Recent Developments and Challenges for the Industrial Implementation of Polyphosphate Kinases

Abstract

Polyphosphate kinases (PPKs) have emerged as valuable candidates to address the unmet need for scalable recycling of the common enzyme cofactor adenosine‐5’‐triphosphate (ATP) because they use cheap, freely available and stable polyphosphate (polyP) salts as the phosphate donor. The aim of this review is not only to present recent efforts in the characterisation of PPKs but also to provide an overview of the challenges associated with their implementation in chemical manufacturing. In assessing the current status of polyP‐driven biocatalysts, we identify gaps that need to be filled for successful adoption of these biocatalysts in industry. Guidelines for a wider adoption of PPKs are also provided.

Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence

“…Uniquely, the class II P. aeruginosa PPK2C has two fused PPK2 domains: a catalytically inactive N-terminal and an active C-terminal [ 36 ]. A comparison of the above crystal structures revealed a class-specific signature residue located after the Walker B motif: class I is asparagine (N153 in S. meliloti PPK2), class II is glycine (G367 in PA3455), and class III is glutamate (E137 in C. hutchinsonii PPK2) [ 46 , 63 ].…”

“…This marks the first time PPK2s have been shown to use all the natural nucleotides—including thymidine—as substrates, highlighting the potential of these so-called universal PPK2s as tools for biotechnological NTP regeneration [ 66 ]. Specifically, universal PPK2s could be a boon for the regeneration of UTP and CTP required for the enzymatic synthesis of complex glycans [ 59 , 67 , 68 ], in addition to more traditional ATP regeneration systems (reviewed in [ 63 ]).…”

“…While the authors of this study concede that their methodology could not exclude the possibility of some sequential monophosphorylation being mistaken for pyrophosphorylation, previous reports that PPK2 polyP utilization is processive [ 34 , 35 , 69 ]—i.e., the polyP chain is not released by the enzyme between reactions—support the possibility that multiple P i units can be transferred from polyP at once. Regardless of the reaction mechanism, class III enzymes offer a streamlined means of recycling both AMP and ADP and are thus beginning to supplant class I enzymes for use in biotechnological ATP regeneration systems [ 63 ].…”

“…Some PPK2 reaction products do not fit squarely within the categories described above. Moreover, the catalytic activities defining the three PPK2 classes should be considered preferences, rather than absolute definitions, as several class I and class II enzymes were recently shown to be capable of AMP phosphorylation to ATP in the presence of large (500 µg/mL) amounts of enzyme [ 63 , 71 ]. Several studies have also described unusual nucleoside phosphate species produced by PPK2s.…”

Electrochemical Recycling of Adenosine Triphosphate in Biocatalytic Reaction Cascades

“…, acetyl phosphate or polyphosphate) , (Figure B). These approaches not only compromise the cost, and atom economy, but also potentially impact the enzyme performance. , Given that electricity is the cheapest alternative energy source and the lowest carbon footprint when produced from renewable sources, here, we report a robust and scalable method to drive the recycling of the ubiquitous cofactor ATP electrochemically (Figure C). In this system, pyruvate is oxidized to CO 2 to produce ATP, effectively providing a simplified electrochemical mimic of the cellular respiration process.…”