Establishing <i>Komagataella phaffii</i> as a Cell Factory for Efficient Production of Cholesterol Sulfate

Cholesterol sulfate (CS), a sulfated derivative of cholesterol, has important applications in the medical field, because of its extensive biological and pharmacological activities. As the supply of CS heavily relies on chemical conversion of cholesterol extracted from wool grease, there is growing interest in engineering microbial cell factories for large-scale production of CS. This study reports microbial production of CS in the yeast, Komagataella phaffii (also known as Pichia pastoris), for the first time. Through combinatorial metabolic engineering strategies, including blocking the competing branches, overexpressing the rate-limiting mevalonate pathway genes, manipulating lipid metabolism, and enhancing the supply of key cofactor 3′-phosphoadenosine-5′-phosphosulfate (PAPS), CS production was increased more than 6.8-fold when compared with the parent strain. The optimal strain (CHS0505) produced CS with titers reaching 249 mg/L in a shake flask and 545 mg/L in a fed-batch fermenter, highlighting the potential of K. phaffii as a cell factory for steroid production. More importantly, the development of a CS-producing cell factory will advance microbial production of glycosaminoglycans and other sulfated natural products via synthetic biology.