Experimental evolution of a new enzymatic function. II. Evolution of multiple functions for ebg enzyme in E. coli

Area of Science:

• Evolutionary biology
• Enzymology
• Microbial genetics

Background:

• The ebgo enzyme in Escherichia coli initially lacks effective hydrolysis capabilities for various galactosides.
• Understanding enzyme evolution is crucial for metabolic engineering and synthetic biology.

Purpose of the Study:

• To investigate the directed evolution of the ebgo enzyme in Escherichia coli.
• To elucidate the evolutionary pathway for acquiring new substrate specificities.
• To determine if gene duplication is necessary for enzyme functional divergence.

Main Methods:

• Directed evolution experiments were performed on Escherichia coli strains with a lacZ deletion.
• Mutations in the ebg genes were introduced and selected in a specific order.
• Comparative analysis of evolved and parental enzyme functions was conducted.

Main Results:

• A specific, ordered series of mutations in the ebg genes created an obligatory evolutionary pathway.
• The evolved ebgo enzyme gained the ability to effectively hydrolyze lactose, lactulose, lactobionate, and galactose-arabinoside.
• Acquisition of new functions sometimes led to loss of old functions, but in some cases, old functions were retained or improved.

Conclusions:

• Enzyme evolution can follow an obligatory pathway, requiring specific sequential mutations.
• Divergence of enzyme functions can occur without the need for gene duplication.
• This provides insights into the mechanisms of enzyme adaptation and innovation.