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.

中文翻译：

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将 Komagataella phaffii 建立为高效生产硫酸胆固醇的细胞工厂


硫酸胆固醇 （CS） 是胆固醇的硫酸化衍生物，由于其广泛的生物和药理活性，在医学领域具有重要的应用。由于 CS 的供应严重依赖于从羊毛脂中提取的胆固醇的化学转化，因此人们对用于大规模生产 CS 的工程微生物细胞工厂的兴趣越来越大。本研究首次报道了酵母 Komagataella phaffii（也称为 Pichia pastoris）中 CS 的微生物产生。通过组合代谢工程策略，包括阻断竞争分支、过表达限速甲羟戊酸通路基因、操纵脂质代谢以及增强关键辅因子 3′-磷酸腺苷-5′-磷酸硫酸盐 （PAPS） 的供应，CS 产生比母株增加了 6.8 倍以上。最佳菌株 （CHS0505） 产生的 CS 在摇瓶中滴度达到 249 mg/L，在补料分批发酵罐中达到 545 mg/L，突出了 K. phaffii 作为类固醇生产细胞工厂的潜力。更重要的是，生产 CS 的细胞工厂的开发将通过合成生物学促进糖胺聚糖和其他硫酸化天然产物的微生物生产。