Using continuous directed evolution to improve enzymes for plant applications

Abstract

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 enzyme’s 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 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 yeast (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.

Continuous directed evolution of a feedback-resistant Arabidopsis arogenate dehydratase in plantized E. coli

“…Variation in growth between replicate clones is expected because their AtADT2 genes are in the eMutaT7 system, and thus subject to ongoing hypermutation and hence to reduction or loss of AtADT2 expression. 8 The S222N benchmark variant was more highly expressed in soluble form than wildtype AtADT2, as were the A208V and R213W variants to a lesser extent ( Figure 4D ). This is unlikely to relate to the observed resistance phenotypes because overexpression of feedback-inhibited enzymes cannot per se confer feedback-resistance, 18–20 and the benchmark S222N variant gives feedback-resistance in planta without being overexpressed.…”

“…9 This strategy is still at the concept stage. Further, for the E. coli systems, the differences between conditions in plant and prokaryote cells (e.g., metabolic pathway architecture, metabolite levels, redox poise, protein-folding and degradation systems 8 ) and the uncertain durability of the hypermutation machinery in long evolution campaigns 4,5,8 are potential roadblocks. Nor is it clear that E. coli CDE systems work in minimal media, which is critical because many selection schemes require auxotrophs.…”

Directed evolution of aerotolerance in sulfide-dependent thiazole synthases

“…We transformed a metl5 Δ thi4 Δ strain with the nuclear ArEc-TDH3 plasmid harboring the error-prone polymerase TP-DNAP1_611, then introduced plasmids p1 and p2 by protoplast fusion. 15 p1 harbors the THI4 target gene that the error-prone polymerase hypermutates ( Figure 2A ). We ran three independent clones of each THI4 through three selection schemes: 15 1, serial passages on thiamin-free medium; 2, initial passages on medium with limiting thiamin, then transfer to thiamin-free medium; and 3, initial passages on medium with luxury thiamin (to build a mutant library), then transfer to scheme 2 ( Figure 2B–D ).…”

“…11,12 In CDE, the enzyme gene is hypermutated in vivo , the enzyme’s activity is coupled to host cell growth, and improved variants are obtained by selecting for faster growth. 12 We chose the yeast (Saccharomyces cerevisiae) OrthoRep CDE system because: (i) it durably mutates a target gene at ~10 5 -fold the natural rate; 13,14 (ii) sulfide-dependent prokaryote THI4s complement a yeast thiazole auxotroph; 8 and (iii) yeast is a closer facsimile of plants than Escherichia coli , 15 the main alternative CDE platform. 12 To avoid overtaxing the initially low activity of the target THI4s, we supported their function by using a host strain (met15Δ ) with a high internal sulfide level.…”

“…12 To avoid overtaxing the initially low activity of the target THI4s, we supported their function by using a host strain (met15Δ ) with a high internal sulfide level. 16 Since cytosolic conditions in aerobic yeast, as in plants, differ from those in anaerobic bacteria with respect to, e.g., NADH/NAD + ratio 17–19 and protein folding and degradation systems, 15 selection in yeast might improve adaptation to such factors as well as to oxygen. Structure modeling and comparative genomics were applied to distinguish these possibilities.…”