Perdeuteration of cholesterol for neutron scattering applications using recombinant Pichia pastoris

Deuteration of biomolecules has a great impact on both quality and scope of neutron scattering experiments. Cholesterol is a major component of mammalian cells, where it plays a critical role in membrane permeability, rigidity and dynamics, and contributes to specific membrane structures such as lipid rafts. Cholesterol is the main cargo in low and high-density lipoprotein complexes (i.e. LDL, HDL) and is directly implicated in several pathogenic conditions such as coronary artery disease which leads to 17 million deaths annually. Neutron scattering studies on membranes or lipid-protein complexes exploiting contrast variation have been limited by the lack of availability of fully deuterated biomolecules and especially perdeuterated cholesterol. The availability of perdeuterated cholesterol provides a unique way of probing the structural and dynamical properties of the lipoprotein complexes that underly many of these disease conditions. Here we describe a procedure for in vivo production of perdeuterated recombinant cholesterol in lipid-engineered Pichia pastoris. Using flask and fed-batch fermenter cultures in deuterated minimal medium perdeuteration of the purified cholesterol was verified by mass spectrometry and its use in a neutron scattering study was demonstrated using neutron reflectometry

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

Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase

Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases

Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis

Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy

The impact of deuteration on natural and synthetic lipids : A neutron diffraction study

The structural investigation of cellular membranes requires access to model systems where the molecular complexity is representative of the cellular environment and that allow for the exploitation of structural techniques. Neutron scattering, and in particular neutron diffraction can provide unique and detailed information on the structure of lipid membranes. However, deuterated samples are desirable to fully exploit this powerful method. Recently, the extraction of lipids from microorganisms grown in deuterated media was demonstrated to be both an attracting route to obtain complex lipid mixtures resembling the composition of natural membranes, and to producing deuterated molecules in a very convenient way. A full characterization of these deuterated extracts is hence pivotal for their use in building up model membrane systems. Here we report the structural characterization of lipid extracts obtained from Pichia pastoris by means of neutron diffraction measurements. In particular, we compare the structure of membranes extracted from yeast cells grown in a standard culture medium and in a corresponding deuterated culture medium. The results show that the different molecular composition of the deuterated and protiated lipid extracts induce different structural organization of the lipid membranes. In addition, we compare these membranes composed of extracted yeast lipids with stacked bilayers prepared from synthetic lipid mixtures

The Production of Matchout-Deuterated Cholesterol and the Study of Bilayer-Cholesterol Interactions

The deuteration of biomolecules provides advanced opportunities for neutron scattering studies. For low resolution studies using techniques such as small-angle neutron scattering and neutron reflection, the level of deuteration of a sample can be varied to match the scattering length density of a specific DO/HO solvent mixture. This can be of major value in structural studies where specific regions of a complex system can be highlighted, and others rendered invisible. This is especially useful in analyses of the structure and dynamics of membrane components. In mammalian membranes, the presence of cholesterol is crucial in modulating the properties of lipids and in their interaction with proteins. Here, a protocol is described for the production of partially deuterated cholesterol which has a neutron scattering length density that matches that of 100% DO solvent (hereby named matchout cholesterol). The level of deuteration was determined by mass spectrometry and nuclear magnetic resonance. The cholesterol match-point was verified experimentally using small angle neutron scattering. The matchout cholesterol was used to investigate the incorporation of cholesterol in various phosphatidylcholine supported lipid bilayers by neutron reflectometry. The study included both saturated and unsaturated lipids, as well as lipids with varying chain lengths. It was found that cholesterol is distributed asymmetrically within the bilayer, positioned closer to the headgroups of the lipids than to the middle of the tail core, regardless of the phosphatidylcholine species

Nanoscale Structure and Dynamics of Model Membrane Lipid Raft Systems, Studied by Neutron Scattering Methods

Quasi-elastic neutron scattering (QENS) and small angle neutron scattering (SANS), in combination with isotopic contrast variation, have been used to determine the structure and dynamics of three-component lipid membranes, in the form of vesicles, comprising an unsaturated [palmitoyl-oleoyl-phosphatidylcholine (POPC) or dioleoyl-phosphatidylcholine (DOPC)], a saturated phospholipid (dipalmitoyl-phosphatidylcholine (DPPC)), and cholesterol, as a function temperature and composition. SANS studies showed vesicle membranes composed of a 1:1:1 molar ratio of DPPC:DOPC:cholesterol and a 2:2:1 molar ratio of DPPC:POPC:cholesterol phase separated, forming lipid rafts of ∼18 and ∼7 nm diameter respectively, when decreasing temperature from 308 to 297 K. Phase separation was reversible upon increasing temperature. The larger rafts observed in systems containing DOPC are attributed to the greater mis-match in lipid alkyl chains between DOPC and DPPC, than for POPC and DPPC. QENS studies, over the temperature range 283–323K, showed that the resulting data were best modelled by two Lorentzian functions: a narrow component, describing the “in-plane” lipid diffusion, and a broader component, describing the lipid alkyl chain segmental relaxation. The overall “in-plane” diffusion was found to show a significant reduction upon increasing temperature due to the vesicle membranes transitioning from one containing rafts to one where the component lipids are homogeneously mixed. The use of different isotopic combinations allowed the measured overall reduction of in-plane diffusion to be understood in terms of an increase in diffusion of the saturated DPPC lipid and a corresponding decrease in diffusion of the unsaturated DOPC/POPC lipid. As the rafts are considered to be composed principally of saturated lipid and cholesterol, the breakdown of rafts decreases the exposure of the DPPC to cholesterol whilst increasing the exposure of cholesterol to unsaturated lipid. These results show the sensitivity of lipid diffusion to local cholesterol concentration, and the importance of considering the local, rather that the global composition of a membrane when understanding the diffusion processes of lipids within the membrane. The novel combination of SANS and QENS allows a non-intrusive approach to characterize the structure and dynamics occurring in phase-separated model membranes which are designed to mimic the lateral heterogeneity of lipids seen in cellular membranes–a heterogeneity that can have pathological consequences