Amine-catalyzed Racemization of the Imidazoline Derivatives

Protoplast fusion was carried out between a saké brewer’s yeast strain, Saccharomyces cerevisiae Kyokai 7, and a lactose utilizing yeast strain, Kluyveromyces lactis T396. A stable hybrid, PN 13, which was selected from the many resultant fusants, showed physiologically complemented traits with respect to sugar utilization, vitamin requirements and so on. Biochemical investigations also revealed that fusant PN 13 was an intermediate hybrid between the parental strains. In glucose and lactose media, moreover, the fusant grew and produced ethanol at higher rates than K. lactis T396.     

Efficiency of genetically engineered yeast in the production of ethanol from dextrinized cassava starch

Summary The ability of a polyploid/aneuploidSaccharomyces diastaticus spheroplast fusion product and a diploidSaccharomyces diastaticus hybridization product, to produce ethanol from dextrinized cassava starch with varying amounts of supplemented glucoamylase (amyloglucosidase), was investigated. It was found that the added glucoamylase could be reduced by over 50% using these glucoamylase producing strains as compared to a commercially availableSaccharomyces cerevisiae strain commonly used in ethanol producing industries.     

Sorbitol production from lactose by engineered Lactobacillus casei deficient in sorbitol transport system and mannitol-1-phosphate dehydrogenase

Sorbitol is a sugar alcohol largely used in the food industry as a low-calorie sweetener. We have previously described a sorbitol-producing Lactobacillus casei (strain BL232) in which the gutF gene, encoding a sorbitol-6-phosphate dehydrogenase, was expressed from the lactose operon. Here, a complete deletion of the ldh1 gene, encoding the main l-lactate dehydrogenase, was performed in strain BL232. In a resting cell system with glucose, the new strain, named BL251, accumulated sorbitol in the medium that was rapidly metabolized after glucose exhaustion. Reutilization of produced sorbitol was prevented by deleting the gutB gene of the phosphoenolpyruvate: sorbitol phosphotransferase system (PTS^Gut) in BL251. These results showed that the PTS^Gut did not mediate sorbitol excretion from the cells, but it was responsible for uptake and reutilization of the synthesized sorbitol. A further improvement in sorbitol production was achieved by inactivation of the mtlD gene, encoding a mannitol-1-phosphate dehydrogenase. The new strain BL300 (lac::gutF Δldh1 ΔgutB mtlD) showed an increase in sorbitol production whereas no mannitol synthesis was detected, avoiding thus a polyol mixture. This strain was able to convert lactose, the main sugar from milk, into sorbitol, either using a resting cell system or in growing cells under pH control. A conversion rate of 9.4% of lactose into sorbitol was obtained using an optimized fed-batch system and whey permeate, a waste product of the dairy industry, as substrate.     

Rapid ethanol production from sucrose without by-product formation

Summary Non-sorbitol-producing Zymomonas mobilis ACM 3963 was developed from Z. mobilis UQM 2716. This strain was co-immobilised with invertase in alginate and incubated on sucrose-based media. This combination allowed theoretical yields of ethanol to be produced from 100 and 150 g/l sucrose, using both semi-defined media and sugar-cane syrup. No sorbitol or fructo-oligosaccharides were formed in either fermentation. Increased biomass concentrations immobilised in alginate reduced the batch fermentation times of 100 and 150 g/l sucrose by 50–70%, to 3 and 5 hours respectively. This strain also improved the efficiency of the fed-batch fermentation of sucrose.     

Production of sorbitol and ethanol from Jerusalem artichokes by Saccharomyces cerevisiae ATCC 36859

Summary This study shows the possibile use of Jerusalem artichokes for the production of sorbitol and ethanol by Saccharomyces cerevisiae ATCC 36859. Ethanol was produced from the beginning of the process, while sorbitol production started after glucose had been entirely consumed from Jerusalem artichoke (J.a.) juice. The importance of yeast extract and inoculum concentrations on the production of sorbitol from the above raw material was demonstrated. With a low initial biomass concentration sorbitol was not produced in pure J.a. juice. When the juice was supplemented with 3% yeast extract, the concentration of sorbitol was 4.6%. The sorbitol, ethanol and biomass yields (gram of product produced per gram of sugars consumed) were 0.259, 0.160 and 0.071 at the end of the process respectively. Adding glucose to increase its concentration to about 9% in the J.a. juice with 3% yeast extract had a positive effect on the production of ethanol, while commencement of the production of sorbitol was delayed and its final concentration was less than 50% of its concentration in the medium without added glucose. The effect of glucose was much stronger when it was added during the process than when added at the beginning of the process. Offprint requests to: Z. Duvnjak     

Osmotic pressure effects and intracellular accumulation of ethanol in yeast during fermentation

Summary The intracellular accumulation of ethanol in yeast and its potential effects on growth and fermentation have been topics of controversy for the past several years. The determination of intracellular ethanol based on the exclusion of [¹⁴C]sorbitol to estimate aqueous cell volume was used to examine the question of intracellular ethanol accumulation. An intracellular accumulation of ethanol inSaccharomyces cerevisiae was observed during the early stages of fermentation. However, as fermentation continued, the intracellular and extracellular concentrations of ethanol became similar. Increasing the osmotic pressure of the medium with glucose or sorbitol was observed to cause an increase in the intracellular ethanol concentration. Associated with this was a decrease in yeast growth and fermentation rates. In addition, increasing the osmotic pressure of the medium was observed to cause an increase in glycerol production. Supplementation of the media with excess peptone, yeast extract, magnesium sulfate and potassium phosphate was found to relieve the detrimental effects of high osmotic pressure. Under these conditions, though, no effect on the intracellular and extracellular ethanol distribution was observed. These results indicate that nutrient limitation, and not necessarily intracellular ethanol accumulation, plays a key role during yeast fermentations in media of high osmolarity.     

Engineering high-gravity fermentations for ethanol production at elevated temperature with Saccharomyces cerevisiae

Thermal damage, high osmolarity, and ethanol toxicity in the yeast Saccharomyces cerevisiae limit titer and productivity in fermentation to produce ethanol. We show that long-term adaptive laboratory evolution at 39.5°C generates thermotolerant yeast strains, which increased ethanol yield and productivity by 10% and 70%, in 2% glucose fermentations. From these strains, which also tolerate elevated-osmolarity, we selected a stable one, namely a strain lacking chromosomal duplications. This strain (TTY23) showed reduced mitochondrial metabolism and high proton efflux, and therefore lower ethanol tolerance. This maladaptation was bolstered by reestablishing proton homeostasis through increasing fermentation pH from 5 to 6 and/or adding potassium to the media. This change allowed the TTY23 strain to produce 1.3–1.6 times more ethanol than the parental strain in fermentations at 40°C with glucose concentrations ~300 g/L. Furthermore, ethanol titers and productivities up to 93.1 and 3.87 g·L ⁻¹·hr ⁻¹ were obtained from fermentations with 200 g/L glucose in potassium-containing media at 40°C. Albeit the complexity of cellular responses to heat, ethanol, and high osmolarity, in this study we overcome such limitations by an inverse metabolic engineering approach.     

Use of the somatic fusion method to introduce the flocculation property into Saccharomyces diastaticus

Summary Spheroplasts of a petite mutant of the amylolitic Saccharomyces diastaticus 1376 yeast strain were successfully fused with spheroplasts of a flocculent and respiratory competent Saccharomyces cerevisiae 1161 yeast strain.Flocculent and non-flocculent stable recombinants were recovered after regeneration of the cell walls all of which formed halos around their colonies in media containing starch/dextrin as carbon source. The sporulation ability varied in some of the fusion products and the possible influence of genetic instability is discussed.     

Transcriptome analysis identifies genes involved in ethanol response of Saccharomyces cerevisiae in Agave tequilana juice

During ethanol fermentation, yeast cells are exposed to stress due to the accumulation of ethanol, cell growth is altered and the output of the target product is reduced. For Agave beverages, like tequila, no reports have been published on the global gene expression under ethanol stress. In this work, we used microarray analysis to identify Saccharomyces cerevisiae genes involved in the ethanol response. Gene expression of a tequila yeast strain of S. cerevisiae (AR5) was explored by comparing global gene expression with that of laboratory strain S288C, both after ethanol exposure. Additionally, we used two different culture conditions, cells grown in Agave tequilana juice as a natural fermentation media or grown in yeast-extract peptone dextrose as artificial media. Of the 6368 S. cerevisiae genes in the microarray, 657 genes were identified that had different expression responses to ethanol stress due to strain and/or media. A cluster of 28 genes was found over-expressed specifically in the AR5 tequila strain that could be involved in the adaptation to tequila yeast fermentation, 14 of which are unknown such as yor343c, ylr162w, ygr182c, ymr265c, yer053c-a or ydr415c. These could be the most suitable genes for transforming tequila yeast to increase ethanol tolerance in the tequila fermentation process. Other genes involved in response to stress (RFC4, TSA1, MLH1, PAU3, RAD53) or transport (CYB2, TIP20, QCR9) were expressed in the same cluster. Unknown genes could be good candidates for the development of recombinant yeasts with ethanol tolerance for use in industrial tequila fermentation.     

NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae

Saccharomyces cerevisiae alcohol dehydrogenases responsible for NADH-, and NADPH-specific reduction of the furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural have previously been identified. In the present study, strains overexpressing the corresponding genes (mut-ADH1 and ADH6), together with a control strain, were compared in defined medium for anaerobic fermentation of glucose in the presence and absence of HMF. All strains showed a similar fermentation pattern in the absence of HMF. In the presence of HMF, the strain overexpressing ADH6 showed the highest HMF reduction rate and the highest specific ethanol productivity, followed by the strain overexpressing mut-ADH1. This correlated with in vitro HMF reduction capacity observed in the ADH6 overexpressing strain. Acetate and glycerol yields per biomass increased considerably in the ADH6 strain. In the other two strains, only the overall acetate yield per biomass was affected. When compared in batch fermentation of spruce hydrolysate, strains overexpressing ADH6 and mut-ADH1 had five times higher HMF uptake rate than the control strain and improved specific ethanol productivity. Overall, our results demonstrate that (1) the cofactor usage in the HMF reduction affects the product distribution, and (2) increased HMF reduction activity results in increased specific ethanol productivity in defined mineral medium and in spruce hydrolysate.     

Formation and degradation of gluconate by Zymomonas mobilis

Summary Zymomonas mobilis ATCC 29191 is able to degrade gluconate but cannot use it as a single carbon and energy source. Gluconate is phosphorylated by a gluconate kinase (EC 2.7.1.12) and the resulting 6-phosphogluconate is further catabolized to yield about 0.8 mol ethanol per mol of gluconate, considerable amounts of acetate and acetoin. This product spectrum agrees with the theoretical yield of only one reduction equivalent if gluconate is phosphorylated by a kinase and subsequently metabolized via the Entner-Doudoroff pathway.Furthermore, Z. mobilis contains a membrane-bound enzyme system which is able to oxidize glucose to gluconate. Cell-free extracts were active in an assay system with Wurster's blue as electron acceptor, and various aldoses as well as maltose, mannitol and sorbitol could be oxidized. The affinity for sorbitol was very low (K _m =330 mM) but reasonable for glucose (K _m =2.8 mM). The pH optimum for the glucose-oxidizing reaction was 6.5, while that for sorbitol oxidation was 5.5.Dedicated to Prof. Dr. H. Dörfel on the occasion of his 60th birthday     

Alcoholic fermentation of starch containing media using yeast protoplast fusion products

Summary Fermentation of starch based industrial media was tested with yeast fusion products previously described, from a Baker's yeastSaccharomyces cerevisiae and Saccharomyces diastaticus and from a highly flocculentSaccharomyces cerevisiae andSaccharomyces diastaticus. The (somatic) fusion products were capable to produce more ethanol than parental strains after 96 h of batch fermentation. The aim of this work was to reduce the amount of enzyme used in saccharification by using good fermenting amylolytic yeast strains.     

Ethanol production and fermentation characteristics of recombinant saccharomyces cerevisiae strains grown on starch

The production of ethanol from starch has been investigated in three genetically modified Saccharomyces cerevisiae strains (YPG/AB, YPG/MM, and YPB-G). Two of the three strains produce the Aspergillus awamori glucoamylase together with either the Bacillus subtilis (YPG/AB) or the mouse (YPG/MM) α-amylase as separately secreted polypeptides. YPB-G, on the other hand, secretes a bifunctional fusion protein that contains both the B. subtilis α-amylase and the A. awamori glucoamylase activities. Substrate utilization, biomass growth, and ethanol production were all studied in both starch- and glucose-containing media. Much higher growth rates were found when any of the three strains were grown on glucose. YPG/AB showed the most efficient utilization of starch for ethanol production with the lowest levels of reducing sugars accumulating in the medium. The superior performance of YPG/AB as compared to YPB-G was found to correlate with its higher level of α-amylase activity. The ethanol production levels of YPG/AB in starch- and glucose-containing media were found to be comparable. YPB-G, which secretes the bifunctional fusion protein, could produce ethanol in media with starch concentrations above 100 g l⁻¹ while YPG/MM did not produce ethanol from starch because of its negligible secretion of glucoamylase.     

Intergeneric yeast fusants with efficient ethanol production from cheese whey powder solution: Construction of a Kluyveromyces marxianus and Saccharomyces cerevisiae AY‐5 hybrid

Due to its high content of lactose and abundant availability, cheese whey powder (CWP) has received much attention for ethanol production in fermentation processes. However, lactose‐fermenting yeast strains including Kluyveromyces marxianus can only produce alcohol at a relatively low level, while the most commonly used distiller yeast strain Saccharomyces cerevisiae cannot ferment lactose since it lacks both β‐galactosidase and the lactose permease system. To combine the unique aspects of these two yeast strains, hybrids of K. marxianus TY‐22 and S. cerevisiae AY‐5 were constructed by protoplast fusion. The fusants were screened and characterized by DNA content, β‐galactosidase activity, ethanol tolerance, and ethanol productivity. Among the genetically stable fusants, the DNA content of strain R‐1 was 6.94%, close to the sum of the DNA contents of TY‐22 (3.99%) and AY‐5 (3.51%). The results obtained by random‐amplified polymorphic DNA analysis suggested that R‐1 was a fusant between AY‐5 and TY‐22. During the fermentation process with CWP, the hybrid strain R‐1 produced 3.8% v/v ethanol in 72 h, while the parental strain TY‐22 only produced 3.1% v/v ethanol in 84 h under the same conditions.     

Elimination of glycerol and replacement with alternative products in ethanol fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is a major by-product of ethanol fermentation by Saccharomyces cerevisiae and typically 2–3% of the sugar fermented is converted to glycerol. Replacing the NAD⁺-regenerating glycerol pathway in S. cerevisiae with alternative NADH reoxidation pathways may be useful to produce metabolites of biotechnological relevance. Under fermentative conditions yeast reoxidizes excess NADH through glycerol production which involves NADH-dependent glycerol-3-phosphate dehydrogenases (Gpd1p and Gpd2p). Deletion of these two genes limits fermentative activity under anaerobic conditions due to accumulation of NADH. We investigated the possibility of converting this excess NADH to NAD⁺ by transforming a double mutant (gpd1∆gpd2∆) with alternative oxidoreductase genes that might restore the redox balance and produce either sorbitol or propane-1,2-diol. All of the modifications improved fermentative ability and/or growth of the double mutant strain in a self-generated anaerobic high sugar medium. However, these strain properties were not restored to the level of the parental wild-type strain. The results indicate an apparent partial NAD⁺ regeneration ability and formation of significant amounts of the commodity chemicals like sorbitol or propane-1,2-diol. The ethanol yields were maintained between 46 and 48% of the sugar mixture. Other factors apart from the maintenance of the redox balance appeared to influence the growth and production of the alternative products by the genetically manipulated strains.     

Effects of “media osmotic agents” on the growth and morphogenesis ofActinidia deliciosa cv “hayward” callus

Summary The influence of various osmotic agents (carbohydrates) on the morphogenesis and growth of callus ofActinidia deliciosa cv Hayward was studied. Sucrose supported the highest level of growth and the lowest was supported by the sugar alcohols used in the experiments (glycerol, mannitol, sorbitol). The growth and survival of callus were evaluated with different osmotic sources in media containing glycerol, mannitol, or sorbitol at a concentration of 0.2M each for an extended period of eight subcultures (360 days). Two crucial points were identified: until the third subculture (135 days) the vitality seemed to be elevated; whereas the fifth (225 days) seemed to be a “point of no return” for tissues grown in glycerol and mannitol. Pretreatment with osmotic carbohydrates was shown to increase the magnitude of the morphogenetic events of callus subsequently transferred to sucrose-containing medium. Callus grown in the presence of mannitol and sorbitol showed a similar frequency of morphogenetic response. With respect to the media containing glycerol and sucrose, these induced more intense regeneration of shoots. When glycerol was present in the medium, however, we observed a synchronization of the morphogenetic response. Our results suggest that it is possible both to stimulate and to synchronize morphogenesis utilizing osmotic conditioning subcultures.     

<Emphasis Type="Italic">In vitro</Emphasis> germplasm conservation of <Emphasis Type="Italic">Podophyllum peltatum</Emphasis> L. under slow growth conditions

Germplasm conservation of Podophyllum peltatum L. was attempted by using synthetic seed technology and media supplemented with osmotic agents. Excised buds from in vitro cultures were encapsulated in calcium alginate beads and cultured on different substrates then stored at 5, 10, and 25°C for up to 8 mo. Survival and vigor in re-growth were the parameters used to evaluate the germplasm storage conditions. Vigor in re-growth was measured by number of buds induced after storage, which was achieved on a substrate containing water solidified with 1% w/v agar under 10°C. In vitro storage of shoot cultures was also evaluated by supplementing osmotic agents, mannitol, or sorbitol to the media. Such treatment had a negative impact on post-storage re-growth (at 25°C), even though the inclusion of 2% w/v sorbitol and mannitol each to the media increased plantlet survival during 10°C storage treatment. A deleterious effect was noticed among cultures in re-growth when higher concentrations of these supplements were added to the media. Genetic stability was assessed following 8 mo of storage using a PCR-based multilocus DNA fingerprinting technique, amplified fragment-length polymorphism. No differences in the DNA fragment patterns were observed using eight primer combinations in stored clones. However, a polymorphic band was noticed in the accession that served as explant source, suggesting that the mutation has occurred prior to this study perhaps during the 9 years of in vitro cultivation.     

Coordinate induction of dissimilatory ammonification and fermentative pathways in rhizobia

Dissimilatory ammonification was indicated as the common feature of ten rhizobial strains representing six species and three genera. In the absence of external electron acceptors, all investigated strains were capable of ethanolic fermentation. However, induction of anaerobic nitrite reduction was shown to be coupled with a shift of fermentation towards acetate in all the strains tested. Three metabolic groups could be distinguished with regard to nitrite regulation of ethanolic fermentation. It was shown for Bradyrhizobium sp. strain USDA 3045 that nitrite is the signal for switching between fermentative pathways although both ammonia and acetate excretion could not accelerate until nitrate had been utilized first. In the absence of N oxyanions, ethanol was indicated as the main product of mannitol fermentation, five-fold more abundant than acetate. An inverse composition was found in nitrite-amended cultures, due to a four-fold increase in acetate excretion whereas ethanol was kept at low level. Nitrite-supported fermentation towards acetate has not been previously reported for rhizobia. This benefit of this pathway was a two-fold shorter doubling time on 1% mannitol and 2.5 mM nitrite compared to no-nitrite media variants but also enabled fermentation of the more reduced carbon compound glycerol.     

A genome shuffling-generated <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> isolate that ferments xylose and glucose to produce high levels of ethanol

Genome shuffling is an efficient approach for the rapid improvement of industrially important microbial phenotypes. This report describes optimized conditions for protoplast preparation, regeneration, inactivation, and fusion using the Saccharomyces cerevisiae W5 strain. Ethanol production was confirmed by TTC (triphenyl tetrazolium chloride) screening and high-performance liquid chromatography (HPLC). A genetically stable, high ethanol-producing strain that fermented xylose and glucose was obtained following three rounds of genome shuffling. After fermentation for 84 h, the high ethanol-producing S. cerevisiae GS3-10 strain (which utilized 69.48 and 100% of the xylose and glucose stores, respectively) produced 26.65 g/L ethanol, i.e., 47.08% higher than ethanol production by S. cerevisiae W5 (18.12 g/L). The utilization ratios of xylose and glucose were 69.48 and 100%, compared to 14.83 and 100% for W5, respectively. The ethanol yield was 0.40 g/g (ethanol/consumed glucose and xylose), i.e., 17.65% higher than the yield by S. cerevisiae W5 (0.34 g/g).     

Production of polyols and ethanol by the osmophilic yeastZygosaccharomyces rouxii

Summary The yeastZygosaccharomyces rouxii ATCC 12572 was selected for its ability to produce appreciable levels of ethanol and of various polyols from concentrated glucose media (20 %, w/v).Z. rouxii was shown to yield large quantities of glycerol and of the mixture arabitol + mannitol. Good agitation combined with appropriate aeration (1 vvm) allowedZ. rouxii to utilize glucose readily leading to high polyol production. Depending on the fermentation conditions used,Z. rouxii ATCC 12572 will give either ethanol or various polyols as main fermentation product(s).