Behaviour of the yeast Kluyveromyces marxianus var. marxianus during its autolysis

The lactic yeast Kluyveromyces marxianus var.marxianus (formerly K. fragilis) autolyzates at faster rate than Saccharomyces cerevisiae. During K. marxianus autolysis, quite similar release kinetics were observed for intracellular space markers (potassium ions, nucleotides), cell-wall components (polysaccharides, N-acetyl-D-Glucosamine) and non specific products (amino nitrogen). By Scanning Electronic Microscopy examination, no cell burst was observed, but a variation in cell shape (from ellipsoidal to cylindrical), as well as a 43% decrease in the internal volume were observed. The mechanism proposed for S. cerevisiae autolysis appeared also likely for K. marxianus.Abbreviations NacGlc N-acetyl-D-glucosamine - x total biomass (dry cellular weight) concentration     

The yeast Kluyveromyces marxianus and its biotechnological potential

Strains belonging to the yeast species Kluyveromyces marxianus have been isolated from a great variety of habitats, which results in a high metabolic diversity and a substantial degree of intraspecific polymorphism. As a consequence, several different biotechnological applications have been investigated with this yeast: production of enzymes (beta-galactosidase, beta-glucosidase, inulinase, and polygalacturonases, among others), of single-cell protein, of aroma compounds, and of ethanol (including high-temperature and simultaneous saccharification-fermentation processes); reduction of lactose content in food products; production of bioingredients from cheese-whey; bioremediation; as an anticholesterolemic agent; and as a host for heterologous protein production. Compared to its congener and model organism, Kluyveromyces lactis, the accumulated knowledge on K. marxianus is much smaller and spread over a number of different strains. Although there is no publicly available genome sequence for this species, 20% of the CBS 712 strain genome was randomly sequenced (Llorente et al. in FEBS Lett 487:71-75, 2000). In spite of these facts, K. marxianus can envisage a great biotechnological future because of some of its qualities, such as a broad substrate spectrum, thermotolerance, high growth rates, and less tendency to ferment when exposed to sugar excess, when compared to K. lactis. To increase our knowledge on the biology of this species and to enable the potential applications to be converted into industrial practice, a more systematic approach, including the careful choice of (a) reference strain(s) by the scientific community, would certainly be of great value.     

Modes of lactose uptake in the yeast species Kluyveromyces marxianus

Twelve lactose-assimilating strains of the yeast species Kluyveromyces marxianus and its varieties marxianus, lactis and bulgaricus were studied with respect to transport mechanisms for lactose, glucose and galactose, fermentation of these sugars and the occurrence of extracellular lactose hydrolysis. The strains fell into three groups. Group I (two strains): Fermentation of lactose, glucose and galactose, extracellular lactose hydrolysis, apparent facilitated diffusion of glucose and galactose; Group II (two strains): Lactose not fermented, glucose and galactose fermented and transported by an apparent proton symport, extracellular hydrolysis of lactose present (one strain) or questionable; Group III (eight strains): Lactose, glucose and galactose fermented, lactose transported by an apparent proton symport mechanism, extracellular hydrolysis of lactose and transport modes for glucose and galactose variable.     

Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source

Growth, substrate consumption, metabolite formation, biomass composition and respiratory parameters of Kluyveromyces marxianus ATCC 26548 were determined during aerobic batch and chemostat cultivations, using mineral medium with glucose as the sole carbon source, at 30 degrees C and pH 5.0. Carbon balances closed within 95-101% in all experiments. A maximum specific growth rate of 0.56 h(-1), a biomass yield on glucose of 0.51 g g(-1), and a maximum specific consumption of oxygen of 11.1 mmol g(-1) h(-1) were obtained during batch cultures. The concentration of excreted metabolites was very low at the culture conditions applied, representing 6% of the consumed carbon at most. Acetate and pyruvate were excreted to a larger extent than ethanol under the batch conditions, and the protein content accounted for 54.6% of the biomass dry weight. Steady states were obtained during chemostats at dilution rates of 0.1, 0.25 and 0.5 h(-1). At the two former dilution rates, cells grew at carbon limitation and the biomass yield on glucose was similar to that obtained under the batch conditions. Metabolite formation was rather low, accounting for a total of 0.005 C-mol C-mol(-1) substrate. At 0.5 h(-1), although the biomass yield on glucose was similar to the value obtained under the above-mentioned conditions, the cultivation was not under carbon limitation. Under this condition, 2-oxoglutarate, acetate, pyruvate and ethanol were the prevalent metabolites excreted. Total metabolite formation only accounted to 0.056 C-mol C-mol(-1) of substrate. A very high protein and a low carbohydrate content (71.9% and 9.6% of biomass dry weight, respectively) were measured in cells under this condition. It is concluded that K. marxianus aligns with the so-called aerobic-respiring or Crabtree-negative yeasts. Furthermore, it has one of the highest growth rates among yeasts, and a high capacity of converting sugar into biomass, even when carbon is not the limiting nutrient. These results provide useful data regarding the future application of K. marxianus in processes aimed at the production of biomass-linked compounds, with high yields and productivities.     

Ethanol adaptation in a thermotolerant yeast strain Kluyveromyces marxianus IMB3

The maximum ethanol concentration produced from glucose in defined media at 45°C by the thermotolerant yeast Kluyveromyces marxianus IMB3 was 44 g L⁻¹. Acclimatisation of the strain through continuous culture at ethanol concentrations up to 80 g L⁻¹, shifted the maximum ethanol concentration at which growth was observed from 40 g L⁻¹ to 70 g L⁻¹. Four isolates were selected from the continuous culture, only one of which produced a significant increase in final ethanol concentration (50 ± 0.4 g L⁻¹), however in subsequent fermentations, following storage on nutrient agar plates, the maximum ethanol concentration was comparable with the original isolate. The maximum specific ethanol production rates (approximately 1.5 g (gh)⁻¹) were also comparable with the original strain except for one isolate (0.7 g (gh)⁻¹). The specific ethanol productivity decreased with ethanol concentration; this decrease correlated linearly (rval 0.92) with cell viability. Due to the transience of induced ethanol tolerance in the strain it was concluded that this was not a valid method for improving final ethanol concentrations or production rates. Received 18 July 1997/ Accepted in revised form 19 February 1998     

Morphological characterization of the dimorphic yeast Kluyveromyces marxianus var. marxianus NRRLy2415 by semi-automated image analysis

A semiautomatic image analysis method has been developed to characterize the morphology of the dimorphic yeast Kluyveromyces marxianus var. marxianus (formerly fragilis) NRRLy2415 undergoing alcoholic fermentation of cheese whey permeate. The method is capable of separating cells into six defined categories, varying from simple ovoid yeast cells to branched mycelial cells. A sample size of 300 cells was found to be sufficient to obtain a statistically significant categorization. The processing time for a sample was found to be approximately 90 min. In addition to qualitative characterization, the method permits the measurement of geometric properties such as the width, length, and volume of individual cells or clusters of cells. When the cells analyzed by the automatic method were categorized on a manual basis, the error level in the automatic routine was found to be less than 3%.     

Application of the Cre-loxP system for multiple gene disruption in the yeast Kluyveromyces marxianus

The yeast Kluyveromyces marxianus presents several interesting features that make this species a promising industrial yeast for the production of several compounds. In order to take full advantage of this yeast and its particular properties, proper tools for gene disruption and metabolic engineering are needed. The Cre-loxP system is a very versatile tool that allows for gene marker rescue, resulting in mutant strains free of exogenous selective markers, which is a very important aspect for industrial application. As the Cre-loxP system works in some non-conventional yeasts, namely Kluyveromyces lactis, we wished to know whether it also works in K. marxianus. Here, we report the validation of this system in K. marxianus CBS 6556, by disrupting two copies of the LAC4 gene, which encodes a beta-galactosidase activity.     

Extraction, purification and characterization of ADH1 from the budding yeast Kluyveromyces marxianus

The enzyme ADH1 has been extracted and purified from the budding yeast Kluyveromyces marxianus, and its enzymatic activity has been compared, with the ADH1 extracted and purified in the same way from the well known yeast Saccharomyces cerevisiae. K. marxianus ADH1 has an optimal temperature higher than the S. cerevisiae enzyme (45-50 degrees vs 35 degrees C), a better stability to pH variations in the oxidative reaction (pH optimum 7.5), a lower Michaelis constant for acetaldehyde, and a good catalytic activity both for fermentative and oxidative reactions. In fact, while in Saccharomyces the constants ratio (velocity constant fermentation/velocity constant oxidation) is about 20,000, in Kluyveromyces the same ratio is only 15. Even if these two Genera are quite related (they belong to the same subfamily) it seems that their ADH1s possess different catalytic properties.     

Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus

Kluyveromyces marxianus is a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. Unlike Saccharomyces cerevisiae, K. marxianus grows on low-cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluated K. marxianus for the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2-pyrone synthase (2-PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high-stability 2-PS mutant and a promoter substitution increased titer four-fold. 2-PS expression from a multi-copy pKD1-based plasmid improved TAL titers a further five-fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed-batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.     

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.     

Listeria monocytogenes Scott A transports glucose by high-affinity and low-affinity glucose transport systems.

Listeria monocytogenes transported glucose by a high-affinity phosphoenolpyruvate-dependent phosphotransferase system and a low-affinity proton motive force-mediated system. The low-affinity system (Km = 2.9 mM) was inhibited by 2-deoxyglucose and 6-deoxyglucose, whereas the high-affinity system (Km = 0.11 mM) was inhibited by 2-deoxyglucose and mannose but not 6-deoxyglucose. Cells and vesicles artificially energized with valinomycin transported glucose or 2-deoxyglucose at rates greater than those of de-energized cells, indicating that a membrane potential could drive uptake by the low-affinity system.     

Protoplast transformation of Kluyveromyces marxianus

The dairy yeast Kluyveromyces marxianus is a promising cell factory for producing bioethanol and heterologous proteins, as well as a robust synthetic biology platform host, due to its safe status and beneficial traits, including fast growth and thermotolerance. However, the lack of high-efficiency transformation methods hampers the fundamental research and industrial application of this yeast. Protoplast transformation is one of the most commonly used fungal transformation methods, but it yet remains unexplored in K. marxianus. Here, we established the protoplast transformation method of K. marxianus for the first time. A series of parameters on the transformation efficiency were optimized: cells were collected in the late-log phase and treated with zymolyase for protoplasting; the transformation was performed at 0 °C with carrier DNA, CaCl₂, and PEG; after transformation, protoplasts were recovered in a solid regeneration medium containing 3–4% agar and 0.8 m sorbitol. By using the optimized method, plasmids of 10, 24, and 58 kb were successfully transformed into K. marxianus. The highest efficiency reached 1.8 × 10⁴ transformants per μg DNA, which is 18-fold higher than the lithium acetate method. This protoplast transformation method will promote the genetic engineering of K. marxianus that requires high-efficiency transformation or the introduction of large DNA fragments.     

Characterisation of a novel thermotolerant yeast, Kluyveromyces marxianus var marxianus: development of an ethanol fermentation process

The fermentation characteristics of the novel, thermotolerant, isolate Kluyveromyces marxianus var marxianus were determined to evaluate its aptitude for use in an ethanol production process. Sustainable growth was not observed under anaerobic conditions, even in the presence of unsaturated fatty acid and sterol. A maximum ethanol concentration of 40 g L⁻¹ was produced at 45°C, with an initial specific ethanol production rate of 1.7 g g⁻¹ h⁻¹. This was observed at ethanol concentrations below 8 g L⁻¹ and under oxygen-limited conditions. The low ethanol tolerance and low growth under oxygen-limited conditions required for ethanol production implied that a simple continuous process was not feasible with this yeast strain. Improved productivity was achieved through recycling biomass into the fermenter, indicating that utilising an effective cell retention method such as cell recycle or immobilisation, could lead to the development of a viable industrial process using this novel yeast strain. Received 14 February 1998/ Accepted in revised form 19 May 1998     

Characterization of Kluyveromyces marxianus as a potential feed additive for ruminants

Aim: The aim of this study was to find suitable yeast isolates as potential microbial feed additives for ruminants. Methods and Results: Yeast isolates from traditional fermented food (tapai) and home‐made wine were selected based on their tolerance to volatile fatty acids (VFA) mixture of acetic, propionic and butyric acids and to pH and temperature according to the rumen condition. The ability to grow in and produce ethanol was determined in yeast extract peptone glucose broth supplemented with a VFA mixture (VFA‐YEPG medium). Fifty‐five isolates showed OD_660nm values between 0·35–0·6, and 27 isolates showed ethanol production in the range of 0·17–0·30% (v/v). All selected isolates were identified as Kluyveromyces marxianus base on biochemical tests (BioLog kit; Biolog Inc., Hayward, CA) and molecular techniques. The best isolate in terms of ethanol production (K. marxianus WJ1) significantly (P < 0·01) improved in vitro apparent dry matter (DM) digestibility of alfalfa (Medicago sativa), guinea grass (Panicum maximum) and timothy (Phleum pretense) hay by rumen microbes. Conclusion: Yeast isolates from tapai and wine were able to grow in VFA‐YEPG medium, and K. marxianus WJ1 improved in vitro DM digestibility of plant substrates. Significance and Impact of the Study: This study indicated the possibility of using K. marxianus as a microbial feed additive.