Engineering Yarrowia lipolytica for de novo production of tetraacetyl phytosphingosine

J Appl Microbiol. 2021 Jun;130(6):1981-1992. doi: 10.1111/jam.14931. Epub 2020 Nov 30.

Abstract

Aims: To genetically engineer the oleaginous yeast Yarrowia lipolytica for de novo production of tetraacetylphytosphingosine (TAPS), a precursor of phytosphingosine, and optimization of fermentation conditions for high yield.

Methods and results: We successfully constructed a TAPS-producing Y. lipolytica CE3 strain by co-expression of Wickerhamomyces ciferrii-derived acetyl transferases, Sli1p and Atf2p. Next, we optimized several environmental factors including temperature, initial pH and C/N ratio for TAPS production in a shake culture. Deletion of LCB4 in CE3 strain increased the volumetric TAPS titre and cell-specific yield to 142·1 ± 10·7 mgTAPS l-1 and 3·08 ± 0·11 mgTAPS gDCW -1 , respectively, in a shake flask culture incubated for 120 h at 28°C with glycerol as the carbon source. Finally, we developed a 5-l fed-batch process with NaOH-mediated pH control and olive oil as a carbon source, exhibiting 650 ± 24 mgTAPS l-1 of TAPS production within 56 h of the fermentation.

Conclusions: The introduction of codon-optimized Sli1p and Atf2p, deletion of LCB4 gene and sexual hybridization, accompanied by specific fermentation conditions, enhanced TAPS yield in Y. lipolytica.

Significance and impact of the study: Our results highlight Y. lipolytica as a promising candidate for the industrial production of TAPS, an important component of cosmetic formulations.

Keywords: ATF2; SLI1; Yarrowia lipolytica; ceramides; optimization; sphingolipid; tetraacetyl phytosphingosine; tetraacetylphytosphingosine.

MeSH terms

  • Batch Cell Culture Techniques
  • Fermentation
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Industrial Microbiology
  • Metabolic Engineering
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomycetales / enzymology
  • Saccharomycetales / genetics
  • Sphingosine / analogs & derivatives*
  • Sphingosine / analysis
  • Sphingosine / biosynthesis
  • Yarrowia / genetics*
  • Yarrowia / metabolism*

Substances

  • Fungal Proteins
  • Recombinant Proteins
  • tetraacetyl-phytosphingosine
  • phytosphingosine
  • Sphingosine

Supplementary concepts

  • Wickerhamomyces ciferrii