Whole-cell (+)-ambrein production in the yeast Pichia pastoris

Abstract

The triterpenoid (+)-ambrein is a natural precursor for (-)-ambrox, which constitutes one of the most sought-after fragrances and fixatives for the perfume industry. (+)-Ambrein is a major component of ambergris, an intestinal excretion of sperm whales that is found only serendipitously. Thus, the demand for (-)-ambrox is currently mainly met by chemical synthesis. A recent study described for the first time the applicability of an enzyme cascade consisting of two terpene cyclases, namely squalene-hopene cyclase from Alicyclobacillus acidocaldarius (AaSHC D377C) and tetraprenyl-β-curcumene cyclase from Bacillus megaterium (BmeTC) for in vitro (+)-ambrein production starting from squalene. Yeasts, such as Pichia pastoris, are natural producers of squalene and have already been shown in the past to be excellent hosts for the biosynthesis of hydrophobic compounds such as terpenoids. By targeting a central enzyme in the sterol biosynthesis pathway, squalene epoxidase Erg1, intracellular squalene levels in P. pastoris could be strongly enhanced. Heterologous expression of AaSHC D377C and BmeTC and, particularly, development of suitable methods to analyze all products of the engineered strain provided conclusive evidence of whole-cell (+)-ambrein production. Engineering of BmeTC led to a remarkable one-enzyme system that was by far superior to the cascade, thereby increasing (+)-ambrein levels approximately 7-fold in shake flask cultivation. Finally, upscaling to 5 L bioreactor yielded more than 100 mg L−1 of (+)-ambrein, demonstrating that metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-ambrein.

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

“…Product analysis of strain ERG9 ‐t HMG expressing Bme TC‐D373C confirmed the clear advantage of this enzyme variant over the two‐enzyme cascade that we had also already observed in P. pastoris (Moser et al, ). This one amino acid exchange, which actually corresponds to the D377C mutation that changes the product spectrum of Aa SHC from hopene to 3‐deoxyachilleol, leads to a versatile enzyme that is able to catalyse the conversions from squalene to the intermediate 3‐deoxyachilleol and further (+)‐ambrein far more efficiently.…”

“…confirmed the clear advantage of this enzyme variant over the twoenzyme cascade that we had also already observed in P. pastoris (Moser et al, 2018). This one amino acid exchange, which actually corresponds to the D377C mutation that changes the product spectrum of AaSHC FIGURE 4 Quantification of intracellular triterpenoid levels in strains ERG9-tHMG AaSHC-D377C BmeTC and ERG9-tHMG BmeTC-D373C by GC-FID after biomass accumulation for 48 hr and induction of heterologous protein expression for 72 hr in SD-Ura medium in baffled shake flasks.…”

“…TABLE 2 Titers (mg L −1 ) and specific yields (mg g −1 DCW) produced by the best S. cerevisiae strain of this study, ERG9-tHMG BmeTC-D373C (+6 μg mL −1 of terbinafine) in comparison with triterpenoid production of the best-performing P. pastoris strain from Moser et al (2018), P PIS1 -ERG1 BmeTC-D373C (+0.1 μg mL −1 of terbinafine) Abbreviation: dry cell weight. a specific yields were not given but calculated for this study based on our own data.…”

“…We have recently published a study describing the engineering of a P. pastoris for the production of (+)-ambrein. In addition to heterologous expression of the same terpene cyclases described here, namely AaSHC-D377C/BmeTC or BmeTC-D373C, the respective strain had been optimised by exchanging the native promoter of ERG1 to the zinc-regulatable PIS1 promoter that allowed, especially in combination with terbinafine, a clear reduction of squalene flux towards sterol biosynthesis, thereby improving precursor supply for (+)-ambrein biosynthesis (Moser et al, 2018).…”

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

“…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] .…”