Metabolic engineering uses enzymes as parts to build biosystems for specified tasks. Although a part’s working life and failure modes are key engineering performance indicators, this is not yet so in metabolic engineering because it is not known how long enzymes remain functional in vivo or whether cumulative deterioration (wear-out), sudden random failure, or other causes drive replacement. Consequently, enzymes cannot be engineered to extend life and cut the high energy costs of replacement. Guided by catalyst engineering, we adopted catalytic cycles until replacement (CCR) as a metric for enzyme functional life span in vivo. CCR is the number of catalytic cycles that an enzyme mediates in vivo before failure or replacement, i.e., metabolic flux rate/protein turnover rate. We used estimated fluxes and measured protein turnover rates to calculate CCRs for ∼100–200 enzymes each from Lactococcus lactis, yeast, and Arabidopsis. CCRs in these organisms had similar ranges (10⁷) but different median values (3–4 × 10⁴ in L. lactis and yeast versus 4 × 10⁵ in Arabidopsis). In all organisms, enzymes whose substrates, products, or mechanisms can attack reactive amino acid residues had significantly lower median CCR values than other enzymes. Taken with literature on mechanism-based inactivation, the latter finding supports the proposal that 1) random active-site damage by reaction chemistry is an important cause of enzyme failure, and 2) reactive noncatalytic residues in the active-site region are likely contributors to damage susceptibility. Enzyme engineering to raise CCRs and lower replacement costs may thus be both beneficial and feasible. Significance The continuous replacement of enzymes and other proteins appropriates up to half the maintenance energy budget in microorganisms and plants. High enzyme replacement rates therefore cut the productivity of biosystems ranging from microbial fermentations to crops. However, yardsticks to assess what drives enzyme protein replacement and guidelines on how to reduce it are lacking. Accordingly, we compared enzymes’ life spans across kingdoms using a new yardstick (catalytic cycles until replacement [CCR]) and related CCR to enzyme reaction chemistry. We concluded that 1) many enzymes fail due to collateral damage from the reaction they catalyze, and 2) such damage and its attendant enzyme replacement costs are mitigable by engineering and are therefore promising targets for synthetic biology.

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.

Abiotic stresses reduce crop growth and yield in part by disrupting metabolic homeostasis and triggering responses that change the metabolome. Experiments designed to understand the mechanisms underlying these metabolomic responses have usually not used agriculturally relevant stress regimes. We therefore subjected maize plants to drought, salt, or heat stresses that mimic field conditions and analyzed leaf responses at metabolome and transcriptome levels. Shared features of stress metabolomes included synthesis of raffinose, a compatible solute implicated in tolerance to dehydration. In addition, a marked accumulation of amino acids including proline, arginine, and γ-aminobutyrate combined with depletion of key glycolysis and tricarboxylic acid cycle intermediates indicated a shift in balance of carbon and nitrogen metabolism in stressed leaves. Involvement of the γ-aminobutyrate shunt in this process is consistent with its previously proposed role as a workaround for stress-induced thiamin-deficiency. Although convergent metabolome shifts were correlated with gene expression changes in affected pathways, patterns of differential gene regulation induced by the three stresses indicated distinct signaling mechanisms highlighting the plasticity of plant metabolic responses to abiotic stress.

The PDH bypass and the GABA shunt serve to maintain mainline metabolic fluxes during episodes of organellar thiamin diphosphate deficiency.

The whole field of synthetic biology (SynBio) is only about 20 years old, and plant SynBio is younger still. Nevertheless, within that short time, SynBio in general has drawn more scientific, philosophical, government, and private-sector interest than anything in biology since the recombinant DNA revolution. Plant SynBio, in particular, is now drawing more and more interest in relation to plants’ potential to help solve planetary problems such as carbon capture and storage and replacing fossil fuels and feedstocks. As plant SynBio is so young and so fast-developing, we felt it was too soon to try to analyze its history. Instead, we set out to capture the essence of plant SynBio's origins and early development through interviews with 8 of the field's founders, representing 5 countries and 3 continents. We then distilled these founders’ personal recollections and reflections into this review, centering the narrative on timelines for pivotal events, articles, funding programs, and quoting from interviews. We have archived the interview recordings and documented timeline entries. This work provides a resource for future historical scholarship.