Spontaneous formation of a mannitol-producing variant of Leuconostoc pseudomesenteroides grown in the presence of fructose.

We report the spontaneous formation of a stable mannitol-producing variant of Leuconostoc pseudomesenteroides. The mannitol-producing variant showed mannitol dehydrogenase activity which was absent in the parental strain. It was also able to use fructose and glucose simultaneously, whereas the parental strain showed diauxic growth with these sugars. A possible explanation of these observations is discussed.

Properties, modifications and applications of biopolyesters.

Poly(hydroxyalkanoates) (PHAs), of which poly(hydroxybutyrate) (PHB) is the most common, can be accumulated by a large number of bacteria as energy and carbon reserve. Due to their biodegradability and biocompatibility these optically active biopolyesters may find industrial applications. A general overview of the physical and material properties of PHAs, alongside with accomplished applications and new developments in this field is presented in this chapter. The properties of PHAs are dependent on their monomer composition and therefore it is of great interest that recent research has revealed that, in addition to PHB, a large variety of PHAs can be synthesized microbially. The monomer composition of PHAs depends on the nature of the carbon source and microorganism used. PHB is a typical highly crystalline thermoplastic whereas medium chain length PHAs are elastomers with low melting points and a relatively lower degree of crystallinity. By (chemical) modification of the PHAs, the ultimate properties of the materials can be adjusted even further, when necessary. Applications that have been developed from PHB and related materials (e.g. Biopol) can be found in very different application areas and cover packaging, hygienic, agricultural and biomedical products. Recent application developments based on medium chain length PHAs range from high solid alkyd-like paints to pressure sensitive adhesives, biodegradable cheese coatings and biodegradable rubbers. Technically, the prospects for PHAs are very promising. When the price of these materials can be further reduced, application of biopolyesters will also become economically very attractive.

Development of environmentally friendly coatings and paints using medium-chain-length poly(3-hydroxyalkanoates) as the polymer binder.

Unsaturated medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs) produced by Pseudomonas putida from linseed oil fatty acids (LOFA) and tall oil fatty acids (TOFA), were used as the polymer binder in the formulation of high solid alkyd-like paints. The relatively high concentration of unsaturated alkyl side chains incorporated into the PHA resins resulted in oxidative drying PHA paints having excellent coating properties. The homogeneously pigmented PHA coatings yielded high-gloss, smooth and strong films upon curing and showed an excellent flexibility, a good adhesion to different substrates, cohesive film properties and resistance to chipping.

High-cell-density cultivation of yeasts on disaccharides in oxygen-limited batch cultures.

Many facultatively fermentative yeast species exhibit a "Kluyver effect": even under oxygen-limited growth conditions, certain disaccharides that support aerobic, respiratory growth are not fermented, even though the component monosaccharides are good fermentation substrates. This article investigates the applicability of this phenomenon for high-cell-density cultivation of yeasts. In glucose-grown batch cultures of Candida utilis CBS 621, the onset of oxygen limitation led to alcoholic fermentation and, consequently, a decrease of the biomass yield on sugar. In maltose-grown cultures, alcoholic fermentation did not occur and oxygen-limited growth resulted in high biomass concentrations (90 g dry weight L(-1) from 200 g L(-1) maltose monohydrate in a simple batch fermentation). It was subsequently investigated whether this principle could also be applied to Kluyveromyces species exhibiting a Kluyver effect for lactose. In oxygen-limited, glucose-grown chemostat cultures of K. wickerhamii CBS 2745, high ethanol concentrations and low biomass yields were observed. Conversely, ethanol was absent and biomass yields on sugar were high in oxygen-limited chemostat cultures grown on lactose. Batch cultures of K. wickerhamii grown on lactose exhibited the same growth characteristics as the maltose-grown C. utilis cultures: absence of ethanol formation and high biomass yields. Within the species K. marxianus, the occurrence of a Kluyver effect for lactose is known to be strain dependent. Thus, K. marxianus CBS 7894 could be grown to high biomass densities in lactose-grown batch cultures, whereas strain CBS 5795 produced ethanol after the onset of oxygen limitation and, consequently, yielded low amounts of biomass. Because the use of yeast strains exhibiting a Kluyver effect obviates the need for controlled substrate-feeding strategies to avoid oxygen limitation, such strains should be excellently suited for the production of biomass and growth-related products from low-cost disaccharide-containing feedstocks. (c) 1996 John Wiley & Sons, Inc.

Coordination of sucrose uptake and respiration in the yeast Debaryomyces yamadae.

Screening in batch cultures identified Debaryomyces yamadae as a yeast that exhibits the Kluyver effect for sucrose: this disaccharide can be respired but, even under oxygen-limited conditions, alcoholic fermentation of sucrose does not occur. Ethanol, glycerol and arabitol were the main fermentation products during oxygen-limited growth on glucose in chemostat cultures. None of these fermentation products were produced in oxygen-limited chemostat cultures grown on sucrose and the fraction of the sucrose that could not be respired remained unused in the culture medium. This absence of alcoholic fermentation was not due to repression of the key fermentative enzymes pyruvate decarboxylase and alcohol dehydrogenase. In contrast to some other yeasts that exhibit a Kluyver effect, D. yamadae did not exhibit a preference for ethanol in batch cultures grown on mixtures of ethanol and sucrose. Sucrose metabolism in D. yamadae involves intracellular hydrolysis by an alpha-glucosidase. Incubation of weakly buffered cell suspensions with sucrose led to a rapid transient alkalinization, indicating the presence of a sucrose-proton symport system. The apparent substrate saturation constant of the sucrose-uptake system was 0.2 mmol l-1. Sucrose-dependent alkalinization rates were much lower in samples from oxygen-limited cultures than in samples from aerobic cultures. Transient responses of D. yamadae to oxygen limitation were investigated by applying a sudden decrease in the oxygen feed to aerobic sugar-limited chemostat cultures. In glucose-grown cultures, this led to alcoholic fermentation and no significant accumulation of sugar occurred after the switch. In sucrose-limited cultures, sugar accumulation occurred instantaneously after the switch, and ethanol formation was virtually absent.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient responses of Candida utilis to oxygen limitation: regulation of the Kluyver effect for maltose.

The facultatively fermentative yeast Candida utilis exhibits the Kluyver effect for maltose: this disaccharide is respired and assimilated but, in contrast to glucose, it cannot be fermented. To study the mechanism of the Kluyver effect, metabolic responses of C. utilis to a transition from aerobic, sugar-limited growth to oxygen-limited conditions were studied in chemostat cultures. Unexpectedly, the initial response of maltose-grown cultures to oxygen limitation was very similar to that of glucose-grown cultures. In both cases, alcoholic fermentation occurred after a lag phase of 1 h, during which glycerol, pyruvate and D-lactate were the main fermentation products. After ca. 10 h the behaviour of the maltose- and glucose-grown cultures diverged: ethanol disappeared from the maltose-grown cultures, whereas fermentation continued in steady-state, oxygen-limited cultures grown on glucose. The disappearance of alcoholic fermentation in oxygen-limited chemostat cultures growing on maltose was not due to a repression of the synthesis of pyruvate decarboxylase and alcohol dehydrogenase. The results demonstrate that the Kluyver effect for maltose in C. utilis does not reflect an intrinsic inability of this yeast to ferment maltose, but is caused by a regulatory phenomenon that affects a key enzyme in maltose metabolism, probably the maltose carrier. The observed kinetics indicate that this regulation occurs at the level of enzyme synthesis rather than via modification of existing enzyme activity.

Chemostat cultivation as a tool for studies on sugar transport in yeasts.

Chemostat cultivation enables investigations into the effects of individual environmental parameters on sugar transport in yeasts. Various means are available to manipulate the specific rate of sugar uptake (qs) in sugar-limited chemostat cultures. A straightforward way to manipulate qs is variation of the dilution rate, which, in substrate-limited chemostat cultures, is equal to the specific growth rate. Alternatively, qs can be varied independently of the growth rate by mixed-substrate cultivation or by variation of the biomass yield on sugar. The latter can be achieved, for example, by addition of nonmetabolizable weak acids to the growth medium or by variation of the oxygen supply. Such controlled manipulation of metabolic fluxes cannot be achieved in batch cultures, in which various parameters that are essential for the kinetics of sugar transport cannot be controlled. In sugar-limited chemostat cultures, yeasts adapt their sugar transport systems to cope with the low residual sugar concentrations, which are often in the micromolar range. Under the conditions, yeasts with high-affinity proton symport carriers have a competitive advantage over yeasts that transport sugars via facilitated-diffusion carriers. Chemostat cultivation offers unique possibilities to study the energetic consequences of sugar transport in growing cells. For example, anaerobic, sugar-limited chemostat cultivation has been used to quantify the energy requirement for maltose-proton symport in Saccharomyces cerevisiae. Controlled variation of growth conditions in chemostat cultures can be used to study the differential expression of genes involved in sugar transport and as such can make an important contribution to the ongoing studies on the molecular biology of sugar transport in yeasts.

Is the Kluyver effect in yeasts caused by product inhibition?

Candida utilis CBS 621 exhibits the Kluyver effect for maltose, i.e. this yeast can respire maltose and is able to ferment glucose, but is unable to ferment maltose. When glucose was pulsed to a maltose-grown, oxygen-limited chemostat culture of C. utilis, ethanol formation from glucose started almost instantaneously, indicating that the enzymes needed for alcoholic fermentation are expressed in maltose-grown cells. However, the addition of glucose inhibited maltose metabolism. To eliminate a possible catabolite inhibition and/or repression of enzyme activities involved in maltose metabolism, the effect of simultaneously feeding glucose and maltose to an oxygen-limited, maltose-grown chemostat culture was studied. In this case, the glucose concentration in the culture remained below 0.1 mM, which makes glucose catabolite repression unlikely. Nevertheless, maltose metabolism appeared to cease when the culture was switched to the mixed feed. Based on the outcome of the mixed-substrate studies, it was postulated that the Kluyver effect may be caused by feedback inhibition of maltose utilization by ethanol, the product of fermentative maltose metabolism. If ethanol suppresses the utilization of non-fermentable disaccharides, this would provide a phenomenological explanation for the occurrence of the Kluyver effect: accumulation would then not occur and the rate of maltose metabolism would be tuned to the culture's respiratory capacity. This hypothesis was tested by studying growth of C. utilis CBS 621 and Debaryomyces castellii CBS 2923 in aerobic batch cultures on mixtures of sugars and ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of oxygen limitation on sugar metabolism in yeasts: a continuous-culture study of the Kluyver effect.

Growth and metabolite formation were studied in oxygen-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 growing on glucose or maltose at a dilution rate of 0.1 h-1. With either glucose or maltose S. cerevisiae could be grown under dual limitation of oxygen and sugar. Respiration and alcoholic fermentation occurred simultaneously and the catabolite fluxes through these processes were dependent on the magnitude of the oxygen feed. C. utilis could also be grown under dual limitation of glucose and oxygen. However, at very low oxygen feed rates (i.e. below 4 mmol l-1 h-1) growth was limited by oxygen only, as indicated by the high residual glucose concentration in the culture. In contrast to S. cerevisiae, C. utilis could not be grown anaerobically at a dilution rate of 0.1 h-1. With C. utilis absence of oxygen resulted in wash-out, despite the presence of ergosterol and Tween-80 in the growth medium. The behaviour of C. utilis with respect to maltose utilization in oxygen-limited cultures was remarkable: alcoholic fermentation did not occur and the amount of maltose metabolized was dependent on the oxygen supply. Oxygen-limited cultures of C. utilis growing on maltose always contained high residual sugar concentrations. These observations throw new light on the so-called Kluyver effect. Apparently, maltose is a non-fermentable sugar for C. utilis CBS 621, despite the fact that it can serve as a substrate for growth of this facultatively fermentative yeast. This is not due to the absence of key enzymes of alcoholic fermentation. Pyruvate decarboxylase and alcohol dehydrogenase were present at high levels in maltose-utilizing cells of C. utilis grown under oxygen limitation. It is concluded that the Kluyver effect, in C. utilis growing on maltose, results from a regulatory mechanism that prevents the sugar from being fermented. Oxygen is not a key factor in this phenomenon since under oxygen limitation alcoholic fermentation of maltose was not triggered.

Identification of the maltose transport protein of Saccharomyces cerevisiae.

Identification of the maltose transport protein of Saccharomyces cerevisiae was attempted by searching for maltose-inducible proteins in isolated plasma membranes. Membranes from maltose-grown cells contained two proteins that were absent in glucose-grown cells. The proteins differed in size, but peptide sequence analysis indicated a high degree of homology. The amino-terminal and internal sequences of the largest protein, with an apparent molecular mass of 64 kDa, were determined. These sequences were identical to predicted amino acid sequences in the MAL61 gene product. It is concluded that this protein is the inducible maltose permease of Saccharomyces cerevisiae.

Energetics and kinetics of maltose transport in Saccharomyces cerevisiae: a continuous culture study.

In Saccharomyces cerevisiae, maltose is transported by a proton symport mechanism, whereas glucose transport occurs via facilitated diffusion. The energy requirement for maltose transport was evaluated with a metabolic model based on an experimental value of YATP for growth on glucose and an ATP requirement for maltose transport of 1 mol.mol-1. The predictions of the model were verified experimentally with anaerobic, sugar-limited chemostat cultures growing on a range of maltose-glucose mixtures at a fixed dilution rate of 0.1 h-1. The biomass yield (grams of cells.gram of sugar-1) decreased linearly with increasing amounts of maltose in the mixture. The yield was 25% lower during growth on maltose than during that on glucose, in agreement with the model predictions. During sugar-limited growth, the residual concentrations of maltose and glucose in the culture increased in proportion to their relative concentrations in the medium feed. From the residual maltose concentration, the in situ rates of maltose consumption by cultures, and the Km of the maltose carrier for maltose, it was calculated that the amount of this carrier was proportional to the in situ maltose consumption rate. This was also found for the amount of intracellular maltose. These two maltose-specific enzymes therefore exert high control over the maltose flux in S. cerevisiae in anaerobic, sugar-limited, steady-state cultures.

Kinetics of growth and sugar consumption in yeasts.

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts. Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called 'Crabtree effect' probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect in S. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast. S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions. 'Non-Saccharomyces' yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeast Candida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.

Purification and characterization of an NAD(+)-linked formaldehyde dehydrogenase from the facultative RuMP cycle methylotroph Arthrobacter P1.

When Arthrobacter P1 is grown on choline, betaine, dimethylglycine or sarcosine, an NAD(+)-dependent formaldehyde dehydrogenase is induced. This formaldehyde dehydrogenase has been purified using ammonium sulphate fractionation, anion exchange- and hydrophobic interaction chromatography. The molecular mass of the native enzyme was 115 kDa +/- 10 kDa. Gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular mass of the subunit was 56 kDa +/- 3 kDa, which is consistent with a dimeric enzyme structure. After ammonium sulphate fractionation the partially purified enzyme required the addition of a reducing reagent in the assay mixture for maximum activity. The enzyme was highly specific for its substrates and the Km values were 0.10 and 0.80 mM for formaldehyde and NAD+, respectively. The enzyme was heat-stable at 50 degrees C for at least 10 min and showed a broad pH optimum of 8.1 to 8.5. The addition of some metal-binding compounds and thiol reagents inhibited the enzyme activity.

Maltose/proton co-transport in Saccharomyces cerevisiae. Comparative study with cells and plasma membrane vesicles.

Maltose/proton co-transport was studied in intact cells and in plasma membrane vesicles of the yeast Saccharomyces cerevisiae. In order to determine uphill transport in vesicles, plasma membranes were fused with proteoliposomes containing cytochrome c oxidase as a proton-motive force-generating system. Maltose accumulation, dependent on the electrical and pH gradients, was observed. The initial uptake velocity and accumulation ratio in vesicles proved to be dependent on the external pH. Moreover, kinetic analysis of maltose transport showed that Vmax. values greatly decreased with increasing pH, whereas the Km remained virtually constant. These observations were in good agreement with results obtained with intact cells, and suggest that proton binding to the carrier proceeds with an apparent pK of 5.7. The observation with intact cells that maltose is co-transported with protons in a one-to-one stoichiometry was ascertained in the vesicle system by measuring the balance between proton-motive force and the chemical maltose gradient. These results show that maltose transport in vesicles prepared by fusion of plasma membranes with cytochrome c oxidase proteoliposomes behaves in a similar way as in intact cells. It is therefore concluded that this vesicle model system offers a wide range of new possibilities for the study of maltose/proton co-transport in more detail.