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Cyanobacteria can produce isoprene (C5H8) by fusing isoprene synthase (IspS) with phycocyanin (cpcB). A cpcB*L7*IspS fusion strain significantly boosted isoprene production by increasing enzyme accumulation, despite lower specific activity.

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Area of Science:

  • Metabolic Engineering
  • Synthetic Biology
  • Cyanobacterial Biotechnology

Background:

  • Renewable isoprene (C5H8) production from CO2 and H2O via cyanobacterial photosynthesis is hindered by low enzyme expression and activity.
  • Isoprene synthase (IspS) is key for isoprene biosynthesis, but its heterologous production in cyanobacteria faces challenges.

Purpose of the Study:

  • To overcome expression barriers for isoprene synthase (IspS) in cyanobacteria.
  • To engineer cpcB*IspS fusion constructs to enhance isoprene hydrocarbon production.
  • To investigate the impact of linker amino acids on IspS activity and enzyme accumulation.

Main Methods:

  • Constructed various cpcB*IspS fusion proteins with different linker lengths (0, 7, 10, 16, 65 amino acids).
  • Utilized the highly expressed cpcB protein as a leader sequence to improve IspS expression.
  • Quantified isoprene production, enzyme accumulation, and specific activity in engineered cyanobacterial strains.

Main Results:

  • Fusion constructs increased transgene expression by 61 to 275-fold compared to unfused IspS.
  • Specific activity of IspS was reduced in all fusion strains, with variations based on linker composition.
  • The cpcB*L7*IspS strain exhibited a 27-fold improvement in isoprene to biomass yield (5.4 mg g-1) due to a 254-fold increase in enzyme accumulation.

Conclusions:

  • Fusion of cpcB with IspS effectively enhances heterologous expression in cyanobacteria.
  • Optimizing linker amino acids in fusion proteins is crucial for balancing enzyme accumulation and specific activity.
  • The cpcB*L7*IspS fusion strategy represents a significant advancement in renewable isoprene production using cyanobacteria.