Influence of Water Potential on γ-Decalactone Production by the Yeast Sporidiobolus salmonicolor

The influence of water potential on γ-decalactone production by the yeast Sporidiobolus salmonicolor cultivated in a liquid medium was evaluated by gas-chromatographic analysis. Modifications in water potential led to a number of variations in the aroma production. Maximum extracellular production occurred at water activity (a_w) with a value of 0.99. Further analyses revealed an important phenomenon of cellular accumulation of aroma for a_w values between 0.97 and 0.99.     

Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-Decalactone by Yarrowia lipolytica

We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δpox3, which produced 220 mg of γ-decalactone per liter after 24 h. The Δpox2 Δpox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Δpox2 Δpox3 Δpox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Δpox2 Δpox3 Δpox4 Δpox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into γ-decalactone, demonstrating that Aox4p is essential for the biotransformation.     

The Production of γ-Decalactone by Fermentation of Castor Oil

A microbial process for the production of optically-active γ-decalactone from the ricinoleic acid present as triglycerides in castor oil has been developed, γ-decalactone (γDL) is a component of some fruit flavours, being an important organoleptic component of peach flavours. Screening showed two red yeast microorganisms, Rhodotorula glutinis and Sporobolomyces odortts to be especially suitable for this biotransformation. The process involves lipase-mediated hydrolysis of the castor oil to give free ricinoleic acid, uptake of the acid by the cells and aerobic fermentation to achieve abbreviated β-oxidation of the ricinoleic acid (12-hydroxyoleic acid) into 4-hydroxydecanoic acid (4HDA), lactonisation of the acid into γ-DL, followed by solvent extraction and distillation. γ-DL broth concentrations of 0.5-1.2g · 1^-t were obtained after 3-5 days from fermentation media containing 10 g · 1^-1 castor oil, representing an 8.3-20.0% theoretical yield. Intermediates detected were consistent with the operation of the β-oxidation pathway. Appreciable amounts of novel metabolites identified as cis and trans isomers of a tetrahydrofuran (C₁₀) were also produced. Their formation from 4HDA appeared to be non-enzymic and was favoured by anaerobic conditions. Yields of γ-DL were inversely proportional to the concentration of castor oil present in the medium, indicating that substrate inhibition takes place. The highest yields of γ-DL were obtained when castor oil was present from the beginning of the fermentation, rather than when added once the fermentation had become established, demonstrating that the β-oxidation pathway and/or transport system require continual induction. Significant amounts of γ-DL were not produced from other fatty acids, including ricinelaidic acid, the trans isomer of ricinoleic acid. γ-DL formation was dramatically inhibited by antibiotic inhibitors of oxidative phosphorylation, indicating the importance of intact β-oxidation pathways, whereas inhibitors of protein synthesis and cell-wall synthesis had much less marked effects. Selective extraction of 4HDA from the fermentation broths, and of γDL from broth lactonised by heating at low pH, could be achieved by adsorption to Amberlite XAD-1 and XAD-7 resins respectively. Some product could be recovered from the exit gases of the fermenter by passing through propylene glycol traps. This pathway is unusual in that it is a rare example of the truncated β-oxidation of a fatty acid by microorganisms. This effect probably occurs because of partial inhibition of one or more enzymes of the β-oxidation pathway by the C₁₀ hydroxylated fatty acid intermediate(s) allowing intracellular accumulation of the 4HDA, followed by leakage out of the cell; although further metabolism of this C₁₀ intermediate does take place slowly.     

Production of Lycopene in the Non-Carotenoid-Producing Yeast Yarrowia lipolytica

The codon-optimized genes crtB and crtI of Pantoea ananatis were expressed in Yarrowia lipolytica under the control of the TEF1 promoter of Y. lipolytica. Additionally, the rate-limiting genes for isoprenoid biosynthesis in Y. lipolytica, GGS1 and HMG1, were overexpressed to increase the production of lycopene. All of the genes were also expressed in a Y. lipolytica strain with POX1 to POX6 and GUT2 deleted, which led to an increase in the size of lipid bodies and a further increase in lycopene production. Lycopene is located mainly within lipid bodies, and increased lipid body formation leads to an increase in the lycopene storage capacity of Y. lipolytica. Growth-limiting conditions increase the specific lycopene content. Finally, a yield of 16 mg g⁻¹ (dry cell weight) was reached in fed-batch cultures, which is the highest value reported so far for a eukaryotic host.     

Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail

Platelet agonists increase the affinity state of integrin α_IIbβ₃, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. β₃-Endonexin is a novel 111–amino acid protein that binds selectively to the β₃ tail. Since β₃-endonexin is present in platelets, we asked whether it can affect α_IIbβ₃ function. When β₃-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor α_IIbβ₃ affinity state in transfected cells by flow cytometry. Cells transfected with GFP and α_IIbβ₃ bound little or no PAC1. However, those transfected with GFP/β₃-endonexin and α_IIbβ₃ bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β₃-endonexin did not affect levels of surface expression of α_IIbβ₃ nor did it modulate the affinity of an α_IIbβ₃ mutant that is defective in binding to β₃-endonexin. Affinity modulation of α_IIbβ₃ by GFP/β₃-endonexin was inhibited by coexpression of either a monomeric β₃ cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β₃-endonexin can modulate the affinity state of α_IIbβ₃ in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein–protein interactions at the level of the cytoplasmic tails of α_IIbβ₃.     

Dual Lipid Modification of the Yeast Gγ Subunit Ste18p Determines Membrane Localization of Gβγ

The pheromone response in the yeast Saccharomyces cerevisiae is mediated by a heterotrimeric G protein. The Gbetagamma subunit (a complex of Ste4p and Ste18p) is associated with both internal and plasma membranes, and a portion is not stably associated with either membrane fraction. Like Ras, Ste18p contains a farnesyl-directing CaaX box motif (C-terminal residues 107 to 110) and a cysteine residue (Cys 106) that is a potential site for palmitoylation. Mutant Ste18p containing serine at position 106 (mutation ste18-C106S) migrated more rapidly than wild-type Ste18p during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic mobility of wild-type Ste18p (but not the mutant Ste18p) was sensitive to hydroxylamine treatment, consistent with palmitoyl modification at Cys 106. Furthermore, immunoprecipitation of the Gbetagamma complex from cells cultured in the presence of [(3)H]palmitic acid resulted in two radioactive species on nonreducing SDS-PAGE gels, with molecular weights corresponding to Ggamma and Gbetagamma. Substitution of serine for either Cys 107 or Cys 106 resulted in the failure of Gbetagamma to associate with membranes. The Cys 107 substitution also resulted in reduced steady-state accumulation of Ste18p, suggesting that the stability of Ste18p requires modification at Cys 107. All of the mutant forms of Ste18p formed complexes with Ste4p, as assessed by coimmunoprecipitation. We conclude that tight membrane attachment of the wild-type Gbetagamma depends on palmitoylation at Cys 106 and prenylation at Cys 107 of Ste18p.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

The β-Glucosidase of the Yeast Cell Surface

There are two distinct components of the system which limits the rate at which intact cells of S. cerevisiae C hydrolyze external β-glucosides; one component requires metabolic energy and the other is stereospecific for β-glucosides. The stereospecific component is localized at the cell membrane, as shown by its sensitivity to heavy metal inhibitors which did not penetrate the cell under the conditions used. It was shown that cellobiose-grown cells were able to remove cellobiose from the medium in which they were incubated, and that the cellobiose uptake system was identical to that which limits the patent β-glucosidase activity. In order to test the hypothesis that the system in question was a transport system, for β-glucosides the ability of cellobiose-grown cells to take up ¹⁴C-labeled methyl-β-glucoside (MBG) was studied. The induced cells were able to take up MBG-¹⁴C and the label could be partially chased out by cold MBG and cellobiose; glucose-grown cells could not incorporate label. However, induced cells could not take up label when incubated with ¹⁴C-MBG, thus excluding the hypothesis of transport of intact β-glucosides. It was concluded that the stereospecific membrane component was actually a β-glucosidase, coupled to an energy-dependent transport system for the glucose moiety; the function of the latter was rate-limiting in the over-all activity of the entire system.     

Synthesis of Polyhydroxyalkanoate in the Peroxisome of Saccharomyces cerevisiae by Using Intermediates of Fatty Acid β-Oxidation

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the β-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.     

Inducible γ-Butyrobetaine-Degrading Enzymes in Pseudomonas Species AK 1

Activities of gamma-butyrobetaine hydroxylase and carnitine dehydrogenase were low in cells of Pseudomonas sp. AK 1 grown in the absence of their respective substrates.     

Control of Lipid Accumulation in the Yeast Yarrowia lipolytica

A genomic comparison of Yarrowia lipolytica and Saccharomyces cerevisiae indicates that the metabolism of Y. lipolytica is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the GUT2 gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in Y. lipolytica in this study. This Δgut2 mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to β-oxidation. Y. lipolytica contains six acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX6 genes, that catalyze the limiting step of peroxisomal β-oxidation. Additional deletion of the POX1 to POX6 genes in the Δgut2 strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.     

Physiological Role of β-Phosphoglucomutase in Lactococcus lactis

A β-phosphoglucomutase (β-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of β-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h⁻¹, while the deletion of β-PGM resulted in a maximum specific growth rate of 0.05 h⁻¹ on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as β-glucose 1-phosphate in the medium. Furthermore, the β-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of α-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the β-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded β-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

Assembly and Intracellular Targeting of the βγ Subunits of Heterotrimeric G Proteins

The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent α, β, and γ subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact. Monoclonal anti-β antibodies (ARC5 and ARC9), raised against immunoaffinity purified βγ complexes, recognize β subunits when not associated with γ and can thus be used to monitor assembly of βγ complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of βγ complexes with the plasma membrane fraction starts between 15–30 min of chase. Three pools of β subunits can be distinguished based on their association with γ subunits, their localization, and their detergent solubility. Association of β and α subunits with detergent-insoluble domains occurs within 1 min of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase. Brefeldin A treatment does not interfere with delivery of βγ subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane.     

Protection from Free β-Tubulin by the β-Tubulin Binding Protein Rbl2p

Free beta-tubulin not in heterodimers with alpha-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free beta-tubulin. This study focused specifically on the function of Rbl2p, which, like alpha-tubulin, can rescue cells from free beta-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of beta-tubulin relative to alpha-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of beta-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to beta-tubulin. The data suggest that Rbl2p binds transiently to free beta-tubulin, which then passes into an aggregated form that is not toxic.     

Production of the Carotenoids Lycopene, β-Carotene, and Astaxanthin in the Food Yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, β-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promoters and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, β-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.     

Lytic Action of β(1-3)-Glucanase on Yeast Cells

Candida utilis, Saccharomyces cerevisiae, S. fragilis, Pichia polymorpha, and Hansenula anomala yeast cells, harvested in the early logarithmic phase, were attacked with purified beta(1-3)-glucanase from Micromonospora chalcea, which resulted in the liberation of protoplasts. The treated cells were observed under the electron microscope before the protoplasts were liberated. Differences in the cell walls of the enzyme-treated and untreated cells were observed. The action of the glucanase was also tested against isolated walls of C. utilis. The enzyme attacked the S. cerevisiae cell wall in a uniform manner. The attack on S. fragilis was located in certain zones of the cell wall, where breakage occurred and through which the protoplast emerged. On the other three yeasts, an intermediate attack was observed, not as definitely located as in S. fragilis, yet less uniformly than in S. cerevisiae.