Engineering of Saccharomyces cerevisiae for the production of (+)‐ambrein

Abstract

The triterpenoid (+)-ambrein is the major component of ambergris, a coprolite of the sperm whale that can only be rarely found on shores. Upon oxidative degradation of (+)-ambrein, several fragrance molecules are formed, amongst them (−)-ambrox, one of the highest valued compounds in the perfume industry. In order to generate a Saccharomyces cerevisiae whole-cell biocatalyst for the production of (+)-ambrein, intracellular supply of the squalene was enhanced by overexpression of two central enzymes in the mevalonate and sterol biosynthesis pathway, namely the Nterminally truncated 3-hydroxy-3-methylglutaryl-CoA reductase 1 (tHMG) and the squalene synthase (ERG9). In addition, another key enzyme in sterol biosynthesis, squalene epoxidase (ERG1) was inhibited by an experimentally defined amount of the inhibitor terbinafine in order to reduce flux of squalene towards ergosterol biosynthesis while retaining sufficient activity to maintain cell viability and growth. Heterologous expression of a promiscuous variant of Bacillus megaterium tetraprenyl-β-curcumene cyclase (BmeTC-D373C), which has been shown to be able to catalyse the conversion of squalene to 3-deoxyachillol and then further to (+)-ambrein resulted in production of these triterpenoids in S. cerevisiae for the first time. Triterpenoid yields are comparable with the best microbial production chassis described in literature so far, the methylotrophic yeast Pichia pastoris. Consequently, we discuss similarities and differences of these two yeast species when applied for whole-cell (+)-ambrein production.

Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein

“…2b). However, as the single enzyme system of BmeTC D373C has been shown to be more advantageous for producing of 1 in yeast (Supplementary Table 1) 17,18 , the present study further improved upon BmeTC D373C .…”

“…2c). In a bioreactor, the production of 1 by BmeTC D373C in yeast Pichia pastoris reached a maximum titer of 105 mg L -1 of culture medium 17 , which was higher than that produced using other host systems or with the two enzyme system involving SHC D377C and BmeTC WT (Supplementary Table 1) [17][18][19] .…”

Applying the auxin-based degron system for the inducible, reversible and complete protein degradation in Komagataella phaffii

“…Moser et al. published that, in contrast to S. cerevisiae , there is barely any squalene found in wild-type K. phaffii cells, presumably owing to the quick conversion of this intermediate by Erg1 ( Moser et al., 2018 , 2020 ). Although AID∗-tagging of Erg1 slightly increased protein levels compared to Erg1-3HA by ∼45% ( Figure S4 ), it also led to an increase of squalene even in the absence of auxin ( Figure 4 D), which indicates that C-terminal AID∗-tagging of Erg1 may influence its activity, function or localization.…”

Synthesis of Cyclic Fragrances via Transformations of Alkenes, Alkynes and Enynes: Strategies and Recent Progress

“…The cascade cyclization of multifunctional linear substrates is a step-economic way to gain access to chemical complexity from simplicity, which is a maneuverable strategy as part of Nature’s synthetic power for synthesis of fragrances. For example, the biosynthesis of terpenoids and their derivatives, such as santalol and sclareol, was deemed to include a cationic cascade cyclization and the rearrangement of a linear polyene precursor, which can also be achieved in microbial cell factories [ 103 , 147 , 148 , 149 , 150 , 151 ]. Since Johnson’s landmark synthesis of progesterone [ 152 ], the biomimetic domino cyclization of polyenes and enynes has emerged as one of the most powerful strategies for the construction of polycyclic scaffolds [ 153 ].…”