A universal polyphosphate kinase powers in vitro transcription

Area of Science:

• Biochemistry
• Synthetic Biology
• Enzyme Engineering

Background:

• Polyphosphate kinases (PPKs) are enzymes that transfer phosphoryl groups from polyphosphates to nucleotides.
• Polyphosphates offer a cost-effective phosphorylating source, making PPKs valuable for nucleotide production.
• Current PPKs have limited substrate specificity, restricting their use in applications requiring diverse nucleotide utilization.

Purpose of the Study:

• To engineer a universal polyphosphate kinase (PPK) with broad substrate specificity.
• To demonstrate the enzyme's capability in phosphorylating all eight common ribonucleotides (purines and pyrimidines, mono- and diphosphates) to triphosphates.
• To showcase the biotechnological potential of the universal PPK in a one-pot in vitro transcription assay.

Main Methods:

• Enzyme engineering to broaden substrate specificity of polyphosphate kinases.
• Characterization of the universal PPK's activity across various ribonucleotide substrates.
• Development of a coupled one-pot assay integrating the universal PPK for in vitro transcription.

Main Results:

• The engineered universal PPK efficiently phosphorylates all eight common ribonucleotides to triphosphates.
• Approximately 70% triphosphate conversion was achieved for each substrate under optimal conditions.
• Successful demonstration of PPK-powered in vitro transcription in a one-pot system.

Conclusions:

• A universal PPK has been developed, significantly expanding the utility of polyphosphate-dependent phosphorylation.
• This engineered enzyme overcomes previous substrate limitations, enabling more versatile nucleotide synthesis.
• The universal PPK serves as a powerful tool for synthetic biology, facilitating complex in vitro reaction systems and mimicking principles of primitive metabolism.