Controlling the fatty acid hydroxylation regioselectivity of CYP152A1 (P450Bsb) by active site engineering

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 regioselectivity of the enzyme. Regioselective in-chain 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 are well described for hydroxylating fatty acids selectively in the chemically more distinct a- or w-position. In contrast, selective in-chain hydroxylation of fatty acids lacks precedence. The peroxygenase CYP152A1 (P450Bsb) is a family member that displays fatty acid hydroxylation at both, the a- and b-position. Please click Additional Files below to see the full abstract

Total synthesis of (-)-neplanocin A from L-ribulose

(-)-Neplanocin A (9) has been synthesized from L-ribulose (1) in 14 steps and in 15 % overall yield. The key step involves an intramolecular nitrone [2 + 3] cycloaddition reaction

Bryostatins - the asymmetric-synthesis of C1-C9 and C11-C16 fragments

The fragments C1-C-9 19 and C-11-C-16 26 of the bryostatins are constructed in an enantioselective and highly diastereoselective fashion from respectively D-pantolactone (2) and L-erythrulose (3) as chiral templates

L-ribulose : a novel chiral pool compound

The unnatural keto-sugar L-ribulose, presently more readily available, is an interesting “chiral pool” product. Some key-intermediates for its transformation are described

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

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: Pichia pastoris, Metabolic engineering, Terpene cyclase, Triterpenoid, Squalene, (+)-ambrei