Metabolic transition step from ethanol consumption to sugar/ethanol

The metabolic pathway shift between only ethanol consumption to both sugar/ethanol consumption was measured by on-line analysis of respiratory quotient of a Saccharomyces cerevisiae. The experiments were carried out in a fed-batch culture under aerobic conditions. During the transition phase, respiratory quotient (RQ) profile shows that sugar can be metabolized through the fermentative pathway even to values of RQ lower than 1.     

Kinetics of growth and sugar consumption in yeasts

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts.Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called Crabtree effect probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect inS. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast.S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions.Non-Saccharomyces yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeastCandida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.     

Crabtree-negative characteristics of recombinant xylose-utilizing Saccharomyces cerevisiae

For recombinant xylose-utilizing Saccharomyces cerevisiae, ethanol yield and productivity is substantially lower on xylose than on glucose. In contrast to glucose, xylose is a novel substrate for S. cerevisiae and it is not known how this substrate is recognized on a molecular level. Failure to activate appropriate genes during xylose-utilization has the potential to result in sub-optimal metabolism and decreased substrate uptake. Certain differences in fermentative performance between the two substrates have thus been ascribed to variations in regulatory response. In this study differences in substrate utilization of glucose and xylose was analyzed in the recombinant S. cerevisiae strain TMB3400. Continuous cultures were performed with glucose and xylose under carbon- and nitrogen-limited conditions. Whereas biomass yield and substrate uptake rate were similar during carbon-limited conditions, the metabolic profile was highly substrate dependent under nitrogen-limited conditions. While glycerol production occurred in both cases, ethanol production was only observed for glucose cultures. Addition of acetate and 2-deoxyglucose pulses to a xylose-limited culture was able to stimulate transient overflow metabolism and ethanol production. Application of glucose pulses enhanced xylose uptake rate under restricted co-substrate concentrations. Results are discussed in relation to regulation of sugar metabolism in Crabtree-positive and -negative yeast.     

Direct ethanol fermentation from lignocellulosic biomass by Antarctic basidiomycetous yeast Mrakia blollopis under a low temperature condition

Antarctic basidiomycetous yeast Mrakia blollopis SK-4 has unique fermentability for various sugars under a low temperature condition. Hence, this yeast was used for ethanol fermentation from glucose and also for direct ethanol fermentation (DEF) from cellulosic biomass without/with Tween 80 at 10 °C. Maximally, 48.2 g/l ethanol was formed from 12% (w/v) glucose. DEF converted filter paper, Japanese cedar and Eucalyptus to 12.2 g/l, 12.5 g/l and 7.2 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80, ethanol concentration increased by about 1.1–1.6-fold compared to that without Tween 80. This is the first report on DEF using cryophilic fungi under a low temperature condition. We consider that M. blollopis SK-4 has a good potential for ethanol fermentation in cold environments.     

Metabolic diversity of Saccharomyces cerevisiae strains from spontaneously fermented grape musts

One hundred and fifteen Saccharomyces cerevisiae strains from Aglianico del Vulture, a red wine produced in Southern Italy, were characterized for the production of some secondary compounds involved in the aroma and taste of alcoholic beverages. The strains exhibited a uniform behaviour in the production levels of n-propanol, active amyl alcohol and ethyl acetate, whereas isobutanol, isoamyl alcohol and acetaldehyde were formed with a wide variability. Only five strains produced wines close to the reference Aglianico del Vulture wine for the traits considered. Of these, two strains were selected, underwent to tetrad analysis and the single spore cultures were tested in grape must fermentation. The progeny of one strain showed a significant metabolic variability, confirming the necessity to test starter cultures for the segregation of traits of technological interest. Our findings suggest the selection of specific strains for specific fermentations as a function of the vine variety characteristics in order to take the major advantage from the combination grape must/S. cerevisiae strain.     

Fermentation of sugar cane bagasse hemicellulosic hydrolysate and sugar mixtures to ethanol by recombinant Escherichia coli KO11

Escherichia coli KO11, carrying the ethanol pathway genes pdc (pyruvate decarboxylase) and adh (alcohol dehydrogenase) from Zymomonas mobilis integrated into its chromosome, has the ability to metabolize pentoses and hexoses to ethanol, both in synthetic medium and in hemicellulosic hydrolysates. In the fermentation of sugar mixtures simulating hemicellulose hydrolysate sugar composition (10.0 g of glucose/l and 40.0 g of xylose/l) and supplemented with tryptone and yeast extract, recombinant bacteria produced 24.58 g of ethanol/l, equivalent to 96.4% of the maximum theoretical yield. Corn steep powder (CSP), a byproduct of the corn starch-processing industry, was used to replace tryptone and yeast extract. At a concentration of 12.5 g/l, it was able to support the fermentation of glucose (80.0 g/l) to ethanol, with both ethanol yield and volumetric productivity comparable to those obtained with fermentation media containing tryptone and yeast extract. Hemicellulose hydrolysate of sugar cane bagasse supplemented with tryptone and yeast extract was also readily fermented to ethanol within 48 h, and ethanol yield achieved 91.5% of the theoretical maximum conversion efficiency. However, fermentation of bagasse hydrolysate supplemented with 12.5 g of CSP/l took twice as long to complete. This revised version was published online in November 2006 with corrections to the Cover Date.     

Aerobic sugar metabolism in the spoilage yeast Zygosaccharomyces bailii

Despite the importance of some Zygosaccharomyces species as agents causing spoilage of food, the carbon and energy metabolism of most of them is yet largely unknown. This is the case with Zygosaccharomyces bailii. In this study the occurrence of the Crabtree effect in the petite-negative yeast Z. bailii ATCC 36947 was investigated. In this yeast the aerobic ethanol production is strictly dependent on the carbon source utilised. In glucose-limited continuous cultures a very low level of ethanol was produced. In fructose-limited continuous cultures ethanol was produced at a higher level and its production increased with the dilution rate. As a consequence, on fructose the onset of respiro-fermentative metabolism caused a reduction in biomass yield. An immediate aerobic alcoholic fermentation in Z. bailii was observed during the transition from sugar limitation to sugar excess, both on glucose and on fructose. The analysis of some key enzymes of the fermentative metabolism showed a high level of acetyl-CoA synthetase in Z. bailii growing on fructose. At high dilution rates, the activities of glucose- and fructose-phosphorylating enzymes, as well as of pyruvate decarboxylase and alcohol dehydrogenase, were higher in cells during growth on fructose than on glucose.     

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6 g/l, and 14.6 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5 U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.     

OsPDR在酵母中异源表达增强了酵母对钴的耐受性

在通过RNA-Seq技术得到的镉响应转录组图谱中,用50 μmol/L Cd处理24 h后,一个镉响应金属离子转运蛋白OsPDR被鉴定出其在水稻(Oryza sativa ssp. japonica cv. Nipponbare)茎中的表达量显著上调．本研究中,从水稻(Oryza sativa cv. Nipponbare)中分离了OsPDR基因,并对其金属离子转移活性进行了分析．金属耐受性实验结果表明,过表达OsPDR能提高酵母对Co的耐受性,但对Zn、Ni和Cd的耐受性不强,并且经电感耦合等离子体质谱法(ICP-MS)测定Co含量后,与空载体转化酵母相比,过表达OsPDR的酵母中Co的积累更高．利用共聚焦显微镜观察发现,EGFP-OsPDR融合蛋白定位于液泡膜上．这些数据表明OsPDR可能在Co稳态中起着重要作用．OsPDR在植物中的作用,还需要进一步的研究．     

苹果渣乙醇提取物抗菌活性及防腐作用研究

为提高苹果渣资源利用率,探究苹果渣乙醇提取物的抗菌活性和防腐性能,该文采用微波辅助提取法制取苹果渣乙醇提取物,用抑菌圈实验测定其抗菌活性,并研究了其防腐作用.结果表明:(1)苹果渣乙醇提取物对酵母菌抑制作用不明显(抑菌圈直径<1 mm),对金黄色葡萄球菌和大肠杆菌的抑菌作用较明显(抑菌圈直径为6～9 mm),最佳抑菌浓...     

Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol

The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37°C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose.     

Fermentation of high concentrations of lactose to ethanol by engineered flocculent Saccharomyces cerevisiae

The development of microorganims that efficiently ferment lactose has a high biotechnological interest, particularly for cheese whey bioremediation processes with simultaneous bio-ethanol production. The lactose fermentation performance of a recombinant Saccharomyces cerevisiae flocculent strain was evaluated. The yeast consumed rapidly and completely lactose concentrations up to 150 g l⁻¹ in either well- or micro-aerated batch fermentations. The maximum ethanol titre was 8% (v/v) and the highest ethanol productivity was 1.5–2 g l⁻¹ h⁻¹, in micro-aerated fermentations. The results presented here emphasise that this strain is an interesting alternative for the production of ethanol from lactose-based feedstocks.     

Metabolic engineering of sugar catabolism in lactic acid bacteria

Lactic acid bacteria are characterized by a relatively simple sugar fermentation pathway that, by definition, results in the formation of lactic acid. The extensive knowledge of traditional pathways and the accumulating genetic information on these and novel ones, allows for the rerouting of metabolic processes in lactic acid bacteria by physiological approaches, genetic methods, or a combination of these two. This review will discuss past and present examples and future possibilities of metabolic engineering of lactic acid bacteria for the production of important compounds, including lactic and other acids, flavor compounds, and exopolysaccharides.     

Oleic acid delays and modulates the transition from respiratory to fermentative metabolism in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> after exposure to glucose excess

This work aimed to study the transition from respiratory to fermentative metabolism in Saccharomyces cerevisiae CEN.PK 113-7D and more specifically to evaluate the implication of the acetyl-coenzymeA-derived carbon transport from cytosol to mitochondria in the onset of the metabolic shift. The strategy consisted in introducing, during aerobic glucose-limited chemostat (D = 0.16 h¹), a local perturbation around the step to be studied by the addition of cosubstrate and in analyzing the consequences of such a perturbation on the metabolic transition. Oleic acid and l-carnitine were among the tested cosubstrates because they were known to stimulate enzymes implicated in the acetyl-coenzymeA transport between the different cell compartments, such as the carnitine acetyl transferases. The metabolic transition was then comparatively quantified in sole glucose and in glucose/oleic acid chemostats in presence/absence of l-carnitine after a pulse of glucose. Feeding the culture with oleic acid (D _ole = 0.0041 and 0.0073 h¹) led to a delay in the onset of the metabolic shift (up to 15 min), a 33% decrease in the ethanol production and a redirection of the carbon flux toward biomass production. The data clearly showed a modulation of the carbon distribution among respiration and fermentation, in favor of a decrease in the “short-term” Crabtree effect by the oleic acid. David Feria-Gervasio and Jean-Roch Mouret worked equally on this project and should be considered both as first authors. An erratum to this article can be found at     

Comparisons of SPORL and dilute acid pretreatments for sugar and ethanol productions from aspen

This study reports comparative evaluations of sugar and ethanol production from a native aspen (Populus tremuloides) between sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) and dilute acid (DA) pretreatments. All aqueous pretreatments were carried out in a laboratory wood pulping digester using wood chips at 170°C with a liquid to oven dry (od) wood ratio (L/W) of 3:1 at two levels of acid charge on wood of 0.56 and 1.11%. Sodium bisulfite charge on od wood was 0 for DA and 1.5 or 3.0% for SPORL. All substrates produced by both pretreatments (except DA with pretreatment duration of 0) had good enzymatic digestibility of over 80%. However, SPORL produced higher enzymatic digestibility than its corresponding DA pretreatment for all the experiments conducted. As a result, SPORL produced higher ethanol yield from simultaneous saccharification and fermentation of cellulosic substrate than its corresponding DA pretreatment. SPORL was more effective than its corresponding DA pretreatment in reducing energy consumption for postpretreatment wood chip size-reduction. SPORL, with lower energy input and higher sugar and ethanol yield, produced higher sugar and ethanol production energy efficiencies than the corresponding DA pretreatment.     

两粤黄檀乙醇提取物化学成分分析及抗炎活性研究

为了研究壮药两粤黄檀的化学成分及其抗炎活性,阐明其药理作用机制,该研究采用小鼠二甲苯致炎耳廓肿胀法、角叉菜胶致炎足肿胀急性炎症模型,观察不同剂量两粤黄檀乙醇提取物对小鼠耳廓肿胀度、肿胀率及足肿胀度的影响,并利用液相色谱-质谱(LC-MS)、热裂解气相色谱-质谱(PY-GC/MS)等技术分析提取物的化学成分,结合网络药理...     

Metabolic engineering of a Lactobacillus plantarum double ldh knockout strain for enhanced ethanol production

Lactobacillus plantarum ferments glucose through the Embden–Meyerhof–Parnas pathway: the central metabolite pyruvate is converted into lactate via lactate dehydrogenase (LDH). By substituting LDH with pyruvate decarboxylase (PDC) activity, pyruvate may be redirected toward ethanol production instead of lactic acid fermentation. A PDC gene from the Gram-positive bacterium Sarcina ventriculi (Spdc) was introduced into an LDH-deficient strain, L. plantarum TF103, in which both the ldhL and ldhD genes were inactivated. Four different fusion genes between Spdc and either the S. ventriculi promoter or three Lactococcus lactis promoters in pTRKH2 were introduced into TF103. PDC activity was detected in all four recombinant strains. The engineered strains were examined for production of ethanol and other metabolites in flask fermentations. The recombinant strains grew slightly faster than the parent TF103 and produced 90–130 mM ethanol. Although slightly more ethanol was observed, carbon flow was not significantly improved toward ethanol, suggesting that a further understanding of this organism’s metabolism is necessary.     

猪不同肠段微生物体外培养对蛋氨酸的代谢特性

【目的】旨在通过微生物体外发酵技术,以回肠微生物为参照,研究猪盲肠及结肠微生物对在小肠微生物中代谢率较低的蛋氨酸的代谢特性。【方法】采集4头健康100 kg左右杜×长×大杂交猪的盲肠、结肠与回肠食糜作为接种物,分别接种于10 mmol/L蛋氨酸的培养基中,37°C体外培养24 h。分别设含蛋基酸溶液和含各肠段食糜接种物的空白对照组。【结果】(1)不同肠段微生物以蛋氨酸为底物体外发酵,盲肠组蛋氨酸消失率(21.9%)显著高于结肠组(16.7%)与回肠组(16.3%)(P0.05)。盲肠组总SCFA量显著高于结肠与回肠组(P0.05),伴随着p H值下降程度最高;盲肠组MCP产量也显著高于结肠与回肠组(P0.05);在产气量与NH3-N浓度上,盲肠组与结肠组均显著低于回肠组(P0.05)。(2)以蛋氨酸为底物体外发酵,门水平上,总菌、厚壁菌门含量在各肠段组间无显著差异(P0.05),拟杆菌门含量在盲肠组最高;与不加蛋氨酸底物的对照组比较,三个肠段试验组总菌、厚壁菌门含量均显著高于对照组(P0.05),而拟杆菌门含量在试验组与对照组间差异不显著(P0.05)。属水平上,盲肠组和结肠组大肠杆菌属数量显著低于回肠组(P0.05),而柔嫩梭菌属和梭菌XIV属数量在盲肠组和结肠组均高于回肠组;各肠段组间双歧杆菌数量无显著差异(P0.05)。【结论】以蛋氨酸为底物,体外培养猪盲肠微生物对蛋氨酸代谢率高于回肠微生物,伴随着其他发酵参数的变化,并且发酵产生更多的菌体蛋白。相比于回肠微生物发酵,大肠微生物发酵后,柔嫩梭菌属和梭菌XIV属数量较高,而大肠杆菌属数量较低。     

Metabolic engineering of Saccharomyces cerevisiae for increased bioconversion of lignocellulose to ethanol

The absence of pentose-utilizing enzymes in Saccharomyces cerevisiae is an obstacle for efficiently converting lignocellulosic materials to ethanol. In the present study, the genes coding xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) from Pichia stipitis were successfully engineered into S. cerevisae. As compared to the control transformant, engineering of XYL1 and XYL2 into yeasts significantly increased the microbial biomass (8.1 vs. 3.4 g/L), xylose consumption rate (0.15 vs. 0.02 g/h) and ethanol yield (6.8 vs. 3.5 g/L) after 72 h fermentation using a xylose-based medium. Interestingly, engineering of XYL1 and XYL2 into yeasts also elevated the ethanol yield from sugarcane bagasse hydrolysate (SUBH). This study not only provides an effective approach to increase the xylose utilization by yeasts, but the results also suggest that production of ethanol by this recombinant yeasts using unconventional nutrient sources, such as components in SUBH deserves further attention in the future.     

代谢工程改造大肠杆菌合成己二酸

己二酸是一种具有重要应用价值的二元羧酸,是合成尼龙-66的关键前体。目前,生物法生产己二酸存在生产周期长、生产效率低的问题。本研究选择一株野生型高产琥珀酸菌株大肠杆菌(Escherichia coli) FMME N-2为底盘细胞,首先通过引入逆己二酸降解途径的关键酶,成功构建了可合成0.34 g/L己二酸的E. coli JL00菌株;接着,对合成路径限速酶进行表达优化,使E. coli JL01菌株在摇瓶发酵条件下产量达到0.87 g/L;随后,通过敲除sucD基因、过表达acs基因和突变lpd基因的组合策略平衡己二酸合成前体的供应,优化菌株E. coli JL12己二酸产量进一步提升至1.51 g/L;最后,在5 L发酵罐上对己二酸发酵工艺进行优化。工程菌株经72 h分批补料发酵,己二酸的产量达到22.3 g/L,转化率为0.25 g/g,生产强度为0.31 g/(L·h),具备了一定的应用潜力。本研究可为包括己二酸在内的多种二元羧酸细胞工厂的构建提供理论依据和技术基础。