Chain length engineering

We have been pursuing the goal of de novo synthesis of defined fatty acids for many years, beginning with our initial study on the binding of cerulenin to yeast FAS (1). Building on this foundation, we described the mechanisms of chain-length control in fatty acid synthesis by bacterial type I FAS. Through combined enzyme kinetics and quantitative computational modeling, we gained a molecular understanding of the parameters governing iterative catalysis in FAS (2).

This knowledge enabled us to produce short-chain fatty acids (SCFAs) in yeast strains carrying rationally engineered FAS variants. A key feature of our design strategy has always been to introduce only essential, minimal mutations. In the yeast FAS, as few as five targeted mutations were sufficient to enable SCFA synthesis. Even without additional metabolic pathway engineering, these strains produced predominantly hexanoic (C6) and octanoic (C8) acids at total titers of up to 464 mg/l (3,4).

We have subsequently extended this approach to engineer mammalian FAS (mFAS) variants capable of producing short- and medium-chain fatty acids (SMCFAs). By introducing targeted amino acid substitutions, we achieved control over product specificity—primarily by reducing the efficiency of the ketoacyl synthase (KS) domain in elongating long-chain acyl intermediates (5) and incorporating a thioesterase (TE) with broad chain-length tolerance. In these mFAS variants, the promiscuous TE preferentially hydrolyzes short- and medium-chain acyl intermediates, which are poor KS substrates, thereby releasing free SMCFAs. This dual optimization enables efficient synthesis of SMCFAs with tunable enzymatic properties, allowing selective production of defined chain-length subsets.

To explore the full potential of our platform, we further optimized C12-FA biosynthesis by metabolic engineering of a yeast strain. Under fed-batch fermentation conditions, the strain - equipped with mFAS and metabolically engineering - achieved high yields (Ludig and Zhai et al., accepted; further details forthcoming). Medium-chain fatty acids (MCFAs), particularly C12-FA, are currently sourced mainly from palm kernel oil. To serve as a sustainable alternative to existing plant-based oleochemical technologies, microbial production platforms must achieve selective synthesis of short- and medium-chain oleochemicals at competitive yields. Our work demonstrates that fatty acid synthases are powerful biosynthetic machines, well-suited as robust and tunable de novo platforms for the production of SMCFAs.

1. Johansson, P. et al. Inhibition of the fungal fatty acid synthase type I multienzyme complex. Proc Natl Acad Sci U S A. 105, 12803–12808 (2008).
2. Gajewski, J. et al. Engineering fatty acid synthases for directed polyketide production. Nat Chem Biol 13, 363–365 (2017).
3. Gajewski, J., Pavlovic, R., Fischer, M., Boles, E. & Grininger, M. Engineering fungal de novo fatty acid synthesis for short chain fatty acid production. Nat Commun 8, 14650 (2017).
4. Gusenda, C., Calixto, A. R., Da Silva, J. R., Fernandes, P. A. & Grininger, M. The Kinetics of Carbon‐Carbon Bond Formation in Metazoan Fatty Acid Synthase and Its Impact on Product Fidelity. Angew. Chem. Int. Ed. e202412195 (2024) doi:10.1002/anie.202412195.
5. Heil, C. S., Wehrheim, S. S., Paithankar, K. S. & Grininger, M. Fatty Acid Biosynthesis: Chain-Length Regulation and Control. Chembiochem : a European journal of chemical biology 20, 2298–2321 (2019).