Biosynthesis of beta-glucans by cell-free extracts from Saccharomyces cerevisiae.

Cell-free extracts from Saccharomyces cerevisiae catalyzed the incorporation of glucosyl residues from UDP-[U-14C]glucose into beta-1,3-glucans which contained a significant proportion of beta-1,6-glycosidic linkages. When GDP-[U-14C]glucose was used as substrate only trace amounts of glucose were incorporated. Activity of beta-glucan synthetase was distributed among membrane and cell wall fractions, specific activity being higher in this latter. Beta-glucan synthesized by membrane and cell wall fractions contained 0.6% and 2.5% of beta-1,6-glycosidic linkages respectively. A marked decrease in the activity of beta-glucan synthetase occurred as the cells aged. Significant activity of glycogen synthetase was detected only in cells which had reached the stationary phase of growth.     

Modelling of ethanol production by Saccharomyces cerevisiae from a glucose and maltose mixture

Summary A model for ethanol production from a glucose-maltose mixture has been proposed, which includes a term representing the glucose repression effect on maltose consumption. The model parameters were estimated from batch experimental data. Results of sensitivity analysis on the Monod constants for glucose and maltose, and the repression constant, showed that ±10% changes in these three parameters caused no significant effect on data fitting.     

High-level production of ethanol during fed-batch ethanol fermentation with a controlled aeration rate and non-sterile glucose powder feeding of Saccharomyces cerevisiae

In this study, we utilized a unique strategy for fed-batch fermentation using ethanol-tolerant Saccharomyces cerevisiae to achieve a high-level of ethanol production that could be practically applied on an industrial scale. During this study, the aeration rate was controlled at 0.0, 0.13, 0.33, and 0.8 vvm to determine the optimal aeration conditions for the production of ethanol. Additionally, non-sterile glucose powder was fed during fed-batch ethanol fermentation and corn-steep liquor (CSL) in the medium was used as an organic N-source. When aeration was conducted, the ethanol production and productivity were superior to that when aeration was not conducted. Specifically, the maximum ethanol production reached approximately 160 g/L, when the fermentor was aerated at 0.13 vvm. These findings indicate that the use of a much less expensive C-source may enable the fermentation process to be directed towards the improvement of overall ethanol production and productivity in fermentors that are aerated at 0.13 vvm. Furthermore, if a repeated fed-batch process in which the withdrawal and fill is conducted prior to 36 h can be employed, aeration at a rate of 0.33 and/or 0.8 vvm may improve the overall ethanol productivity     

Improved ethanol production from xylose with glucose isomerase and Saccharomyces cerevisiae using the respiratory inhibitor azide

Summary Ethanol was produced from xylose, using the enzyme glucose isomerase (xylose isomerase) and Saccharomyces cerevisiae. The influence of aeration, pH, enzyme concentration, cell mass and the concentration of the respiratory inhibitor sodium azide on the production of ethanol and the formation of by-products was investigated. Anaerobic conditions at pH 6.0, 10 g/l enzyme, 75 g/l dry weight cell mass and 4.6 mM sodium azide were found to be optimal. Under these conditions theoretical yields of ethanol were obtained from 42 g/l xylose within 24 hours.In a fed-batch culture, 62 g/l ethanol was produced from 127 g/l xylose with a yield of 0.49 and a productivity of 1.35 g/l·h.     

Improvements in ethanol production from xylose by mating recombinant xylose-fermenting Saccharomyces cerevisiae strains

To improve the ability of recombinant Saccharomyces cerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due to chromosome doubling as well as the increase in the copy number of xylose assimilation genes. Moreover, compared to the parent strain, the MN8140XX strain exhibited higher ethanol production under elevated temperatures (38 °C) and acidic conditions (pH 3.8). Thus, the simple hybridization technique facilitated an increase in the xylose fermentation activity.     

Study of the production of fructose and ethanol from sucrose media by Saccharomyces cerevisiae

The production of ethanol and enriched fructose syrups from a synthetic medium with various sucrose concentrations using the mutant Saccharomyces cerevisiae ATCC 36858 was investigated. In batch tests, fructose yields were above 90% of theoretical values for the sucrose concentrations between 35 g/l and 257 g/l. The specific growth rates and biomass yields were from 0.218 to 0.128 h(-1) and from 0.160 to 0.075 g biomass/g of glucose and fructose consumed, respectively. Ethanol yields were in the range of 72 to 85% of theoretical value when sucrose concentrations were above 81 g/l. The volumetric ethanol productivity was 2.23 g ethanol/(l h) in a medium containing 216 g/l sucrose. Fructo-oligosaccharides and glycerol were also produced in the process. A maximum fructo-oligosaccharides concentration (up to 9 g/l) was attained in the 257 g/l sucrose medium in the first 7 h of the fermentation. These sugars started to be consumed when the concentrations of sucrose in the media were less than 30% of its initial values. The fructo-oligosaccharides mixture was composed of 6-kestose (61.5%), neokestose (29.7%) and 1-kestose (8.8%). The concentration of glycerol produced in the process was less than 9 g/l. These results will be useful in the production of enriched fructose syrups and ethanol using sucrose-based raw materials.     

Influence of acetate on growth kinetics and production control of Saccharomyces cerevisiae on glucose and ethanol

Summary The production and assimilation of acetate during the growth of Saccharomyces cerevisiae on glucose and ethanol have been studied. Acetate inhibits growth and causes decreased yields on both substrates. The usual respiratory quotient based policy for fed batch control cannot be used for highly acetate producing yeast because of compensation between O₂ over-consumption (due to acetate) and CO₂ over-production (due to ethanol) in case of glucose over-feeding.     

Translation initiation factor-dependent extracts from Saccharomyces cerevisiae

Translation initiation factor 4A- and 4E-dependent extracts were developed from Saccharomyces cerevisiae and used to study factor requirements for translation of individual mRNAs in vitro. Whereas all mRNAs tested required eIF-4A, mRNAs devoid of secondary structure in their 5' untranslated region did not require exogenous eIF-4E for translation. The latter included alfalfa mosaic virus RNA4, mRNA containing the untranslated region of tobacco mosaic virus RNA and mRNA containing part of the untranslated region of poliovirus RNA. Furthermore, initiation of translation on mRNAs containing part of the untranslated region of poliovirus RNA is most likely internal.     

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.     

Effects of xylulokinase activity on ethanol production from D-xylulose by recombinant Saccharomyces cerevisiae

AIMS: Recombinant Saccharomyces cerevisiae strains harbouring different levels of xylulokinase (XK) activity and effects of XK activity on utilization of xylulose were studied in batch and fed-batch cultures. METHODS AND RESULTS: The cloned xylulokinase gene (XKS1) from S. cerevisiae was expressed under the control of the glyceraldehyde 3-phosphate dehydrogenase promoter and terminator. Specific xylulose consumption rate was enhanced by the increased specific XK activity, resulting from the introduction of the XKS1 into S. cerevisiae. In batch and fed-batch cultivations, the recombinant strains resulted in twofold higher ethanol concentration and 5.3- to six-fold improvement in the ethanol production rate compared with the host strain S. cerevisiae. CONCLUSIONS: An effective conversion of xylulose to xylulose 5-phosphate catalysed by XK in S. cerevisiae was considered to be essential for the development of an efficient and accelerated ethanol fermentation process from xylulose. SIGNIFICANCE AND IMPACT OF THE STUDY: Overexpression of the XKS1 gene made xylulose fermentation process accelerated to produce ethanol through the pentose phosphate pathway.     

A factor in cell-free extracts inactivating sexual agglutinability of a mating type in Saccharomyces cerevisiae

Cell-free extracts of both a and a mating-type strains of Saccharomycescerevisiae contained a substance which irreversibly inactivatedsexual agglutinability of a cells, but not that of a cells. ¹ Present address: Department of Pharmacology, Osaka Collegeof Pharmacy, 2-10-65 Kawai-cho, Matsubara, Osaka 580, Japan. (Received January 9, 1976; )     

The role of nisin in fuel ethanol production with Saccharomyces cerevisiae

Aims: To investigate the effects of nisin on lactobacilli contamination of yeast during ethanol fermentation and to determine the appropriate concentration required to control the growth of selected lactobacilli in a YP/glucose media fermentation model. Methods and Results: The lowest concentration of nisin tested (5 IU ml^?1) effectively controlled the contamination of YP/glucose media with 10⁶ CFU ml^?1 lactobacilli. Lactic acid yield decreased from 5·0 to 2·0 g l^?1 and potential ethanol yield losses owing to the growth and metabolism of Lactobacillus plantarum and Lactobacillus brevis were reduced by 11 and 7·8%, respectively. Approximately, equal concentrations of lactic acid were produced by Lact. plantarum and Lact. brevis in the presence of 5 and 2 IU ml^?1 nisin, respectively, thus demonstrating the relatively higher nisin sensitivity of Lact. brevis for the strains in this study. No differences were observed in the final ethanol concentrations produced by yeast in the absence of bacteria at any of the nisin concentrations tested. Conclusions: Metabolism of contaminating bacteria was reduced in the presence of 5 IU ml^?1 nisin, resulting in reduced lactic acid production and increased ethanol production by the yeast. Significance and Impact of the Study: Bacteriocins represent an alternative to the use of antibiotics for the control of bacterial contamination in fuel ethanol plants and may be important in preventing the emergence of antibiotic‐resistant contaminating strains.     

Enhanced ethanol production from industrial lignocellulose hydrolysates by a hydrolysate-cofermenting Saccharomyces cerevisiae strain

Industrial production of lignocellulosic ethanol requires a microorganism utilizing both hexose and pentose, and tolerating inhibitors. In this study, a hydrolysate-cofermenting Saccharomyces cerevisiae strain was obtained through one step in vivo DNA assembly of pentose-metabolizing pathway genes, followed by consecutive adaptive evolution in pentose media containing acetic acid, and direct screening in biomass hydrolysate media. The strain was able to coferment glucose and xylose in synthetic media with the respective maximal specific rates of glucose and xylose consumption, and ethanol production of 3.47, 0.38 and 1.62 g/g DW/h, with an ethanol titre of 41.07 g/L and yield of 0.42 g/g. Industrial wheat straw hydrolysate fermentation resulted in maximal specific rates of glucose and xylose consumption, and ethanol production of 2.61, 0.54 and 1.38 g/g DW/h, respectively, with an ethanol titre of 54.11 g/L and yield of 0.44 g/g. These are among the best for wheat straw hydrolysate fermentation through separate hydrolysis and cofermentation.

     

Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain.     

Application of Saccharomyces cerevisiae and Pichia stipitis karyoductants to the production of ethanol from xylose

Karyoductants of Saccharomyces cerevisiae V30 and Pichia stipitis CCY 39501 with the ability to ferment D-xylose to ethanol were isolated. The ability of these isolates to assimilate different sugars, ethanol tolerance and ethanol production from D-xylose was investigated. Karyoductants didn't grow on starch, lactose and cellobiose, like S. cerevisiae, but showed good growth on xylose and L-arabinose, like P. stipitis. All isolates fermented xylose to ethanol slower than P. stipitis and with lower yields, 0.09 - 0.16 g/g. They secreted also about 3.4 - 7.1 g/dm3 of xylitol to the culture medium (P. stipitis only 0.06 g/dm3). The karyoductants showed an average tolerance to ethanol when compared with the parent strains and fermented glucose in the presence of 6% alcohol whereas parent strain S. cerevisiae and P. stipitis showed exogenic ethanol tolerance of 9% and 3%, respectively.     

Improved ethanol production from whey with Saccharomyces cerevisiae using permeabilized cells of Kluyveromyces marxianus

Permeabilized cells of Kluyveromyces marxianus CCY eSY2 were tested as the source of lactase in the ethanol fermentation of concentrated deproteinized whey (65–70 g/l lactose) by Saccharomyces cerevisiae CCY 10–13–14. Rapid lactose hydrolysis by small amounts of permeabilized cells following the fermentation of released glucose and galactose by S. cerevisiae resulted in a twofold enhancement of the overall volumetric productivity (1.03 g/l × h), compared to the fermentation in which the lactose was directly fermented by K. marxianus.