Continuous Directed Evolution of a Plant Histidinol Dehydrogenase to Extend Lifespan

Enzyme protein turnover accounts for about half the maintenance energy budget in plants. Slowing turnover - i.e., extending lifespan - of short-lived enzymes is thus a rational strategy to conserve energy and carbon, and raise crop productivity. Arabidopsis histidinol dehydrogenase (HDH) is a short-lived enzyme that can sustain life-shortening damage from its aminoaldehyde reaction intermediate. We used the yeast OrthoRep continuous directed evolution system in a his4Δ; strain to raise HDH protein abundance (a proxy for lifespan) by selecting for growth rate while tapering histidinol concentration and escalating that of the inhibitor histamine. Improved HDHs carried diverse nonsynonymous mutations and ranged 20-fold in level. Improved HDH performance was associated with higher HDH abundance in some cases and with greater catalytic efficiency or histamine resistance in others. These findings indicate that OrthoRep-based directed evolution can extend enzyme lifespan in vivo in addition to, as expected, altering kinetic properties.