Stable and Promiscuous Galactose Oxidases Engineered by Directed Evolution, Atomistic Design, and Ancestral Sequence...

Area of Science:

• Biocatalysis and enzyme engineering
• Synthetic chemistry
• Sustainable chemistry

Background:

• Galactose oxidase (GOase) is a versatile biocatalyst with applications in synthetic chemistry and bioelectrochemical devices.
• Previous engineering yielded the M-RQW mutant with novel C6-OH oxidation activity on glucose and promiscuity for secondary alcohols.
• The M-RQW mutant's backbone facilitated broader substrate acceptance, paving the way for further enzyme optimization.

Purpose of the Study:

• To engineer highly thermostable galactose oxidase (GOase) variants with high production titers in yeast.
• To develop enzymes with latent activities suitable for sustainable chemistry.
• To leverage directed evolution combined with computational methods for rapid enzyme improvement.

Main Methods:

• Utilized the M-RQW GOase mutant as a starting point for further engineering.
• Incorporated one-shot computational mutagenesis (PROSS algorithm) and ancestral sequence reconstruction into the directed evolution workflow.
• Employed Pichia pastoris for high-titer fed-batch bioreactor production of engineered GOases.

Main Results:

• Achieved g/L production levels of engineered GOases in Pichia pastoris.
• Engineered GOases demonstrated significant resistance to pH and high temperatures, with thermal stability (T50) increases up to 27 °C over the M-RQW parent.
• The designed GOases exhibited latent activity against glucose and various bulky secondary aromatic alcohols.

Conclusions:

• The synergetic approach of directed evolution and computational mutagenesis rapidly generated highly stable and producible GOase variants.
• These engineered enzymes possess latent activities valuable for sustainable chemical synthesis.
• The developed GOases serve as a strong foundation for future industrial enzyme engineering efforts.