Mirages in Continuous Directed Enzyme Evolution: A Cautionary Case Study with Plantized Bacterial THI4 Enzymes

Continuous directed evolution (CDE) improves enzyme characteristics by hypermutating the enzyme gene in vivo in a microbial platform, linking enzyme activity to growth, and selecting for growth rate. Combined with genome editing, CDE can expand the gene pool for plant breeding. THI4 enzymes, essential for thiamin synthesis, are ideal targets for plant CDE. Plant THI4s are inefficient; their replacement by efficient bacterial THI4s could boost biomass yield by up to 4%. However, bacterial THI4s are O2-sensitive and unsuited to plants. Previous CDE campaigns in the yeast OrthoRep system adapted bacterial THI4s for plant-like conditions, achieving success with Mucinivorans hirudinis THI4 (MhTHI4), which acquired beneficial mutations that improved growth. Here, we increased selection pressure on MhTHI4 by reducing its expression, leading to faster-growing populations with new nonsynonymous and synonymous mutations. Surprisingly, the synonymous mutations appeared largely responsible for the growth rate improvements, providing a cautionary example for other OrthoRep CDE projects. ### Competing Interest Statement The authors have declared no competing interest.