Transformation of maltose into prebiotic isomaltooligosaccharides by a novel α-glucosidase from Xantophyllomyces dendrorhous

The transglycosylation activity of a novel α-glucosidase from the basidiomycetous yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) was studied using maltose as glucosyl donor. The enzyme synthesized oligosaccharides with α-(1 → 2), α-(1 → 4) and α-(1 → 6) bonds. Using 200 g/l maltose, the yield of oligosaccharides was 53.8 g/l, with prebiotic oligosaccharides containing at least one α-(1 → 6) linkage (panose, 6-O-α-glucosyl-maltotriose and 6-O-α-isomaltosyl-maltose) being the major products (47.1 g/l). The transglycosylatying yield was 3.6 times higher than the observed with the α-glucosidase from Saccharomyces cerevisiae (53.8 vs. 14.7 g/l). Moreover, when increasing the maltose concentration up to 525 g/l, the maximum production of tri- and tetrasaccharides reached 167.1 g/l, without altering the percentage of oligosaccharides in the mixture. Compared with other microbial α-glucosidases in which the main transglycosylation product is a disaccharide, the enzyme from X. dendrorhous yields a final product enriched in trisaccharides and tetrasaccharides.     

Cell lysis induced by membrane-damaging detergent saponins from Quillaja saponaria

This paper presents the results of a study to determine the effect of Quillaja saponaria saponins on the lysis of industrial yeast strains. Cell lysis induced by saponin from Q. saponaria combined with the plasmolysing effect of 5% NaCl for Saccharomyces cerevisiae, Kluyveromyces marxianus yeasts biomass was conducted at 50 °C for 24–48 h. Membrane permeability and integrity of the yeast cells were monitored using fluorescent techniques and concentrations of proteins, free amino nitrogen (FAN) and free amino acids in resulting lysates were analyzed. Protein release was significantly higher in the case of yeast cell lysis promoted with 0.008% Q. saponaria and 5% NaCl in comparison to plasmolysis triggered by NaCl only.     

Engineering of Saccharomyces cerevisiae for the production of dihydroxyacetone (DHA) from sugars: A proof of concept

Dihydroxyacetone (DHA) has numerous industrial applications. In this work, we pursue the idea to produce DHA from sugars in the yeast Saccharomyces cerevisiae, via glycerol as an intermediate. Firstly, three glycerol dehydrogenase (GDH) genes from different microbial sources were expressed in yeast. Among them, the NAD⁺-dependent GDH of Hansenula polymorpha showed the highest glycerol-oxidizing activity. DHA concentration in shake-flask experiments was roughly 100 mg/l DHA from 20 g/l glucose, i.e. five times the wild-type level. This level was achieved only when cultures were subjected to osmotic stress, known to enhance glycerol production and accumulation in S. cerevisiae. Secondly, DHA kinase activity was abolished to prevent conversion of DHA to dihydroxyacetone phosphate (DHAP). The dak1Δdak2Δ double-deletion mutant overexpressing H. polymorpha gdh produced 700 mg/l DHA under the same conditions. Although current DHA yield and titer still need to be optimized, our approach provides the proof of concept for producing DHA from sugars in yeast.     

Optimization of the medium for glutathione production in Saccharomyces cerevisiae

As a powerful statistical experimental design, uniform design (UD) method has been successfully applied in various fields such as fermentation industry, pharmaceuticals, and others. In this paper, UD was applied to optimize the medium composition for glutathione production in shake-flask culture of Saccharomyces cerevisiae T65. The experiments of nine factors (glucose, yeast extract, peptone, malt extract, molasses, MgSO₄, ZnSO₄, (NH₄)₂HPO₄ and thiamine) and nine levels were carried out according to the uniform design table U₂₇(9⁹). The experimental data was analyzed to obtain the regression model and the optimal medium composition was achieved by optimization with UD 3.0 software. The optimal medium consisted of 70 g/L glucose, 3 g/L yeast extract, 5 g/L peptone, 70 g/L malt extract, 20 g/L molasses, 5.6 g/L MgSO₄, 16 mg/L ZnSO₄, 7 g/L (NH₄)₂HPO₄ and 0.2 mg/L thiamine. The GSH yield at the optimal point achieved 74.6 mg/L, which was 1.81 times higher than that of the control. The application of UD method resulted in enhancement in GSH production.     

Optimization of culture medium for cordycepin production using Cordyceps militaris mutant obtained by ion beam irradiation

The effect of medium components on cordycepin production by Cordyceps militaris mutant obtained by ion beam irradiation was investigated. According to the response surface analysis using a central composite design for the prospective mutant G81-3, the predicted optimal concentrations of glucose as the carbon source and the yeast extract as the nitrogen source were 86.2 g/l and 93.8 g/l, respectively, and 6.84 g/l cordycepin was obtained. To date, this is the highest value for cordycepin production. The optimal concentrations of glucose and yeast extract for cordycepin production of the mutant was much higher than that of control (wild strain) and the cordycepin production was 2.79 times higher. Therefore, this new mutant will be a promising strain for future higher cordycepin production at industrial levels.     

Effect of amino acids addition and feedback control strategies on the high-cell-density cultivation of Saccharomyces cerevisiae for glutathione production

Controlling glucose feeding rate, according to the change of ethanol concentration and respiratory quotient (RQ), has been studied in high-cell-density cultivation of Saccharomyces cerevisiae for glutathione (GSH). GSH yield and dry cell weight reached 1620 mg/L and 140 g/L, respectively, after 52 h of cultivation. In addition, optimized single shot addition of precursor amino acids (performed in both flask and fermentor experiments) at 32 h could result in the GSH yield reached 2020 mg/L after 38 h of cultivation. The yield and productivity of GSH increased 25% and 70%, respectively. Moreover, cultivation time was reduced to 38 h, compared with results without adding the precursor amino acids.     

Redox potential-driven repeated batch ethanol fermentation under very-high-gravity conditions

When Saccharomyces cerevisiae was cultivated under ～200 g glucose/l condition, the time point at which glucose was completely utilized coincided with the moment at which the slope of a redox potential profile changed from negative or zero to positive. Based on this feature, a redox potential-driven glucose-feeding fermentation operation was developed, and resulted in a self-cycling period of 14.25 ± 0.4 h. The corresponding ethanol concentration was maintained at 88.4 ± 1.0 g/l with complete glucose conversion, and the cell viabilities increased from 80% in the transition period to 97.2 ± 1.1%, implying the occurrence of yeast acclimatization. In contrast, a pre-determined 36-h manually adjusted period was chosen to oscillate yeast cells under ～250 g glucose/l conditions, which resulted in 106.76 ± 0.7 g ethanol/l and 15.19 ± 1.3 g glucose/l remaining at the end of each cycle. Compared to the equivalent batch and continuous ethanol fermentation processes, the annual ethanol productivity of the reported fermentation operation is 2.4% and 13.2% greater, respectively in ～200 g feeding glucose/l conditions.     

Inulinase overproduction by a mutant of the marine yeast Pichia guilliermondii using surface response methodology and inulin hydrolysis

In this study, in order to isolate inulinase overproducers from the marine yeast Pichia guilliermondii, its cells were treated by using UV light and LiCl. The mutant M-30 with enhanced inulinase production was obtained and was found to be stable after cultivation for 20 generations. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant M-30 in liquid fermentation. Inulin, yeast extract, NaCl, temperature, pH for maximum inulinase production by the mutant M-30 were found to be 20.0 g/l, 5.0 g/l, 20.0 g/l, 28 °C and 6.5, respectively. Under the optimized conditions, 127.7 U/ml of inulinase activity was reached in the liquid culture of the mutant M-30 whereas the predicted maximum inulinase activity of 129.8 U/ml was derived from RSM regression. Under the same conditions, its parent strain only produced 48.1 U/ml of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far. We also found that inulin could be actively converted into monosaccharides by the crude inulinase.     

Molecular characterization and molasses fermentation performance of a wild yeast strain operating in an extremely wide temperature range

Molasses fermentation performance by both a cryotolerant and a thermophilic yeast (strain AXAZ-1) isolated from grapes in Greece was evaluated in an extremely wide temperature range (3–40 °C). Sequence analysis of the 5.8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions assigned isolate to Saccharomyces cerevisiae. Restriction fragment length polymorphism of the mitochondrial DNA showed that strain AXAZ-1 is genetically divergent compared to other wild strains of Greek origin or commercial yeast starters. Yeast cells growing planktonically were capable of fermentation in a wide temperature spectrum, ranging from 3 °C to 38 °C. Immobilization of yeast on brewer’s spent grains (BSG) improved the thermo-tolerance of the strain and enabled fermentation at 40 °C. Time to complete fermentation with the immobilized yeast ranged from 20 days at 3 to 38 h at 40 °C. The daily ethanol productivity reached maximum (58.1 g/L) and minimum (2.5 g/L) levels at 30 and 3 °C, respectively. The aroma-related compounds’ profiles of immobilized cells at different fermentation temperatures were evaluated by using solid phase microextraction (SPME) gas chromatography–mass spectrometry (GC–MS). Molasses fermentation resulted in a high quality fermentation product due to the low concentrations of higher and amyl alcohols at all temperatures tested. Strain AXAZ-1 is very promising for the production of ethanol from low cost raw materials, as it was capable to perform fermentations of high ethanol concentration and productivities in both low and high temperatures.     

Exploration of a natural reservoir of flocculating genes from various Saccharomyces cerevisiae strains and improved ethanol fermentation using stable genetically engineered flocculating yeast strains

Flocculating yeast strains with good fermentation ability are desirable for brewing industry as well as for fuel ethanol production, however, the genetic diversity of the flocculating genes from natural yeast strains is largely unexplored. In this study, FLO1, FLO5, FLO9, FLO10 and FLO11 PCR products were obtained from 16 yeast strains from various sources, and the PCR product amplified from FLO1 of the self-flocculating yeast strain SPSC01 was used for the construction of expression cassette flanked by homologous fragments of the endonuclease gene HO for chromosome integration. A genetically engineered flocculating yeast BHL01 with good fermentation performance was obtained by transforming an industrial strain Saccharomyces cerevisiae 4126 with the expression cassette. The fermentation performances of SPSC01 and BHL01 in flask fermentation were compared using 208 g/L glucose. BHL01 completed the fermentation 8 h earlier than SPSC01, while no significant difference between BHL01 and S. cerevisiae 4126 was observed. In very high gravity repeated batch ethanol fermentation using 255 g/L glucose, BHL01 maintained stable flocculation for at least over 24 batches, while SPSC01 displayed severe deflocculation under the same conditions. The natural reservoir of flocculating genes from yeast strains may represent an unexplored gene source for the construction of new flocculating yeast strains for improved ethanol production.     

Direct ethanol production from N-acetylglucosamine and chitin substrates by Mucor species

Chitin, which is a polymer of β-(1–4) linked N-acetyl-d-glucosamine (GlcNAc) residues, is one of the most abundant renewable resources in nature, after cellulose. In this study, we found some native Mucor strains, which can use GlcNAc and chitin substrates as carbon sources for growth and ethanol production. One of these strains, M. circinelloides NBRC 6746 produced 18.6 ± 0.6 g/l of ethanol from 50 g/l of GlcNAc after 72 h and the maximum ethanol production rate was 0.75 ± 0.1 g/l/h. Furthermore, M. circinelloides NBRC 4572 produced 6.00 ± 0.22 and 0.46 ± 0.04 g/l of ethanol from 50 g/l of colloidal chitin and chitin powder after 16 and 12 days, respectively. We also found an extracellular chitinolytic enzyme producing strain M. ambiguus NBRC 8092, and successfully improved ethanol productivity of NBRC 4572 from colloidal chitin using crude chitinolytic enzyme derived from NBRC 8092. The ethanol titer reached 9.44 ± 0.10 g/l after 16 days. These results were the first bioethanol production from GlcNAc and chitin substrates by native organisms, and also suggest that these Mucor strains have great potential for the simultaneous saccharification and fermentation (SSF) of chitin biomass.     

Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway

Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP⁺-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway.     

Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae

Xylose is the main pentose and second most abundant sugar in lignocellulosic feedstocks. To improve xylose utilization, necessary for the cost-effective bioconversion of lignocellulose, several metabolic engineering approaches have been employed in the yeast Saccharomyces cerevisiae. In this study, we describe the rational metabolic engineering of a S. cerevisiae strain, including overexpression of the Piromyces xylose isomerase gene (XYLA), Pichia stipitis xylulose kinase (XYL3) and genes of the non-oxidative pentose phosphate pathway (PPP). This engineered strain (H131-A3) was used to initialize a three-stage process of evolutionary engineering, through first aerobic and anaerobic sequential batch cultivation followed by growth in a xylose-limited chemostat. The evolved strain H131-A3-AL^CS displayed significantly increased anaerobic growth rate (0.203±0.006 h^?1) and xylose consumption rate (1.866 g g^?1 h^?1) along with high ethanol conversion yield (0.41 g/g). These figures exceed by a significant margin any other performance metrics on xylose utilization and ethanol production by S. cerevisiae reported to-date. Further inverse metabolic engineering based on functional complementation suggested that efficient xylose assimilation is attributed, in part, to the elevated expression level of xylose isomerase, which was accomplished through the multiple-copy integration of XYLA in the chromosome of the evolved strain.     

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP⁺ for acetyl-CoA production. After 24 h of cultivation, a 3.7-fold increase in NADPH/NADP⁺ ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48 h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2–3-fold over the base strain (up to 0.8 g/L), and in combination to 1.4 g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6 g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16 g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.     

Simultaneous production of single cell protein and killer toxin by Wickerhamomyces anomalus HN1-2 isolated from mangrove ecosystem

The yeast Wickerhamomyces anomalus (the previous name was Pichia anomala) HN1-2 isolated from the mangrove ecosystem was found to be able to produce high level of both killer toxin and single cell protein. When the killer yeast cells were grown by batch cultivation in 5-l fermentor, crude protein in the cells, cell mass, reducing sugar, and diameter of the inhibition zone reached 56.0 g per 100 g of cell dry weight, 7.3 g per liter, 9.5 g per liter, and 19.0 mm, respectively within 12 h and this yeast synthesized a large amount of the essential amino acids, such as lysine (7.8%), methionine (1.8%), and leucine (9.0%). The crude killer toxin produced by the killer yeast isolate HN1-2 could kill the cells of Lodderomyces elongisporus, Candida albicans, Metschnikowia bicuspidata, Pichia guilliermondii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Kluyveromyces aestuarii, which were widely distributed in natural marine environments. The results also showed that the undesirable yeast could be avoided during cell growth of the killer yeast.     

Effects of magnetic fields on biomass and glutathione production by the yeast Saccharomyces cerevisiae

The effect of magnetic fields (MF) on glutathione (GSH) production by Saccharomyces cerevisiae ATCC 7754 was studied. For this purpose, a factorial design of experiments was used to determine the influence of the time of exposure (8–16 h) and MF induction (25.0–34.3 mT), in GSH and biomass production. Additionally, control experiments (CE), without the application of MF, were performed. The results indicated the existence of favourable alterations in GSH and biomass concentrations due to the application of MF. In all experiments, the amount of biomass produced was higher than in CE and, with regard to GSH yield, in all the experiments at 24 and 48 h it was higher and in three experiments at 72 h of culture. The highest specific GSH yield (20.9 mg_GSH/g_biomass), GSH yield (340.0 mg/L) and biomass (16.26 g/L) were obtained using a MF induction of 25.0 mT for 16 h. These results were 16.1%, 39.0% and 19.6% higher than in the CE, respectively. Through statistical analysis it was found that the MF induction was a significant factor in GSH yield, and also it was observed that, within the range of the experimental conditions used, the lower MF induction, the higher the GSH yield.     

Evaluation of cysteine ethyl ester as efficient inducer for glutathione overproduction in Saccharomyces spp.

Economical yeast based glutathione (GSH) production is a process that is influenced by several factors like raw material and production costs, biomass production and efficient biotransformation of adequate precursors into the final product GSH. Nowadays the usage of cysteine for the microbial conversion into GSH is industrial state of practice. In the following study, the potential of different inducers to increase the GSH content was evaluated by means of design of experiments methodology. Investigations were executed in three natural Saccharomyces strains, S. cerevisiae, S. bayanus and S. boulardii, in a well suited 50 ml shake tube system. Results of shake tube experiments were confirmed in traditional baffled shake flasks and finally via batch cultivation in lab-scale bioreactors under controlled conditions. Comprehensive studies showed that the usage of cysteine ethyl ester (CEE) for the batch-wise biotransformation into GSH led up to a more than 2.2 times higher yield compared to cysteine as inducer. Additionally, the intracellular GSH content could be significantly increased for all strains in terms of 2.29 ± 0.29% for cysteine to 3.65 ± 0.23% for CEE, respectively, in bioreactors. Thus, the usage of CEE provides a highly attractive inducing strategy for the GSH overproduction.     

A novel membrane-surface liquid co-culture to improve the production of laccase from Ganoderma lucidum

In this work, a laccase producer, Ganoderma lucidum, was separated and identified according to its morphological characteristics and phylogenetic data. A 4000 U/l and 8500 U/l of laccase activity was obtained in 500 ml flask by submerged culture and biomembrane-surface liquid culture (BSLC), respectively. Furthermore, the novel biomembrane-surface liquid co-culture (BSLCc) was developed by adding Saccharomyces cerevisiae to reactor in order to shorten the fermentation period and improve laccase production. Laccase activity obtained by BSLCc, 23 000 U/l, is 5.8 and 2.7 times of that obtained by submerged culture and BSLC, respectively. In addition, laccase production by BSLCc was successfully scaled-up to 100 l reactor, and 38 000 U/l of laccase activity was obtained on day 8. The mechanism of overproducing laccase by BSLCc was investigated by metabolism pathway analysis of glucose. The results show glucose limitation in fermentation broth induces the secretion of laccase. The addition of S. cerevisiae, on one hand, leads to an earlier occurrence of glucose limitation state, and thus shortens the fermentation time; on the other hand, it also results in the appearance of a series of metabolites of the yeast including organic acids, ethanol, glycerol and so forth in fermentation broth, and both polyacrylamide gel electrophoresis analysis and enzyme activity detection of laccase show that these metabolites contribute to the improvement of laccase activity.