Evolutionary Engineering Improves Tolerance for Replacement Jet Fuels in Saccharomyces cerevisiae

Area of Science:

• Biotechnology
• Synthetic Biology
• Microbial Engineering

Background:

• Monoterpenes are valuable hydrocarbons with diverse applications, including biofuels.
• Toxicity of monoterpenes hinders their microbial synthesis, posing a significant challenge.
• Developing robust microbial strains is crucial for sustainable production of these compounds.

Purpose of the Study:

• To evolve Saccharomyces cerevisiae strains with enhanced tolerance to limonene.
• To identify genetic modifications responsible for improved monoterpene tolerance.
• To assess the potential of engineered strains for biofuel production.

Main Methods:

• Directed evolution of Saccharomyces cerevisiae in the presence of limonene.
• Genomic sequencing of evolved strains to identify mutations.
• Genomic reconstruction of key mutations in the parent strain.
• Phenotypic analysis of tolerance to various monoterpenes and biofuel blends.

Main Results:

• Evolutionary time course identified mutations in tricalbin proteins Tcb2p and Tcb3p.
• Truncation of Tcb3p (tTcb3p(1-989)) conferred a 9-fold increase in limonene fitness.
• The truncated protein enhanced tolerance to β-pinene (11-fold) and myrcene (8-fold).
• Engineered strain showed a 4-fold increase in tolerance to a biojet fuel blend (AMJ-700t).

Conclusions:

• Truncation of Tcb3p is sufficient to confer significant monoterpene tolerance in yeast.
• This engineering strategy overcomes phase tolerance limitations for toxic alkene production.
• Opens new avenues for the microbial synthesis of C10 alkenes for biofuel applications.