Enzyme protein turnover accounts for about half the maintenance energy budget in plants. Slowing turnover─i.e., extending the effective working life (Catalytic Cycles till Replacement, CCR)─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 cumulative HDH function and, by proxy, lifespan as functional enzymes, by selecting for growth rate while tapering histidinol concentration and escalating that of the inhibitor histamine. Improved HDH variants 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 working life in vivo in addition to, as expected, altering kinetic properties.
Keywords: directed evolution; enzyme performance; histidinol dehydrogenase; protein turnover.