Abstract
Most fungal fatty acid synthases assemble from two multidomain subunits, α and β, into a heterododecameric FAS complex. It has been recently shown that the complex assembly occurs in a cotranslational manner and is initiated by an interaction between the termini of α and β subunits. This initial engagement of subunits may be the rate-limiting phase of the assembly and subject to cellular regulation. Therefore, we hypothesized that bypassing this step by genetically fusing the subunits could be beneficial for biotechnological production of fatty acids. To test the concept, we expressed fused FAS subunits engineered for production of octanoic acid in Saccharomyces cerevisiae. Collectively, our data indicate that FAS activity is a limiting factor of fatty acid production and that FAS fusion proteins show a superior performance compared to their split counterparts. This strategy is likely a generalizable approach to optimize the production of fatty acids and derived compounds in microbial chassis organisms.
Highlights
Most fungal fatty acid synthases assemble from two multidomain subunits, α and β, into a heterododecameric FAS complex
Construction and functionality of fused FAS subunits. To synthesize both S. cerevisiae FAS subunits as a single polypeptide (“fusFAS”), FAS1 and FAS2 (α subunit) open reading frames (ORFs) were connected by a sequence encoding a linker derived from the single-chain Ustilago maydis FAS9 (Fig. 1)
In accordance with previous observations[12], the mutated constructs conferred slower growth rates compared to the wildtype fusFAS (Fig. 2A), due to their reduced ability to synthesize the essential long chain (C16 and C18) FA12 and cytotoxicity of the produced OA19
Summary
Most fungal fatty acid synthases assemble from two multidomain subunits, α and β, into a heterododecameric FAS complex. It has been recently shown that the complex assembly occurs in a cotranslational manner and is initiated by an interaction between the termini of α and β subunits. Our data indicate that FAS activity is a limiting factor of fatty acid production and that FAS fusion proteins show a superior performance compared to their split counterparts. As an evolutionary late event, gene splitting was non-invasive to the overall structure, and did not affect the assembly pathway, which was already established before on the single-gene variant[3,6] This view is in line with recent findings proposing that protein complexes are under strong evolutionary selection for ordered assembly pathways[7]. Since upstream pathway engineering can unfold its full potential only if FAS has sufficient capacity to process the precursor molecules, FAS genes are usually overexpressed, e.g. by using strong promoters and/or plasmids[12,15,16]
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