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Article Dans Une Revue Applied Microbiology and Biotechnology Année : 2016

Modulation of gluconeogenesis and lipid production in an engineered oleaginous Saccharomyces cerevisiae transformant

Résumé

We previously created an oleaginous Saccharomyces cerevisiae transformant as a dga1 mutant overexpressing Dga1p lacking 29 amino acids at the N-terminal (Dga1a dagger Np). Because we have already shown that dga1 disruption decreases the expression of ESA1, which encodes histone acetyltransferase, the present study was aimed at exploring how Esa1p was involved in lipid accumulation. We based our work on the previous observation that Esa1p acetylates and activates phosphoenolpyruvate carboxykinase (PEPCK) encoded by PCK1, a rate-limiting enzyme in gluconeogenesis, and subsequently evaluated the activation of Pck1p by yeast growth with non-fermentable carbon sources, thus dependent on gluconeogenesis. This assay revealed that the a dagger dga1 mutant overexpressing Dga1a dagger Np had much lower growth in a glycerol-lactate (GL) medium than the wild-type strain overexpressing Dga1a dagger Np. Moreover, overexpression of Esa1p or Pck1p in mutants improved the growth, indicating that the a dagger dga1 mutant overexpressing Dga1a dagger Np had lower activities of Pck1p and gluconeogenesis due to lower expression of ESA1. In vitro PEPCK assay showed the same trend in the culture of the a dagger dga1 mutant overexpressing Dga1a dagger Np with 10 % glucose medium, indicating that Pck1p-mediated gluconeogenesis decreased in this oleaginous transformant under the lipid-accumulating conditions introduced by the glucose medium. The growth of the a dagger dga1 mutant overexpressing Dga1a dagger Np in the GL medium was also improved by overexpression of acetyl-CoA synthetase, Acs1p or Acs2p, indicating that supply of acetyl-CoA was crucial for Pck1p acetylation by Esa1p. In addition, the a dagger dga1 mutant without Dga1a dagger Np also showed better growth in the GL medium, indicating that decreased lipid accumulation was enhancing Pck1p-mediated gluconeogenesis. Finally, we found that overexpression of Ole1p, a fatty acid a dagger 9-desaturase, in the a dagger dga1 mutant overexpressing Dga1a dagger Np improved its growth in the GL medium. Although the exact mechanisms leading to the effects of Ole1p were not clearly defined, changes of palmitoleic and oleic acid contents appeared to be critical. This observation was supported by experiments using exogenous palmitoleic and oleic acids or overexpression of elongases. Our findings provide new insights on lipid accumulation mechanisms and metabolic engineering approaches for lipid production.
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Dates et versions

hal-01531703 , version 1 (01-06-2017)

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Yasushi Kamisaka, Kazuyoshi Kimura, Hiroshi Uemura, Rodrigo Ledesma Amaro. Modulation of gluconeogenesis and lipid production in an engineered oleaginous Saccharomyces cerevisiae transformant. Applied Microbiology and Biotechnology, 2016, 100 (18), pp.8147-8157. ⟨10.1007/s00253-016-7662-x⟩. ⟨hal-01531703⟩
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