Biology subjects

Van Gelder, K.

Publications and source records attributed to Van Gelder, K..

4 recordsLinked to original sources

bioRxiv2025

Comparative genomic insights into the action of suicide Thi4 thiazole synthases

Suicide thiazole synthases (Thi4) are mononuclear metal enzymes that form the thiazole moiety of thiamin from NAD+, glycine, and a sulfur atom that is stripped from an active-site cysteine residue, causing enzyme inactivation. Comparative genomic analysis indicates that prokaryotic Thi4 genes often cluster on the chromosomal regions encoding ThiS, ThiF, and other proteins that can produce, relay, or use persulfide or thiocarboxylate sulfur. This genomic evidence suggests that certain suicide Thi4s might use a persulfide or thiocarboxylate as sulfur donor instead of the active-site cysteine - i.e., that they can operate in a non-suicide mode - and that a metal cofactor reservoir supports Thi4 function. To explore these possibilities, we performed proof-of-concept experiments using Escherichia coli as a heterologous platform. A representative bacterial Thi4 that clustered with thiS and thiF complemented an E. coli {Delta}thiG (thiazole auxotroph) single mutant better than a {Delta}thiG {Delta}thiF {Delta}thiS triple mutant, consistent with predicted interactions with the host sulfide transfer chain. Collectively, this evidence indicates that suicide Thi4s may not necessarily operate suicidally and highlights genomic and structural clues that warrant deeper biochemical investigation.

biochemistry↗

bioRxiv2024

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.

synthetic biology↗

bioRxiv2022

Directed evolution of aerotolerance in sulfide-dependent thiazole synthases

Sulfide-dependent THI4 thiazole synthases could potentially be used to replace plant cysteine-dependent suicide THI4s, whose high protein turnover rates make thiamin synthesis exceptionally energy-expensive. However, sulfide-dependent THI4s are anaerobic or microoxic enzymes and hence unadapted to the aerobic conditions in plants; they are also slow enzymes (kcat <1 h-1). To improve aerotolerance and activity, we applied continuous directed evolution under aerobic conditions in the yeast OrthoRep system to two sulfide-dependent bacterial THI4s. Six beneficial single mutations were identified, of which five lie in the active-site cleft predicted by structural modeling and two recapitulate features of naturally aerotolerant THI4s. That single mutations gave substantial improvements suggests that further advance under selection will be possible by stacking mutations. This proof-of-concept study established that the performance of sulfide-dependent THI4s in aerobic conditions is evolvable and, more generally, that yeast OrthoRep provides a plant-like bridge to adapt nonplant enzymes to work better in plants.

synthetic biology↗

bioRxiv2021

Using Continuous Directed Evolution to Improve Enzymes for Plant Applications

Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzymes activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful new way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the Saccharomyces cerevisiae OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt ( plantize) enzymes from prokaryotes - especially exotic prokaryotes - to function well in mild, plant-like conditions. One-sentence summaryContinuous directed evolution using the yeast OrthoRep system is a powerful new way to improve enzymes for use in plant engineering as illustrated by plantizing a bacterial thiamin synthesis enzyme..

synthetic biology↗