Efficient bioethanol production from xylose by recombinant saccharomyces cerevisiae requires high activity of xylose reductase and moderate xylulokinase activity

We varied the promoter strength of xylose reductase (XR) gene and the copy number of xylulokinase (XK) gene to determine how XR and XK activities affect the xylose-fermenting abilities of recombinant Saccharomyces cerevisiae expressing xylitol dehydrogenase (XDH). The most enhanced ethanol yield and lowered xylitol yield occurred in strain I-PGK/AUR, which has high activity of both XR and XDH and moderate XK activity.     

Bioethanol production from rice straw by a sequential use of Saccharomyces cerevisiae and Pichia stipitis with heat inactivation of Saccharomyces cerevisiae cells prior to xylose fermentation

In order to establish an efficient bioethanol production system from rice straw, a new strategy to ferment the mixture of glucose and xylose by a sequential application of Saccharomyces cerevisiae and Pichia stipitis was developed, in which heat inactivation of S. cerevisiae cells before addition of P. stipitis was employed. The results showed that heating at 50°C for 6h was sufficient to give high xylose fermentation efficiency. By application of the inactivation process, 85% of the theoretical yield was achieved in the fermentation of the synthetic medium. At the same time, the xylitol production was reduced by 42.4% of the control process. In the simultaneous saccharification and fermentation of the lime-pretreated and CO(2)-neutralized rice straw, the inactivation of S. cerevisiae cells enabled the full conversion of glucose and xylose within 80 h. Finally, 21.1g/l of ethanol was produced from 10% (w/w) of pretreated rice straw and the ethanol yield of rice straw reached 72.5% of the theoretical yield. This process is expected to be useful for the ethanol production from lignocellulosic materials in the regions where large-scale application of recombinant microorganisms was restricted.     

Ethanol production from carob pods by Saccharomyces cerevisiae

The production of ethanol from carob pods extract by Saccharomyces cerevisiae in static and shake flask fermentation was investigated. Shake flask fermentation proved to be a better fermentation system for the production of ethanol than static fermentation. The external addition of nutrients into the carob pods extract did not improve the production of ethanol. The maximum concentration of ethanol (75 g/l) was obtained at an inoculum amount of 0.3%, a pH of 4.5, 30°C and an initial sugar concentration of 200 g/1. Under the same fermentation conditions both sterilized and non‐sterilized carob pods extract gave the same final ethanol concentration.     

Ethanol from lactose in salted cheese whey by recombinant Saccharomyces cerevisiae

Seven yeast recombinants were selected from 57 fusant colonies on the basis of higher DNA content, nuclear diameter and ethanol yield compared to parental Saccharomyces cerevisiae ATCC 4126 and Kluyveromyces lactis CBS 683. Six recombinants out of the above colonies revealed growth on lactose and sucrose, indicating that they are S. cerevisiae with transformed β-galactosidase systems. The fusant colonies were investigated with respect to their capacity to convert lactose in salted cheese whey into ethanol. Among these recombinants that showed high tolerance towards sodium chloride and higher ethanol yield than lactose fermenting parental K. lactis CBS 683, SK-1 exhibited high tolerance up to 4 g dl^–1 sodium chlonride with an ethanol yield of 4.66 ml dl^–1 (v/v) , SK-23 tolerated 6 g dl^–1 sodium chloride with an ethanol yield of 4.14 ml dl^–1 (v/v) and SK-26 showed resistance towards 8 g dl^–1 sodium chloride and give an ethanol yield of 3 ml dl^–1 (v/v).     

Effect of pre-fermentation on the production of cutinase by a recombinant Saccharomyces cerevisiae

The importance of controlling the expression of heterologous cutinase in a recombinant Saccharomyces cerevisiae SU50 strain was investigated. Maximum specific growth rate and the biomass yield increased 1.91 and 1.16 fold, respectively, when cutinase production was induced by galactose in a pre-fermentation step. However, only 19% of specific cell activity was obtained in comparison to other fermentations following a pre-fermentation step without induction of cutinase expression. Thus, the pre-fermentation step was performed using a selective medium not containing galactose, and the fermentation was performed with a cheaper and complex non-selective medium containing galactose. Under these conditions, and with the aim of maximising the specific cutinase activity, a pre-fermentation with low volume and high density of viable cells must be used. However, due to the low pre-fermentation volume, low yeast cell concentrations and low specific cell activities were obtained after 96 h of fermentation. Otherwise, when the aim was to maximise cutinase yield and productivity, a pre-fermentation volume of 10% (v/v) in relation to fermentation and in the exponential growth phase with a cell concentration between 1.1 and 1.8 g dcw/l should be used. A higher pre-fermentation volume, such as 20% (v/v), would still be economical in the case of a pre-fermentation with low cell density or low cell viability.     

Ethanol from lactose in salted cheese whey by recombinant Saccharomyces cerevisiae

 Seven yeast recombinants were selected from 57 fusant colonies on the basis of higher DNA content, nuclear diameter and ethanol yield compared to parental Saccharomyces cerevisiae ATCC 4126 and Kluyveromyces lactis CBS 683. Six recombinants out of the above colonies revealed growth on lactose and sucrose, indicating that they are S. cerevisiae with transformed β-galactosidase systems. The fusant colonies were investigated with respect to their capacity to convert lactose in salted cheese whey into ethanol. Among these recombinants that showed high tolerance towards sodium chloride and higher ethanol yield than lactose fermenting parental K. lactis CBS 683, SK-1 exhibited high tolerance up to 4 g dl^–1 sodium chlonride with an ethanol yield of 4.66 ml dl^–1 (v/v) , SK-23 tolerated 6 g dl^–1 sodium chloride with an ethanol yield of 4.14 ml dl^–1 (v/v) and SK-26 showed resistance towards 8 g dl^–1 sodium chloride and give an ethanol yield of 3 ml dl^–1 (v/v).     

Efficient production of L-lactic acid from xylose by a recombinant Candida utilis strain

Efficient L-lactic acid production from xylose was achieved using a pyruvate decarboxylase-deficient Candida utilis strain expressing an L-lactate dehydrogenase, an NADH-preferring mutated xylose reductase (XR), a xylitol dehydrogenase and a xylulokinase. The recombinant strain showed 53% increased L-lactic acid production compared with the reference strain expressing native XR (NADPH-preferring).     

重组酿酒酵母发酵生产瓦伦西亚烯及其衍生物

以酿酒酵母为宿主菌株,重构其体内代谢途径,生物合成瓦伦西亚烯及其衍生物。该研究在酿酒酵母菌株中引入来源于纯天仙子的细胞色素P450单加氧酶(cytochrome P450 monooxygenase,HPO)、来源于黄扁柏的瓦伦西亚烯氧化酶(valencene oxidase,Cn VO),并构建了它们对应的4个突变体HPOM、Cn VO-3、Cn VO-4、Cn VO-34,与来源于拟南芥的细胞色素还原酶(cytochrome P450 reductase,At CPR)组合表达,通过静息细胞试验,筛选得到表达HPO和HPOM的菌株催化瓦伦西亚烯的效果最优,选取HPOM作为后续试验的基础。进一步引入来自黄扁柏的瓦伦西亚烯合成酶(valencene synthase,Cn VS),过表达醇脱氢酶(alcohol dehydrogenase,ADH1),截短的3-羟基-3-甲基戊二酰辅酶A还原酶(truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase,t HMG1)。最终获得1株原位生产瓦伦西亚烯及其衍生物的重组酵母菌株PK2-24,进行3 L发酵罐的发酵试验,158 h后总萜产量达310. 94 mg/L,较原始菌株提高了111倍。该研究为利用酵母规模化生产瓦伦西亚烯及其衍生物奠定了重要基础。     

酿酒酵母醛酮还原酶的克隆表达和酶学性质研究

从酿酒酵母(Saccharom yces cerevisiae)CICC 1002基因组中克隆获得醛酮还原酶基因,并在大肠杆菌BL21 (DE3)中实现过量表达.重组醛酮还原酶经Ni-NTA亲和层析分离纯化获得高纯度目的蛋白,并对其进行酶学性质表征.该重组酶经SDS-PAGE检测为单一条带,分子量为38 kDa.该酶的最适pH和最适温度分别为6.0,60℃,且具有良好的稳定性.1 mmol/L金属离子C02+或Ni2+显著提高酶活力.底物特异性分析表明:该重组酶对邻位二酮具有较高活力,其中对酮基泛解酸内酯的比酶活可达20.53 U/mg.     

Stable disruption of ethanol production by deletion of the genes encoding alcohol dehydrogenase isozymes in Saccharomyces cerevisiae

We analyzed the effects of the deletions of genes encoding alcohol dehydrogenase (ADH) isozymes of Saccharomyces cerevisiae. The decrease in ethanol production by ADH1 deletion alone could be partially compensated by the upregulation of other isozyme genes, while the deletion of all known ADH isozyme genes stably disrupted ethanol production.     

响应面法优化酿酒酵母乙醛脱氢酶和乙醇脱氢酶酶活检测方法

从酿酒酵母前处理方式方面对ALD和ADH酶活测定条件进行了优化,采用单因素试验对前处理方式进行了分析研究,结果表明破壁时间、超声波功率、超声时间、反应温度和缓冲液pH值对酶活测定值产生一定的影响.通过Box-Behnken中心组合设计和响应面分析法,确定了测定这2种酶的最理想酵母前处理条件,即超声功率420W,超声时间为11min,破碎时间为12min.     

Optimization of honey-must preparation and alcoholic fermentation by Saccharomyces cerevisiae for mead production

Mead fermentation is a time-consuming process, often taking several months to complete. Despite of the use of starter cultures several problems still persist such as lack of uniformity of the final products, slow or premature fermentation arrest and the production of off-flavors by yeast. Thus the aim of this study was to optimize mead production through the use of an appropriate honey-must formulation to improve yeast performance alcoholic fermentation and thereby obtain a high quality product. Honey-must was centrifuged to reduce insoluble solids, pasteurized at 65 °C for 10 min, and then subjected to different conditions: nitrogen supplementation and addition of organic acids. Although the addition of diammonium phosphate (DAP) reduced fermentation length, it did not guarantee the completeness of the fermentation process, suggesting that other factors could account for the reduced yeast activity in honey-must fermentations. Sixteen yeast-derived aroma compounds which contribute to the sensorial quality of mead were identified and quantified. Global analysis of aromatic profiles revealed that the total concentration of aroma compounds in meads was higher in those fermentations where DAP was added. A positive correlation between nitrogen availability and the levels of ethyl and acetate esters, associated to the fruity character of fermented beverages, was observed whereas the presence of potassium tartrate and malic acid decreased, in general, their concentration.This study provides very useful information that can be used for improving mead quality.     

Development of a fed-batch cultivation strategy for the enhanced production and secretion of cutinase by a recombinant Saccharomyces cerevisiae SU50 strain

Saccharomyces cerevisiae SU50 strain was cultivated with different concentrations of glucose and galactose with the aim of increasing cutinase activity, cutinase yield on the carbon source, and bioreactor productivity. Cultivations in shake flasks with galactose as the sole carbon source, with sugar concentrations between 10 and 40 g/l, exhibited growth-associated cutinase production and a constant specificity of cutinase secretion. Furthermore, as the galactose concentration increased to values higher than 15 g/l, a progressively higher maximum specific galactose consumption rate and a consequent higher alcoholic fermentation occurred, resulting in progressively lower biomass yields on the carbon source and cutinase yields on biomass. Using high glucose and galactose concentrations in a well-aerated bioreactor resulted in a high biomass productivity (0.5 g dcw/l/h), a high cutinase yield on biomass (21.5 U/mg dew), a final high cutinase secretion efficiency (97%), and plasmid stability (99%). Based on these studies, a two phase fed-batch cultivation strategy was developed. A batch phase with high glucose and galactose concentrations, followed by a fed-batch with a constant feed rate with galactose as the sole carbon source in order to minimize the repression of the GAL 7 promoter, were established. The feed rate was estimated to maintain a pre-determined concentration of galactose (20 g/l) on the culture medium in order to maximize the efficiency of cutinase secretion and minimize the galactose alcoholic fermentation. By this cultivation strategy, enhancements of 3.6-fold in cutinase activity, 1.2-fold in cutinase yield on the carbon source, and 8.7-fold culture productivity were obtained in relation to a batch cultivation performed in shake flasks with 20 g/l of galactose.     

Reduced pyruvate decarboxylase and increased glycerol-3-phosphate dehydrogenase [NAD+] levels enhance glycerol production in Saccharomyces cerevisiae

This investigation deals with factors affecting the production of glycerol in Saccharomyces cerevisiae. In particular, the impact of reduced pyruvate-decarboxylase (PDC) and increased NAD-dependent glycerol-3-phosphate dehydrogenase (GPD) levels was studied. The glycerol yield was 4·7 times (a pdc mutant exhibiting 19% of normal PDC activity) and 6·5 times (a strain exhibiting 20-fold increased GPD activity resulting from overexpression of GPD1 gene) that of the wild type. In the strain carrying both enzyme activity alterations, the glycerol yield was 8·1 times higher than that of the wild type. In all cases, the substantial increase in glycerol yield was associated with a reduction in ethanol yield and a higher by-product formation. The rate of glycerol formation in the pdc mutant was, due to a slower rate of glucose catabolism, only twice that of the wild type, and was increased by GPD1 overexpression to three times that of the wild-type level. Overexpression of GPD1 in the wild-type background, however, led to a six- to seven-fold increase in the rate of glycerol formation. The experimental work clearly demonstrates the rate-limiting role of GPD in glycerol formation in S. cerevisiae.     

Glucose production from cellulose through biological simultaneous enzyme production and saccharification using recombinant bacteria expressing the β-glucosidase gene

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Favorable effect of very low initial KLa value on xylitol production from xylose by a self-isolated strain of Pichia guilliermondii

Xylitol production from xylose by a self-isolated furfural and 5-hydroxymethyl furfural assimilating Pichia guilliermondii was studied under oxygen limitation. An extremely low initial volumetric oxygen transfer coefficient (0.075 h^− 1) was found most favorable to the xylitol production with yield of 0.61 g g^− 1. Related enzymes activities were also investigated and discussed.     

Disruption of seven hypothetical aryl alcohol dehydrogenase genes from Saccharomyces cerevisiae and construction of a multiple knock-out strain

By in silicio analysis, we have discovered that there are seven open reading frames (ORFs) in Saccharomyces cerevisiae whose protein products show a high degree of amino acid sequence similarity to the aryl alcohol dehydrogenase (AAD) of the lignin-degrading fungus Phanerochaete chrysosporium. Yeast cultures grown to stationary phase display a significant aryl alcohol dehydrogenase activity by degrading aromatic aldehydes to the corresponding alcohols. To study the biochemical and the biological role of each of the AAD genes, a series of mutant strains carrying deletion of one or more of the AAD-coding sequences was constructed by PCR-mediated gene replacement, using the readily selectable marker kanMX. The correct targeting of the PCR-generated disruption cassette into the genomic locus was verified by analytical PCR and by pulse-field gel electrophoresis (PFGE) followed by Southern blot analysis. Double, triple and quadruple mutant strains were obtained by classical genetic methods, while the construction of the quintuple, sextuple and septuple mutants was achieved by using the marker URA3 from Kluyveromyces lactis, HIS3 from Schizosaccharomyces pombe and TRP1 from S. cerevisiae. None of the knock-out strains revealed any mutant phenotype when tested for the degradation of aromatic aldehydes using both spectrophotometry and high performance liquid chromatography (HPLC). Specific tests for changes in the ergosterol and phospholipids profiles did not reveal any mutant phenotype and mating and sporulation efficiencies were not affected in the septuple deletant. Compared to the wild-type strain, the septuple deletant showed an increased resistance to the anisaldehyde, but there is a possibility that the nutritional markers used for gene replacement are causing this effect.     

The influence of CDTA and DPTA on ethanol production from sugar cane molasses using Saccharomyces cerevisiae

Effects of complexing agents on the sensitivity of ethanol production using Saccharomyces cerevisiae were investigated using trans-1,2-diamino-cyclohexane-N,N,N',N'-tetraacetic acid (CDTA) and diethylene triamine pentaacetic acid (DPTA). Addition of 600 ppm CDTA during inoculation produces a maximum stimulation; ethanol production at this concentration was 1.5% (v/v) more than the control cultures (approximately 6%). 1000 ppm CDTA produces maximum effect during propagation which was 2.3% more than the control cultures. When DPTA was introduced during inoculation, 800 ppm DPTA produces a maximum effect, which was 2.9% more than the control cultures. 200 ppm DPTA produces a maximum stimulatory effect of 1.0% more than the control cultures. No significant effect was observed when DPTA was added during fermentation but 700 ppm CDTA increased ethanol production by 1.3% more than the control cultures.     

Extraction and rapid inactivation of proteins from Saccharomyces cerevisiae by trichloroacetic acid precipitation

Methods currently used for the extraction of proteins from yeast involve relatively long time periods between sampling cells from a culture and analysis of their proteins by polyacrylamide gel electrophoresis-sodium dodecylsulphate. Often it is desirable to inactivate cellular metabolism rapidly after sampling and here we show that trichloroacetic acid precipitation techniques, often used for rapid extraction and inactivation of proteins from higher eukaryotes, can be adapted for use with organisms which have cell walls.