Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

Abstract

Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN+). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN+ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN+ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA+), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)+.

Coenzyme Engineering of Glucose-6-phosphate Dehydrogenase on a Nicotinamide-Based Biomimic and Its Application as a Glucose Biosensor

“…mobilis G6PDH, an important dehydrogenase for the regeneration of reduced coenzyme, was engineered from NADP + -specific to NADP + /NMN + -compatible, wherein the coenzyme specificity values ([ k cat / K m ] NMN + /[ k cat / K m ] NAD + and [ k cat / K m ] NMN + /[ k cat / K m ] NADP + ) of the R4 mutant were ∼4.7 × 10 3 and 2.6 × 10 3 -fold of the WT enzyme, respectively (Table ). Although the catalytic efficiency of ZmG6PDH R4 to NMN + could not outperform that of WT ZmG6PDH to NADP + , considering the cost of NAD + and NMN + (approximately $2.8 × 10 3 /Kg and $1.5 × 10 3 /Kg at the 100 kg scale offered by the manufacturers, respectively) and the higher stability of NMN + than NAD­(P) + , using a stable, inexpensive cofactor instead of a natural and expensive cofactor is a useful strategy, particularly in the application of oxidoreductases in vitro at an industrial scale, similar to NMN + -based cost-effective biomanufacturing in numerous studies. ,, Furthermore, here we revealed structural information regarding the enhanced catalytic efficiency on NMN + of the R4 mutant by resolving the crystal structures of apo-WT and R4:NMN + and performing MD simulations (Figures –). Finally, a glucose biosensor with satisfactory bioelectrochemical performance was constructed based on a bioelectrode immobilized with GK and R4 ZmG6PDH, thus highlighting the prospects of the findings of this study in manufacturing glucose detection products without cryopreservation (Figure ).…”

“…In addition, there exists a large space in the active pocket of the enzyme that was initially used for binding with the AMP portion of NADP + . It is expected that filling this space with large side-chain groups will help enhance the enzyme activity of G6PDH against NMN + , and maintain the channel to be large enough for NMN + entry simultaneously. This replacement may improve the binding affinity of ZmG6PDH to NMN + (reduce the K m value) and concurrently reduce the binding affinity of ZmG6PDH to NADP + due to the increased steric hindrance, increasing catalytic efficiency under a relatively low NMN + loading amount in R4-mediated enzymatic biocatalysis.…”

“…However, efficient utilization of BNCs by oxidoreductases constitutes the major obstacle to their biocatalytic application of BNCs. 6,7,9 Numerous natural enzymes, such as old yellow enzymes, 10−12 diaphorase, 13 nitroreductase, 14,15 monooxygenase, 16 and NADH oxidase, 17 have been reportedly utilized to reduce BNCs in reduction reactions. However, all of them possess a prosthetic group containing flavin adenine dinucleotide (FAD) that nonselectively facilitates electron transfer.…”

“…The strategy of using BNCs as the coenzymes can potentially decrease the processing cost of oxidoreductase-catalyzed biotransformation. However, efficient utilization of BNCs by oxidoreductases constitutes the major obstacle to their biocatalytic application of BNCs. ,, …”

Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase

“…While this method by Huang et al 16 greatly enhanced the throughput of screening for NCB activity (10 5 per round), the required 70 °C heat treatment excludes application for mesophilic enzymes. Recent expansions of redox-based growth selections highlight the practical use of these platforms for engineering diverse cofactor-dependent enzymes with cell growth as an easy readout of activity 17 – 25 . While these selections can increase throughput to >10 6 , a link between the desired enzyme activity and a life-essential function is needed.…”