Functional replacement of the Saccharomyces cerevisiae fatty acid synthase with a bacterial type II system allows flexible product profiles. Issue 12 (4th September 2015)

• Record Type: Journal Article
• Authors: Fernandez‐Moya, Ruben; Leber, Christopher; Cardenas, Javier; Da Silva, Nancy A.
• Is Part Of: Biotechnology and bioengineering. Volume 112:Issue 12(2015:Dec.)
• Page Start: 2618
• Page End: 2623
• Publication Date: 2015-09-04
• Subjects: Saccharomyces cerevisiae -- E. coli fatty acid synthase -- medium‐chain fatty acids -- thioesterase -- biorenewable chemicals; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.25679
• Languages: English
• ISSNs: 0006-3592

Abstract

ABSTRACT: The native yeast type I fatty acid synthase (FAS) is a complex, rigid enzyme, and challenging to engineer for the production of medium‐ or short‐chain fatty acids. Introduction of a type II FAS is a promising alternative as it allows expression control for each discrete enzyme and the addition of heterologous thioesterases. In this study, the native Saccharomyces cerevisiae FAS was functionally replaced by the Escherichia coli type II FAS (eFAS) system. The E. coli acpS + acpP (together), fabB, fabD, fabG, fabH, fabI, fabZ, and tesA were expressed in individual S. cerevisiae strains, and enzyme activity was confirmed by in vitro activity assays. Eight genes were then integrated into the yeast genome, while tesA or an alternate thioesterase gene, fatB from Ricinus communis or TEII from Rattus novergicus, was expressed from a multi‐copy plasmid. Native FAS activity was eliminated by knocking out the yeast FAS2 gene. The strains expressing only the eFAS as de novo fatty acid source grew without fatty acid supplementation demonstrating that this type II FAS is able to functionally replace the native yeast FAS. The engineered strain expressing the R. communis fatB thioesterase increased total fatty acid titer 1.7‐fold and shifted the fatty acid profile towards C14 production, increasing it from <1% in the native strain to more than 30% of total fatty acids, and reducing C18 production from 39% to 8%. Biotechnol. Bioeng. 2015;112: 2618–2623. © 2015 Wiley Periodicals, Inc. Abstract : The native Saccharomyces cerevisiae type I fatty acid synthase (FAS) was functionally replaced by the E. coli type II FAS (eFAS). Strains expressing only eFAS grew without fatty acid supplementation. An engineered yeast strain expressing eFAS and a heterologous C14 ‐specific thioesterase increased total fatty acid titer 1.7‐fold and shifted the fatty acid profile towards C14 production, increasing it from <1% in the native strain to more than 30% of total fatty acids, and reducing C18 levels substantially.