Active Inclusion Bodies in the Multienzymatic Synthesis of UDP-N-acetylglucosamine

Area of Science:

• Biotechnology
• Biocatalysis
• Protein Engineering

Background:

• Bacterial inclusion bodies (IBs) are typically viewed as waste products in recombinant protein production.
• Recent research suggests IBs can be utilized as immobilized enzymes for in vivo biocatalysis.
• Enzymatic cascades for valuable product synthesis require efficient cofactor regeneration, often necessitating expensive enzyme purification.

Purpose of the Study:

• To investigate the potential of active inclusion bodies (aIBs) as a compromise between purified enzymes and cell lysates for biocatalysis.
• To demonstrate the efficacy of engineered aIBs for cofactor regeneration in multienzymatic reactions.
• To explore the application of aIBs in the synthesis of valuable compounds like UDP-N-acetylglucosamine.

Main Methods:

• Engineered bacterial inclusion bodies (aIBs) were created to immobilize enzymatic activity.
• Two polyphosphate kinases (PPKs) were co-expressed in aIBs to achieve ATP regeneration.
• PPKs were combined with other enzymes for the synthesis of UDP-N-acetylglucosamine.

Main Results:

• The combination of PPKs within aIBs resulted in a nearly 10-fold increase in ATP regeneration.
• 100% UTP utilization was achieved without degradation into adenosine or uridine.
• A multienzyme cascade using aIBs successfully synthesized UDP-N-acetylglucosamine.

Conclusions:

• Active inclusion bodies (aIBs) offer a viable strategy for immobilizing enzymes and achieving efficient cofactor regeneration.
• This approach provides a cost-effective alternative to enzyme purification for multienzymatic syntheses.
• Engineered aIBs hold promise for various biocatalytic applications, including the synthesis of valuable nucleotide-based compounds.