Production of oils and fats by oleaginous microorganisms with an emphasis given to the potential of the nonconventional yeast Yarrowia lipolytica

Recently, there has been a great upsurge of interest in studies related to several aspects of microbial lipid production, which is one of the top topics in relevant research fields due to the high demand of these fatty materials in food, medical, oleochemical and biofuel industries. Lipid accumulation by the so-called “oleaginous microorganisms” can generate more than 20% w/w of oil in dry biomass and is governed by a plethora of parameters, such as medium pH, incubation temperature, nutrient limitation and C/N (carbon/nitrogen) ratio, which drastically affect the lipid production bioprocess. Until now, considerable work has been undertaken to find the cheapest substrate to enable lipid fermentation by oleaginous microorganisms. This review principally details information regarding microbial lipids, suitable production conditions and focuses attention on using the yeast Yarrowia lipolytica to achieve these objectives. Lipid production by this yeast is discussed and the necessary conditions and suitable substrates are reviewed.     

Production of succinic acid at low pH by a recombinant strain of the aerobic yeast Yarrowia lipolytica

Biotechnological production of weak organic acids such as succinic acid is most economically advantageous when carried out at low pH. Among naturally occurring microorganisms, several bacterial strains are known to produce considerable amounts of succinic acid under anaerobic conditions but they are inefficient in performing the low‐pH fermentation due to their physiological properties. We have proposed therefore a new strategy for construction of an aerobic eukaryotic producer on the basis of the yeast Yarrowia lipolytica with a deletion in the gene coding one of succinate dehydrogenase subunits. Firstly, an original in vitro mutagenesis‐based approach was proposed to construct strains with Ts mutations in the Y. lipolytica SDH1 gene. These mutants were used to optimize the composition of the media for selection of transformants with the deletion in the Y. lipolytica SDH2 gene. Surprisingly, the defects of each succinate dehydrogenase subunit prevented the growth on glucose but the mutant strains grew on glycerol and produced succinate in the presence of the buffering agent CaCO₃. Subsequent selection of the strain with deleted SDH2 gene for increased viability allowed us to obtain a strain capable of accumulating succinate at the level of more than 45 g L^?1 in shaking flasks with buffering and more than 17 g L^?1 without buffering. The possible effect of the mutations on the utilization of different substrates and perspectives of constructing an industrial producer is discussed. Biotechnol. Bioeng. 2010;107:673–682. © 2010 Wiley Periodicals, Inc.     

Combining substrate dynamics, binding statistics, and energy barriers to rationalize regioselective hydroxylation of octane and lauric acid by CYP102A1 and mutants

Hydroxylations of octane and lauric acid by Cytochrome P450-BM3 (CYP102A1) wild-type and three active site mutants--F87A, L188Q/A74G, and F87V/L188Q/A74G--were rationalized using a combination of substrate orientation from docking, substrate binding statistics from molecular dynamics simulations, and barrier energies for hydrogen atom abstraction from quantum mechanical calculations. Wild-type BM3 typically hydroxylates medium- to long-chain fatty acids on subterminal (omega-1, omega-2, omega-3) but not the terminal (omega) positions. The known carboxylic anchoring site Y51/R47 for lauric acid, and hydrophobic interactions and steric exclusion, mainly by F87, for octane as well as lauric acid, play a role in the binding modes of the substrates. Electrostatic interactions between the protein and the substrate strongly modulate the substrate's regiodependent activation barriers. A combination of the binding statistics and the activation barriers of hydrogen-atom abstraction in the substrates is proposed to determine the product formation. Trends observed in experimental product formation for octane and lauric acid by wild-type BM3 and the three active site mutants were qualitatively explained. It is concluded that the combination of substrate binding statistics and hydrogen-atom abstraction barrier energies is a valuable tool to rationalize substrate binding and product formation and constitutes an important step toward prediction of product ratios.     

Inhibition of CYP2B4 by 2-ethynylnaphthalene: evidence for the co-binding of substrate and inhibitor within the active site

2-Ethynylnaphthalene (2EN) is an effective mechanism-based inhibitor of CYP2B4. There are two inhibitory components: (1) irreversible inactivation of CYP2B4 (a typical time-dependent inactivation), and (2) a reversible component. The reversible component was unusual in that the degree of inhibition was not simply a characteristic of the enzyme-inhibitor interaction, but dependent on the size of the substrate molecule used to monitor residual activity. The effect of 2EN on the metabolism of seven CYP2B4 substrates showed that it was not an effective reversible inhibitor of substrates containing a single aromatic ring; substrates with two fused rings were competitively inhibited by 2EN; and larger substrates were non-competitively inhibited. Energy-based docking studies demonstrated that, with increasing substrate size, the energy of 2EN and substrate co-binding in the active site became unfavorable precisely at the point where 2EN became a competitive inhibitor. Hierarchical docking revealed potential allosteric inhibition sites separate from the substrate binding site.     

Generation of a homology model for the human cytochrome P450, CYP24A1, and the testing of putative substrate binding residues by site-directed mutagenesis and enzyme activity studies

A systematic analysis of conserved H-bonding patterns and tertiary structural motifs from 13 crystal structures was used to create a homology model for the human multicatalytic cytochrome P450, CYP24A1, involved in catabolism of 1alpha,25-dihydroxyvitamin D3. The substrate was docked in the active site and used to identify potential substrate contact residues in the B' helix, B'/C loop, F-helix and the beta-5 hairpin. Seven CYP24A1 mutants were created and studied by mammalian cell transfection and CYP24A1 activity assay. Mutants showed reduced metabolic rates and altered metabolite patterns compared to wild-type. We conclude that: Ile-131 positions substrate via A-ring and cis-triene contacts; Trp-134 and Gly-499 are determinants of substrate access; Leu-148 contacts the substrate side-chain; Met-246 is important in mediating regioselectivity. Our findings validate the new model of CYP24A1, which can now be used to predict structural modifications for rational vitamin D drug design.