High-level ethanol production from starch by a flocculent <Emphasis Type="Italic">Saccharomyces cerevisiae </Emphasis>strain displaying cell-surface glucoamylase

A Strain of host yeast YF207, which is a tryptophan auxotroph and shows strong flocculation ability, was obtained from SaccharomYces diastaticus ATCC60712 and S. cerevisiae W303-1B by tetrad analysis. The plasmid pGA11, which is a multicopy plasmid for cell-surface expression of the Rhyzopus oryzae glucoamylase/alpha-agglutinin fusion protein, was then introduced into this flocculent yeast strain (YF207/pGA11). Yeast YF207/pGA11 grew rapidly under aerobic condition (dissolved oxygen 2.0 ppm), using soluble starch. The harvested cells were used for batch fermentation of soluble starch to ethanol under anaerobic condition and showed high ethanol production rates (0.71 g h(-1) l(-1)) without a time lag, because glucoamylase was immobilized on the yeast cell surface. During repeated utilization of cells for fermentation, YF207/pGA11 maintained high ethanol production rates over 300 h. Moreover, in fed-batch fermentation with YF207/pGA11 for approximately 120 h, the ethanol concentration reached up to 50 g l(-1). In conclusion, flocculent yeast cells displaying cell-surface glucoamylase are considered to be very effective for the direct fermentation of soluble starch to ethanol.     

自絮凝酵母SPSC01在组合反应器系统中酒精连续发酵的研究

建立了一套由四级磁力搅拌发酵罐串联组成、总有效容积4000mL的小型组合生物反应器系统 ,其中一级罐作为种子培养罐。以脱胚脱皮玉米粉双酶法制备的糖化液为种子培养基和发酵底物 ,进行了自絮凝颗粒酵母酒精连续发酵的研究。种子罐培养基还原糖浓度为100g L ,添加 (NH₄)₂HPO₄ 和KH₂PO₄ 各 20g L ,以0.017h^-1 的恒定稀释速率流加 ,并溢流至后续酒精发酵系统。发酵底物初始还原糖浓度 220g/L ,添加 (NH₄)₂HPO₄ 15g/L和KH₂PO₄2 5g/L ,流加至第一级发酵罐 ,稀释速率分别为 0.017、0.025、0.033、0.040和0.05 0h^-1。实验数据表明 ,自絮凝颗粒酵母在各发酵罐中呈部分固定化状态 ,在稀释速率0.040h^-1 条件下 ,发酵系统呈一定的振荡行为 ,其他四个稀释速率实验组均能够达拟稳态。当稀释速率不超过 0 0 33h^-1 ,流出末级发酵罐的发酵液中酒精浓度可以达到 12 % (V/V)以上 ,残还原糖和残总糖分别在 0 11%和 0 35 % h^-1,流出末级发酵罐的发酵液中酒精浓度可以达到12%（V/V）以上,残还原糖和残总糖分别在0.11%和0.35%（W/V）以下。在稀释速率为0.033h^-1时,计算发酵系统酒精的设备生产强度指标为3.32（g·L^-1·h^-1）,与游离酵母细胞传统酒精发酵工艺相比,增加约1倍。     

Effect of Dissolved Oxygen, Temperature, Initial Cell Count, and Sugar Concentration on the Viability of Saccharomyces cerevisiae in Rapid Fermentations

By using 7 x 10(8) cells of Saccharomyces cerevisiae per ml with which 25 degrees Brix honey solutions were fermented to 9.5% (wt/vol; 12% vol/vol) ethanol in 2.5 to 3 h at 30 C, i.e., rapid fermentation, the death rate was found to be high, with only 2.1% of the yeast cells surviving at the end of 3 h under anaerobic conditions. As the dissolved oxygen in the medium was increased from 0 to 13 to 20 to 100% in rapid fermentations at 30 C, there was a progressive increase in the percentage of cells surviving. The ethanol production rate and total were not seriously affected by a dissolved oxygen concentration of 13%, but fermentation was retarded by 20% dissolved oxygen and still further decreased as the dissolved oxygen content reached 100%. When the fermentation temperature was decreased to 15 C (at 13% dissolved oxygen), the rate of fermentation decreased, and the fermentation time to 9.5% ethanol (wt/vol) increased to 6 h. It was found that the higher the temperature between 15 and 30 C, the greater the rate of death as initial cell counts were increased from 1.1 x 10(7) to 7.8 x 10(8) cells per ml. At the lowest level of inoculum, 1.1 x 10(7) cells per ml, there was actual multiplication, even at 30 C; however, the fermentation was no longer rapid. The addition of 15% sugar, initially followed after an hour by the remaining 10%, or addition of the sugar in increments of 2.5 or 5% yielded a better survival rate of yeast cells than when the fermentation was initiated with 25% sugar.     

Effect of air supplement on the performance of continuous ethanol fermentation system

For the purpose of improving ethanol productivity, the effect of air supplement on the performance of continuous ethanol fermentation system was studied. The effect of oxygen supplement on yeast concentration, cell yield, cell viability, extracellular ethanol concentration, ethanol yield, maintenance coefficient, specific rates of glucose assimilation, ethanol production, and ethanol productivity have been evaluated, using a high alcohol tolerant Saccharomyces cerevisiae STV89 strain and employing a continuous fermentor equipped with an accurate air metering system in the flow rate range 0-11 mL air/L/h. It was found that, when a small amount of oxygen up to about 80mu mol oxygen/L/h was supplied, the ethanol productivity was significantly enhanced as compared to the productivity of the culture without any air supplement. It was also found that the oxygen supplement improved cell viability considerably as well as the ethanol tolerance level of yeast. As the air supply rate was increased, from 0 to 11 mL air/L/h while maintaining a constant dilution rate at about 0.06 h(-1), the cell concentration increased from 2.3 to 8.2 g/L and the ethanol productivity increased from 1.7 to 4.1 g ethanol/L/h, although the specific ethanol production rate decreased slightly from 0.75 to 0.5 g ethanol/g cell/h. The ethanol yield was slightly improved also with an increase in air supply rate, from about 0.37 to 0.45 ethanol/g glucose. The maintenance coefficient increased by only a small amount with the air supplement. This kind of air supplement technique may very well prove to be of practical importance to a development of a highly productive ethanol fermentation process system especially as a combined system with a high density cell culture technique.     

The multiple functions of coenzyme Q

The coenzyme function of ubiquinone was subject of extensive studies in mitochondria since more than 40 years. The catalytic activity of ubiquinone (UQ) in electron transfer and proton translocation in cooperation with mitochondrial dehydrogenases and cytochromes contributes essentially to the bioenergetic activity of ATP synthesis. In the past two decades UQ was recognized to exert activities which differ from coenzyme functions in mitochondria. From extraction/reincorporation experiments B. Chance has drawn the conclusion that redox-cycling of mitochondrial ubiquinone supplies electrons for univalent reduction of dioxygen. The likelihood of O2(.-) release as normal byproduct of respiration was based on the existence of mitochondrial SOD and the fact that mitochondrial oxygen turnover accounts for more than 90% of total cellular oxygen consumption. Arguments disproving this concept are based on results obtained from a novel noninvasive, more sensitive detection method of activated oxygen species and novel experimental approaches, which threw light into the underlying mechanism of UQ-mediated oxygen activation. Single electrons for O2(.-) formation are exclusively provided by deprotonated ubisemiquinones. Impediment of redox-interaction with the bc1 complex in mitochondria or the lack of stabilizing interactions with redox-partners are promotors of autoxidation. The latter accounts for autoxidation of antioxidant-derived ubisemiquinones in biomembranes, which do not recycle oxidized ubiquinols. Also O2(.-)-derived H2O2 was found to interact with ubisemiquinones both in mitochondria and nonrecycling biomembranes when ubiquinol was active as antioxidant. The catalysis of reductive homolytic cleavage of H2O2, which contributes to HO. formation in biological systems was confirmed under defined chemical conditions in a homogenous reduction system. Apart from dioxygen and hydrogen peroxide we will provide evidence that also nitrite may chemically interact with the ubiquinol/bc1 redox couple in mitochondria. The reaction product NO was reported elsewhere to be a significant bioregulator of the mitochondrial respiration and O2 activation. Another novel finding documents the bioenergetic role of UQ in lysosomal proton intransport. A lysosomal chain of redox couples will be presented, which includes UQ and which requires oxygen as the terminal electron acceptor.     

Ethanol production by Kluyveromyces lactis immobilized cells in copolymer carriers produced by radiation polymerization

The conditions for batch and continuous production of ethanol, using immobilized growing yeast cells of Kluyveromyces lactis, have been optimized. Yeast cells have been immobilized in hydrogel copolymer carriers composed of polyvinyl alcohol (PVA) with various hydrophilic monomers, using radiation copolymerization technique. Yeast cells were immobilized through adhesion and multiplication of yeast cells themselves. The ethanol production of immobilized growing yeast cells with these hydrogel carriers was related to the monomer composition of the copolymers and the optimum monomer composition was hydroxyethyl methacrylate (HEMA). In this case by using batch fermentation, the superior ethanol production was 32.9 g L(-1) which was about 4 times higher than that of cells in free system. The relation between the activity of immobilized yeast cells and the water content of the copolymer carriers was also discussed. Immobilized growing yeast cells in PVA: HEMA (7%: 10%, w/w) hydrogel copolymer carrier, were used in a packed-bed column reactor for the continuous production of ethanol from lactose at different levels of concentrations (50, 100 and 150) g L(-1). For all lactose feed concentrations, an increase in dilution rates from 0.1 h(-1) to 0.3 h(-1) lowered ethanol concentration in fermented broth, but the volumetric ethanol productivity and volumetric lactose uptake rate were improved. The fermentation efficiency was lowered with the increase in dilution rate and also at higher lactose concentration in feed medium and a maximum of 70.2% was obtained at the lowest lactose concentration 50 g L(-1).     

Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process

Several bottlenecks in the alcoholic fermentation process must be overcome to reach a very high and competitive performance of bioethanol production by the yeast Saccharomyces cerevisiae. In this paper, a nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30 degrees C. This performance was achieved by implementing exponential feeding of vitamins throughout the fermentation process. In comparison to an initial addition of a vitamin cocktail, an increase in the amount of vitamins and an exponential vitamin feeding strategy improved the final ethanol titre from 126 g l(-1) to 135 g l(-1) and 147 g l(-1), respectively. A maximum instantaneous productivity of 9.5 g l(-1) h(-1) was reached in the best fermentation. These performances resulted from improvements in growth, the specific ethanol production rate, and the concentration of viable cells in response to the nutritional strategy.     

表达血管生长抑制素的重组毕赤酵母在诱导阶段混合碳源的流加

为进行高密度发酵并实现外源基因的高表达,在表型为Mut^S的重组毕赤酵母（Pichia pastoris）表达人血管生长抑制素的诱导阶段,采用了甘油甲醇混合补料的培养方式。以溶氧水平作为甘油代谢指针来控制甘油限制性流加既可维持一定菌体生长,又不会发生发酵液中残余甘油及有害代谢产物（乙醇）阻遏蛋白表达。当表达阶段的菌体平均比生长速率控制于0.012h^-1,菌体浓度达150 g/L,血管生长抑制素浓度最高达到108 mg/L,血管生长抑制素的平均比生产速率为0.02 mg/(g·h),菌体关于甘油的表观得率为0.69 g/g,菌体关于甲醇的表观得率为0.93g/g,较没有采用甘油限制性流加时都有所提高。     

Alcohol fermentation of starch by a genetic recombinant yeast having glucoamylase activity

Alcohol fermentation of starch was investigated using a direct starch fermenting yeast, Saccharomyces cerevisiae SR93, constructed by integrating a glucoamylase-producing gene (STA1) into the chromosome of Saccharomyces cerevisiae SH1089. The glucoamylase was constitutively produced by the recombinant yeast. The ethanol concentration produced by the recombinant yeast was 14.3 g/L which was about 1.5-fold higher than by the conventional mixed culture using an amylolytic microorganism and a fermenting microorganism. About 60% of the starch was converted into ethanol by the recombinant yeast, and the ethanol yield reached its maximum value of 0.48 at the initial starch concentration of 50 g/L. The fed-batch culture, which maintains the starch concentration in the range of 30 to 50 g/L, was used to produce a large amount of ethanol from starch. The amount of ethanol produced in the fed-batch culture increased about 20% compared to the batch culture. (c) 1997 John Wiley & Sons, Inc.     

Alcohol production from cheese whey permeate using genetically modified flocculent yeast cells.

Alcoholic fermentation of cheese whey permeate was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus enabling for lactose metabolization. Data on yeast fermentation and growth on cheese whey permeate from a Portuguese dairy industry is presented. For cheese whey permeate having a lactose concentration of 50 gL(-1), total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. Using a continuously operating 5.5-L bioreactor, ethanol productivity near 10 g L(-1) h(-1) (corresponding to 0.45 h(-1) dilution rate) was obtained, which raises new perspectives for the economic feasibility of whey alcoholic fermentation. The use of 2-times concentrated cheese whey permeate, corresponding to 100 gL(-1) of lactose concentration, was also considered allowing for obtaining a fermentation product with 5% (w/v) alcohol.     

Breeding of flocculent industrial alcohol yeast strains by self-cloning of the flocculation gene FLO1 and repeated-batch fermentation by transformants

A nonflocculent industrial polyploid yeast strain, Saccharomyces cerevisiae 396-9-6V, was converted to a flocculent one by introducing a functional FLO1 gene at the URA3 locus. The flocculent strain FSC27 obtained was a so-called self-cloned strain, having no bacterial DNA. FSC27 cells could be easily recovered for reuse from fermentation mash without any physical energy. The strain produced a concentration of alcohol as high as 396-9-6V, although the fermentation rate of FSC27 was slightly lower than that of 396-9-6V. When uracil was added to the medium or when URA3 was reintroduced into FSC27 (named FSCU-L18), the fermentation rate and the growth rate increased, and the ethanol concentration produced was higher than that produced by the parent strain. The stable flocculation and high ethanol productivity were observed by using FSCU-L18 during 10 cycles of repeated-batch fermentation test.     

Expression of various genes to enhance ubiquinone metabolic pathway in Agrobacterium tumefaciens

Ubiquinone (UQ), a lipid-soluble component, acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system in many organisms, and has been widely used in pharmaceuticals due to its antioxidant property. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study, dps gene, which encodes decaprenyl diphosphate synthase, involved in ubiquinone biosynthesis from Agrobacterium tumefaciens, and coq2 gene of Saccharomyces cerevisiae, ppt1 gene of Schizosaccahromyces pombe and ubiA gene of Escherichia coli, all of them encoding 4-hydroxybenzoate:polyprenyl diphosphate (4-HB:PPP) transferase, were reconfigured into an operon under the control of a single promoter to yield various plasmids including pBIV-dps, pBIV-dpsq, pBIV-dpsp and pBIV-dpsca. The recombinant A. tumefaciens containing dps-ubiC-ubiA gene showed the highest level ubiquinone production than that of the other recombinants and the nonrecombinant bacterium. In an aerobic fed-batch fermentation, A. tumefaciens containing the pBIV-dpsca plasmid produced 25.2 mg of ubiquinone-10 per liter which was 1.68 times higher than that of nonrecombinant type. While in microaerobic fed-batch fermentation, recombinant cell pBIV-dpsca produced 30.8 mg L⁻¹ of ubiquinone-10. Compared to the original A. tumefaciens, the ubiquinone-10 yield and productivities of the recombinant bacterium pBIV-dpsca increased 88.9% and 77.7%, respectively, under microaerobic fed-batch conditions.     

代谢工程与全基因组重组构建酿酒酵母抗逆高产乙醇菌株

将酿酒酵母海藻糖代谢工程与全基因组重组技术相结合,改良工业酿酒酵母菌株的抗逆性和乙醇发酵性能。对来源于二倍体出发菌株Zd4的两株优良单倍体Z1和Z2菌株进行杂交获得基因组重组菌株Z12,并对Z1和Z2先进行(1)过表达海藻糖-6-磷酸合成酶基因 (TPS1) ,(2)敲除海藻糖水解酶基因 (ATH1), (3)同时过表达 TPS1和敲除ATH1, 经此三种基因工程操作后再进行杂交获得代谢工程菌株的全基因组重组菌株Z12ptps1、Z12 Δath1和Z12pTΔA。与亲株Zd4相比,Z12及结合代谢工程获得的菌株在高糖、高乙醇浓度与高温条件下生长与乙醇发酵性能都有不同程度的改进。对比研究结果表明:在高糖发酵条件下,同时过表达 TPS1和敲除ATH1 的双基因操作工程菌株胞内海藻糖积累、乙醇主发酵速率和乙醇产量相对于亲株的提高幅度要大于只过表达 TPS1,或敲除ATH1 的工程菌。结合了全基因组重组后获得的二倍体工程菌株Z12pTΔA,与原始出发菌株Zd4及重组子Z12相比,主发酵速率分别提高11.4%和6.3%,乙醇产量提高7.0%和4.1%,与其胞内海藻糖含量高于其它菌株、在胁迫条件下具有更强耐逆境能力相一致。结果证明,海藻糖代谢工程与杂交介导的全基因组重组相结合,是提高酿酒酵母抗逆生长与乙醇发酵性能的有效策略与技术途径。     

Inhibition of ubiquinone synthesis in isolated rat heart under an ischemic condition

1. The biosynthesis of ubiquinone (UQ) in isolated rat heart under ischemic and hypoxic conditions was investigated. 2. Under ischemic perfusion, a greater amount of biosynthetic intermediates, 3-nonaprenyl and 3-decaprenyl-4-hydroxybenzoate (PPHBs) was accumulated and a smaller amount of UQ-9 and -10 was synthesized when compared with normal conditions. 3. The accumulation of PPHBs was observed without forming UQs during anaerobic perfusion. 4. Hydroxylation which is the following reaction of PPHBs for the biosynthesis of UQ in rat heart, was proceeded by the monooxygenase(s) depending upon the oxygen concentrations.     

Ethanol tolerance of Pichia stipitis and Candida shehatae strains in fed-batch cultures at controlled low dissolved oxygen levels

Summary Fed-batch cultivations of Pichia stipitis and strains of Candida shehatae with d-xylose or d-glucose were conducted at controlled low dissolved oxygen tension (DOT) levels. There were some marked differences between the strains. In general growth was inhibited at lower ethanol concentrations than fermentation, and ethanol levels of up to 47 g·l^-1 were produced at 30°C. Ethanol production was mainly growth associated. The yeast strains formed small amounts of monocarboxylic acids and higher alcohols, which apparently did not enhance the ethanol toxicity. The maximum ethanol concentration obtained on d-xylose could not be increased by using a high cell density culture, nor by using d-glucose as substrate. The latter observation suggested that the low ethanol tolerance of these xylose-fermenting yeast strains was not a consequence of the metabolic pathway used during pentose fermentation. In contrast with the C. shehatae strains, it was apparent with P. stipitis CSIR-Y633 that when the ethanol concentration reached about 28 g·l^-1, ethanol assimilation exceeded ethanol production, despite cultivation at a low DOT of 0.2% of air saturation. Discontinuing the aeration enabled ethanol accumulation to proceed, but with concomitant xylitol production and cessation of growth.     

Muscle ubiquinone in healthy physically active males

Thirty-five (35) healthy physically active males had muscle biopsies taken from their vastus lateralis muscle to analyze for ubiquinone (vitamin Q, UQ), oxidative (muscle fiber types expressed as %ST and citrate synthase activity, CS) and fermentative (lactate dehydrogenase, LD) profiles. Graded cycle ergometer exercise to determine the intensities corresponding to onset of blood lactate accumulation set to 4.0 mmol × 1^–1 (W_OBLA) and symptom limited exercise (maximal, W_SL) were also undertaken. Eleven (11) subjects had also recently participated in a marathon race. UQ was positively related to CS (r = 0.67, p < 0.001) and %ST (r = 0.60, p < 0.001) but not to LD. UQ was also positively related to exercise capacity and/or marathon performance (e.g. W_OBLA × kg^–1 BW, r = 0.70, p < 0.001). It was suggested that muscle UQ allocation in man was related to variables describing molecular oxygen availability, respiratory activity and oxidative energy releasing processes but not to fermentation activity. UQ allocation to ST fibers/CS activity was suggested to be due to the double role of UQ: 1) as a mitochondrial coenzyme (CoQ₁₀) and 2) as a nonspecific antioxidant.     

Controlled fed-batch fermentation of recombinant Saccharomyces cerevisiae to produce hepatitis B surface antigen

We have performed controlled fed-batch fermentation experiments to compare the production level of hepatitis B surface antigen (HBsAg) by recombinant yeast Saccharomyces cerevisiae strains (YNN27/pYBH-1, YNN27/ p2mu-S11, YNN27/pDCB-S2) containing plasmid vector with alcohol dehydrogenase (ADH1), acid phosphatase (PHO5), and glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, respectively. Yeast cell concentrations of 15-35 g dry cell weight/L were obtained. By limiting phosphorous concentration, HBsAg expression level for the YNN27/p2mu-S11 strain with inducible PHO5 promoter reached 0.2-0.3 mg/L. By controlling nutrient addition rate and dissolved oxygen concentration, HBsAg concentrations of 3-10 mg/L were achieved in 60-70 h fermentation using the YNN27/pDCB-S2 strain with the constitutive GPD promoter.     

Cytochrome P-450 accumulation and loss as controlled by growth phase of Saccharomyces cerevisiae: relationship to oxygen, glucose and ethanol concentrations

Ethanol induced small amounts of cytochrome P-450 in Saccharomyces cerevisiae NCYC 754 under conditions in which it is not normally detectable. Moreover, in non-growing yeast the existing cytochrome P-450 content was increased by 50% at a limited range of glucose concentrations (8-12% in 0.1 M-potassium phosphate buffer, pH 7.0), in which ethanol is produced by fermentation, possibly at an optimum concentration for induction of cytochrome P-450. Added alkanols, other than ethanol, caused rapid degradation of cytochrome P-450 in non-growing yeast; the rate of loss was directly related to the lipid solubility of the alkanol. Ethanol therefore favoured the accumulation of cytochrome P-450 in yeast; this may be related to an important putative role of one of the isoenzymes in ethanol-tolerance of the yeast, by the oxidative removal of ethanol from the endoplasmic reticulum of the cell. It is the accumulation of dissolved oxygen, rather than ethanol, that occurs on cessation of yeast growth that is likely to trigger the rapid disappearance of cytochrome P-450 observed at this time.