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 N‐terminally 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.
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.
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•Squalene accumulation in P. pastoris for triterpenoid biosynthesis.•First whole-cell biosynthesis of (+)-ambrein in yeast.•Cell and enzyme engineering improved (+)-ambrein yield to> 100 mg L−1 culture.
Simple and efficient: Protonation of Ru(1,2:5,6‐η‐cod)(η6‐cot) (cod=1,5‐cyclooctadiene, cot=1,3,5‐cyclooctatriene) with HBF4⋅Et2O and then reaction with chiral bisphosphane ligands ($_{\rm ...PP}^{\frown }$=Me‐DuPHOS, BINAP, Tol‐BINAP, JOSIPHOS) affords the corresponding Ru($_{\rm PP}^{\frown }$)(H)(η6‐cot)+ or Ru($_{\rm PP}^{\frown }$)(1,2,3,4,5‐η‐C8H11′)+ (C8H11′=2,4‐cyclooctadienyl; see scheme). Exposure of these cations to H2 in solvents (sol) such as acetone, methanol, and THF affords Ru($_{\rm PP}^{\frown }$)(H)(sol)3+, which are catalysts for (amongst other things) enantioselective hydrogenations of alkenes.
Prototypes of new families of precatalysts and catalysts, Ru((−)‐Me‐DuPHOS)(H)(η6‐1,3,5‐cyclooctatriene)(BF4) and the derived “Ru((−)‐Me‐DuPHOS)(H)(sol)(BF4)”, are presented. They are used in an ...industrial, catalytic, enantioselective hydrogenation that leads to (+)‐cis‐methyl dihydrojasmonate Eq. (1). This stereoisomer is the odorant component of an important, large volume perfumery chemical. P−P=Diphosphane ligand (for example, Me‐DuPHOS=1,2‐bis((2R,5R)‐2,5‐dimethylphospholanyl)benzene); sol=solvent.
Regioselective hydroxylation on inactivated C−H bonds is among the dream reactions of organic chemists. Cytochrome P450 enzymes (CYPs) perform this reaction in general with high regio‐ and ...stereoselectivity (e. g. for steroids as substrates). Furthermore, enzyme engineering may allow to tune the properties of the enzyme. Regioselective hydroxylation of shorter or linear molecules (fatty acids), however, remains challenging even with this enzyme class, due to the high similarity of the substrate's backbone carbons and their conformational flexibility. CYPs hydroxylating fatty acids selectively in the chemically more distinct α‐ or ω‐ position are well described. In contrast, selective in‐chain hydroxylation of fatty acids lacks precedence. The peroxygenase CYP152A1 (P450Bsβ) is a family member that displays fatty acid hydroxylation at both, the α‐ and β‐position, with preference for the α‐position. Herein we report the influence of hydrophobic active site residues on the hydroxylation pattern of this enzyme. By site directed mutagenesis and combination of the libraries, double and triple mutation variants were identified, which hydroxylated decanoic acid (C10) with improved regio‐selectivity in the β‐position. Variants were identified with a 10‐fold increase of the β‐regioselectivity (expressed as α/β‐ratio) compared to the wild type. In total 103 variants of CYP152A1 (P450Bsβ) were investigated.
Taking control of place. The regioselectivity of the peroxygenase P450Bsβ was shifted to increase the hydroxylation in either the α‐ or β‐position.
Simple and efficient: Protonation of Ru(1,2:5,6-η-cod)(η
-cot) (cod=1,5-cyclooctadiene, cot=1,3,5-cyclooctatriene) with HBF
⋅Et
O and then reaction with chiral bisphosphane ligands ($_{\rm ...PP}^{\frown }$=Me-DuPHOS, BINAP, Tol-BINAP, JOSIPHOS) affords the corresponding Ru($_{\rm PP}^{\frown }$)(H)(η
-cot)
or Ru($_{\rm PP}^{\frown }$)(1,2,3,4,5-η-C
H
')
(C
H
'=2,4-cyclooctadienyl; see scheme). Exposure of these cations to H
in solvents (sol) such as acetone, methanol, and THF affords Ru($_{\rm PP}^{\frown }$)(H)(sol)
, which are catalysts for (amongst other things) enantioselective hydrogenations of alkenes.
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