Abstract:Ceramides play critical roles in maintaining skin barrier functions and exhibit anti-inflammatory and antioxidant properties; thus, their biosynthesis has substantial market potential. Ceramide synthesis has primarily been achieved using Saccharomyces cerevisiae, whereas there have been no reports on ceramide synthesis using the important industrial host Pichia pastoris. This research represents the first instance of ceramide synthesis via genetically modified P. pastoris GS115. The key genes PAS_chr4_0427 and PAS_chr2-2_0408 were sequentially targeted for deletion using CRISPR-Cas9 technology, leading to the construction of the mutant strains GS115-O and GS115-OC. The maximum biomasses of GS115-O and GS115-OC were reduced by 17.03% and 24.06%, respectively, compared with that of GS115, whereas the relative contents of ceramides in these mutants were increased by 3.80- and 4.75-fold, respectively. The analysis revealed the synthesis of 67 ceramide species, including 28 phytoceramides, using GS115-O and GS115-OC, marking an additional five species than that produced using GS115. Notably, the relative contents of 28 ceramide species increase by more than 100-fold, with the highest reaching 8533.98-fold. Furthermore, the contents of phosphatidic acid, diacylglycerols, triacylglycerols, and particularly inositol phosphoceramide were significantly increased in the mutants, with the inositol phosphoceramide content showing the most substantial increase. These results indicate that remarkable lipid metabolic alterations occur, accompanied by increased conversion of ceramides into complex sphingolipids. GS115-O and GS115-OC show great potential for ceramide production, including in industrial applications.
