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.     

转木糖还原酶基因XYL1酿酒酵母的构建及产木糖醇能力研究

将人工合成的树干毕赤酵母（Pichia stipitis）的木糖还原酶基因XYL1插入酿酒酵母（Saccharomyces cerevisiae）表达载体pYES2中,然后将重组质粒pYES2-XYL1导入酿酒酵母INVSc1中,构建转木糖还原酶基因XYL1酿酒酵母菌株INVSc1/pYES2-XYL1,最后采用营养缺陷培养基筛选转木糖还原酶基因酿酒酵母并对其产木糖醇的能力进行检测。结果表明,成功获得2株转木糖还原酶基因XYL1酿酒酵母菌株INVSc1/pYES2-XYL1-01、INVSc1/pYES2-XYL1-02,当两菌株以50 g/L木糖及10 g/L半乳糖为碳源发酵5 d后,木糖醇产量分别高达（13.68±2.37）g/L、（12.09±1.45）g/L,显著高于非转基因酿酒酵母INVSc1的木糖醇产量（1.08±0.37）g/L（P＜0.05）,说明XYL1基因的导入显著提高了酿酒酵母INVSc1生产木糖醇的能力（P＜0.05）。为采用基因工程酿酒酵母制备食用木糖醇提供了理论及技术基础。     

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.     

Endogenous NADPH-dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae

Introduction of the xylose pathway from Pichia stipitis into Saccharomyces cerevisiae enables xylose utilization in recombinant S. cerevisiae. However, xylitol is a major by-product. An endogenous aldo-keto reductase, encoded by the GRE3 gene, was expressed at different levels in recombinant S. cerevisiae strains to investigate its effect on xylose utilization. In a recombinant S. cerevisiae strain producing only xylitol dehydrogenase (XDH) from P. stipitis and an extra copy of the endogenous xylulokinase (XK), ethanol formation from xylose was mediated by Gre3p, capable of reducing xylose to xylitol. When the GRE3 gene was overexpressed in this strain, the xylose consumption and ethanol formation increased by 29% and 116%, respectively. When the GRE3 gene was deleted in the recombinant xylose-fermenting S. cerevisiae strain TMB3001 (which possesses xylose reductase and XDH from P. stipitis, and an extra copy of endogenous XK), the xylitol yield decreased by 49% and the ethanol yield increased by 19% in anaerobic continuous culture with a glucose/xylose mixture. Biomass was reduced by 31% in strains where GRE3 was deleted, suggesting that fine-tuning of GRE3 expression is the preferred choice rather than deletion.     

Functional analysis of structural genes for NAD(+)-dependent formate dehydrogenase in Saccharomyces cerevisiae

Co-consumption of formate by aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK 113-7D led to an increased biomass yield relative to cultures grown on glucose as the sole carbon and energy substrate. In this respect, this strain differed from two previously investigated S. cerevisiae strains, in which formate oxidation did not lead to an increased biomass yield on glucose. Enzyme assays confirmed the presence of a formate-inducible, cytosolic and NAD(+)-dependent formate dehydrogenase. To investigate whether this enzyme activity was entirely encoded by the previously reported FDH1 gene, an fdh1Delta null mutant was constructed. This mutant strain still contained formate dehydrogenase activity and remained capable of co-consumption of formate. The formate dehydrogenase activity in the mutant was demonstrated to be encoded by a second structural gene for formate dehydrogenase (FDH2) in S. cerevisiae CEN.PK 113-7D. FDH2 was highly homologous to FDH1 and consisted of a fusion of two open reading frames (ORFs) (YPL275w and YPL276w) reported in the S. cerevisiae genome databases. Sequence analysis confirmed that, in the database genetic background, the presence of two single-nucleotide differences led to two truncated ORFs rather than the full-length FDH2 gene present in strain CEN.PK 113-7D. In the latter strain background an fdh1Deltafdh2Delta double mutant lacked formate dehydrogenase activity and was unable to co-consume formate. Absence of formate dehydrogenase activity did not affect growth on glucose as sole carbon source, but led to a reduced biomass yield on glucose-formate mixtures. These findings are consistent with a role of formate dehydrogenase in the detoxification of exogenous formate.     

Bioethanol production from sugar beet molasses and thick juice by free and immobilised Saccharomyces cerevisiae

The aim of this study was the investigation and comparison of the potential of sugar beet molasses and thick juice as raw materials for bioethanol production, as renewable and sustainable energy sources. Ethanol fermentation of a wide range of initial sugar concentrations (100–300 g/L) was performed using either free or immobilised Saccharomyces cerevisiae in calcium alginate beads in the absence of any added nutrients. In general, immobilised cells showed better fermentative performance, enhanced ethanol productivity, stability and cell viability compared with free cells, under the same fermentation conditions. The high concentration of non‐sugar components contained in molasses affected yeast fermentation performance and viability. Maximum ethanol concentration in fermented media of 84.6 and 109.5 g/L were obtained by immobilised cells for initial sugar concentrations of 200 and 250 g/L for molasses and thick juice, respectively. However, the highest ethanol yields of 31.7 L per 100 kg of molasses and 37.6 L per 100 kg of thick juice were obtained by immobilised cells at an initial sugar concentration of 175 g/L. In the high gravity fermentation process, thick juice resulted in a higher ethanol yield per mass of raw material compared with molasses. This study shows the advantage of immobilised yeast for the efficient production of high gravity bioethanol from thick juice, which was a more favourable raw material than molasses. © 2018 The Institute of Brewing & Distilling     

The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains

Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wild-type level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD(+) into NADP(+) and NADH, and TH-overproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization. Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulose-derived inhibitors. Low G6PDH-activity strains were also more sensitive to H(2)O(2) than the control strain TMB3001.     

Characterization of the recombinant succinic semi‐aldehyde dehydrogenase from Saccharomyces cerevisiae

The yeast succinic semi‐aldehyde dehydrogenase gene (SSADH; EC 1.2.1.16) was cloned and overexpressed in Escherichia coli. Based on SDS–PAGE, the molecular mass of the subunit was around 54 kDa, and the purified recombinant enzyme had a tetrameric molecular mass of ca. 200 kDa. The specific activity of the recombinant enzyme was 1.90 µm NADH formed/min/mg, and showed maximal activity at pH 8.4. The recombinant protein was highly specific for succinate semi‐aldehyde (K_m = 15.48 ± 0.14 µm ) and could use both NAD⁺ and NADP⁺ as co‐factors, with K_m values of 579.06 ± 30.1 µm and 1.017 ± 0.46 mm, respectively. Initial velocity studies showed that NADH was a competitive inhibitor with respect to NAD⁺ (K_i = 129.5 µm ) but a non‐competitive inhibitor with respect to succinate semi‐aldehyde. Adenine nucleotides of AMP, ADP and ATP inhibited yeast SSADH activity with K_i = 1.13–10.2 mm, and showed competitive inhibition with respect to NAD⁺ and mixed‐competitive, non‐competitive and non‐competitive inhibition, respectively, with respect to succinate semi‐aldehyde. The kinetic data suggest a 'ping‐pong' mechanism. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering

A Saccharomyces cerevisiae screening strain was designed by combining multiple genetic modifications known to improve xylose utilization with the primary objective of enhancing xylose growth and fermentation in xylose isomerase (XI)-expressing strains. Strain TMB 3045 was obtained by expressing the XI gene from Thermus thermophilus in a strain in which the GRE3 gene coding for aldose reductase was deleted, and the genes encoding xylulokinase (XK) and the enzymes of the non-oxidative pentose phosphate pathway (PPP) [transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI) and ribulose 5-phosphate epimerase (RPE)] were overexpressed. A xylose-growing and fermenting strain (TMB 3050) was derived from TMB 3045 by repeated cultivation on xylose medium. Despite its low XI activity, TMB 3050 was capable of aerobic xylose growth and anaerobic ethanol production at 30 degrees C. The aerobic xylose growth rate reached 0.17 l/h when XI was replaced with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes expressed from a multicopy plasmid, demonstrating that the screening system was functional. Xylose growth had not previously been detected in strains in which the PPP genes were not overexpressed or when overexpressing the PPP genes but having XR and XDH genes chromosomally integrated. This demonstrates the necessity to simultaneously increase the conversion of xylose to xylulose and the metabolic steps downstream of xylulose for efficient xylose utilization in S. cerevisiae.     

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).     

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).     

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。     

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).     

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.     

Improved cellulase production in recombinant Saccharomyces cerevisiae by disrupting the cell wall protein-encoding gene CWP2

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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.