Yeast Kluyveromyces Marxianus Research Articles

Modeling of a simultaneous saccharification and fermentation process for ethanol production from lignocellulosic wastes by kluyveromyces marxianus

This paper presents the modeling of the main dynamics of a Simultaneous Saccharification and Fermentation (SSF) process using lignocellulosic wastes as substrate. SSF experiments were carried out using the yeast Kluyveromyces marxianus as the inoculum and oil palm wastes as the substrate, in order to obtain glucose and ethanol concentration data. The experimental data were used for the parameter identification and model validation. The resulting model predictsthe dynamic behavior of glucose and ethanol concentrations very closely. Performing a sensitivity analysis, parameters which have a higher effect in the modelpredictions are recognized, so the model can be re-optimized in particular cases with low computational requirements. The re-optimization strategy improves the model capacity to predict the dynamics of the SSF process.

Deletion of a KU80 homolog enhances homologous recombination in the thermotolerant yeast Kluyveromyces marxianus.

Targeted gene replacement in the thermotolerant yeast Kluyveromyces marxianus KCTC 17555 has been hampered by its propensity to non-homologous end joining (NHEJ). To enhance homologous recombination (HR) by blocking NHEJ, we identified and disrupted the K. marxianus KU80 gene. The ku80 deletion mutant strain (Kmku80∆) of K. marxianus KCTC 17555 did not show apparent growth defects under several conditions with the exception of exposure to tunicamycin. The targeted disruption of the three model genes, KmLEU2, KmPDC1, and KmPDC5, was increased by 13-70 % in Kmku80∆, although the efficiency was greatly affected by the length of the homologous flanking fragments. In contrast, the double HR frequency was 0-13.7 % in the wild-type strain even with flanking fragments 1 kb long. Therefore, Kmku80∆ promises to be a useful recipient strain for targeted gene manipulation.

Sulfuric acid hydrolysis and detoxification of red alga Pterocladiella capillacea for bioethanol fermentation with thermotolerant yeast Kluyveromyces marxianus.

One-step sulfuric acid saccharification of the red alga Pterocladiella capillacea was optimized, and various detoxification methods (neutralization, overliming, and electrodialysis) of the acid hydrolysate were evaluated for fermentation with the thermotolerant yeast Kluyveromyces marxianus. A proximate composition analysis indicated that P. capillacea was rich in carbohydrates. A significant galactose recovery of 81.1 ± 5% was also achieved under the conditions of a 12% (w/v) biomass load, 5% (v/v) sulfuric acid, 121°C, and hydrolysis for 30 min. Among the various detoxification methods, electrodialysis was identified as the most suitable for fermentable sugar recovery and organic acid removal (100% reduction of formic and levulinic acids), even though it failed to reduce the amount of the inhibitor 5-HMF. As a result, K. marxianus fermentation with the electrodialyzed acid hydrolysate of P. capillacea resulted in the best ethanol levels and fermentation efficiency.

Enhancing bioethanol production from delactosed whey permeate by upstream desalination techniques

Industrial cheese whey processing comprises generally the isolation of proteins and lactose, but the economic use for the residual molasses, the so‐called delactosed whey permeate (DWP), is still to be improved. One possibility to maximize valorization and to minimize waste water treatment is the conversion of the remaining lactose in the DWP to ethanol by the yeast Kluyveromyces marxianus. This fermentation process depends strongly on the total ash content of the DWP, as high salt concentrations inhibit yeast metabolism. Here, three different approaches were tested to lower the DWP salt content: (i) simple dilution; (ii) nanofiltration; and (iii) electrodialysis. Lactose consumption, ethanol production and time‐dependent yields were compared between the three methods. A dilution of DWP to 60% v/v led to fermentation taking less than 80 h and yield above 7% AbV (alcohol by volume). After nanofiltration, 7.5% AbV was produced in about 80 h, and after electrodialysis, 11% AbV was produced in about 52 h. On the one hand the technical treatments (nanofiltration and electrodialysis) led to enhanced productivity in the fermentations, but, on the other hand, elaborate and extensive preprocessing is needed. Overall, ethanol production from DWP could be enhanced by prior partial desalination.

Effect of oxygenation and temperature on glucose-xylose fermentation in Kluyveromyces marxianus CBS712 strain

BackgroundThe yeast Kluyveromyces marxianus features specific traits that render it attractive for industrial applications. These include production of ethanol which, together with thermotolerance and the ability to grow with a high specific growth rate on a wide range of substrates, could make it an alternative to Saccharomyces cerevisiae as an ethanol producer. However, its ability to co-ferment C5 and C6 sugars under oxygen-limited conditions is far from being fully characterized.ResultsIn the present study, K. marxianus CBS712 strain was cultivated in defined medium with glucose and xylose as carbon source. Ethanol fermentation and sugar consumption of CBS712 were investigated under different oxygen supplies (1.75%, 11.00% and 20.95% of O2) and different temperatures (30°C and 41°C). By decreasing oxygen supply, independently from the temperature, both biomass production as well as sugar utilization rate were progressively reduced. In all the tested conditions xylose consumption followed glucose exhaustion. Therefore, xylose metabolism was mainly affected by oxygen depletion. Loss in cell viability cannot explain the decrease in sugar consumption rates, as demonstrated by single cell analyses, while cofactor imbalance is commonly considered as the main cause of impairment of the xylose reductase (KmXR) - xylitol dehydrogenase (KmXDH) pathway. Remarkably, when these enzyme activities were assayed in vitro, a significant decrease was observed together with oxygen depletion, not ascribed to reduced transcription of the corresponding genes.ConclusionsIn the present study both oxygen supply and temperature were shown to be key parameters affecting the fermentation capability of sugars in the K. marxianus CBS712 strain. In particular, a direct correlation was observed between the decreased efficiency to consume xylose with the reduced specific activity of the two main enzymes (KmXR and KmXDH) involved in its catabolism. These data suggest that, in addition to the impairment of the oxidoreductive pathway being determined by the cofactor imbalance, post-transcriptional and/or post-translational regulation of the pathway enzymes contributes to the efficiency of xylose catabolism in micro-aerobic conditions. Overall, the presented work provides novel information on the fermentation capability of the CBS712 strain that is currently considered as the reference strain of the genus K. marxianus.

Potential of some yeast strains in the stereoselective synthesis of (R)-(−)-phenylacetylcarbinol and (S)-(+)-phenylacetylcarbinol and their reduced 1,2-dialcohol derivatives

Whole cells of different yeast species have been widely used for a number of asymmetric transformations. In the present study, the screening of several yeast strains revealed the utility of Debaryomyces etchellsii in acyloin condensation for (R)-(-)-phenylacetylcarbinol production. Some conditions for the efficient biotransformation of benzaldehyde and minimization in the production of by-products were explored: pH of the reaction medium, use of additives (ethanol or acetonitrile), temperature, time, and substrate concentration and dosing. The optimal conditions found allowed the transformation of up to 10g/L of the starting material in reactions carried out at high scale. Furthermore, the yeast Kluyveromyces marxianus was seen to be a convenient biocatalyst to carry out the kinetic resolution by the bioreduction of racemic (+/-)-phenylacetylcarbinol, resulting in (S)-(+)-phenylacetylcarbinol with excellent stereoselectivity. Finally, the ketone reduction of both isolated stereoisomers (R and S) by D. etchellsii allowed the obtainment of two of the four diastereoisomers of 1-phenyl-1,2-propanediol. All these compounds are key precursors for the production of interesting pharmaceutical and chemical products.

Direct fermentation of raw starch using a Kluyveromyces marxianus strain that expresses glucoamylase and Alpha‐amylase to produce ethanol

Raw starch and raw cassava tuber powder were directly and efficiently fermented at elevated temperatures to produce ethanol using the thermotolerant yeast Kluyveromyces marxianus that expresses α-amylase from Aspergillus oryzae as well as α-amylase and glucoamylase from Debaryomyces occidentalis. Among the constructed K. marxianus strains, YRL 009 had the highest efficiency in direct starch fermentation. Raw starch from corn, potato, cassava, or wheat can be fermented at temperatures higher than 40°C. At the optimal fermentation temperature 42°C, YRL 009 produced 66.52 g/L ethanol from 200 g/L cassava starch, which was the highest production among the selected raw starches. This production increased to 79.75 g/L ethanol with a 78.3% theoretical yield (with all cassava starch were consumed) from raw cassava starch at higher initial cell densities. Fermentation was also carried out at 45 and 48°C. By using 200 g/L raw cassava starch, 137.11 and 87.71 g/L sugar were consumed with 55.36 and 32.16 g/L ethanol produced, respectively. Furthermore, this strain could directly ferment 200 g/L nonsterile raw cassava tuber powder (containing 178.52 g/L cassava starch) without additional nutritional supplements to produce 69.73 g/L ethanol by consuming 166.07 g/L sugar at 42°C. YRL 009, which has consolidated bioprocessing ability, is the best strain for fermenting starches at elevated temperatures that has been reported to date.

Production and Characterization of β-Glucanase Secreted by the Yeast Kluyveromyces marxianus

An extracellular β-glucanase secreted by Kluyveromyces marxianus was identified for the first time. The optimal conditions for the production of this enzyme were evaluated by response surface methodology. The optimal conditions to produce β-glucanase were a glucose concentration of 4% (w/v), a pH of 5.5, and an incubation temperature of 35 °C. Response surface methodology was also used to determine the pH and temperature required for the optimal enzymatic activity. The highest enzyme activity was obtained at a pH of 5.5 and a temperature of 55 °C. Furthermore, the enzyme was partially purified and sequenced, and its specificity for different substrates was evaluated. The results suggest that the enzyme is an endo-β-1,3(4)-glucanase. After optimizing the conditions for β-glucanase production, the culture supernatant was found to be effective in digesting the cell wall of the yeast Saccharomyces cerevisiae, showing the great potential of β-glucanase in the biotechnological production of soluble β-glucan.

Yeasts are essential for cocoa bean fermentation

Cocoa beans (Theobroma cacao) are the major raw material for chocolate production and fermentation of the beans is essential for the development of chocolate flavor precursors. In this study, a novel approach was used to determine the role of yeasts in cocoa fermentation and their contribution to chocolate quality. Cocoa bean fermentations were conducted with the addition of 200ppm Natamycin to inhibit the growth of yeasts, and the resultant microbial ecology and metabolism, bean chemistry and chocolate quality were compared with those of normal (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii and Kluyveromyces marxianus, the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in the control fermentation. In fermentations with the presence of Natamycin, the same bacterial species grew but yeast growth was inhibited. Physical and chemical analyses showed that beans fermented without yeasts had increased shell content, lower production of ethanol, higher alcohols and esters throughout fermentation and lesser presence of pyrazines in the roasted product. Quality tests revealed that beans fermented without yeasts were purplish-violet in color and not fully brown, and chocolate prepared from these beans tasted more acid and lacked characteristic chocolate flavor. Beans fermented with yeast growth were fully brown in color and gave chocolate with typical characters which were clearly preferred by sensory panels. Our findings demonstrate that yeast growth and activity were essential for cocoa bean fermentation and the development of chocolate characteristics.

Non‐homologous end joining‐mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus

The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date.

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

Avaliação sensorial de pães de fermentação natural a partir de culturas starters inovadoras

A fermentação sourdough para a produção de pães é fortemente motivada por seus efeitos benéficos quanto ao sabor, textura, vida de prateleira e pelas propriedades nutricionais dos produtos obtidos. Nesta pesquisa, estudou-se a aplicação dos micro-organismos Kluyveromyces marxianus, Dekkera bruxellensis e Lactobacillus plantarum como culturas starters alternativas na produção de pães. A partir de sete ensaios resultantes de um delineamento de mistura simplex-centroide, foram realizadas nos pães análises sensorial e instrumental. Observou-se que pães produzidos com L. plantarum apresentaram menor volume específico, enquanto que a mistura entre K. marxianus e L. plantarum exerceu influência positiva, possibilitando obter produtos de maior volume específico. A análise sensorial demonstrou a boa aceitabilidade para os pães produzidos com a mistura entre D. bruxellensis e K. marxianus e para a mistura entre os três micro-organismos. Os resultados deste trabalho demonstram a possibilidade do uso de culturas starters definidas para a fermentação sourdough, que é tradicionalmente obtida sem controle de processo.

ChemInform Abstract: Kluyveromyces marxianus CBS 6556 Growing Cells as a New Biocatalyst in the Asymmetric Reduction of Substituted Acetophenones.

Abstract The use of microorganism growing cells is a well recognized methodology in biocatalyzed organic reactions. A non-conventional thermotolerant Kluyveromyces marxianus yeast strain was used for the bio-reduction of different arylketones. Differently substituted ketones were converted into the corresponding ( S )-alcohols with up to 96% enantiomeric excess under very mild reaction conditions. Kluyveromyces marxianus represents a promising biocatalyst for the production of optically active 1-arylethanols.

Simultaneous saccharification and fermentation of Agave tequilana fructans by Kluyveromyces marxianus yeasts for bioethanol and tequila production

Agave tequilana fructans (ATF) constitute a substrate for bioethanol and tequila industries. As Kluyveromyces marxianus produces specific fructanases for ATF hydrolysis, as well as ethanol, it can perform simultaneous saccharification and fermentation. In this work, fifteen K. marxianus yeasts were evaluated to develop inoculums with fructanase activity on ATF. These inoculums were added to an ATF medium for simultaneous saccharification and fermentation. All the yeasts, showed exo-fructanhydrolase activity with different substrate specificities. The yeast with highest fructanase activity in the inoculums showed the lowest ethanol production level (20 g/l). Five K. marxianus strains were the most suitable for the simultaneous saccharification and fermentation of ATF. The volatile compounds composition was evaluated at the end of fermentation, and a high diversity was observed between yeasts, nevertheless all of them produced high levels of isobutyl alcohol. The simultaneous saccharification and fermentation of ATF with K. marxianus strains has potential for industrial application.

Bioethanol Production from Lignocellulosic Biomass by a NovelKluyveromyces marxianusStrain

The yeast Kluyveromyces marxianus is considered as a potential alternative to Saccharomyces cerevisiae in producing ethanol as a biofuel. In this study, we investigated the ethanol fermentation properties of novel K. marxianus strain DMB1, isolated from bagasse hydrolysates. This strain utilized sorbitol as well as various pentoses and hexoses as single carbon sources under aerobic conditions and produced ethanol from glucose in hydrolysates of the Japanese cedar at 42 °C. Reference strains K. marxianus NBRC1777 and S. cerevisiae BY4743 did not assimilate sorbitol or ferment lignocellulosic hydrolysates to ethanol at this temperature. Thus strain DMB1 appears to be optimal for producing bioethanol at high temperatures, and might provide a valuable means of increasing the efficiency of ethanol fermentation.

Ethanol from laccase-detoxified lignocellulose by the thermotolerant yeast Kluyveromyces marxianus—Effects of steam pretreatment conditions, process configurations and substrate loadings

In our previous work, the ability of laccase enzymes to improve the fermentation performance of the thermotolerant yeast Kluyveromyces marxianus CECT 10875 on steam-exploded wheat straw slurry was demonstrated. As a continuation of this study, the present research evaluates different aspects, including pretreatment conditions, process configurations and substrate loadings, with the aim to proceed towards the use of K. marxianus and laccases for second generation ethanol production. For it, two wheat straw slurries resulting from different steam explosion pretreatment conditions (200°C, 2.5min and 220°C, 2.5min) were employed at various substrate loadings [5–14% (w/v)] under two process configurations: SSF (simultaneous saccharification and fermentation) and PSSF (presaccharification and simultaneous saccharification and fermentation). The better performance of K. marxianus was observed on the slurry produced at softer conditions. Its lower inhibitors content allowed to increase the total solids loading up to 10% (w/v) in both process configurations, reaching higher ethanol concentrations (12g/L). Moreover, laccase detoxification improved these results, particularly in SSF processes, increasing the substrate loading up to 12% (w/v) and, consequently, obtaining the highest ethanol concentration (16.7g/L).

Ethanol fermentation from xylose by metabolically engineered strains of Kluyveromyces marxianus

We constructed a xylose-fermenting recombinant strain of thermotolerant yeast Kluyveromyces marxianus, DMB3-7. Both xylose consumption and ethanol production were remarkably increased in DMB3-7 compared to the control strain at 30°C. Furthermore, DMB3-7 produced ethanol from xylose at both 42°C and 45°C, above which xylose metabolic activity decreased.

Improved ethanol production in Jerusalem artichoke tubers by overexpression of inulinase gene in Kluyveromyces marxianus

Ethanol production from Jerusalem artichoke tubers through a consolidated bioprocessing (CBP) strategy using the inulinase-producing yeast Kluyveromyces marxianus is an economical and competitive than that from a grainbased feedstock. However, poor inulinase production under ethanol fermentation conditions significantly prolongs the fermentation time and compromises ethanol productivity. Improvement of inulinase activity appears to be promising for increasing ethanol production from Jerusalem artichoke tubers by CBP. In the present study, expression of the inulinase gene INU with its own promoter in K. marxianus (K/INU2) was explored using the integrative cassette. Overexpression of INU was explored using chromosome integration via the HO locus of the yeast. Inulinase activity and ethanol were determined from inulin and Jerusalem artichoke tubers under fed-batch operation. Inulinase activity was 114.9 U/mL under aerobic conditions for K/INU2, compared with 52.3 U/mL produced by the wild type strain. Importantly, inulinase production was enhanced in K/INU2 under ethanol fermentation conditions. When using 230 g/L inulin and 220 g/L Jerusalem artichoke tubers as substrates, inulinase activities of 3.7 and 6.8 U/mL, respectively, were measured using K/INU2, comparing favorably with 2.4 and 3.1 U/mL, respectively, using the wide type strain. Ethanol concentration and productivity for inulin were improved by the recombinant yeast to 96.2 g/L and 1.34 g/L/h, respectively, vs 93.7 g/L and 1.12 g/L/h, respectively, by the wild type strain. Ethanol concentration and productivity improvements for Jerusalem artichoke tubers were 69 g/L and 1.44 g/L/h, respectively, from the recombinant strain vs 62 g/L and 1.29 g/L/h, respectively, from the wild type strain.

Expression of exoinulinase genes in Saccharomyces cerevisiae to improve ethanol production from inulin sources

To improve inulin utilization and ethanol fermentation, exoinulinase genes from the yeast Kluyveromyces marxianus and the recently identified yeast, Candida kutaonensis, were expressed in Saccharomyces cerevisiae. S. cerevisiae harboring the exoinulinase gene from C. kutaonensis gave higher ethanol yield and productivity from both inulin (0.38 vs. 0.34 g/g and 1.35 vs. 1.22 g l(-1) h(-1)) and Jerusalem artichoke tuber flour (0.47 vs. 0.46 g/g and 1.62 vs. 1.54 g l(-1) h(-1)) compared with the strain expressing the exoinulinase gene from K. marxianus. Thus, the exoinulinase gene from C. kutaonensis is advantageous for engineering S. cerevisiae to improve ethanol fermentation from inulin sources.

β-Galactosidase over-production by a mig1 mutant of Kluyveromyces marxianus KM for efficient hydrolysis of lactose

In this study, the MIG1 gene in the industrial yeast Kluyveromyces marxianus KM was disrupted by integrating the HPT gene (encoding hygromycin B phosphotransferase) into ORF (Open Reading Frame) of the MIG1 gene. The disruptant (mig1 mutant) KM-15 obtained could grow in the media containing hygromycin or 2-deoxy-d-glucose, respectively. β-Galactosidase production and expression of its gene in the disruptant KM-15 were significantly enhanced compared to those in K. marxianus KM. This confirmed that Mig1, the transcriptional repressor, indeed regulated expression of the gene and biosynthesis of the enzyme in K. marxianus KM. Under the optimal conditions, the β-galactosidase activity of 121.0U/mL was produced by the disruptant KM-15 within 60h during 2-L fermentation. 99.2% of lactose in 9.0% of lactose solution, 99.0% of lactose in 12.0% of whey powder suspension and 99.3% of lactose in the milk were hydrolyzed by the crude β-galactosidase within 3h, 4h and 3h, respectively. The end products of the lactose hydrolysis were only glucose and galactose.