Repurposing a Fully Reducing Polyketide Synthase toward 2-Methyl Guerbet-like Lipids

Area of Science:

• Biotechnology
• Synthetic Biology
• Enzyme Engineering

Background:

• Polyketide synthases (PKS) are natural enzyme complexes that synthesize diverse bioactive compounds.
• Reprogramming PKS assembly lines offers potential for generating valuable chemicals like biofuels and platform chemicals.
• Mycobacterium tuberculosis PKS12 is an unorthodox, modularly iterative PKS with potential for engineering.

Purpose of the Study:

• To modify and repurpose the first module of Mycobacterium tuberculosis PKS12 (Mt PKS12) for the synthesis of 2-methyl Guerbet lipids.
• To establish a robust method for recombinant expression and purification of the engineered PKS module.
• To demonstrate the utility of the engineered PKS module in a one-pot biosynthetic cascade for producing valuable lipids.

Main Methods:

• Engineered the first module of Mt PKS12 ([M1*]) for 2-methyl Guerbet lipid formation.
• Developed a method for recombinant expression and purification of the [M1*] module.
• Utilized the thioesterase activity of a neighboring beta-ketoacyl synthase (KS) for product release.
• Coupled Escherichia coli fatty acyl-CoA synthetase FadD to [M1*] for targeted lipid synthesis.

Main Results:

• Successfully expressed and purified the engineered [M1*] PKS module.
• Demonstrated the catalytic ability of [M1*] to produce 2-methyl Guerbet-like lipids (C13-C21).
• Achieved one-pot synthesis of Guerbet-like lipids by leveraging promiscuous thioesterase activity.
• Generated the target compound, 2-methyltetradecanoic acid, from lauric acid using FadD and [M1*].

Conclusions:

• Engineered PKS modules, like [M1*], are biosynthetically useful for generating valuable compounds.
• This approach enables the production of Guerbet-like lipids from inexpensive fatty acids.
• The study highlights the potential of synthetic biology for sustainable chemical production.