Engineering of Saccharomyces cerevisiae as a platform strain for microbial production of sphingosine-1-phosphate

Background: Sphingosine-1-phosphate (S1P) is a multifunctional sphingolipid that has been implicated in regulating cellular activities in mammalian cells. Due to its therapeutic potential, there is a growing interest in developing efficient methods for S1P production. To date, the production of S1P has been achieved through chemical synthesis or blood extraction, but these processes have limitations such as complexity and cost. In this study, we generated an S1P-producing Saccharomyces cerevisiae strain by using metabolic engineering and introducing a heterologous sphingolipid biosynthetic pathway to demonstrate the possibility of microbial S1P production.

Results: To construct the sphingosine-producing S. cerevisiae strain, both the sphingolipid delta 4 desaturase gene (DES1) and the alkaline ceramidase gene (ACER1) derived from Homo sapiens were introduced into the genome of S. cerevisiae by deleting the dihydrosphingosine phosphate lyase gene (DPL1) and the sphingoid long-chain base kinase gene (LCB5) to prevent S1P degradation and byproduct formation, respectively. The sphingosine-producing strain, DDLA, produced sphingolipids containing sphingosine. In flask fed-batch fermentation, the DDLA strain showed a higher production level of sphingosine under aerobic conditions with high initial cell density. The S1P-producing strain was generated by expressing the human sphingosine kinase gene (SPHK1) under the control of the inducible promoter, while deleting the ORM1 gene involved in the regulation of sphingolipid biosynthesis. The S1P-producing strain, DDLAOgS, exhibited the highest sphingosine production level under fed-batch fermentation in a bioreactor, achieving a 2.6-fold increase compared to flask fermentation. S1P biosynthesis in the DDLAOgS strain was verified by qualitative analysis using electrospray ionization mass spectrometry (ESI-MS).

Conclusions: We successfully developed a metabolically engineered S. cerevisiae as a platform strain for microbial production of S1P by introducing an exogenous pathway of sphingolipids metabolism. The engineered yeast strains showed significant capabilities for sphingolipid production, including S1P. To our knowledge, this is the first report demonstrating that engineered S. cerevisiae can be a major platform strain for producing microbial S1P.