Microbial Cell Factories (Jul 2023)

Genetic inactivation of the Carnitine/Acetyl-Carnitine mitochondrial carrier of Yarrowia lipolytica leads to enhanced odd-chain fatty acid production

  • Eugenia Messina,
  • Camilla Pires de Souza,
  • Claudia Cappella,
  • Simona Nicole Barile,
  • Pasquale Scarcia,
  • Isabella Pisano,
  • Luigi Palmieri,
  • Jean-Marc Nicaud,
  • Gennaro Agrimi

DOI
https://doi.org/10.1186/s12934-023-02137-8
Journal volume & issue
Vol. 22, no. 1
pp. 1 – 16

Abstract

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Abstract Background Mitochondrial carriers (MCs) can deeply affect the intracellular flux distribution of metabolic pathways. The manipulation of their expression level, to redirect the flux toward the production of a molecule of interest, is an attractive target for the metabolic engineering of eukaryotic microorganisms. The non-conventional yeast Yarrowia lipolytica is able to use a wide range of substrates. As oleaginous yeast, it directs most of the acetyl-CoA therefrom generated towards the synthesis of lipids, which occurs in the cytoplasm. Among them, the odd-chain fatty acids (OCFAs) are promising microbial-based compounds with several applications in the medical, cosmetic, chemical and agricultural industries. Results In this study, we have identified the MC involved in the Carnitine/Acetyl-Carnitine shuttle in Y. lipolytica, YlCrc1. The Y. lipolytica Ylcrc1 knock-out strain failed to grow on ethanol, acetate and oleic acid, demonstrating the fundamental role of this MC in the transport of acetyl-CoA from peroxisomes and cytoplasm into mitochondria. A metabolic engineering strategy involving the deletion of YlCRC1, and the recombinant expression of propionyl-CoA transferase from Ralstonia eutropha (RePCT), improved propionate utilization and its conversion into OCFAs. These genetic modifications and a lipogenic medium supplemented with glucose and propionate as the sole carbon sources, led to enhanced accumulation of OCFAs in Y. lipolytica. Conclusions The Carnitine/Acetyl-Carnitine shuttle of Y. lipolytica involving YlCrc1, is the sole pathway for transporting peroxisomal or cytosolic acetyl-CoA to mitochondria. Manipulation of this carrier can be a promising target for metabolic engineering approaches involving cytosolic acetyl-CoA, as demonstrated by the effect of YlCRC1 deletion on OCFAs synthesis.

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