Abstract
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.
Original language | English |
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Article number | 108102 |
Number of pages | 14 |
Journal | Biotechnology Advances |
Volume | 63 |
DOIs | |
Publication status | Published - Mar 2023 |
Keywords
- Enzyme engineering
- Growth-coupling
- Metabolic engineering
- Non-canonical redox cofactor
- Redox cofactor
- Redox cofactor auxotrophy