产甘油假丝酵母两种转化方法的比较

为了研究产甘油假丝酵母高产甘油和耐高渗的机制,以Zeocin作为选择标记,考察和比较了两种转化方法,即醋酸锂法和电转化法的转化效果,以建立简单有效的产甘油假丝酵母转化体系。结果表明,细胞生长时期是影响转化率的关键因素。在OD600约为1.3时制备感受态细胞,在电压为1.5 kV时进行电击,可获得较高转化率,为每微克DNA 139个转化子。在OD600约为1.0时制备感受态细胞,醋酸锂预处理细胞1 h,获得的转化率为每微克DNA 154个转化子。综合考虑,醋酸锂法更适合于产甘油假丝酵母的转化。     

甲醇毕赤酵母Pichia methanolica的高效电转化方法

采用电穿孔的方法对甲醇毕赤酵母进行外源DNA的转化。通过调整参数,对影响电转化效率的主要因素进行探索,建立了质粒pMETA-Lip对甲醇毕赤酵母PMAD16的高效电转化方法。结果表明,当采用对数生长中期的菌体制备感受态细胞、质粒DNA浓度为30μg/mL、采用0.2cm电转杯、电压为600 V时,转化率达到最大值,为57个转化子/μg质粒DNA。经抽样鉴定所得到的转化子均为阳性克隆。为外源基因在甲醇毕赤酵母中的表达奠定了基础。     

根癌农杆菌介导酿酒酵母的遗传转化

根癌农杆菌（Agrobacterium tumefaciens）可以将它的Ti质粒的一段T-DNA序列转化到广泛的宿主细胞并能整合到宿主染色体中.作者利用根癌农杆菌转化系统成功地实现了产甘油假丝酵母（Candida glycerinogenes）乳清苷酸脱羧酶基因（Ura3）对酿酒酵母（Saccharamyces cerevisiae）W303-1A的Ura3缺陷遗传互补转化,转化率约为3.2个转化子/10^5个酵母细胞.通过转化子PCR和表型验证说明了T-DNA已经整合进入酵母染色体,并能够稳定地进行遗传表达.     

生物转化法生产木糖醇的细胞增殖培养基优化

以木糖为底物利用酵母细胞转化生产木糖醇,研究细胞增殖培养基中不同种类的碳源、氮源和微量元素及其添加量对酵母细胞生长和木糖转化率的影响。结果表明,当碳源为木糖和葡萄糖添加量分别为1%时,木糖醇浓度为49.6g/L;当无机氮源为蛋白胨且浓度为2%时,木糖转化时间为60h,木糖醇浓度达到58g/L;微量元素为磷酸二氢钾浓度为0.1%时,木糖的转化时间为48h,此时木糖醇浓度达到59g/L。     

啤酒酵母转化次黄嘌呤核苷酸生成三磷酸腺苷的研究

啤酒厂废酵母经培养可转化次黄嘌呤核苷酸(IMP)生成三磷酸腺苷(ATP).研究了初始葡萄糖、磷酸盐、Mg2+、IMP底物浓度、pH值等主要参数对转化率的影响以及纯化过程中洗脱液的选择.利用酵母体内的酶成功地对IMP进行了转化,结果表明,在优化条件下ATP转化率稳定在90%以上,含量超过85%,纯度超过99%.     

α-淀粉酶与面包

１α－淀粉酶的种类及其特性比较表１不同种类的最佳失活温度（℃）温度（℃）pH开始结束麦芽６０～６５５．２～５．７６５８０～９０不稳定虫真菌５５５．５～６．０５５８０稳定细菌７０７０＞１００稳定种类活性稳定性１．２不同种类的α－淀粉酶特性的比较２α－淀粉酶对面包制作的主要影响由于α－淀粉酶对酵母活性有激活作用，使酵母产气量提高。如果有溴酸钾存在，这种作用更为加强。酵母中含有葡萄糖及转化酶，他可以将麦芽糖转化为葡萄糖，麦芽糖含量越高，转化率越高，而当葡萄糖浓度过高时，这种转化率可以减低。α－淀粉酶及…     

玉米秸秆制酒精--秸秆预处理及水解方法的探讨

利用丰富、价廉的玉米秸秆为原料生产酒精已成为必然趋势，而最需要解决的关键问题是玉米秸秆的预处理及水解转化技术。研究结果表明，玉米秸秆先采用蒸汽爆破法或温氧法预处理，使木质素和纤维分离，然后进行酶水解，可使纤维素转化率达85％，再利用酵母发酵生成酒精。     

啤酒废酵母综合破壁法提取酵母味素技术

研究了高压均质、酶法溶胞、温差破壁3种细胞壁破碎法对啤酒废酵母抽提物(酵母味素)的得率、蛋白质的转化率及氨基氮溶出率的影响.结果表明,在适宜的自溶条件下,利用综合破壁法可使酵母抽提物中酵母味素得率、蛋白质的转化率及氨基氮溶出率显著提高.     

酵母对无机锌的富集及其影响因素研究

在培养基中添加无机锌，利用生物转化技术，通过酵母的生命活动，将无机锌有效地富集到细胞内，研究锌离子浓度对酵母生长繁殖、细胞含锌量、锌富集率及酵母发酵力的影响，并通过控制锌离子浓度，使酵母细胞既能富集锌，又对其发酵力不产生显著的影响，以便将仍具有发酵活力的富锌酵母用于食品加工之中。研究结果表明，锌离子浓度对酵母的生长繁殖和富集能力影响较大，浓度越高，酵母细胞含锌量越大。但无机锌对酵母细胞有显著的损伤，且这种损伤是非遗传性的，当无机锌去掉后，酵母细胞形态又恢复正常。     

能量策略对酶法合成谷胱甘肽效率影响

通过比较和调控细胞酶催化前体氨基酸合成谷胱甘肽(GSH)反应过程中的能量类型和添加策略,跟踪反应过程中合成GSH的浓度变化,考察能量种类和添加模式对酶法合成GSH转化效率的影响。结果表明,葡萄糖作为能量碳源对酵母细胞酶催化合成GSH转化率的影响明显,初始反应液添加100 mmol/L葡萄糖酶法合成GSH转化率为27.88%,分批定点流加葡萄糖(第2、3、4小时各添加27.78 mmol/L)酶法合成GSH转化率可达到35.81%;在酶反应过程能量供应策略中,初始反应液中添加0.5 g/L的腺苷时,酶法合成GSH的转化率提高到36.37%;初始反应液添加0.5 g/L腺苷结合分批定点流加葡萄糖(第2、3、4小时各添加27.78 mmol/L),酶法合成GSH转化率达到41.57%。     

High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation

A highly efficient method for transformation of the fission yeast Schizosaccharomyces pombe by electroporation has been developed. Significantly higher transformation efficiency was obtained when intact cells grown in SD medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose) were pretreated with thiol compounds before an electric pulse was applied to the cells. Among the thiol compounds tested, dithiothreitol (DTT) was the most effective for pretreatment. A high transformation efficiency was obtained when the cells were pretreated with 25 mM DTT at 30 degrees C for 15 min in an osmotically adjusted buffer, since the cells were sensitive to osmotic pressure. It was important to exclude glucose from the DTT pretreatment buffer, as it caused a drastic decrease in efficiency. The optimal cell concentration and amount of DNA during the electric pulse were 1x10(9) cells/ml and 10 ng, respectively. The maximum transformation efficiency, 1.2x10(7) transformants/microg plasmid DNA, was obtained when an electric pulse of 11.0 kV/cm was applied for 5 ms. Furthermore, the high competency of cells pretreated with DTT was maintained by freezing them in a non-permeating cryoprotectant such as sorbitol.     

A rapid and simple procedure for high-efficiency lithium acetate transformation of cryopreserved Schizosaccharomyces pombe cells

A rapid, simple, convenient, and highly efficient transformation of the fission yeast Schizosaccharomyces pombe has been developed. Freezing fission yeast cells in glycerol, a permeating cryoprotectant, with lithium acetate improved remarkably the transformation efficiency by one to two orders of magnitude. The optimum concentration of glycerol was found to be 30%, which is higher than that (10-15%) in the conventional cryopreservation of yeast cells. Glycerol not only played a role in cryopreserving the competent cells but also improved the transformation efficiency of the process. The thawed cell suspension with glycerol and lithium acetate was immediately mixed with carrier DNA, plasmid DNA and polyethylene glycol. Next, the mixture was heat shocked and directly spread on a selection plate. This simple procedure yielded more than 10(6) transformants/microg plasmid DNA, reducing the time required to only 20 min in total, including the thawing time. Furthermore, the frozen competent cells were stored long-term for more than 3 months without any significant loss of efficiency.     

Cryopreservation of competent intact yeast cells for efficient electroporation

We have developed a simple method for cryopreserving Schizosaccharomyces pombe and Saccharomyces cerevisiae competent intact cells that permits high transformation efficiency and long-term storage for electroporation. Transformation efficiency is significantly decreased if intact cells are frozen in common permeating cryoprotectants such as glycerol or dimethyl sulphoxide. On the other hand, we found that a high transformation efficiency could be maintained if the cells were frozen in a non-permeating cryoprotectant such as sorbitol. The optimum concentration of sorbitol was found in a hypertonic solution of around 2 M. It was also very important to use S. pombe cells grown in minimal medium and S. cerevisiae cells grown in nutrient medium in the exponential growth phase. A slow freezing rate of 10 degrees C/min and a rapid thawing rate of 200 degrees C/min resulted in the highest transformation efficiency. We also found it necessary to wash the thawed cells with 1.0 M of non-electrolyte sorbitol, since the intracellular electrolytes had leaked as a result of cryoinjury. The frozen competent cells stored at -80 degrees C could be used for more than 9 months without any loss of transformation efficiency. This cryopreservation method for electroporation is simple and useful for routine transformations of intact cells. Frozen competent cells offer the advantages of long-term storage with high efficiency and freedom from the preparation of fresh competent cells for each transformation.     

Enhancement of plasmid DNA transformation efficiencies in early stationary‐phase yeast cell cultures

Chemical‐based methods have been developed for transformation of DNA into log‐phase cells of the budding yeast Saccharomyces cerevisiae with high efficiency. Transformation of early stationary‐phase cells, e.g. cells grown in overnight liquid cultures or as colonies on plates, is less efficient than log‐phase cells but is simpler and more adaptable to high‐throughput projects. In this study we have tested different approaches for transformation of early stationary‐phase cell cultures and identified a method utilizing polyethylene glycol (PEG), lithium acetate and dimethyl sulphoxide (DMSO) as the most efficient. Plasmid DNA transformations using this method could be improved modestly by allowing cells to recover from the chemical treatment in rich broth before plating to selective media. Strong increases in transformation efficiencies were observed when cells were treated briefly with dithiothreitol (DTT). Tests using several different yeast strain backgrounds indicated that DTT treatment could enhance transformation efficiencies by up to 40‐fold. Evaluation of multiple parameters affecting the efficiency of the method led to development of an optimized protocol achieving > 50 000 transformants/µg DNA in most backgrounds tested. Copyright © 2013 John Wiley & Sons, Ltd.     

Extremely simple, rapid and highly efficient transformation method for the yeast Saccharomyces cerevisiae using glutathione and early log phase cells

In the presence of polyethylene glycol (PEG), budding cells of Saccharomyces cerevisiae in the early log phase were transformed by exogenous plasmid DNA without additional specific chemical or physical treatments. This capacity of the yeast cells to become competent was strictly dependent on the growth phase, being induced in the early log phase, becoming maximum between the early and mid log phases and then disappearing rapidly in the mid log phase. The transformation was most efficient at pH 6 and the frequency increased with increasing DNA and cell concentrations. PEGs with average molecular sizes between 1000 and 3500 showed almost the same effects and were used most efficiently at 35%. The transformation frequency of S. cerevisiae was markedly enhanced when the oxidized form of glutathione (GSSG), but not the reduced form, was included in the mixture comprising early log phase cells, plasmid DNA, and PEG, and the transformation system with GSSG could be used as a convenient transformation method for the yeast S. cerevisiae.     

Transformation of Candida albicans by electroporation

In contrast to a variety of other yeasts, Candida albicans has proved difficult to transform with high efficiency. Lithium acetate transformation is fast and simple but provides a very low efficiency of DNA transfer (50-100 transformants/microg DNA), while spheroplast transformation, although more efficient ( approximately 300 transformants/microg integrative DNA and 10(3)-10(4) transformants/microg replicative DNA), is complicated and time-consuming. In this study we applied various yeast transformation techniques to C. albicans and selected an electroporation procedure for further optimization. Transformation efficiencies of up to 300 transformants/microg were obtained for an integrative plasmid and up to 4500 transformants/microg for a CARS-carrying plasmid. This reasonably high transformation efficiency, combined with the ease and speed of electroporation in comparison to alternative techniques, make it the preferred method for transformation of C. albicans.     

Cells of the yeast Saccharomyces cerevisiae are transformable by DNA under non-artificial conditions

Transformants of bakers' yeast (Saccharomyces cerevisiae) can be generated when non-growing cells metabolize sugars (without additional nutrients) in the presence of plasmid DNA. These results suggest that there is a mechanism by which DNA can naturally be taken up by the yeast cell. Natural transformation does not take place in common complete or minimal yeast culture media such as YPD and YNB. The starvation conditions used in our experiments thus seem to be an important prerequisite for such transformation events.     

A rapid technique for the determination of unknown plasmid library insert DNA sequence directly from intact yeast cells

A polymerase chain reaction (PCR)-based technique is described which allows for the determination of library plasmid insert DNA sequence directly and rapidly from intact yeast cells. Yeast spheroplasts are used to template a PCR reaction to amplify the insert sequence. This PCR product is then purified and its sequence directly determined using thermal cycle sequencing. Readable sequence can reproducibly be obtained from multiple yeast colonies in just two days. Uses of this technique in yeast two-hybrid screening as well as other types of yeast library screens are discussed.     

Damage of yeast cells induced by pulsed light irradiation

DNA damage, such as formation of single strand breaks and pyrimidine dimers was induced in yeast cells after irradiation by pulsed light, which were essentially the same as observed with continuous ultraviolet (UV) light. The UV-induced DNA damage is slightly higher than seen with pulsed light. However, increased concentration of eluted protein and structural change in the irradiated yeast cells were observed only in the case of pulsed light. A difference in the inactivation effect between pulsed light and UV light was found and this suggested cell membrane damage induced by pulsed light irradiation. It is proposed that pulsed light can be used as an effective sterilizing method for the yeast Saccharomyces cerevisiae.     

A novel transfection method for mammalian cells using calcium alginate microbeads

The direct transfer of genetic materials into mammalian cells is an indispensable technique. We have developed calcium alginate (CA) microbeads which can deliver plasmid DNAs and yeast artificial chromosomes into plant and yeast cells. In this paper, we demonstrate the effective transfection of mammalian cells by CA microbeads immobilizing plasmid DNAs. The transfection was performed using the pEGFP-C1 plasmid containing the cytomegalovirus (CMV) promoter and enhanced green fluorescent protein (EGFP) gene. The transient expression of EGFP was observed 24 h after transfection. The expression efficiency was maximum when the concentration of sodium alginate was 1% and the amount of plasmid DNA was increased to 100 microg. The expression efficiency of our method using CA microbeads is 2-10 times higher than that of the polyethylene glycol (PEG) method. Our results suggest that the CA microbead mediated transfection of mammalian cells effectively delivers genetic materials into mammalian suspension cells.