Establishing <i>Komagataella phaffii</i> as a Cell Factory for Efficient Production of Sesquiterpenoid α-Santalene

Abstract

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, <i>Komagataella phaffii</i> (also known as <i>Pichia pastoris</i>) 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 (<i>SAS</i>) gene, overexpression of the key mevalonate pathway genes (i.e., <i>tHMG1</i>, <i>IDI1</i>, and <i>ERG20</i>), and multi-copy integration of the <i>SAS</i> 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 <i>K. phaffii</i> cell factories for the production of terpenoids and other natural products.