Microbial synthesis of pinene.

Area of Science:

• Biotechnology
• Synthetic Biology
• Chemical Engineering

Background:

• Current biofuels lack sufficient energy density for demanding applications like aviation and rocketry.
• Pinene and its dimers offer a promising alternative with high volumetric heating values, comparable to tactical fuels like JP-10.
• Sustainable production of pinene is crucial for its viability as a next-generation fuel source.

Purpose of the Study:

• To engineer Escherichia coli for sustainable pinene production.
• To enhance pinene yield through combinatorial expression and protein fusion strategies.
• To investigate factors influencing pinene isomer ratios in engineered microbes.

Main Methods:

• Combinatorial expression of pinene synthases (PS) and geranyl diphosphate synthases (GPPS) in E. coli.
• Construction and testing of GPPS-PS protein fusions to overcome pathway bottlenecks.
• Analysis of pinene isomer profiles resulting from different enzyme combinations and substrate concentrations.

Main Results:

• Achieved ~28 mg/L pinene production with optimal combinatorial enzyme expression.
• Engineered GPPS-PS protein fusions, notably the Abies grandis variant, increased pinene yield to 32 mg/L, a six-fold improvement.
• Demonstrated that both pinene synthase and geranyl diphosphate synthase identity, along with geranyl diphosphate availability, dictate the final pinene isomer ratio.

Conclusions:

• Escherichia coli can be engineered for efficient pinene production, a high-energy-density biofuel.
• Protein fusion engineering is a viable strategy to enhance flux and overcome enzyme inhibition in microbial biosynthesis pathways.
• Precise control over enzyme pairings and substrate availability allows for tuning of pinene isomer profiles, impacting fuel properties.