Xylose fermentation by yeasts

Summary Xylose reductase from the xylose-fermenting yeastPichia stipitis was purified to electrophoretic homogeneity via ion-exchange, gel and affinity chromatography. At physiological pH values the thermodynamic equilibrium constant was determined to be 0.575x10¹⁰ (l·mol^-1). Product inhibiton studies are reported which clearly show that the kinetic mechanism of the xylose reductase is ordered-bi-bi with isomerisation of a stable enzyme form.     

Xylose fermentation by Saccharomyces cerevisiae

We have performed a comparative study of xylose utilization in Saccharomyces cerevisiae transformants expressing two key enzymes in xylose metabolism, xylose reductase (XR) and xylitol dehydrogenase (XDH), and in a prototypic xylose-utilizing yeast, Pichia stipitis. In the absence of respiration (see text), baker's yeast cells convert half of the xylose to xylitol and ethanol, whereas P. stipilis cells display rather a homofermentative conversion of xylose to ethanol. Xylitol production by baker's yeast is interpreted as a result of the dual cofactor dependence of the XR and the generation of NADPH by the pentose phosphate pathway. Further limitations of xylose utilization in S. cerevisiae cells are very likely caused by an insufficient capacity of the non-oxidative pentose phosphate pathway, as indicated by accumulation of sedoheptulose-7-phosphate and the absence of fructose-1,6-bisphosphate and pyruvate accumulation. By contrast, uptake at high substrate concentrations probably does not limit xylose conversion in S. cerevisiae XYL1/XYL2 transformants. Correspondence to: M. Ciriacy     

Xylose fermentation by yeasts. 5. Use of ATP balances for modeling oxygen-limited growth and fermentation of yeast Pichia stipitis with xylose as carbon source

Kinetic studies are presented for the growth and fermentation of the yeast Pichia stipitis with xylose as the carbon source. Ethanol is produced from xylose under anaerobic as well as under oxygen-limiting conditions but only at dissolved oxygen concentrations up to 3 mumol/L Maximum yields and production rates were obtained under oxygen-limiting conditions, where the xylose metabolism may be considered to be consisted of three different components (assimilation, respiration, fermentation). The contribution of each pathway is determined by the availability of oxygen and the energy yield of each pathway. In order to describe the course of oxygen-limited fermentations, a mathematical model has been developed with the assumption that growth is coupled to the energy production. The resulting model requires only four independent parameters (Y(x/O(2) ), Y(ATP) (max), m(ATP), and P/O). These parameters were estimated on the basis of eight separate batch fermentations.     

Inhibition of malolactic fermentation by cryotolerant yeasts

White wines produced by some cryotolerant strains of Saccharomyces cerevisiae are more resistant to malolactic fermentation than those produced by normal strains: e.g. for two months of storage, the wines, inoculated with Leuconostoc oenos or Lactobacillus plantarum, were fully stabilized with levels of 51-65 mg total SO 2 /l and 5.70-5.75 g titratable acidity/l. The use of these yeasts in wine-making can decrease the quantities of sulfites added to stabilize wines.     

D-xylulose fermentation in yeasts

Summary With pure D-xylulose as substrate, Schizosaccharomyces pombe produced ethanol in good yield with low quantities of polyols as by-products. Saccharomyces cerevisiae was found to be a good alcohol producer in glucose but not as good in D-xylulose. Other yeast cultures converted D-xylulose to xylitol, or D-arabitol or both, with lower ethanol yield.     

Xylose and yeasts: A story beyond xylitol production

Six different yeasts were used to study their metabolism of glucose and xylose, and mainly their capacity to produce ethanol and xylitol. The strains used were Candida guilliermondii, Debaryomyces hansenii, Saccharomyces cerevisiae, Kluyveromyces marxianus, Meyerozyma guilliermondii and Clavispora lusitaniae, four isolated from a rural mezcal fermentation facility. All of them produced ethanol when the substrate was glucose. When incubated in a medium containing xylose instead of glucose, only K. marxianus and M. guilliermondii were able to produce ethanol from xylose. On the other hand, all of them could produce some xylitol from xylose, but the most active in this regard were K. marxianus, M. guilliermondii, C. lusitaniae, and C. guilliermondii with the highest amount of xylitol produced. The capacity of all strains to take up glucose and xylose was also studied. Xylose, in different degrees, produced a redox imbalance in all yeasts. Respiration capacity was also studied with glucose or xylose, where C. guilliermondii, D. hansenii, K. marxianus and M. guilliermondii showed higher cyanide resistant respiration when grown in xylose. Neither xylose transport nor xylitol production were enhanced by an acidic environment (pH 4), which can be interpreted as the absence of a proton/sugar symporter mechanism for xylose transport, except for C. lusitaniae. The effects produced by xylose and their magnitude depend on the background of the studied yeast and the conditions in which these are studied.     

油脂酵母发酵木糖生产微生物油脂

目的用斯达油脂酵母(Lipomyces starkeyi)作为发酵菌株,以纯木糖溶液为油脂发酵原料,对L.starkeyi利用木糖积累油脂进行系统研究。方法 L.starkeyi于斜面培养基中活化后,接种于YPD液体培养基,于30℃、200 r/min摇床培养。在摇瓶中培养一段时间后,测定发酵液细胞浓度,离心发酵液收集细胞。将离心后得到的菌体加入木糖溶液重悬,并转接于含50 mL木糖溶液的250 mL摇瓶中进行发酵生产。结果相比一阶段法,两阶段发酵方法可以在更短的时间内达到较高的油脂含量,油脂含量能够达到细胞自身干重的60%以上。实验发现高菌龄酵母产油速度更快;并且初始木糖浓度高达120 g/L时,酵母细胞仍然能够高效合成油脂。结论 L.starkeyi能够有效利用木糖进行发酵产生油脂,是以木质纤维素为原料生产微生物油脂的优良菌种。     

Metabolic engineering for improved fermentation of pentoses by yeasts

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g^–1 sugar consumed, so commercialization seems feasible for some applications.     

A simplified routine method for determining carbohydrate fermentation by yeasts

The use of commercially available materials in a minimal medium contributes to the simplicity, reliability, and reproducibility of a method described to demonstrate carbohydrate fermentation reactions by yeasts. The medium consists of 1% yeast extract and 2% test carbohydrate in distilled water, dispensed in a modified Durham fermentation tube. Carbohydrate fermentation patterns can usually be obtained within a period of 7 days. The ability of yeasts to ferment carbohydrates is determined strictly on the basis of gas production from the substrate. The method proved reliable in reproducing established fermentation patterns for 112 different yeast strains representing 13 separate genera.

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Zusammenfassung Der Gebrauch von handelsüblichen Grundbestandteilen in einem geringen Medium trägt zu der Einfachheit, Zuverlässigkeit und Reproduzierbarkeit der beschriebenen Methode, um die Gärungsreaktionen der Karbohydrate durch Hefen zu veranschaulichen, bei. Das Medium besteht aus einem 1 prozent. Hefenauszug und aus einem 2 prozentigen Testkarbohydrat in destilliertem Wasser, die auf modifiziertes Durham Gärungsreagensglas verteilt sind. Das Karbohydrat-Gärungsmodell kann gewöhnlich innerhalb einer Periode von sieben Tagen erhalten werden. Die Fähigkeit von Hefen, Karbohydrate zu vergären, ist ausschließlich auf Grund der Gasentwicklung aus den Produkten bestimmt. Die Methode erwies sich zuverlässig in der Erzeugung festgestellter Gärungsmodelle aus 112 Hefen, die 13 verschiedene Gattungen darstellten.

     

Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha

Wild-type strains of the thermotolerant methylotrophic yeast Hansenula polymorpha are able to ferment glucose, cellobiose and xylose to ethanol. H. polymorpha most actively fermented sugars to ethanol at 37 degrees C, whereas the well-known xylose-fermenting yeast Pichia stipitis could not effectively ferment carbon substrates at this temperature. H. polymorpha even could ferment both glucose and xylose up to 45 degrees C. This species appeared to be more ethanol tolerant than P. stipitis but more susceptible than Saccharomyces cerevisiae. A riboflavin-deficient mutant of H. polymorpha increased its ethanol productivity from glucose and xylose under suboptimal supply with riboflavin. Mutants of H. polymorpha defective in alcohol dehydrogenase activity produced lower amounts of ethanol from glucose, whereas levels of ethanol production from xylose were identical for the wild-type strain and the alcohol dehydrogenase-defective mutant.     

Xylose fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite liquor

Spent sulfite pulping liquor (SSL) is a high-organic content byproduct of acid bisulfite pulp manufacture which is fermented to make industrial ethanol. SSL is typically concentrated to 240 g/l (22% w/w) total solids prior to fermentation, and contains up to 24 g/l xylose and 30 g/l hexose sugars, depending upon the wood species used. The xylose present in SSL is difficult to ferment using natural xylose-fermenting yeast strains due to the presence of inhibitory compounds, such as organic acids. Using sequential batch shake flask experiments, Saccharomyces cerevisiae 259ST, which had been genetically modified to ferment xylose, was compared with the parent strain, 259A, and an SSL adapted strain, T2, for ethanol production during SSL fermentation. With an initial SSL pH of 6, without nutrient addition or SSL pretreatment, the ethanol yield ranged from 0.32 to 0.42 g ethanol/g total sugar for 259ST, compared to 0.15-0.32 g ethanol/g total sugar for non-xylose fermenting strains. For most fermentations, minimal amounts of xylitol (<1 g/l) were produced, and glycerol yields were approximately 0.12 g glycerol/g sugar consumed. By using 259ST for SSL fermentation up to 130% more ethanol can be produced compared to fermentations using non-xylose fermenting yeast.