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

Sandra Moser; Gernot A Strohmeier; Erich Leitner; Thomas J Plocek; Koenraad Vanhessche; Harald Pichler

doi:10.1016/j.mec.2018.e00077

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

Metab Eng Commun. 2018 Aug 16:7:e00077. doi: 10.1016/j.mec.2018.e00077. eCollection 2018 Dec.

Authors

Sandra Moser¹, Gernot A Strohmeier^{1

2}, Erich Leitner³, Thomas J Plocek⁴, Koenraad Vanhessche⁴, Harald Pichler^{1

5}

Affiliations

¹ Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
² Institute of Organic Chemistry, NAWI Graz, Stremayrgasse 9, 8010 Graz, Austria.
³ Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, NAWI Graz, Stremayrgasse 9, 8010 Graz, Austria.
⁴ ACS International S.A., 184 Route de St-Julien, CH-1228 Plan-les-Ouates, Switzerland.
⁵ Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, BioTechMed Graz, Petersgasse 14/2, 8010 Graz, Austria.

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.

Keywords: (+)-ambrein; AOX1, alcohol oxidase; AaSHC, Alicyclobacillus acidocaldarius squalene-hopene cyclase; BSM, basal salt medium; BmeTC, Bacillus megaterium terpene cyclase; CDW, cell dry weight; FLD1, formaldehyde dehydrogenase 1; HRP, horse radish peroxidase; Metabolic engineering; PTM1, Pichia trace metals; Pichia pastoris; Squalene; Terpene cyclase; Triterpenoid; YNB, yeast nitrogen base; YPD, yeast extract peptone dextrose medium.