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Area of Science:

  • Synthetic biology
  • Metabolic engineering
  • Microbial biotechnology

Background:

  • Stable production of value-added products requires robust microbial biocatalysts.
  • Multicopy expression plasmids are advantageous for high-titer biosynthesis but are often unstable during long-term cultivation.
  • Cupriavidus necator H16 can convert CO2 into products but struggles with plasmid stability, limiting industrial application.

Purpose of the Study:

  • To design and implement plasmid addiction systems to enhance multicopy plasmid stability in Cupriavidus necator H16.
  • To enable stable production of value-added chemicals from CO2 using engineered C. necator H16.

Main Methods:

  • Development of plasmid addiction systems based on the complementation of essential genes.
  • Implementation of a system utilizing the complementation of RubisCO-deficient mutants for CO2 fixation.
  • Engineering the mevalonate pathway operon (MvaES) for mevalonate production.

Main Results:

  • A plasmid addiction system based on RubisCO complementation successfully stabilized a multicopy plasmid in C. necator H16.
  • Expression of the mevalonate pathway operon (MvaES) using the stabilized plasmid yielded approximately 10 g/L mevalonate.
  • Achieved carbon yields of approximately 25% for mevalonate production from CO2.

Conclusions:

  • Plasmid addiction systems are effective for stabilizing multicopy plasmids in C. necator H16 for industrial bioprocesses.
  • This study demonstrates the highest reported titers and yields for C6 compounds from C1 feedstocks (CO2).
  • The developed system enhances the potential of C. necator H16 as a microbial chassis for sustainable chemical production.