不同非氧化磷酸戊糖途径基因的过表达对酿酒酵母木糖发酵性能的影响

以双拷贝过表达木糖代谢上游途径关键酶(木糖还原酶XR、木糖醇脱氢酶XDH和木酮糖激酶XKS)的酿酒酵母菌株为背景,在过表达非氧化磷酸戊糖(PP)途径中转醛酶基因TAL1的基础上,对途径中其他基因TKL1(转酮酶)、RPE1(核酮糖-5-磷酸差向异构酶)和RKI1(核酮糖-5-磷酸异构酶)进行了不同程度的过表达,以研究PP途径基因过表达对酿酒酵母木糖代谢的影响。在不同培养基条件下对重组菌株木糖代谢进行研究,结果显示,在过表达TAL1的基础上不同组合过表达PP途径其他基因不同程度改善了酿酒酵母木糖发酵性能,重组菌株能在36~48 h耗完质量分数(下同)为5%的木糖。其中,过表达PP途径全部基因比其他过表达基因组合表现出明显的优势,在8%木糖发酵条件下其乙醇产量达到了每1 g木糖0.337 g,较对照菌株提高了7.86%。这说明同步过表达PP途径基因更有利于酿酒酵母木糖发酵。     

酿酒酵母采用木糖发酵产乙醇的研究现状

酿酒酵母具有安全性好,高产量和高的抑制剂耐受性等优点,故一直在生物乙醇工业中有重要作用。然而该酵母不能使木糖发酵,而木糖是木质纤维素水解产物中重要的戊糖。为了得到利用木糖高效产乙醇的工程茵,我们通过引入初始木糖的新陈代谢和木糖的运输体来改变细胞内的氧化还原平衡．木酮糖激酶的过表达和磷酸戊糖途径来提高乙醇产率。     

缺失PHO13基因与进化工程构建高效利用木糖酵母

过表达了木糖还原酶、木糖醇脱氢酶和木酮糖激酶基因的重组工业酿酒酵母菌株KAM-6X内缺失了编码对硝基苯磷酸盐磷酸化酶的PHO13基因,接着通过EMS诱变和进化工程筛选,获得了一株高效利用木糖的菌株,命名为PE。有氧条件下,在含50 g/L木糖和100 g/L木糖的YPX中最大比生长速率分别为0.299和0.282 h^-1,分别比出发菌提高了95.43%和102.87%,同时PE菌株能在前24 h内耗掉36.12 g/L木糖,48 h内耗掉70.25 g/L木糖。微好氧条件下副产物产量降低,糖醇转化率最高达到0.382 g/g,证明PE是一株高效利用木糖发酵的工业酵母菌株。     

木糖还原酶基因的克隆及其在酿酒酵母中的定向整合

酿酒酵母(Saccharomyces cerevisiae)是重要的乙醇生产菌株,但因缺少戊糖代谢途径而不能利用木糖,为了改良工业酿酒酵母利用半纤维素发酵生产乙醇的性能,利用分子生物学技术构建能够利用木糖的基因工程酵母。选取酿酒酵母染色体的rDNA重复序列作为外源基因整合位点,依此构建多拷贝染色体整合型载体pUG-LR。采用融合表达策略扩增得到含有酿酒酵母乙醇脱氢酶启动子PADH和树干毕赤酵母木糖还原酶基因xyl1的融合序列,并将其插入pUG-LR载体中,构建成含遗传霉素G418抗性标记的同源重组质粒pUG-LR-XYL1。以工业酿酒酵母ZU-01为宿主,通过优化后的电穿孔法将重组质粒导入经缓冲液处理的酵母细胞,30℃培养。通过提高YEPX复筛培养基G418浓度,得到10株生长较快的优良性状转化子。在不含G418的YEPX培养基上传代8次以上,以转化子基因组DNA为模板,进行PCR检测,均可获得目的基因片段。研究结果表明:木糖还原酶基因xyl1已定向整合于ZU-01染色体DNA上并稳定遗传,为后续构建工业酿酒酵母的木糖代谢通路、利用木糖产酒精的重组菌株奠定了基础。     

低甘油产率的酿酒酵母工程菌构建研究进展

酿酒酵母具有安全、遗传背景清楚、生长速度快、高糖耐受性、高乙醇产量以及高胁迫耐受性等特性,是乙醇生产较为理想的细胞工厂.甘油是酿酒酵母发酵产乙醇过程中的一种主要伴生副产物,过量表达甘油影响糖醇转化率.通过基因工程改造甘油代谢和乙醇生产途径,是降低甘油产量和提高乙醇转化率的有效策略.首先分析改造酿酒酵母细胞中甘油和乙醇代...     

整合型酿酒酵母菌株强化发酵和特性比较

为了提高木糖异构酶基因在重组酿酒酵母体内的稳定性,并比较不同真菌来源的木糖异构酶在木糖或者葡萄糖-木糖培养基的发酵利用特性,分别构建来自Piromyces sp.E2和Orpinomyces sp.的木糖异构酶基因的整合表达载体,利用同源重组将其整合进入呼吸缺陷型菌株的18S rDNA非转录区,结果测得Orpinomyces的木糖异构酶酶活活力为0.72 U/mg,比Piromyces的木糖异构酶酶活高2.8倍。在木糖培养基中发酵获得乙醇的得率分别为0.40 g/g和0.48 g/g。且整合入Orpinomyces的木糖异构酶基因菌株能获得最高酶活和乙醇得率。     

低聚木糖对胃粘膜保护作用的研究

本课题通过实验研究低聚木糖对乙醇造成的胃损伤是否有保护作用。将实验用大鼠设置高、中、低剂量组、水对照组和淀粉对照组,对实验大鼠进行灌胃处理,之后进行无水乙醇灌胃处理,以每鼠的溃疡总面积作为指标,观察各组的胃粘膜损伤情况。实验中低聚木糖的各个剂量组,相对于水对照组和淀粉对照组,都能明显减少胃粘膜的损伤面积。实验结果表明低聚木糖对乙醇造成的胃粘膜损伤有保护作用。     

天然酵母对木糖利用的研究进展

开发能有效利用木糖的微生物是纤维素乙醇生产的关键问题之一,能发酵木糖的天然酵母得到了广泛的研究.对微生物发酵木糖的机理、天然酵母发酵木糖的特性及该领域的有关进展进行了介绍.     

木糖发酵酒精代谢工程的研究进展

木糖发酵是生物转化木质纤维素产生酒精及其他化工产品最为重要的一环,但自然界中缺少能将上述生物质有效转化为乙醇的微生物菌种. 近年来,根据代谢工程原理,利用基因工程技术对酵母和细菌进行遗传改造,或将木糖代谢途径引入传统的酒精发酵菌酿酒酵母及高酒精产生菌运动发酵单胞菌中,从而拓展其底物利用范围;或使原本可以利用多种糖底物的细菌获得选择性产生酒精的能力,构建了各种不同类型的木糖发酵重组菌株. 虽然这些重组菌株在木糖转化酒精方面均显示出良好的应用前景,但仍存在诸多问题. 有必要在对木糖代谢调控机制深入系统研究的基础上,进一步改造现有菌株,并结合生化工程技术对重组菌株发酵条件进行优化,以实现高效生物转化木质纤维素原料制取乙醇. 本工作介绍了近年来代谢工程改造微生物菌种发酵木糖生产酒精的研究进展.     

木糖发酵微生物的研究进展

木糖广泛存在于林业及农业废弃物中,木糖发酵生产乙醇是再生资源的有效利用。简述了木糖发酵微生物和木糖代谢工程菌的种类及研究进展。     

Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review.     

大肠杆菌中核黄素的生物合成途径改造及生产

利用PCR技术从枯草芽孢杆菌(Bacillus subtilis 168)中扩增出3.5 kb的核黄素操纵子,将其分别连接到不同拷贝数的表达载体pSC101、p15A、pBR322,得到重组载体pSC101-BSrib、p15A-BSrib和pBR322-BSrib,并分别转化到大肠杆菌(Escherichia coli K-12 MG1655)。对含有核黄素操纵子的重组大肠杆菌进行摇瓶发酵,结果表明其核黄素合成能力随着质粒拷贝数的增加而增强。随后对E. coli K-12 MG1655 ECX3菌株的诱导剂IPTG浓度和发酵温度进行了优化。结果显示,0.1 mmol·L^-1 IPTG和42 ℃为核黄素生产的最适宜条件。在此条件下,菌株ECX3在LB培养基中核黄素产量达到251.4 mg·L^-1。最后,通过无痕基因操作技术,减弱了工程菌株ECX3核黄素激酶/黄素腺嘌呤二核苷酸氨酰转移酶(ribF)的表达以减少核黄素转化为FMN和FAD,摇瓶中工程菌株ECX4的核黄素产量提高到292.3 mg·L^-1。     

衣康酸共聚物合成工艺的优化及其阻垢性能研究

以衣康酸(IA)、烯丙氧基羟丙基磺酸钠(AHPS)、烯丙基聚氧乙烯醚(APEG)为单体,通过自由基聚合的方式,合成出IA-AHPS-APEG三元共聚物。利用中心复合设计法建立了数学模型,结合静态阻碳酸钙垢测试,对聚合反应的引发剂用量、反应温度和反应时间进行了优化。通过FT-IR技术分析了聚合物IA-AHPS-APEG的分子结构,利用TG、SEM和XRD等技术分析了碳酸钙垢和硫酸钙垢的形貌和晶体结构变化,探究了聚合物IA-AHPS-APEG的阻垢机理。结果表明,优化后的IA-AHPS-APEG可以抑制碳酸钙和硫酸钙晶体的正常生长,并使碳酸钙晶体发生晶格畸变,其对碳酸钙的阻垢率最高可达93.2%,对硫酸钙的阻垢率接近100%。     

Production of fructose and ethanol from media with high sucrose concentrations by a mutant of Saccharomyces cerevisiae

The production of enriched fructose syrups and ethanol from a synthetic medium with high sucrose concentrations was studied in a batch process using Saccharomyces cerevisiae ATCC 36858. The results showed that the fructose yield was above 92% of theoretical values in synthetic media with sucrose concentrations between 180 g dm^?3 and 726 g dm^?3. Ethanol yield was about 82% in media with sucrose concentrations up to 451 g dm^?3. A product containing 178 g dm^?3 fructose, which represents 97% of the total sugar content, and 79 g dm^?3 ethanol was obtained using a medium with 360 g dm^?3 sucrose. The fructose fraction in the carbohydrates content in the produced syrups decreased with increases in the initial sucrose concentration. In a medium with initial sucrose concentration of 574 g dm^?3, the fructose content in the produced broth was 59% of the total carbohydrates. Glycerol and fructo‐oligosaccharides were also produced in this process. The produced fructo‐oligosaccharides started to be consumed when the concentration of sucrose in the media was less than 30% of its initial value. Complete hydrolysis of these sugars was noticed in media with sucrose concentrations below 451 g dm^?3. These findings will be useful in the production of ethanol and high fructose syrups using sucrose‐based raw materials with high concentrations of this carbohydrate. © 2001 Society of Chemical Industry     

乙炔氢氯化过程中铜基催化剂的制备及催化性能

采用球形活性炭为载体,制备了用于乙炔氢氯化反应的载Cu催化剂, 并采用TEM进行了表征。在常压固定床反应器中考察了溶剂、酸洗液、Cu的负载量、焙烧温度对Cu/C催化剂性能的影响。结果表明, 以1 mol/L盐酸为溶剂,1 mol/L H₃PO₄为活性炭酸洗液,Cu的负载量为15%,焙烧温度为500℃时,该催化剂具有较高的分散度和反应活性。空速180 h^-1、V(HCl)/V(C₂H₂)=1.1、Cu的负载量为15%、温度180℃时,乙炔氢氯化反应的转化率可达68%以上, 氯乙烯选择性不低于99.5%,同时具有较好的稳定性; 在空速为540 h^-1时, 其催化活性会因活性组分的团聚结晶而降低。     

铜硅催化剂中层状硅酸铜的形成过程

采用一种在催化剂制备过程中取样的方法,研究了Cu/SiO₂催化剂中层状硅酸铜的形成过程。通过对样品进行N₂-吸附、FTIR、XRD和TEM表征,发现层状硅酸铜主要形成于催化剂制备的蒸氨阶段,而在老化阶段则主要发生了铜氨离子与硅溶胶表面羟基之间的离子交换反应。     

Effects of light wavelength and intensity on the production of ethanol by Saccharomyces cerevisiae in batch cultures

BACKGROUND: Photoreceptors have been identified in Saccharomyces cerevisae, however, the influence of light on the performance of ethanol fermentation of S. cerevisiae is not yet clear. The aims of this study are to elucidate the influence of light wavelength and intensity on the growth and ethanol production of S. cerevisiae and to describe a novel two‐stage LED light process to optimize ethanol fermentation. RESULTS: Experimental results indicated that maximum biomass concentration X_max of the batch under red LED light increased monotonically with light intensity, and the optimal specific product yield Y_p/x was 13.2 g g^?1 at 600 lux. Maximum ethanol concentration P_max of the batch under blue LED light increased monotonically with light intensity, and the optimal Y_p/x was 18.4 g g^?1 at 900 lux. A novel two‐stage LED light process achieved maximum P_max, of 98.7 g dm^?3 resulting in 36% improvement compared with that of the batch in the dark. CONCLUSION: The light wavelength and its intensity significantly affected cell growth and ethanol formation of S. cerevisiae. Red LED light (630 nm) stimulated cell growth but slightly inhibited ethanol formation. In contrast, blue LED light (470 nm) significantly inhibited cell growth but stimulated ethanol formation. A novel two‐stage LED light process has been successfully demonstrated to optimize ethanol fermentation of S. cerevisiae. Copyright © 2009 Society of Chemical Industry