立用原生质体融合技术改善双歧杆菌的耐氧性

利用原生质体融合技术将专性厌氧的短双歧杆菌和典型上面发酵的啤酒酵母进行原生质体细胞融合，通过研究两亲本原生质体的形成与再生，并进行跨界融合，成功初筛出兼具两亲本生物学性状、较稳定的融合了BSF1和BSF2，改善了双歧杆菌的耐氧性。     

双歧杆菌和酿酒酵母原生质体融合子筛选方法的探讨

目的:探讨双歧杆菌(Bifidobacterium)和酿酒酵母(Saccharomycescerevisiae)原生质体融合子的筛选方法。方法:将双歧杆菌和酿酒酵母原生质体进行原生质体融合,采用10%乳糖中性红培养基进行融合子的初筛,并分析融合子传代稳定性、生物学特性,采用双歧杆菌属特异性寡核苷酸探针对亲本菌和融合子进行了荧光原位杂交检测比较。结果:结果表明,筛选得到的融合子具有双歧杆菌的特异性序列和亲本菌的生物学特性。结论:双歧杆菌和酿酒酵母原生质体融合,实现了厌氧菌和酵母的跨界融合,为双歧杆菌生物学功能的开发提供新思路、新途径。     

双歧杆菌原生质体的制备及其转化系统的建立

考察了长双歧杆菌WBL001菌体生长状态、酶解浓度、时长、温度以及不同渗透压稳定剂等因素,获得长双歧杆菌WBL001原生质体制备及再生的优化条件:长双歧杆菌以3%接种量活化至MRS培养基,培养至对数中期(OD600∶1),5μg/mL变溶菌素,37℃孵育30 min,以原生质体稳定液SMM为反应缓冲液,原生质体生成率达到81%,其再生率达到48%;在此基础上,通过聚乙二醇PEG融合,将穿梭表达载体pBAX转入双歧杆菌原生质体,成功建立双歧杆菌转化体系。     

产胸苷磷酸化酶短乳杆菌融合菌株的构建及其特性

利用紫外加热灭活双亲原生质体技术对短乳杆菌原生质体进行融合,考察短乳杆菌原生质体制备、再生及融合的影响因素,并对短乳杆菌融合子产酶能力和遗传稳定性进行了研究。结果表明:短乳杆菌在含0.6%甘氨酸发酵培养液培养至对数生长期,2 mg/mL溶菌酶于37℃恒温水浴酶解脱壁2 h,原生质体制备率和再生率可达95%和48%;20 W紫外灯距离20 cm照射50 min,60℃处理短乳杆菌原生质体60 min,原生质体的灭活率几乎为100%;将双亲灭活的原生质体用40%PEG6000,30℃恒温融合10 min,筛选融合子F15并进行5次传代测试其胸苷磷酸化酶活均在1.500 U/mg湿菌体,较亲本酶活提高了50%。     

灭活原生质体融合技术提高豆豉纤溶酶菌产酶量

以豆豉纤溶酶产生菌株蜡状芽孢杆菌UV-101和MW-101作为亲本菌株,首先对亲本原生质体进行紫外灭活,然后用PEG(6000)作为融合剂对灭活双亲进行原生质体融合.通过对再生平板上长出的融合子进行平板初筛和摇瓶复筛,最终获得一株高产菌株PF-101,其酶活比亲本菌株分别提高了74.47%和86.36%,并且该菌株具有良好的遗传稳定性.     

双亲灭活青霉菌与枯草芽孢杆菌原生质体融合

目的:为选育具有高纤维素酶活性且强抗逆性的新菌株,将高产纤维素酶的青霉菌Penicillium Q5与强抗逆性的枯草芽孢杆菌Bacillus subtilis K3进行原生质体融合。方法:采用双亲灭活原生质体技术对青霉菌与枯草芽孢杆菌进行原生质体融合,考察原生质体制备条件与融合中的影响因素。结果:对亲本菌株进行酶解前处理,随着酶解浓度和酶解时间的增加,原生质体的形成率随之增大,而再生率下降。对融合过程进行正交试验,结果在融合温度38℃,融合时间10 min,PEG质量浓度0.4 g/L的条件下,融合率为3.08×10-7。在172株融合子中筛选出1株R62,保留了亲本的酶活且在p H 8~10条件下有较强的抗逆性。结论:双灭活原生质体融合方法成功选育出具有抗逆性的高产纤维素酶菌株。     

酿酒酵母与粟酒裂殖酵母融合子检出方法的研究

为了快速检出酿酒酵母与粟酒裂殖酵母的融合子,在双亲原生质体融合处理后,利用两亲本再生营养条件的差异及氧化邻苯二胺能力的不同,通过设计选择性底层培养基和鉴定性上层培养基,进行了融合子检出方法的研究,并确定了3株融合子。该方法简单易行,可用于酿酒酵母与粟酒裂殖酵母原生质体融合育种的研究。     

L-缬氨酸产生菌的原生质体融合及抗高糖融合株的筛选

通过原生质体融合技术选育出一株抗高糖的融合株。以黄色短杆菌(Brevibacterium flavum)NV128和黄色短杆菌(B.flavum)JV16(Leu-Ile-Met-)为亲本菌株,通过单灭活原生质体融合、高糖梯度平板筛选及进一步的硫酸二乙酯(DES)诱变,获得了一株L-缬氨酸高产菌NJ-237。同时研究了影响原生质体形成、再生的3种重要条件(青霉素、酶和渗透压稳定剂),并确定了其最佳处理浓度和时间。该融合株经7 L发酵罐培养72 h L-缬氨酸达到46.8 g/L,糖酸转化率为27.7%,比两亲株都有显著提高。     

一株高产3-羟基丁酮枯草芽孢杆菌的构建

以产3-羟基丁酮枯草芽孢杆菌(Bacillus subtilis)TH-49(Val-)和产α-乙酰乳酸脱羧酶地衣芽孢杆菌(Bacillus licheniformis)AD-30为亲本菌株,以聚乙二醇为融合促进剂,进行原生质体融合获得融合子,融合子经再生、筛选等过程,最终获得高产3-羟基丁酮菌株HB-32,该菌株3-羟基丁酮产量高达49.64 g/L,比菌株TH-49(Val-)提高了61.8%,且遗传稳定性良好。进一步采用随机扩增多态性DNA(RAPD)技术,从分子水平上分析了高产3-羟基丁酮菌株HB-32与亲本菌株基因组的变化。结果表明,枯草芽孢杆菌(B. subtilis)TH-49(Val-)和地衣芽孢杆菌(B. licheniformis)AD-30原生质体融合成功,提高了融合子HB-32 3-羟基丁酮产量。     

酿酒酵母与粟酒裂殖酵母融合子检出方法的研究

为了快速检出酿酒酵母与粟酒裂殖酵母的融合子,在双亲原生质体融合处理后,利用两亲本再生营养条件的差异及氧化邻苯二胺能力的不同,通过设计选择性底层培养基和鉴定性上层培养基,进行了融合子检出方法的研究,并确定了3株融合子.该方法简单易行,可用于酿酒酵母与粟酒裂殖酵母原生质体融合育种的研究.     

融合子生料发酵产酒精的研究

构建的融合子AK-2(K氏酵母与黑曲霉原生质体融合)在高细胞密度发酵的前提下,对免蒸煮生料发酵的一些特性指标及抑菌进行了探讨。结果表明:采用融合子AK-2进行高细胞密度生料发酵生产酒精的方法是可行的。     

Improvement of the growth defect in salt- and ethanol-tolerant yeast mutagenized with error-prone DNA polymerization by using backcross cell fusion

Salt- and ethanol-tolerant mutants of Saccharomyces cerevisiae, isolated from the uracil-requiring mutant derived from Taiken No. 396 by proofreading-deficient DNA polymerization, showed less growth than their parent strain. The fusants, between these tolerant mutants and the lysine-requiring mutant from Taiken No. 396 obtained by the protoplast fusion, indicated improved growth.     

Cell fusion of Saccharomyces cerevisiae fragile mutants

Fragile mutants of Saccharomyces cerevisiae are defective in the structure of the cell wall and plasma membrane. The mutant cells lyse in hypotonic solutions but grow exponentially when osmotic stabilizer is included in the medium. These mutants display a general increase in the permeability of the plasma membrane. We show here that fragile yeast cells of the same mating type can fuse without protoplast formation. The frequency of cell x cell fusion is lower than that observed for protoplast x protoplast fusion and can be significantly increased if the cells of one partner are converted to protoplasts. Microscopic observations and genetic analysis demonstrate that the hybrids obtained are fusion products. The fusion between fragile cells is explained in terms of the existence of local defects on their surface where the cell wall is thinner (or even missing), thus allowing a direct contact of cells by means of their plasma membranes.     

聚合酶链反应在融合子鉴定中的应用

以粟酒裂殖酵母和酿酒酵母为出发菌株,进行了原生质体融合试验,并对检出的融合子采用聚合酶链反应(PCR)扩增亲本的特定基因序列,以此达到鉴定融合子的目的.在检出的3株融合子中,均扩增出亲本特定基因,证明了该法在融合子鉴定中的可行性.     

A recombinant Saccharomyces cerevisiae strain for efficient conversion of lactose in salted and unsalted cheese whey into ethanol

For utilization of lactose in salted and unsalted cheese whey, intergeneric protoplast fusion between lactose nonfermenting, salt-tolerant Saccharomyces cerevisiae ATCC4126 and lactose fermenting Kluyveromyces lactis CBS683 was carried out. The fusion process gave rise to new hybrid yeast strains that revealed higher significant DNA contents than parental strains. The recombinants showed growth on either lactose or sucrose. The ethanol yields by some recombinants were 5.55% from sweet whey and 4.66% from salted whey containing up to 6% sodium chloride compared to 4.15 and 2.86% for parental K. lactis CBS683, respectively.     

啤酒酵母融合株QSB—Ⅺ6中试研究

将新选育的酵母融合株ＳａｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅＱＳＢ－Ⅺ６,同本厂生产中使用的酵母菌种ＳａｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅＬＱ１６在２３０Ｌ发酵罐中做对比性发酵试验。试验酒的各项理化指标均已达到国家优质酒标准,其中双乙酰值在００８ｍｇ／ｌ以下,真正发酵度平均为７０２０％,酒体泡沫丰富,清亮透明。口味纯正,风味独特,为使该菌种能更好地应用于生产,奠定了基础。     

关中地区初生婴幼儿肠道双歧杆菌种群多样性分析

旨在探究初生婴幼儿肠道双歧杆菌的种群多样性及其丰度,为婴幼儿健康评价及营养补充设计提供参考依据。本研究利用末端限制性片段长度多态性分析技术与实时荧光定量PCR技术探究陕西关中地区0~6月龄(Ⅰ组)、6~12月龄(Ⅱ组)、12~36月龄(Ⅲ组)婴幼儿肠道中双歧杆菌的种群组成与丰度。结果表明:在24份婴幼儿粪便样品中,3组(阶段)样品中均检出长双歧杆菌、短双歧杆菌、两歧双歧杆菌、动物双歧杆菌及假链状双歧杆菌5个菌种,且在第Ⅲ组样品中还检测到青春双歧杆菌;3组样品中双歧杆菌属的DNA含量均在1010 copies/g左右,Ⅰ组样品中含量(1010.44 copies/g)显著高于Ⅱ、Ⅲ组样品(p<0.05)。Ⅰ、Ⅱ组样品中长双歧杆菌和短双歧杆菌的丰度较高,Ⅰ组中对应的丰度分别为30.12%、38.02%,Ⅱ组中对应的丰度分别为33.89%、43.65%;Ⅲ组中丰度前3位的菌种是长双歧杆菌、短双歧杆菌和两歧双歧杆菌,分别为23.99%、19.95%、24.55%。3个阶段的婴幼儿肠道中双歧杆菌的种群组成差异不明显,但丰度表达存在一定差异(p<0.05)。     

Efficient selection of hybrids by protoplast fusion using drug resistance markers and reporter genes in Saccharomyces cerevisiae

We have developed a selection system for hybrids by protoplast fusion using dominant selective drug resistance markers, Tn601(903) against geneticin and AUR1-C against aureobasidin A, and reporter genes, ADH1p-PHO5-ADH1t and CLN2p-CYC1-lacZ, in Saccharomyces cerevisiae. To examine the effectiveness of this system, plasmids with each marker and reporter gene were introduced into auxotrophic sake yeasts. From the resulting transformants, eight colonies were screened by protoplast fusion in combination with the drug resistance markers and the reporter genes. Among them, seven strains were judged as hybrids between parental strains by analysis of growth on a minimal medium. This selection system was applied to wine yeasts having no genetic markers. Six strains were regarded as hybrids between parental strains by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene and by karyotype analysis using a contour-clamped homogeneous electric field (CHEF). We propose that the plotoplast fusion using dominant selective geneticin- and aureobasidin A-resistance markers and reporter genes is useful for the selection of hybrids from wine yeasts, which are homothallic and have low sporulation ability.