有淀粉糖化酶活性的耐高温酿酒酵母的构建研究

通过诱导Hu—TY一1(耐高温酿酒酵母)产孢及单孢分离得到的单倍体株Hu—TY—l—A(Sa-ccharamyces cerevisiae)与具有STAl、STA2、STA3(淀粉糖化酶)遗传因子的酵母(S．Dias-taticus)单倍体和纯合二倍体进行相同交配型的种间原生质体融合,融合率为3．3×1O^-6-8．7×1O^-6。融合子的细胞形态、大小、DNA含量、生长和发酵特性等方面均不同于亲株。融合株BA-2、BA-3、CA-18、DA-2、CCA-30、其细胞体积与DNA含量为亲株之和,30℃生长速率与生物量为亲株的2—3倍,40℃生长速率与生物量为亲株HU－TY－1－A的1．3－1．6倍,能够利用淀粉,并且在40℃高温葡萄糖发酵酒精产率比糖化酵母高,比HU－TY－1－A低,是一些有淀粉糖化酶、耐高温的酿酒酵母新菌株。     

酿酒酵母与糖化酵母的种间原生质体融合及其融合子的鉴定

本文报道了酒精生产菌株K氏酿酒酵母Sacchormyces cerevisiae var.ellipsoideus的HUK-1(his-,二倍体,但在我们所试的5种产孢培养基上均不产孢)与糖化酵母Sac—charomyces diastaticus 7c(arg-,a)的种间原生质体融合,其营养标记互补的融合频率约是2．07×10^-6—3．40×10^-5。这些融合子曾在选择培养基MMs或Mmo上连续传代10次,以促进两亲核的融合。融合子的酒精发酵特性,细胞形态、体积大小、DNA含量、繁殖速率、发酵强度以及产孢能力等方面的观察和测定结果表明,均不同于双亲菌株。用显微操作器解剖了个别原养型融台子HU—KDF—185的4孢子子囊,在获得的93个单孢株中,其淀粉发酵特性和遗传标记均有双亲类型的分离或重组现象。上述实验结果充分证明了不同倍性的酵母之间可以通过原生质体融合获得种间杂种。     

电诱导酵母属与短梗霉属属间融合的研究

本文报道了经GH一401 型电诱导细胞融合、基因转移仪诱导实现的酿酒酵母(Saccharomyces cerevisiae NK491) 与出芽短梗霉 (Aureobasidium pullulans NKB93—0061eu)的原生质体电诱导融合。在融合小罐中用3个强度为11 Kv／cm,时程为10μs的高压电脉冲处理原生质体,获得的融合子营养互补的融合频率为5.8×10^-5。在选择培养基上连续传代10次,然后在完全培养基上传代20次,最终得到4株稳定的融台子。融合子的细胞形态、大小、核的DNA含量以及酒精发酵等特性的观察和测定表明,4株融合子与双亲均有明显差异。实验结果表明,电诱导原生质体融台对酵母菌的属间融合是一种行之有效的方法,具有较高的融合频率。     

酵母属间原生质体融合改进菌株木糖发酵性能

通过单倍体分离和紫外诱变,获得了１４株树干毕赤酵母（Ｐｉｃｈｉａｓｔｉｐｉｔｉｓ）７１２４和酿酒酵母（Ｓａｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ）１３００的营养缺陷型突变株。用聚乙二醇（ＰＥＧ）和电诱导融合及致死融合等方法,实现了树干毕赤酵母和酿酒酵母的属间原生质体融合。融合子能发酵木糖产生酒精,其厌氧发酵木糖和木糖葡萄糖混合液的能力明显优于亲株,耐酒精的性能也比亲株树干毕赤酵母７１２４有所提高。融合子经ＤＮＡ含量、细胞体积测定和稳定性能实验证明为稳定融合子。     

酵母菌属间原生质体融合构建高温酵母菌株

酿酒酵母(Saccharomyces cerevisiae) A001和克鲁维酵母(Kluyveromyces sp.) Y034属间原生质体融合构建高温酵母菌株。对制备高再生活性原生质体及融合子细胞形态、生理化特征、同工酶性质、遗传稳定性和高温发酵等方面进行了研究。结果表明,融合子AY023和AY680遗传性能稳定,表达了双亲优良性状,获得了在45℃培养条件下产酒率7.4%的属间融合菌株,是目前已见文献报道的产酒率最高的高温(45℃)酵母菌株。     

耐热酵母与酿酒酵母原生质体融合的研究

酿酒酵母(saccharomyces cerevisiae)396是利用甘蔗糖蜜生产酒精的生产菌株,假丝酵母(Candida sp·)C₆是我们从云南温泉底泥中筛选到的一株能在45℃生长良好的耐热酵母,我们应用原生质体融合技术进行了两菌株属间融合的研究。通过亚硝基胍(NTG)诱变得到的营养缺陷型菌株396(arg^－)和C6(lys^-),其融合频率为O．91×10^-5。从检出的融合子中挑选6株进行考核、其细胞体积平均为亲株的1．3倍,DNA含量平均为亲株的1．6倍,培养特征、形态、大小和生理生化特征表现不一,特别是糖类的同化试验。除F₂、F₁₄外,其他4株可以排除异核体形成的可能性。比较了在28℃,40℃和45℃培养条件下出发亲株396、C₆、直接亲株396(arg^-)C₆(lys^-)和融合子F₁、F₇、F₁₂,F₁₃的生长曲线、基质利用率和乙醇产率等,得到一株在40℃培养条件下糖的利用率为94．3％、乙醇产量为59．7g／L的属间融合株F₁₃。     

三角酵母二倍体菌株的选育

由三角酵母(Trigonopsis variabilis)原生质体融合,获得了35株原养型融合子。通过对其中四株进行详绌分析表明,融合子细胞体积和DNA含量为两亲株之和,苏木精染色显示单核,这些结果证明融合子为亲株的二倍体。此外,融合子的生长速度、D-氨基酸氧化酶以及细胞蛋白质含量均明显高于亲株。电镜形态观察：进一步证明融合子细胞大于亲株。     

克鲁维酵母种间原生质体融合的研究

乳酸克鲁维酵母(Kluyueromyces lactis Y12—1)和脆壁克鲁维酵母(K.fragilis8554)是乳糖酶生产菌株。应用原生质体融合技术进行了两菌株种问融合的研究。通过试验．原生质体形成及再生的最佳条件为：对数期的细胞,2％的蜗牛酶．30℃酶解30分钟．原生质体形成率90％以上,再生率20%左右。原生质体融合由聚乙二醇(PEG)诱导。K.lactisY12-l不能旋酵菊糖;K.fragilis 8554不能同化D－松三糖和麦芽糖;利用二菌株自身的营养缺陷性质获得融合子。融合子既能发酵菊糖又能同化D－松三糖和麦芽糖;融合子的DNA含量约为二亲株之和;融合子的菌落形态与亲株相比有一定差别．在以乳糖为碳源的培养基中,融合子的乳糖酶产量提高14一l6％;连续15次传代,融合子稳定。     

电融合技术选育能利用木糖和纤维二糖生产乙醇的菌株

以携带营养缺陷型标记的季也蒙假丝酵母(Candida guilliermondii s 208 Arg-)和酿酒酵母(Saccharomyces cerevisiae 314)为亲本,采用GH-40l型电诱导基因转移／细胞融合仪,用3个强度为18kV／cm,时程为10μs,间隔为1 s的高压电脉冲处理原生质体,营养互补的融合频率为3．6×10^-3。在选择和非选择培养基上连续传代20余次后,对得到的稳定的融合子进行分析比较,结果表明,它们是两亲株融合后形成的融台体。其中有两株融合子能利用木糖和纤维二糖生产乙醇,F－106利用D－木糖(2％)和纤维二糖(2％)生产乙醇的产量分别为3．1g／l和1．05g／l,F－308分别为0．2g／I和2．8g／l。     

啤酒酵母和产朊假丝酵母属间原生质体融合子的筛选

利用不可逆生化抑制剂碘乙酸抑制一亲株细胞的生理活性和利用两亲株细胞间生理性状的差异性,进行啤酒酵母(Saccharomyces cerevisiae)和产朊假丝酵母(Candida utilis)原生质体融合子的筛选,属间原生质体融合率为3.47×10~(-6)。经菌落形态比较,碳化合物的同化和发酵,DNA含量测定,酯酶型分析,细胞核染色,产孢试验和自然的核分离实验证明,融合子分三种类型:87.21%产朊假丝醇母型;9.02%啤酒酵母型;3.77%真正核融合子型。     

Strain improvement of thermotolerant Saccharomyces cerevisiae VS strain for better utilization of lignocellulosic substrates

AIM: Pentose-utilizing yeast development by protoplast fusion and sequential mutations and ethanol fermentation using lignocellulosic substrate. METHODS AND RESULTS: Protoplasts of thermotolerant Saccharomyces cerevisiae and mesophilic, xylose-utilizing Candida shehatae were fused by electrofusion. The fusants were selected based on their growth at 42 degrees C and ability to utilize xylose. The selected best fusant was mutated sequentially and 3 mutant fusants obtained were tested for their stability. The mutant fusant CP11 was found to be stable and used for lignocellulosic fermentation. The Prosopis juliflora wood material was hydrolysed with 1% sulphuric acid initially for 18 h at room temperature and then for 20 min at 121 degrees C. The acid hydrolysate was separated and used for detoxification by ethyl acetate and overliming. The hard cellulosic fraction was hydrolysed with Aspergillus niger crude cellulase enzyme for 18 h at 50 degrees C. The substrate (15% w/v) yielded 84 g l(-1) sugars, representing 80% (w/w) hydrolysis of carbohydrate content present in the lignocellulosic material. The acid and enzyme hydrolysates were then equally mixed and used for fermentation with the developed fusant yeast (CP11). The fusant yeast gave an ethanol yield of 0.459 +/- 0.012 g g(-1), productivity of 0.67 +/- 0.015 g l(-1) h(-1) and fermentation efficiency of 90%. CONCLUSIONS: Protoplast fusion followed by sequential mutations method gave a stable and good performing fusant with maximum utilization of reducing sugars in the media. SIGNIFICANCE AND IMPACT OF THE STUDY: This new method could be applied to develop fusants for better biotechnological applications.     

双亲灭活制备粘质沙雷氏菌和红曲霉的跨界产色素融合子

目的:采用双亲灭活原生质体技术制备粘质沙雷氏菌和红曲霉的跨界产色素融合子,并测定其抑菌活性。方法:经0.2%溶菌酶处理获得粘质沙雷氏菌的原生质体并热灭活;经混合酶(0.8%溶菌酶+1.2%蜗牛酶+1.6%纤维素酶)处理获得红曲霉的原生质体并紫外灭活;用含25%PEG的原生质体融合剂进行促融合与再生。观察融合子的菌落形态和色素合成能力,测定融合子色素提取物对金黄色葡萄球菌的抑制活性。结果:在优化条件下,粘质沙雷氏菌原生质体的形成率为92.58%,红曲霉原生质体形成数约为106个/mL,两菌原生质体灭活率均为100%。共获得13个融合子,9个能产红色素,融合率为1×10-5%。其中8个融合子的95%乙醇提取物对金黄色葡萄球菌表现出不同程度的抑制。结论:采用双亲灭活原生质体技术,能够制备具有抑菌活性的粘质沙雷氏菌和红曲霉的跨界产色素融合子。     

双亲灭活制备粘质沙雷氏菌和红曲霉的跨界产色素融合子

目的：采用双亲灭活原生质体技术制备粘质沙雷氏菌和红曲霉的跨界产色素融合子,并测定其抑菌活性。方法：经0.2%溶菌酶处理获得粘质沙雷氏菌的原生质体并热灭活;经混合酶（0.8%溶菌酶＋1.2%蜗牛酶＋1.6%纤维素酶）处理获得红曲霉的原生质体并紫外灭活;用含25%PEG的原生质体融合剂进行促融合与再生。观察融合子的菌落形态和色素合成能力,测定融合子色素提取物对金黄色葡萄球菌的抑制活性。结果：在优化条件下,粘质沙雷氏菌原生质体的形成率为92.58%,红曲霉原生质体形成数约为106个/mL,两菌原生质体灭活率均为100%。共获得13个融合子,9个能产红色素,融合率为1×10-5%。其中8个融合子的95%乙醇提取物对金黄色葡萄球菌表现出不同程度的抑制。结论：采用双亲灭活原生质体技术,能够制备具有抑菌活性的粘质沙雷氏菌和红曲霉的跨界产色素融合子。     

Construction and Analysis of High-Ethanol-Producing Fusants with Co-Fermentation Ability through Protoplast Fusion and Double Labeling Technology

Double labeling of resistance markers and report genes can be used to breed engineered Saccharomyces cerevisiae strains that can assimilate xylose and glucose as a mixed carbon source for ethanol fermentation and increased ethanol production. In this study Saccharomyces cerevisiae W5 and Candida shehatae 20335 were used as parent strains to conduct protoplast fusion and the resulting fusants were screened by double labeling. High performance liquid chromatography (HPLC) was used to assess the ethanol yield following the fermentation of xylose and glucose, as both single and mixed carbon sources, by the fusants. Interestingly, one fusant (ZLYRHZ7) was demonstrated to have an excellent fermentation performance, with an ethanol yield using the mixed carbon source of 0.424 g g⁻¹, which compares with 0.240 g g⁻¹ (W5) and 0.353 g g⁻¹ (20335) for the parent strains. This indicates an improvement in the ethanol yield of 43.4% and 16.7%, respectively.     

Construction of Lactose-Assimilating and High-Ethanol-Producing Yeasts by Protoplast Fusion

The availability of a yeast strain which is capable of fermenting lactose and at the same time is tolerant to high concentrations of ethanol would be useful for the production of ethanol from lactose. Kluyveromyces fragilis is capable of fermenting lactose, but it is not as tolerant as Saccharomyces cerevisiae to high concentrations of ethanol. In this study, we have used the protoplast fusion technique to construct hybrids between auxotrophic strains of S. cerevisiae having high ethanol tolerance and an auxotrophic strain of lactose-fermenting K. fragilis isolated by ethyl methanesulfonate mutagenesis. The fusants obtained were prototrophic and capable of assimilating lactose and producing ethanol in excess of 13% (vol/vol). The complementation frequency of fusion was about 0.7%. Formation of fusants was confirmed by the increased amount of chromosomal DNA per cell. Fusants contained 8 × 10⁻⁸ to 16 × 10⁻⁸ μg of DNA per cell as compared with about 4 × 10⁻⁸ μg of DNA per cell for the parental strains, suggesting that multiple fusions had taken place.     

Intergeneric yeast fusants with efficient ethanol production from cheese whey powder solution: Construction of a Kluyveromyces marxianus and Saccharomyces cerevisiae AY‐5 hybrid

Due to its high content of lactose and abundant availability, cheese whey powder (CWP) has received much attention for ethanol production in fermentation processes. However, lactose‐fermenting yeast strains including Kluyveromyces marxianus can only produce alcohol at a relatively low level, while the most commonly used distiller yeast strain Saccharomyces cerevisiae cannot ferment lactose since it lacks both β‐galactosidase and the lactose permease system. To combine the unique aspects of these two yeast strains, hybrids of K. marxianus TY‐22 and S. cerevisiae AY‐5 were constructed by protoplast fusion. The fusants were screened and characterized by DNA content, β‐galactosidase activity, ethanol tolerance, and ethanol productivity. Among the genetically stable fusants, the DNA content of strain R‐1 was 6.94%, close to the sum of the DNA contents of TY‐22 (3.99%) and AY‐5 (3.51%). The results obtained by random‐amplified polymorphic DNA analysis suggested that R‐1 was a fusant between AY‐5 and TY‐22. During the fermentation process with CWP, the hybrid strain R‐1 produced 3.8% v/v ethanol in 72 h, while the parental strain TY‐22 only produced 3.1% v/v ethanol in 84 h under the same conditions.     

高效发酵木糖生产乙醇酵母菌株的构建

获得高效发酵木糖生产乙醇的酵母菌株是木质纤维素生物转化生产燃料乙醇的重要前提。在4%乙醇驯化的基础上,选择了乙醇耐性提高的休哈塔假丝酵母（Candida shehatae）CICC1766菌株进一步进行紫外诱变,得到了木糖发酵性能较强的呼吸缺陷型突变体,并与乙醇发酵性能良好的酿酒酵母（Saccharomyces cerevisiae）ATCC4126进行原生质体融合。采用单亲灭活法对休哈塔假丝酵母原生质体进行紫外灭活,在聚乙二醇（PEG）诱导下融合,对得到的融合子进行木糖发酵能力测定,选择到了一株能够更好地利用木糖产乙醇,并且木糖发酵性能比亲本得到明显提高的融合子F6,此融合子发酵50 g/L木糖,最高乙醇浓度达到18.75g/L,乙醇得率为0.375,达到理论转化值0.511的73.4%。与原始出发菌株CICC1766相比,乙醇产量提高了28%。     

Intergeneric protoplast fusion between Kluyveromyces and Saccharomyces cerevisiae – to produce sorbitol from Jerusalem artichokes

The construction of inulin-assimilating and sorbitol-producing fusants was achieved by intergeneric protoplast fusion between Kluyveromyces sp. Y-85 and Saccharomyces cerevisiae E-15. The cells of parental strains were pretreated with 0.1% EDTA (w/v) and 2-mercaptoethanol (0.1%, v/v) and then exposed to 2.0% (w/v) Zymolase at 30 °C for 30–40 min. The optimized fusion condition demonstrated that with the presence of 30% (w/v) polyethylene glycol 6000 (PEG-6000) and 10 mM CaCl₂ for 30 min, the fusion frequency reached 2.64 fusants/10⁶ parental cells. The fusants were screened by different characters between two parental strains and further identified by DNA contents, inulinase activity and sorbitol productivity. One of the genetically stable fusants, Strain F27, reached a maximal sorbitol production of 4.87 g/100 ml under optimal fermentation condition.     

Cell fusion between L-forms and protoplasts of Staphylococcus aureus

Mixtures of various combinations of Lysostaphin protoplasts and stable L-forms of Staphylococcus aureus, which have different markers for drug resistance, were treated with polyethylene glycol (PEG) to examine the development of doubly resistant fusion products (fusants). To recover doubly resistant colonies as L-forms, they were incubated in 4.5% NaCl-brain heart infusion (BHI) broth containing penicillin G (PCG) for enrichment culture and cultured in PCG-4.5% NaCl-BHI agar medium (method 1), while to recover doubly resistant fusants as L-forms and coccal forms, they were grown on reversion medium (R medium) which causes reversion of protoplasts or fusants to parent type cells, and then cultured on assay media, i.e., R medium, BHI agar medium or PCG-4.5% NaCl-BHI agar medium (method 2). Under both experimental conditions, doubly resistant fusants developed as L-form cells by PEG treatment of pairs of protoplasts carrying the chloramphenicol (CP)-resistance plasmid and L-forms having chromosomal resistance to streptomycin (SM). In the reverse combinations, i.e., protoplasts showing chromosomal SM-resistance and L-form cells carrying the CP-resistance plasmid, the first method gave no doubly resistant colonies. By the second method, without enrichment culture on R medium, the latter combination gave doubly resistant fusants as L-form, coccal-type and mixed-type colonial forms, while when the PEG-treated mixture was enriched on R medium, fusants were obtained exclusively as the coccal type on either R medium or BHI agar assay medium. Neither of the methods yielded colonies of doubly resistant fusants on PEG-treatment of pairs of protoplasts and L-forms both of which were chromosomal, but with different drug resistances. These results show that PEG-induced cell fusion between protoplasts and L-forms of S. aureus, unlike the fusion between protoplasts or between L-forms, resulted in transfer of the drug resistance controlled by the plasmid to the fusion products. The fusants obtained were L-forms in method 1, and coccal type in the method 2.     

用CHEF凝胶电泳探测远缘担子菌间原生质体融合后的染色体变异

用电场诱导灰盖鬼伞(Coprinus cinereus Co5104,his~-)和佛罗里达侧耳(Pleurotusflorida Pf67, ade~-)两菌株的原生质体融合,获得了两类属间融合子:一类为不断发生分离的单核体,另一类为稳定的双核异核体。利用等高钳位均匀电场(CHEF)凝胶电泳技术比较融合子与两亲本的染色体长度多型性(CLP),显示出融合子的染色体变异以整条染色体的丢失为主。结合形态观察和DAPI核荧光染色的结果,推测单核融合子在胞质融合后很可能发生了核融合,但来自亲本Pf67的染色体不断丢失,最后融合子的遗传组成以来自Co5104的染色体为主;而双核融合子在质配之后可能并未发生核配,不过,来自各亲本的染色体最终均只有部分得以保留在融合子中。