Engineering of a Novel Substrate Specificity of Biotin Carboxylase by Machine Learning Assisted Directed Evolution
Typ
Examensarbete för masterexamen
Program
Biotechnology (MPBIO), MSc
Publicerad
2021
Författare
Leal Garza, Luis M.
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
The engineering of more efficient CO2 fixation mechanisms is an important target that has been addressed by different approaches. Many of these approaches attempt to tackle the photorespiration process. In plants, photorespiration is a consequence of an oxygenation reaction catalysed by RuBisCO (main enzyme in charge of carbon fixation) instead of a carboxylation. This oxygenation occurs due to atmospheric O2 competing with CO2 for the active site of RuBisCO. In this project, the first step of a multi domain RuBisCO engineering approach is proposed. This first step consists in a substrate walk engineering on biotin carboxylase (accC) from Escherichia coli. Biotin-dependent carboxylases are attractive because they show high specificity for carboxylation and no interaction with atmospheric O2. Biotin-dependent carboxylases have been the target of many different engineering endeavours, most of which focus in the carboxytransferase domain (accA), instead biotin carboxylase (accC). Additionally, many of these approaches focus on random directed evolution. This project uses a combination of Machine Learning-assisted and rational single-site mutagenesis directed evolution approaches to introduce a new-to-nature 2-imidazolidone carboxylase activity. The work presented here represents one of the first attempts to engineer the biotin carboxylase subunit (accC), one of the few to do machine learning assisted evolution, and the only known to the date to attempt a substrate walk in the biotin carboxylase subunit. The Machine Learning Assisted phase of the project managed to incorporate two simultaneous mutations (G163L and G164H) showing for the first time a 2-imidazolidone carboxylase activity 1.5-fold greater than the background. Results show that improvements in the methodology could have reduce the bias introduced into the machine learning models. After an additional round of site-saturation mutagenesis, one subsequent mutation (G83Y) increased the 2-imidazolidone carboxylase activity to be around 4-fold greater than the background. Unexpected ATPase activity was observed in the final mutant (G83Y, G163L, G164H, F279L) leading to an estimated futile ATP use of 4.4 molecules for each 1 molecule of carboxylated 2-imidazolidone. Future work on this enzyme should monitor both, spectrophotometrically and radiometrically to better control the mutant selection. Additionally, computational models could be used to pre-select the region of interest for mutagenesis.
Beskrivning
Ämne/nyckelord
Machine Learning , Directed Evolution , Biotin , Biotin Carboxylase , Desthiobiotin , 2-imidazolidone