Santalene, a major component of the sandalwood essential oil, is a typical representative of sesquiterpenes and has important applications in medicine, food, flavors, and other fields. Due to the limited supply of natural sandalwood resources, there is a growing interest in engineering microbial cell factories for the mass production of santalene. In the present study, Komagataella phaffii (also known as Pichia pastoris) was established as a cell factory for high-level production of α-santalene for the first time. The metabolic fluxes were rewired toward α-santalene biosynthesis through the optimization of promoters to drive the expression of the α-santalene synthase (SAS) gene, overexpression of the key mevalonate pathway genes (i.e., tHMG1, IDI1, and ERG20), and multi-copy integration of the SAS expression cassette. In combination with medium optimization and bioprocess engineering, the optimal strain (STE-9) was able to produce α-santalene with a titer as high as 829.8 ± 70.6 mg/L, 4.4 ± 0.3 g/L, and 21.5 ± 1.6 g/L in a shake flask, batch fermenter, and fed-batch fermenter, respectively. These represented the highest production of α-santalene ever reported, highlighting the advantages of K. phaffii cell factories for the production of terpenoids and other natural products.

中文翻译：

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建立Komagataella phaffii作为高效生产倍半萜类α-檀香烯的细胞工厂

檀香油是檀香精油的主要成分，是倍半萜类的典型代表，在医药、食品、香精等领域有重要应用。由于天然檀香资源的供应有限，人们越来越关注工程微生物细胞工厂以大规模生产檀香。在本研究中，首次建立了Komagataella phaffii（也称为毕赤酵母）作为高水平生产α-檀香烯的细胞工厂。通过优化启动子以驱动 α-檀香烯合酶 ( SAS ) 基因的表达、关键甲羟戊酸途径基因（即tHMG1、IDI1和ERG20 )，以及SAS表达盒的多拷贝整合。结合培养基优化和生物工艺工程，最佳菌株（STE-9）能够生产滴度高达829.8±70.6 mg/L、4.4±0.3 g/L和21.5±1.6 g/的α-檀香萜。 L分别在摇瓶、分批发酵罐和分批补料发酵罐中。这些代表了有史以来最高的 α-檀香烯产量，突出了K. phaffii细胞工厂在生产萜类化合物和其他天然产品方面的优势。