Continuous Directed Evolution of a Short-Lived Plant Histidinol Dehydrogenase

Area of Science:

• Plant Biochemistry
• Enzyme Engineering
• Directed Evolution

Background:

• Enzyme protein turnover is a significant component of plant energy expenditure.
• Short-lived enzymes, like Arabidopsis histidinol dehydrogenase (HDH), are susceptible to damage from reaction intermediates, impacting plant productivity.
• Extending enzyme working life (Catalytic Cycles till Replacement, CCR) is a viable strategy for energy conservation and crop improvement.

Purpose of the Study:

• To enhance the functional lifespan of the short-lived enzyme HDH in Arabidopsis.
• To investigate the potential of directed evolution for increasing enzyme working life in vivo.
• To explore strategies for conserving energy and carbon resources in plants through enzyme engineering.

Main Methods:

• Utilized the yeast OrthoRep continuous directed evolution system.
• Employed a selection strategy involving growth rate optimization under varying histidinol and histamine concentrations in a his4Δ strain.
• Analyzed improved HDH variants for mutations, abundance, catalytic efficiency, and histamine resistance.

Main Results:

• Successfully evolved HDH variants with up to 20-fold improvement in cumulative function and inferred lifespan.
• Identified diverse nonsynonymous mutations across improved HDH variants.
• Observed enhanced HDH performance linked to increased abundance, catalytic efficiency, or histamine resistance.

Conclusions:

• Directed evolution using the OrthoRep system can effectively extend enzyme working life in vivo.
• The strategy successfully improved HDH function and resilience, demonstrating its potential for crop enhancement.
• This approach offers a powerful tool for enzyme engineering beyond altering kinetic properties.