Substrate and energy costs of the production of exocellular enzymes by Bacillus licheniformis

Substrate and energy costs of the production of exocellular enzymes from glucose and citrate by B. Iicheniformis S1684 as well as molar growth yields corrected for these costs of product formation were calculated using data from chemostat experiments. The calculations showed that 1.46-1.73 mol glucose and 2.31-2.77 mol citrate are needed for formation and excretion of 1 mol protein. Consequently, the values of the maximal product yield from substrate (Y(psm') g/mol) are 80 < Y(psm) < 95 when product is formed from glucose and 50 < Y(psm) < 60 when product is formed from citrate. The higher substrate costs for product formation from citrate are due to a higher level of CO(2) production during protein formation and a higher substrate requirement for the energy supply of product formation and excretion than when product is formed from glucose. The theoretical ATP requirement for protein synthesis could be determined reasonably well, but the energy costs of protein excretion could not be determined exactly. The energy costs of protein formation are higher than those of biomass formation or protein excretion. Molar growth yields corrected for the substrate costs of product formation were high, indicating a high efficiency of growth.Growth and production parameters were determined as well from experimental data of recycling fermentor experiments using a parameter optimization procedure based on a mathematical model describing biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of biomass growth rate. The fitting procedure yielded two growth and production domains during glucose limitation. In the first domain the values for the maximal growth yield and maintenance coefficient were in agreement with those found in chemostat experiments at corresponding values of Y(spm). Domain 2 could be described best with linear growth and product formation. In domain 2 the rate of product formation decreased and more substrate became available for biomass formation. As a consequence the specific growth rate increased in the shift from domain 1 to 2. Domain 2 behavior most probably is caused by the rel-status of B. Iicheniformis S1684.     

Sugar transport and potassium permeability in yeast plasma membrane vesicles

Plasma membrane vesicles were isolated from homogenised yeast cells by filtration, differential centrifugation and aggregation of the mitochondrial vesicles at pH 4. As judged by biochemical, cell electrophoretic and electron microscopic criteria a pure plasma membrane vesicle preparation was obtained.The surface charge density of the plasma membrane vesicles is similar to that of intact yeast cells with an isoelectric point below pH 3. The mitochondrial vesicles have a higher negative surface charge density in the alkaline pH range. Their isoelectric point is near pH 4.5, where aggregation is maximal.The yield of vesicles sealed to K⁺ was maximal at pH 4 and accounted for about one third of the total vesicle volume.The plasma membrane vesicles demonstrate osmotic behaviour, they shrink in NaCl solutions when loosing K⁺.As in intact yeast cells the entry and exit of sugars like glucose or galactose in plasma membrane vesicles is inhibited by UO₂²⁺.Counter transport in plasma membrane vesicles with glucose and mannose and iso-counter transport with glucose suggests that a mobile carrier for sugar transport exists in the plasma membrane.After galactose pathway induction in the yeast cells and subsequent preparation of plasma membrane vesicles the uptake of galactose into the vesicles increased by almost 100% over the control value without galactose induction. This increase is explained by the formation of a specific galactose carrier in the plasma membrane.     

Electrochemical potential and ion transport in vesicles of yeast plasma membrane

Vesicles from yeast plasma membrane were prepared according to Franzusoff and Cirillo [1983) J. Biol. Chem. 258, 3608), with slight modifications. When Mg-ATP was added, this preparation was able to generate a membrane potential, that was sensitive to inhibitors of the yeast H+-ATPase and uncouplers, and could be decreased by the addition of permeant anions, as measured by the fluorescence changes of the dye oxonol V. The addition of ATP could also generate a pH gradient, detectable by the fluorescence changes of the monitor aminochloromethoxyacridine. This gradient was sensitive to inhibitors of ATPase and uncouplers, and could be increased by the addition of permeant anions to the incubation mixture. When the vesicles were loaded with KCl, an increased rate of K+ efflux was produced upon the addition of ATP. Cytochrome oxidase from bovine heart could be reconstituted into the vesicles and was shown to generate a membrane potential difference, negative inside, evidenced by the fluorescence quenching of the cyanide dipropylthiacarbocyanine and the uptake of tetraphenylphosphonium. Besides, in these vesicles, K+ and Rb+, but not Na+ or NH+4 could decrease the quenching of fluorescence and the uptake of tetraphenylphosphonium produced when the electron-donor system was present. In the vesicles in which cytochrome oxidase was incorporated, upon the addition of cytochrome c and ascorbate, the uptake of 86Rb+ could be demonstrated also. This uptake was found to be saturable and inhibited by K+, and to a lesser degree by Na+. The results obtained indicate that these vesicles are reasonably sealed and capable of generating and maintaining a membrane potential. The membrane potential could be used to drive ions across the membrane of the vesicles, indicating the presence and functionality of the monovalent cation carrier. The vesicles, in general terms seem to be suitable for studying transport of ions and metabolites in yeast.     

Role of beta-oxidation enzymes in gamma-decalactone production by the yeast Yarrowia lipolytica.

Some microorganisms can transform methyl ricinoleate into gamma-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C(18)) to the C(10) precursor of gamma-decalactone, (ii) accumulation of other lactones (3-hydroxy-gamma-decalactone and 2- and 3-decen-4-olide), and (iii) gamma-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and gamma-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume gamma-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-gamma-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, beta-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the beta-oxidation flux. We also identified mutant strains that produced 26 times more gamma-decalactone than the wild-type parents.     

Marker enzymes in rat liver vesicles involved in transcellular transport

In order to label the vesicles involved in transcellular transfer (transcytosis) through hepatocytes, polymeric IgA (pIgA) was conjugated to horseradish peroxidase (HRP) and injected into rats. The endosomes containing this ligand at 10 or 20 min after injection were isolated by the diaminobenzidine-induced density-shift procedure and their content in various marker enzymes was measured. The endosomes carrying pIgA-HRP 10 min after injection contained only traces of 5'-nucleotidase and low amounts of alkaline phosphodiesterase I. The estimated marker enzyme content is similar to that observed for the particles containing galactosylated bovine serum albumin conjugated to HRP, a ligand degraded in lysosomes. However, 20 min after injection, the transcytotic endosomes showed a marked enrichment in 5'-nucleotidase and especially in alkaline phosphodiesterase I. The results confirm the heterogeneity of rat liver endosomes and substantiate the concept of distinct endosomal compartments.     

Elution of exocellular enzymes from Saccharomyces fragilis and Saccharomyces cerevisiae

Weimberg, Ralph (Northern Regional Research Laboratory, Peoria, Ill.), and William L. Orton. Elution of exocellular enzymes from Saccharomyces fragilis and Saccharomyces cerevisiae. J. Bacteriol. 91:1-13. 1966.-Invertase and acid phosphatase are repressible exocellular enzymes in Saccharomyces fragilis and S. cerevisiae. The conditions for eluting these enzymes from both organisms were compared. Either KCl or beta-mercaptoethanol eluted the enzymes from S. fragilis, and the amounts eluted varied quantitatively according to the physiological age of the organism. In addition to eluting enzymatic activity from the cells, these reagents also caused a large increase in the amount of activity that remained associated with the cells of S. fragilis. Invertase and acid phosphatase were not removed from cells of S. cerevisiae by KCl or beta-mercaptoethanol. These enzymes were separated from S. cerevisiae cells only when there was some degree of cell-wall digestion by snail gut fluid.     

Role of glycosylation in secretion of yeast acid phosphatase

The minimal glycosylation requirement for acid phosphatase secretion and activity was investigated using tunicamycin, an inhibitor of protein glycosylation, and a yeast mutant defective in the synthesis of oligosaccharide outer chains. The results obtained show that outer chain addition is not essential for secretion of active enzyme and that only 4 core chains, out of 8 normally attached to a protein subunit, are sufficient for enzyme transport to the periplasmic space. Enzyme forms with less than 4 chains were retained in membranes of endoplasmic reticulum. Secreted underglycosylated enzyme forms are partially or completely inactive.     

Production of an exocellular acid phosphatase by the yeast Hansenula holstii NCYC 560

1. The yeast Hansenula holstii NCYC 560 produced invertase and an inducible acid phosphatase located betweent the cytoplasmic membrane and the yeast cell wall. 2. These enzymes were also found in the culture medium outside the cell boundaries. 3. The amount of cell wall mannan in cells grown in phosphate-limited medium decreased in comparison with that of cells grown in phospahte-rich medium. 4. It is proposed that the mannan in this yeast is a loose and highly permeable structure, allowing external enzymes to leave the cell boundaries.     

Continuous monitoring of transport by fluorescence on cells and vesicles

Fluorescence techniques are gaining wider applicability in the field of membrane transport due to their high temporal resolution, modest demand for biological material and the kinetic information which is made available by fluorescence tracings. The development of novel fluorescent substrates for particular transport systems and of novel fluorescent indicators for permeant ions, have opened the way for studying transport kinetics and regulation of transport in a variety of cellular and vesicular systems. The various methods of continuous monitoring of transport by fluorescence (CMTF) which are presently in use, are reviewed with emphasis on both analytical and applicative properties.     

Role of exocellular polymer in the design of activated sludge

This research is aimed at understanding the reactor-settler interaction. The reactor operating conditions affect the sludge-settling properties due to (a) production of exocellular polymer that serves as a flocculant in the settler and (b) population shift from flocculating bacteria to filamentous microorganisms that do not settle. A structured kinetic model that accounts for stored substrate and production of polymers, biomass, and inerts was constructed and applied to various open and closed reactor configurations. It is based on our observation that, in a batch reactor, the polymer is produced during endogenous respiration. The model describes well the observed temporal variation in substrate, biomass, and polymer concentration. Application of the model to continuous operation predicts increasing polymer concentration with sludge age, which is in good agreement with one study in the literature. Our study in a once-through reactor, as well as others, suggests an inverse dependence. The difference is probably due to different operating modes. Our study confirmed the role of the polymer in the sedimentation process. Under some conditions, however, a slow population shift to filamentous growth becomes the dominant factor.     

Role of sugars in phosphate transport in baker's yeast

Sugar substrates which depress the intracellular level of inorganic phosphate in baker's yeast (d-glucose,d-fructose,d-mannose, sucrose, as well as maltose andd-galactose after appropriate induction) also make transmembrane flux of phosphate anions possible. Acetate and ethanol, although readily oxidized, as well as nonmetabolized sugars, do not produce the effect. Phosphate uptake in whole cells (but not in protoplasts) is accelerated by preincubation with substrate either aerobically or anaerobically but the actual presence of substrate in the incubation medium is required for transport to take place. Starved cells take up phosphate from the medium with aK _m of 3mm, the half-activation concentration by glucose being 18mm, the amount taken up being constant under given conditions (40 μmol/g dry wt. here). Phosphate-rich cells lose phosphate to the medium in the presence of a suitable substrate. The uptake process is characterized by an activation energy of 13400 cal/mol at 10⁻⁶ m phosphate and of 9400 cal/mol at 10⁻³ m phosphate. The process shows two optima at pH 5.0 and 7.0. A short-lived intermediate of fermentative sugar metabolism is postulated as essential for the translocation of phosphate across the yeast membrane.     

Proton transport by gastric membrane vesicles

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H⁺ uptake into an osmotically sensitive space. The apparent K_m for ATP was 1.7 · 10^?4 M in the absence of a K⁺ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺ and is inhibited by ATPase inhibitors such as N,N′-dicylclohexylcarbodiimide. Maximal H⁺ uptake occurs with an outward K⁺ gradient but the minimal apparent K_A is found in the absence of a K⁺ gradient. The pH optimum for H⁺ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphorylation of the enzyme, but is considerably less than the pH maximum of the K⁺ dependent dephosphorylation. In the presence of an inward K^? gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H⁺ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K⁺ conductance but a measurable H⁺ conductance, hence a K⁺ gradient can produce an H⁺ gradient in the presence of valinomycin. The uptake and spontaneous leak of H⁺ are temperature sensitive skin with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H⁺ uptake by these vesicles is probably due to a dimeric (H⁺ + K⁺)-ATPase and is probably non-electrogenic.     

Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes

In order for secretion to progress, ER-derived transport vesicles must target to, and fuse with the cis-Golgi compartment. These processes have been reconstituted using highly enriched membrane fractions and partially purified soluble components. The functionally active yeast Golgi membranes that have been purified are highly enriched in the cis- Golgi marker enzymes alpha 1,6 mannosyltransferase and GDPase. Fusion of transport vesicles with these membranes requires both GTP and ATP hydrolysis, and depends on cytosolic and peripheral membrane proteins. At least two protein fractions from yeast cytosol are required for the reconstitution of ER-derived vesicle fusion. Soluble fractions prepared from temperature-sensitive mutants revealed requirements for the Ypt1p, Sec19p, Sly1p, Sec7p, and Uso1 proteins. A model for the sequential involvement of these components in the targeting and fusion reaction is proposed.     

Inactivation of active glucose transport in Candida wickerhamii is triggered by exocellular glucose

Abstract Under conditions of derepression the yeast Candida wickerhamii formed a high-affinity glucose proton symport. Glucose and glucose analogues induced inactivation of the glucose proton symport and its interconversion into a low-affinity facilitated diffusion system. The specific inactivation rate increased with the concentration of the inactivating sugar and did not obey saturation kinetics. This dependence was still pronounced at sugar concentrations far above saturation of the glucose transport systems. This suggested that the inactivation and interconversion mechanism was triggered by interaction of the inactivating sugar with receptor sites located on the cell surface.     

Proton-motive force-driven D-galactose transport in plasma membrane vesicles from the yeast Kluyveromyces marxianus.

Galactose transport was studied in membrane vesicles, prepared by fusion of plasma membranes from the yeast Kluyveromyces marxianus with proteoliposomes containing beef heart cytochrome c oxidase as a proton-motive force-generating system. Sugar transport studies performed under nonenergized conditions revealed that, even at high protein to phospholipid ratios, not all vesicles contained a D-galactose-specific transporter. The amount of vesicles containing an active carrier proved to be proportional to the amount of plasma membrane protein present in the fusion mixture. By addition of a suitable electron donor system a proton-motive force of -160 mV could be generated, inside alkaline and negative. Moreover, D-galactose accumulation was observed. It was found that D-galactose accumulation was highly dependent on the phospholipid composition of the vesicles, whereas generation of a proton-motive force was not. Best results were obtained with vesicles prepared with Escherichia coli phospholipid, giving a galactose accumulation of 14 times. Uphill transport could be established under conditions where only the pH gradient or the electrical gradient was present. Moreover, kinetic analysis of the galactose transport activity in energized vesicles revealed influx with a Km value of 540 microM, which is in good agreement with the apparent affinity constant obtained with whole cells. These results establish that galactose transport of K. marxianus is a proton-motive force-driven process. Moreover it demonstrates that plasma membrane vesicles co-reconstituted with cytochrome c oxidase are a valuable resource for the analysis of proton-motive force-driven sugar transport systems of yeast.