Establishing Komagataella phaffii 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, 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 multicopy 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.

Advanced Strategies for the Efficient Production of Squalene by Microbial Fermentation

“…TN22 can reach 95.30 mg/L. 66,67 Moreover, the C/N ratio also has an important effect on the squalene production. Various C/N ratios including 10:1, 20:1, 40:1, 60:1 and 80:1 were studied to investigate their effects on squalene production.…”

“…It was found that when YPD medium was supplemented with 0.14% vitamin (B1, B6, and B12), the squalene titer by using Throstochytrium sp. TN22 can reach 95.30 mg/L. , Moreover, the C/N ratio also has an important effect on the squalene production. Various C/N ratios including 10:1, 20:1, 40:1, 60:1 and 80:1 were studied to investigate their effects on squalene production.…”

Overproduction of Patchoulol in Metabolically Engineered Komagataella phaffii

“…13 The sesquiterpene αsantalene was also synthesized in K. phaffii using combinatorial strategies. 14 Dammarenediol-II is a nuclear molecule of dammarane-type ginsenosides with various pharmacological activities. The production of dammarenediol-II was enhanced 25-fold by improving the supply and reducing the competitive consumption of the precursor 2,3-oxidosqualene.…”

“…Strategies for improving terpenoid production in K. phaffii mainly focus on increasing the supply and decreasing the consumption of precursors. , HMG-CoA reductase, a rate-limiting enzyme of the MVA pathway, and IPP-DMAPP isomerase, which catalyzes an essential activation step in the MVA pathway, are often overexpressed to enhance the precursor supply in the MVA pathway. ,− In a previous study, (+)-nootkatone production was efficiently improved by overexpressing the truncated HMG1 gene ( tHMG1 ) encoding the catalytic domain of HMG1. Many studies have shown that overexpression of tHMG1 alone or in combination with other metabolic engineering strategies could significantly elevate terpenoid production in yeast. ,, In addition, FPP synthase, which is encoded by ERG20 and catalyzes the formation of FPP, was overexpressed to guarantee a sufficient FPP supply in many studies related to terpenoid synthesis .…”

“…Consequently, it has been employed as a potential microbial cell factory to produce various highly valued compounds. 3−6 More specifically, many terpenoids, including carotenoids, 7 (+)-nootkatone, 8 dammarenediol-II, 9,10 lycopene, 11,12 α-farnesene, and α-santalene, 13,14 were produced by engineered K. phaffii. However, there are no reports about the heterologous production of patchoulol by engineering K. phaffii with methanol as the sole carbon source.…”