR tractable to protein engineering. Chapter 2 explores the applicability of multiple ASR methods to the engineering of a carboxylic acid reductase (CAR) biocatalyst. Despite the family emerging only 500 million years ago, ancestors presented considerable improvements in thermostability over their modern counterparts. We proceed to thoroughly characterise the ancestral enzymes for their inclusion into the CAR biocatalytic toolbox. Chapter 3 explores why ASR derived proteins may be thermostable despite a mesophilic history. An in silico toolbox for tracking models of protein stability over simulated evolutionary time at the sequence, protein and population level is built. We provide considerable evidence that the sequence alignments of simulated protein families that evolved at marginal stability are saturated with stabilising residues. ASR therefore derives sequences from a dataset biased toward stabilisation. Importantly, while ASR is accessible, it still requires a steep learning curve based on its requirements of phylogenetic expertise. In chapter 4, we utilise the evolutionary model produced in chapter 3 to develop a highly simplified and accessible ASR protocol. This protocol was then applied to engineer CAR enzymes that displayed dramatic increases in thermostability compared to both modern CARs and the thermostable AncCARs presented in chapter 2.