Growth-coupled enzyme engineering through manipulation of redox cofactor regeneration.

Enzymes need to be efficient, robust, and highly specific for their effective use in commercial bioproduction. These properties can be introduced using various enzyme engineering techniques, with random mutagenesis and directed evolution (DE) often being chosen when there is a lack of structural information -or mechanistic understanding- of the enzyme. The screening or selection step of DE is the limiting part of this process, since it must ideally be (ultra)-high throughput, specifically target the catalytic activity of the enzyme and have an accurately quantifiable metric for said activity. Growth-coupling selection strategies involve coupling a desired enzyme activity to cellular metabolism and therefore growth, where growth (rate) becomes the output metric. Redox cofactors (NAD+/NADH and NADP+/NADPH) have recently been identified as promising target molecules for growth coupling, owing to their essentiality for cellular metabolism and ubiquitous nature. Redox cofactor oxidation or reduction can be disrupted through metabolic engineering and the use of specific culturing conditions, rendering the cell inviable unless a 'rescue' reaction complements the imposed metabolic deficiency. Using this principle, enzyme variants displaying improved cofactor oxidation or reduction rates can be selected for through an increased growth rate of the cell. In recent years, several E. coli strains have been developed that are deficient in the oxidation or reduction of NAD+/NADH and NADP+/NADPH pairs, and of non-canonical redox cofactor pairs NMN+/NMNH and NCD+/NCDH, which provides researchers with a versatile toolbox of enzyme engineering platforms. A range of redox cofactor dependent enzymes have since been engineered using a variety of these strains, demonstrating the power of using this growth-coupling technique for enzyme engineering. This review aims to summarize the metabolic engineering involved in creating strains auxotrophic for the reduced or oxidized state of redox cofactors, and the resulting successes in using them for enzyme engineering. Perspectives on the unique features and potential future applications of this technique are also presented.

Rubisco Adaptation Is More Limited by Phylogenetic Constraint Than by Catalytic Trade-off.

Rubisco assimilates CO2 to form the sugars that fuel life on earth. Correlations between rubisco kinetic traits across species have led to the proposition that rubisco adaptation is highly constrained by catalytic trade-offs. However, these analyses did not consider the phylogenetic context of the enzymes that were analyzed. Thus, it is possible that the correlations observed were an artefact of the presence of phylogenetic signal in rubisco kinetics and the phylogenetic relationship between the species that were sampled. Here, we conducted a phylogenetically resolved analysis of rubisco kinetics and show that there is a significant phylogenetic signal in rubisco kinetic traits. We re-evaluated the extent of catalytic trade-offs accounting for this phylogenetic signal and found that all were attenuated. Following phylogenetic correction, the largest catalytic trade-offs were observed between the Michaelis constant for CO2 and carboxylase turnover (∼21-37%), and between the Michaelis constants for CO2 and O2 (∼9-19%), respectively. All other catalytic trade-offs were substantially attenuated such that they were marginal (<9%) or non-significant. This phylogenetically resolved analysis of rubisco kinetic evolution also identified kinetic changes that occur concomitant with the evolution of C4 photosynthesis. Finally, we show that phylogenetic constraints have played a larger role than catalytic trade-offs in limiting the evolution of rubisco kinetics. Thus, although there is strong evidence for some catalytic trade-offs, rubisco adaptation has been more limited by phylogenetic constraint than by the combined action of all catalytic trade-offs.