Suicide prevention for enzymes using continuous directed evolution

Plant enzymes have evolved through natural selection, glacially inching toward favorable outcomes within their environment and the functional demands of the plant cell. However, natural selection does not guarantee or even require a superior catalyst. Cue the hubris of humankind and our belief that we can improve plant enzymes through directed evolution (DE). This method steers protein sequences toward a functional goal by subjecting a gene to repeated cycles of mutagenesis and selection. Continuous DE improves on classical methods, which rely on separate in vitro gene diversification followed by transformation into cells and selection. The three-step process is now rolled into one, occurs in vivo, and requires minimal input. The target gene is incorporated into microbial hosts, along with the specific means of its replication—a faulty DNA polymerase, which generates in vivo gene diversification. The corresponding protein, metabolic or otherwise, executes a function tied to the growth of the cells. For example, if an enzyme synthesizes an essential nutrient (e.g. an amino acid), its performance will determine the growth rate. Thereby, random nonsynonymous mutations that improve protein function will accumulate within the target gene as it evolves to surpass its peers (Morrison et al., 2020). In this issue of Plant Physiology, García-García et al. (2021) use the OrthoRep platform for continuous DE, modifying an enzyme to perform efficiently in plants. OrthoRep is a DNA polymerase-plasmid pair in yeast (Saccharomyces cerevisiae) that stably and specifically mutates the target gene ∼100,000-fold faster than the host genome (Ravikumar et al., 2018). This orthogonal replication system works independently of the host and does not interfere with endogenous DNA replication. The OrthoRep platform has three main components: (1) a faulty DNA polymerase (TP-DNAP1) borne on a circular nuclear plasmid; (2) a linear cytoplasmic plasmid harboring the target gene (p1); (3) a second linear cytoplasmic plasmid (p2) carrying components needed for transcription of genes on p1, an RNA polymerase (RNAP), and the means of its replication (TP-DNAP2). The two DNA polymerases recognize their replication origins by terminal proteins covalently attached to the 5′ ends of p1 and p2, respectively, thereby ensuring specificity. However, this adds a slight quirk to the system, requiring p1 and p2 to be introduced via protoplast fusion instead of regular yeast transformation. The authors deployed the OrthoRep platform to tackle the case of thiazole synthase (THI4), an enzyme involved in thiamine biosynthesis. Thiamine, also known as vitamin B1, is an essential nutritional supplement for humans and is synthesized by various organisms, from bacteria to plants. It is comprised of a pyrimidine and thiazole moiety that are biosynthesized separately. The thiazole constituent is a heterocyclic compound, i.e., it contains nitrogen and sulfur in a five-member ring, along with three carbon atoms. The genesis of the sulfur atom is notable and at the heart of this exercise in enzyme evolution. Thiazole biosynthesis is chemically complex and varies amongst organisms. In plants, fungi, and some prokaryotes, THI4 forms the thiazole moiety from nicotinamide adenine dinucleotide (NAD), glycine, and a sulfur atom (Chatterjee et al., 2011). THI4 also serves as a co-substrate in this reaction, stripping a sulfur atom from its active site Cys residue. Loss of sulfur leaves behind a dehydroalanine residue and irreversibly inactivates the enzyme. In this manner, THI4 is more of a kamikaze enzyme, serving an inherently fatal function. A single-turnover enzyme needs to be degraded and resynthesized, making suicidal Cys-THI4s very costly for the organism. In its place, a catalytic enzyme, securing sulfur from a nonprotein source, could especially help crop plants reallocate energy and building blocks toward gains in yield. However, it should be noted that researchers have speculated on secondary functions for these spent THI4s (post-sulfur-loss). For example, yeast THI4 has been implicated in DNA protection and stress-related pathways (Machado et al., 1997; Medina-Silva et al., 2006). Some prokaryotes have THI4s that proceed by a more traditionally catalytic route. These variants use sulfide as a sulfur donor and often have His in place of Cys in the active site. Catalytic THI4s were initially characterized in strictly anaerobic, O2-sensitive archaea bacteria from hydrothermal vents. These non-Cys THI4s rely on millimolar sulfide levels to carry out thiazole biosynthesis and would be poor or nonfunctional in the plant cellular context. Thus, they represent excellent candidates for continuous DE to complement a thiazole auxotroph (a yeast strain with Δthi4 knockout that cannot produce thiazole). There is evidence to suggest that certain cereal plants have noncanonical THI4-like homologs, which function catalytically; however, their activity appears to be drastically lower than their prokaryotic counterparts (Joshi et al., 2020). The authors applied continuous DE to a catalytic THI4 from Mucinivorans hirudinis, anaerobic bacteria from the leech gut. The goal was to plantify this enzyme and make it amenable to aerobic and low-sulfide conditions, for which the yeast cell provides a decent proxy (Figure 1). The authors turned the screw on the enzyme using three strategies: (1) cold-turkey, (2) gradual, or (3) variant building/inclusive. These approaches describe the thiamine concentration in the media (0, 10, or 300 nM) and whether cells start without the nutrient or are weaned off supplementation over several passages. In the inclusive mode, the cells are first cultured under nonselective conditions and allowed neutral drift, generating an extensive library of mutant sequences. This method can help bypass suboptimal fitness peaks that are facile (within reach of a few adaptive mutations) and trap evolutionary populations. Taking a catalytic THI4 (MhTHI4) from a prokaryotic, anaerobic, and high-sulfide environment and plantifying it within the yeast (S. cerevisiae) cellular context. MhTHI4 is not optimized to function in the low-sulfide and high-oxygen cells of plants or yeast. However, its catalytic nature represents a significant improvement over the endogenous suicidal, single-turnover THI4. Employing a continuous evolution platform, researchers are attempting multiple rounds of in vivo hypermutation and screening to improve performance and ascend the fitness peak. Taking a catalytic THI4 (MhTHI4) from a prokaryotic, anaerobic, and high-sulfide environment and plantifying it within the yeast (S. cerevisiae) cellular context. MhTHI4 is not optimized to function in the low-sulfide and high-oxygen cells of plants or yeast. However, its catalytic nature represents a significant improvement over the endogenous suicidal, single-turnover THI4. Employing a continuous evolution platform, researchers are attempting multiple rounds of in vivo hypermutation and screening to improve performance and ascend the fitness peak. The result of continuous evolution for MhTHI4, presented by García-García et al., is encouraging but reminds us of the limitations of this system. A beneficial mutation V124A was sufficient to increase the activity of this enzyme from anaerobic bacteria to match that of native suicidal yeast THI4. This mutation was achieved 5 times independently. To go beyond this activity level, researchers will have to make it work harder by either tuning down enzyme expression or inhibiting its function. The platform is also limited by the difficulty in assessing the benefits of multiple mutations—there were 24 other nonsynonymous mutations in the target gene and promoter combined. Causality is obscured by the in vivo coupling of mutation and screening. Attaining those greater heights of activity will require a longer trail through the fitness landscape. As the Nobel laureate Frances Arnold said in her acceptance lecture, “we can … read, write, and edit any sequence of DNA, but we cannot compose it.” Here, the authors have applied the OrthoRep system to bring forth the marginal activity of catalytic THI4s from anaerobic, methanogenic bacteria and adapt it to aerobic, low-sulfide conditions. However, it is still a far cry from evolving wholly new activities, and it has not yet surpassed the low bar of thiazole biosynthesis set by endogenous suicidal THI4s. In practice, evolved, catalytic MhTHI4 could benefit crops, even at their current output levels, shifting resources from expressing suicidal variants to agronomic yield. Such applications of synthetic biology and DE will undoubtedly empower the next set of advances in agriculture. Conflict of interest statement. No conflict of interest to declare.

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