细菌纤维素高产菌株的紫外诱变育种研究

以木醋杆菌(Acetobacter xylinum)C5为出发菌株,对其进行紫外诱变,将照射时间3min作为紫外诱变剂量,经过初筛和复筛,获得1株细菌纤维素高产菌株A.xylinumC544,该突变株的产量是原始菌株的1.5倍。     

产γ-氨基丁酸的乳酸菌株的诱变选育

为获得γ-氨基丁酸(γ-aminobutyric acid,GABA)的高产菌株,对乳酸片球菌进行紫外线和亚硝基胍诱变及二者的复合诱变处理。以正突变为标准确定的最佳诱变条件为:紫外照射距离25cm,照射时间20s;NTG终浓度为0.3mg/mL,处理时间60min。诱变后获得了1株突变株UN-27,经连续传代10次,遗传性状稳定,平均GABA产量为5.017g/L,较初始菌株的1.062g/L提高了4.72倍。     

UV处理筛选耐高浓度乙醇酿酒酵母菌株

通过对酒精酵母进行紫外诱变及2,3,5-氯化三苯基四氮唑(TTC)的鉴别培养,获得了10株呈红色菌落表型的突变株。诱变条件为:紫外(灯管功率20W)波长254nm,照射距离20cm,照射时间10s、60s和80s,获得正突变菌株Z8,可以耐受12%vol酒精。同时表明了酒精高产和酒精耐性有一定的相关性。     

蛹虫草高产胞外虫草素和虫草多糖的诱变育种

通过诱变获得高产胞外虫草素和虫草多糖的蛹虫草菌株.采用紫外线诱变(UV)、化学诱变(LiCl)、复合诱变(UV-LiCl) 3种方式对蛹虫草孢子进行诱变;发酵检测存活菌株的胞外虫草素和虫草多糖的含量.结果:以胞外虫草素为指标,3种诱变方式的最大正突变率分别为化学突变(29.2％)＞紫外突变(28.6％)＞复合诱变(26.5％);以胞外多糖为指标,最大正突变率分别为紫外诱变(35.7％)＞复合诱变(33.3％)＞化学诱变(27.0％).紫外诱变突变株Z-5-1胞外虫草素产量达0.842g/L,比出发菌株高311％;紫外诱变突变株Z-4-7胞外虫草多糖产量达5.250g/L,比出发菌株高148％.在连续培养5代后,仍具有较好的遗传稳定性.紫外诱变能获得较高的蛹虫草正突变率,同时能获得高产虫草素、虫草多糖的突变株.     

谷氨酰胺转胺酶高产菌株的选育

通过对谷氨酰胺转胺酶生产菌株灰轮丝链轮丝菌ZC03(酶活0.58U/mL)的孢子进行紫外线及He-Ne激光复合诱变,筛选获得一株性状稳定、产酶能力显著提高的突变株ZC60(酶活0.91U/mL),诱变条件为:紫外灯功率15W,距离30cm,照射时间60s,He-Ne激光波长632.8nm,功率1.5mW,距离30cm,照射时间为10min。     

激光复合诱变选育那西肽高产菌及其代谢特性的研究

出发菌株经紫外照射诱变后,再采用激光复合诱变方式进行的筛选,并对其传代稳定性进行研究,以期进一步获得稳产高产那西肽产生菌突变株。结果表明紫外照射100 s得到的NXT-U19放瓶效价比出发菌株(546.17μg/mL)提高了44.22%,达到了787.71μg/mL。激光-紫外复合诱变后筛选出6株产量较高的菌株,其中以NXT-U19-J2效价最高,比原始菌株提高了65.74%,达到了905.22μg/mL。经传代培养分析,NXT-U19-J2诱变株的遗传稳定性较好,该结果表明,激光复合诱变是获得高产那西肽产生菌的有效途径。     

紫外-硫酸二乙酯复合诱变选育耐高渗(高糖)酵母菌

从内蒙古锡林郭勒酸马奶中分离筛选出一株酵母菌A9,通过A9的紫外(UV)诱变、硫酸二乙酯(DES)单因素实验,确定最佳诱变参数。结果表明,最佳紫外诱变参数为于30 W紫外灯下、距灯30 cm处、紫外照射75 s时,紫外诱变的致死率为78.9%;最佳的硫酸二乙酯诱变参数为将加入1%DES的菌悬液、在35℃的水浴恒温振荡箱中、振荡15 min,硫酸二乙酯诱变的致死率为76.1%。经过3轮连续复合诱变,筛选出4株突变菌株,与出发菌株A9共同进行耐高渗(高糖)实验,当耐高渗(高糖)培养基中的葡萄糖浓度为65%时,出发菌株A9的生长完全被抑制,而突变菌株A9-2在此条件下依旧生长,A9-2的耐高渗(高糖)性能明显高于A9,最终筛选出A9-2为最佳耐高渗(高糖)菌株。通过酒精发酵实验,突变菌株A9-2的10代发酵液,其CO2的总失重量为13.20 g,残糖量为13.9°Bé,酒精度5.1%(v/v),与3代发酵液各项指标相似,确定遗传性能稳定,且菌株发酵力、酒精度明显高于出发菌株A9。     

葡萄糖氧化酶产生菌的紫外诱变及发酵条件优化

利用紫外诱变黑曲霉的方法产生葡萄糖氧化酶并研究酶的最适发酵条件。以酶活力、蓝紫色透明圈的直径为主要指标,确定了紫外诱变法最佳诱变条件为:15 W的紫外灯在照射时间为8 min,照射距离20 cm处,获得1株优良的正向突变株,其产葡萄糖氧化酶的能力是出发菌株的11.8倍。并且通过单因素实验,确定酶的最适发酵条件。实验结果表明:在30℃、5%接种量、转速160 r/min条件下恒温培养72 h,为酶的最适发酵条件,优化后酶活力是优化前的9.56倍。     

γ-聚谷氨酸产生菌的诱变选育

以Bacillus subtilis GXA-28为出发菌株,采用紫外、微波、氯化锂3种单一诱变及紫外-氯化锂,微波-氯化锂两种复合诱变方式进行诱变育种.经过5轮诱变,13次初筛,8次复筛,总共初筛选3554株,复筛选236株突变菌株.获得1株底物(谷氨酸钠)耐受性为出发菌株的2倍;γ-聚谷氨酸产量达24.00g/L左右(比出发菌株高出约33％)的菌株,命名为U-59.经过6代遗传稳定性试验,该菌株性状稳定.     

紫外诱变球毛壳霉选育木聚糖酶高产菌株

木聚糖酶具有重要的潜在应用价值,为获得一株遗传性状稳定的木聚糖酶高产菌株,以木聚糖酶的酶活为评价指标,采用紫外线诱变的方法,利用单因素试验确定紫外线照射时间,通过含木聚糖的初筛培养基对紫外诱变后的菌株进行初筛,然后采用DNS法测定木聚糖酶活性对诱变菌株进行复筛.将筛选出的木聚糖酶活力最高的诱变菌株进行传代培养,测定其酶活,探讨其遗传稳定性.试验结果表明:紫外灯功率20W,照射距离30cm,照射时间120s,球毛壳霉致死率为92.35％.在此条件下进行菌株的紫外诱变获得高产木聚糖酶的正突变株15株,其中1株高产菌株Z30 - 56的酶活为9850IU/mL,比出发菌株提高53.7％,并且遗传性能稳定.     

常压室温等离子体诱变选育生香型Kluyveromyces marxianus

非酿酒酵母(non-Saccharomyces cerevisiae)生长代谢可以为葡萄酒贡献更多风味物质,但发酵能力弱,耐受性差这一特点使其不能过多参与酒精发酵。针对这一问题,该研究采用常压室温等离子体(atmospheric room temperature plasma, ARTP)诱变技术对从葡萄皮表面筛选的马克斯克鲁维酵母(Kluyveromyces marxianus)YK进行诱变,通过诱变菌株耐受性初筛试验和产香复筛试验获得2株耐受性高,产香能力强的诱变菌株,对诱变菌株传代5次测试其传代遗传稳定性。研究结果表明,2株诱变菌株YK-1,YK-29具有较强的遗传稳定性,将2株诱变菌株应用到葡萄酒发酵中产酒精能力较原始菌株YK分别提高了149%、169%。通过定量描述分析,诱变菌株发酵的葡萄酒较原始菌株具有明显的浆果香和水果香气。该研究结论可以为后期非酿酒酵母与酿酒酵母(Saccharomyces cerevisiae)混合发酵提供理论支撑,有望打破葡萄酒品质单一化局面,具有广泛地应用前景。     

超高压诱变选育富锌啤酒酵母菌株的研究

以啤酒酵母为出发菌株,采用超高压诱变方法诱变选育高富集锌的酵母菌株。优化了超高压处理的工艺条件,并基于菌株对锌离子的耐受性和富集性,对菌株进行筛选。结果表明,培养10 h的啤酒酵母菌处于对数生长期,适宜于诱变;超高压处理压力250 MPa、保压时间20 min为诱变的最佳工艺条件;成功选育出一株高富集锌的突变菌株,其细胞锌含量达2.75 mg/g,是出发菌的6.55倍。     

不同酿酒酵母发酵对红葡萄酒中花色苷组成的影响

比较了不同酿酒酵母菌株发酵对红葡萄酒中花色苷组分的影响,为本土酵母的开发应用提供依据。采用本土自筛酿酒酵母菌株Y17和进口商业酵母菌株F15分别酿造干红葡萄酒,并利用高效液相色谱-质谱联用技术(HPLC-MS)对葡萄酒中的花色苷成分进行检测。结果表明,不同酵母发酵葡萄酒中的花色苷成分种类大体相同,但各种花色苷的含量具有较大差别,本土酵母菌株Y17在发酵生产高花色苷含量葡萄酒方面具有优势。     

Effect of gene disruptions of the TCA cycle on production of succinic acid in Saccharomyces cerevisiae

Succinate is the main taste component produced by yeasts during sake (Japanese rice wine) fermentation. The pathway leading to accumulation of succinate was examined in liquid culture in the presence of a high concentration (15%) of glucose under aerobic and anaerobic conditions using a series of Saccharomyces cerevisiae strains in which various genes that encode the expression of enzymes required in TCA cycle were disrupted. When cultured in YPD medium containing 15% glucose under aerobic conditions, the KGD1 (alpha-ketoglutarate dehydrogenase) gene disrupted mutant produced a lower level of succinate than the wild-type strain, while the SDH1 (succinate dehydrogenase) gene-disrupted mutant produced an increased level of succinate. On the other hand, the FUM1 (fumarase) gene disrupted mutant produced significantly higher levels of fumarate but did not form malate at all. These results indicate that succinate, fumarate and malate are mainly synthesized through the TCA cycle (oxidative direction) even in the presence of glucose at a concentration as high as 15%. When the growth condition was shifted from aerobic to anaerobic, the increased level of succinate in SDH1 disruptants was no longer observed, whereas the decreased level of succinate in the KGD1 diruptant was still observed. A double mutant of the two fumarate reductase isozyme genes (OSM1 and FRDS) showed a succinate productivity of 50% as compared to the parent when cells were incubated in glucose-buffered solution. These results indicate that succinate could be synthesized through two pathways, namely, alpha-ketoglutarate oxidation via the TCA cycle and fumarate reduction under anaerobic conditions.     

23 株酿酒酵母ISSR指纹图谱分析及SCAR标记的建立

目的：构建酿酒酵母菌株的简单重复序列间多态性指纹图谱数据库并建立序列特异性扩增区（sequencecharacterized amplified region,SCAR）标记技术,为酿酒酵母菌株的分类、遗传亲缘关系鉴定及菌种专利保护提供可靠的DNA分子标记技术依据。方法：在简单重复序列间多态性（inter-simple sequence repeat,ISSR）指纹数据分析基础上进行聚类分析并对菌种进行分类鉴定,同时将酿酒酵母菌株9号和15号中扩增获得的ISSR特异性DNA带转化为可以直接用于菌株快速鉴定的SCAR分子标记。结果：构建23 株酿酒酵母的ISSR指纹图谱,并在相似系数为0.85水平上将23 个供试菌株分为3大类,其中,1、2、4、7、15、16、17、19、20、21、23聚为第一类;10、11、12、13、14、18号菌株聚为第二类且10号和11号菌为同一菌株;3、5、6、8、9、22号菌聚为第三类且属于同一菌株。此外,利用所获得的2 个特异性条带成功转化为序列特异性扩增区分子标记。结论：在生产上酿酒酵母菌株遗传背景差异不大,常存在同物异名现象,而采用ISSR指纹及其SCAR分子标记技术快速鉴定酿酒酵母菌株在工业生产上具有重要意义。     

A DNA region of Torulaspora delbrueckii containing the HIS3 gene: sequence, gene order and evolution

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae his3 mutant strain. DNA sequence analysis revealed that the fragment contained two complete ORFs, which share a high similarity with S. cerevisiae His3p and Mrp51p, respectively. The cloned TdHIS3 gene fully complemented the his3 mutation of S. cerevisiae, confirming that it encodes for the imidazoleglycerol-phosphate dehydrate of T. delbrueckii. Two additional ORFs, with a high homology to S. cerevisiae PET56 and DED1 genes, were mapped upstream and downstream from TdHIS3 and TdMRP51, respectively. This genetic organization is analogous to that previously found in Saccharomyces kluyveri and Zygosaccharomyces rouxii. The evolutionary significance of gene order in this chromosomal region is analysed and discussed.     

Isolation of a novel mutant strain of Saccharomyces cerevisiae by an ethyl methane sulfonate-induced mutagenesis approach as a high producer of bioethanol

In order to obtain mutant strains showing higher bioethanol production than wild-type strains, a commercial Saccharomyces cerevisiae type was subjected to mutagenesis using ethyl methane sulfonate (EMS). After adding EMS to a shaken yeast suspension, the viability of yeast cells was assessed by diluted sample inoculation to solid yeast-extract peptone glucose (YEPG) medium at 15-min intervals. At 45 min, the viability of yeast cells was estimated to be about 40%. Mutagenized cells were recovered from YEPG broth after incubation at 30 degrees C for 18 h. After this period, EMS-treated yeast cells were grown on solid aerobic low-peptone (ALP) medium containing 2-12% (v/v) ethanol. All plates were incubated at 30 degrees C for 2-6 d in order to form colonies. The mutant strains that tolerated high concentrations of ethanol were selected for bioethanol production in microfuge tubes containing fermentation medium. Formation of bioethanol in small tubes was detected by the distillation-colorimetric method. In addition, trehalose content and invertase activity were determined in each mutant strain. Among many isolated mutant strains, there were six isolated colonies that grew on ALP medium supplemented with 10% (v/v) ethanol and one of them produced bioethanol 17.3% more than the wild type.     

Ynl038wp (Gpi15p) is the Saccharomyces cerevisiae homologue of human Pig-Hp and participates in the first step in glycosylphosphatidylinositol assembly.

Glycosylphosphatidylinositols (GPIs) are found in all eukaryotes and are synthesized in a pathway that starts with the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol (PI). This reaction is carried out by a protein complex, three of whose subunits in humans, hGpi1p, Pig-Cp and Pig-Ap, have sequence and functional homologues in the Saccharomyces cerevisiae Gpi1, Gpi2 and Gpi3 proteins, respectively. Human GlcNAc-PI synthase contains two further subunits, Pig-Hp and PigPp. We report that the essential YNL038w gene encodes the S. cerevisiae homologue of Pig-Hp. Haploid YNL038w-deletion strains were created, in which Ynl038wp could be depleted by repressing YNL038w expression using the GAL10 promoter. Depletion of Ynl038wp from membranes virtually abolished in vitro GlcNAc-PI synthetic activity, indicating that Ynl038wp is necessary for GlcNAc-PI synthesis in vitro. Further, depletion of Ynl038wp in an smp3 mutant background prevented the formation of the trimannosylated GPI intermediates that normally accumulate in this late-stage GPI assembly mutant. Ynl038wp is therefore required for GPI synthesis in vivo. Because YNL038w encodes a protein involved in GPI biosynthesis, we designate the gene GPI15. Potential Pig-Hp/Gpi15p counterparts are also encoded in the genomes of Schizosacchomyces pombe and Candida albicans.     

微卫星在酿酒酵母研究中的应用

微卫星因其在真核生物基因组中分布广泛、多态性丰富、呈选择中性和共显性遗传等特点,已成为继RFLP之后的第2代分子遗传标记,广泛用于酿酒酵母菌株的分类鉴定、菌株间的亲缘关系分析和酿酒酵母群体遗传多样性分析。本文综述微卫星标记的应用原理及其在酿酒酵母研究中的应用进展。     

Role of the sulphate assimilation pathway in utilization of glutathione as a sulphur source by Saccharomyces cerevisiae.

Mutants unable to grow on medium containing glutathione as a sole source of sulphur (GSH medium) were isolated from Saccharomyces cerevisiae strains carrying met17(deficiency of O-acetylserine and O-acetylhomoserine sulphydrylase). They were defective in the high-affinity glutathione transport system, GSH-P1. Newly acquired mutations belonged to the same complementation group, gsh11. However, it became apparent that gsh11 conferred the mutant phenotype not by itself but in collaboration with met17. Moreover, mutations conferring the defect in sulphate assimilation made the cell unable to grow on GSH medium in collaboration with gsh11. From this finding, we propose that the sulphate assimilation pathway acts as a sulphur-recycling system and that this function is especially vital to the cell when the supply of glutathione is limited.