羟酸还原异构酶基因在啤酒工业酵母中的整合表达

采用PCR技术扩增啤酒工业酵母QY中的羟酸还原异构酶基因ILV5,构建了整合载体YIp 5C,整合转化QY,通过铜抗性筛选转化子,所得的转化子的羟酸还原异构酶的活力明显高于对照菌株QY,转化子产生的双乙酰的量比原始菌株降低了40%.     

低双乙酰啤酒酵母工程菌的构建

利用PCR技术以啤酒酵母QY的染色体为模板扩增出含有乙酰羟酸合成酶（AHAS）基因ILV2的片段，将ILV2基因的内部EcoRI片段连接到整合载体YIp5上，并在该载体的Bam HI-SalI位点插入铜抗性基因CUP1－MT1，构建了YIpCE质粒，将其转化啤酒酵母QY，所得到的转化子AHAS酶的活力比受体菌QY降低75％左右， 在发酵测试中，转化了产生双乙酰的量比原始菌株降低30％。     

瑞氏木霉内切葡聚糖酶基因在酿酒酵母中的表达研究

将PCR合成的瑞氏木霉 (Trichodermareesei) β 内切葡聚糖酶I(EGI)cDNA基因片断分别插入酵母met1 0和 pgk1启动子和终止子序列之间 ,构建了在不同启动子控制下 ,具有不同拷贝数的eg1表达分泌质粒pRS41 5ME、pRS41 5PE和 pRS42 5PE。通过电转化使重组质粒转移至实验室酿酒酵母H1 5 8菌株中 ,分别得到了 3株酵母转化子H1 p、H2p和H1m。在 3株酵母转化子中 ,重组 β 内切葡聚糖酶I都能在酶自身信号肽序列引导下进行分泌型表达。在YPD培养基中 3株重组酵母生长速率大致相同 ,H1m ,H1 p与H2 p的内切葡聚糖酶活力分别为 70 .4,1 2 6.7和 1 2 5 .0U/mL。     

木糖发酵酵母Pichia stipitis木糖还原酶基因XYL1在酿酒酵母中的表达

通过PCR方法克隆得到树干毕赤氏酵母木糖还原酶 (XR)基因XYL1。将该基因连入酵母表达载体pYX2 12的强启动子磷酸丙糖异构酶 (TPI)启动子下 ,得到融合表达载体pYX XYL1。通过电转化方法将 pYX XYL1转入酿酒酵母SaccharonmycescerevisiaeW 30 3-1A中 ,酶活测定表明 ,在酿酒酵母中树干毕赤氏酵母木糖还原酶 (XR)基因XYL1得到活性表达 ,2酿酒酵母转化子粗酶液中木糖还原酶活分别为 0 .89U/mg(蛋白 )和 0 .83U/mg(蛋白 ) ,为供体菌的 1 5倍多。与基因供体菌不同 ,木糖还原酶基因在酿酒酵母中表达不需木糖诱导 ,为组成型表达。树干毕赤氏酵母木糖还原酶 (XR)基因XYL1的成功表达为后续的利用木糖的酿酒酵母菌株的构建奠定了基础     

用基因重组技术构建可降解淀粉和产生酒精的酵母工程菌

将酵母Ty转座子的δ序列 ,黑曲霉糖化酶cDNA及G418抗性基因neo重组进酵母整合型质粒YIplac12 8获得含LEU2 ,neo双标记基因 ,糖化酶cDNA的高整合型表达载体YIp12 8D 17N ,转化GRF18后获得整合型酵母转化子GRF18(YIp12 8D 17N)糖化酶基因在该菌株获得高效表达 ,产物分泌到胞外。Southern印迹分析证明 ,糖化酶基因已整合进工程菌染色体。GRF18(YIp12 8D 17N)在含 2 0 %可溶性淀粉的培养基中培养 2 4h ,淀粉水解率在98%以上 ,酒精浓度达到 9 5 % (v/v)在 2 5 %淀粉中酒精浓度达到 10 2 %。     

山河陈醋生产中酵母菌产酒产酯特性研究

为提高山河陈醋的品质,从山河陈醋大曲、酒醪中分离得到12株酵母菌,根据其菌落形态特征、生理生化实验,鉴定出2株汉逊酵母属(Hansenula)、2株克勒克酵母属(Kloeckera)、3株酵母属(Saccharomyces)、3株假丝酵母属(Candida)、1株毕赤酵母属(Pichia)、1株裂殖酵母属(Schizosaccharomyces)。进行了酵母菌株的耐酒精性实验和产酒、产酯性能研究。其中,菌株QY15,LY14,LY27产酒性能好;菌株QY13,QY18产酯性能突出。采用气相色谱-质谱联用仪(GC-MS)对总酯含量高于0.65g/dL的5株酵母高粱发酵液中挥发性成分进行分析,以大曲高粱发酵液为对照。结果表明:菌株QY18高粱发酵液中有7种食醋特征的香气成分物质。     

青岛啤酒酵母和高浓酵母原生质体融合选育高浓酿造菌株

以青岛啤酒酵母和高浓酵母为供试菌株,通过原生质体融合得到融合子。对融合子利用铜抗性初筛,利用耐压和发酵性能为指标进行实验室和100 L发酵复筛,并对融合子进行鉴定及遗传稳定性实验。结果表明：通过原生质体融合选育出的高浓菌株与青岛啤酒酵母菌株相比,表现出酵母数峰值高、降糖和还原双乙酰快的优势,且代谢风味物质组成与青岛啤酒酵母接近;经过连续使用8 代后,其总染色体DNA指纹图谱保持一致,证明该菌株的遗传稳定性高。     

树干毕赤酵母木糖醇脱氢酶基因表达载体构建及在酿酒酵母中的过表达

针对树干毕赤酵母xyl2基因设计相应引物。以p-AKRX为骨架载体,通过酶切连接的方式构建1个酿酒酵母工业菌株的表达载体p-AKRX2-2。将该表达载体线性转化酿酒酵母后,测定转化子SUN-Ⅰ木糖醇脱氢酶(XDH)及其表达情况。与原始菌株相比,重组酿酒酵母中的XDH活力是原始菌株的3.65倍。该重组质粒载体的构建可有效弥补酿酒酵母缺少代谢木糖关键基因的缺陷,为利用木糖高产乙醇酿酒酵母基因工程菌株的构建奠定基础。     

通过啤酒酵母控制联二酮含量ILV5型基因和ILV3型基因的扩增对联二酮含量和ILV酶活性的影响

在发酵过程中通过加速啤酒酵母 ILV 的生物合成途径可使联二酮的前驱体物质含量减到最小量。啤酒酵母中含 ILV5、ILV3、及 ILV5+ILV3基因串联物的质粒扩增可分别使联二酮含量减少70%、40%和60%,经其作用后啤酒的风味和发酵度都很好。ILV5型基因的扩增对减少联二酮总含量是非常有效的。     

稳定高效表达木糖还原酶基因工业酿酒酵母的构建及木糖醇发酵的初步研究

将树干毕赤氏酵母(Pichia stipitis)木糖还原酶基因XYL1连接到适用于酿酒酵母工业菌株的多拷贝整合载体pYMIKP中,构建得到表达质粒pYMIKP-XYL1,转化酿酒酵母工业菌株Saccharomyces cerevisiae6508。在G418平板上筛选转化子,得到含高拷贝木糖还原酶基因的酿酒酵母重组菌株XGH2,,该菌株的木糖还原酶比活力为0.8 U/mg(蛋白),比出发菌株提高了80倍以上,表明外源基因在工业菌株中实现了高效表达。摇瓶发酵结果显示,重组菌株XGH2木糖消耗为27.9 g/L,木糖消耗率为51%;木糖醇产量为30.2 g/L,木糖醇的转化率大于1.0 g/g木糖。     

Construction of self-cloning industrial brewing yeast with high-glutathione and low-diacetyl production

Self‐cloning strains of industrial brewing yeast were constructed, in which one allele of α‐acetohydroxyacid synthase (AHAS) gene (ILV2) was disrupted by integrating Saccharomyces cerevisiae genes, γ‐glutamylcysteine synthetase gene (GSH1) and copper resistant gene (CUP1) into the locus of ILV2. The self‐cloning strains were selected for their resistance to CuSO₄ and identified by PCR amplification. The results of AHAS and glutathione (GSH) assay from fermentation with the self‐cloning strains in 500‐mL conical flask showed that AHAS activity decreased and GSH content increased compared with that of host yeasts. The results of pilot scale brewing in 5‐L fermentation tank also indicated that GSH content in beer fermented with self‐cloning strains T5‐3 and T31‐2 was 1.3 fold and 1.5 fold of that of host QY5 and QY31, respectively; and diacetyl content decreased to 64% and 58% of their hosts, respectively. The self‐cloning strains do not contain any heterologous DNA, they may be more acceptable to the public.     

Vitamin B1 and B2 ratio as a method of brewer's and food yeast identification

The data on vitamins B1 and B2 content in dry brewer's and baker's yeast are submitted. Their significance as these nutrients source is evaluated. Brewer's yeast are mainly the source of vitamin B1 while baker's yeast--the source of vitamin B2. 5 g of yeast give 10 per cent of vitamin B1 and B2 daily recommended allowance correspondingly. Vitamin B1 and B2 ratio has been proposed as a identification criteria of brewer's and baker's yeast. Ratio more than 1.0 gives evidence that this is brewer's yeast. Vitamin B1 content 4 mg per 100 g or more and at the same time vitamin B1 and B2 ratio exceeding 3.0 demonstrate high quality of brewer's yeast.     

Construction of self‐cloning bottom‐fermenting yeast with low vicinal diketone production by the homo‐integration of ILV5

The vicinal diketones (VDK), such as diacetyl and 2,3‐pentandione, impart an unpleasant butter‐like flavour to beer. Typically, these are required to be reduced below the flavour thresholds during the maturation (lagering) stages of the brewing process. To shorten beer maturation time, we constructed a self‐cloning, bottom‐fermenting yeast with low VDK production by integrating ILV5, a gene encoding a protein that metabolizes α‐acetolactate and α‐aceto‐α‐hydroxybutyrate (precursors of VDK). A DNA fragment containing Saccharomyces cerevisiae‐type ILV5 was inserted upstream of S. cerevisiae‐type ILV2 in bottom‐fermenting yeast to construct self‐cloning strains with an increased copy number of ILV5. Via transformation, ILV2 was replaced with the sulfometuron methyl (SM) resistance gene SMR1B, which differs by a single nucleotide, to create SM‐resistant transformants. The wort fermentation test, using the SC‐ILV5‐homo inserted transformant, confirmed a consecutive reduction in VDK and a shortening period during which VDK was reduced to within the threshold. The concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, 1‐propanol, isobutyl alcohol and active isoamyl alcohol (flavour components) were not changed when compared with the parent strain. We successfully constructed self‐cloning brewer's yeast in which SC‐ILV5 was homo‐inserted. Using the transformed yeast, the concentration of VDK in fermenting wort was reduced, whereas the concentrations of flavour components were not affected. This genetically stable, low VDK‐producing, self‐cloning bottom‐fermenting yeast would contribute to the shortening of beer maturation time without affecting important flavour components produced by brewer's yeast. Copyright © 2012 John Wiley & Sons, Ltd.     

富硒啤酒酵母自溶条件的研究

本文研究了富硒啤酒酵母的自溶条件,并利用酶制剂改进其自溶条件,确定了富硒啤酒酵母的最适水解条件。     

Beer brewing using a fusant between a sake yeast and a brewer's yeast

Beer brewing using a fusant between a sake yeast (a lysine auxotrophic mutant of sake yeast K-14) and a brewer's yeast (a respiratory-deficient mutant of the top fermentation yeast NCYC1333) was performed to take advantage of the beneficial characteristics of sake yeasts, i.e., the high productivity of esters, high tolerance to ethanol, and high osmotolerance. The fusant (F-32) obtained was different from the parental yeasts regarding, for example, the assimilation of carbon sources and tolerance to ethanol. A brewing trial with the fusant was carried out using a 100-l pilot-scale plant. The fusant fermented wort more rapidly than the parental brewer's yeast. However, the sedimentation capacity of the fusant was relatively low. The beer brewed using the fusant contained more ethanol and esters compared to that brewed using the parental brewer's yeast. The fusant also obtained osmotolerance in the fermentation of maltose and fermented high-gravity wort well.     

啤酒废酵母制备1,6-二磷酸果糖的研究

啤酒废酵母生产1,6-二磷酸果糖发酵工艺进行了试验性探讨。正交试验结果表明,啤酒废酵母发酵生产FDP的最佳工艺参数为:发酵体系中葡萄糖的加量为0.5mol/L、无机磷的浓度为0.4mol/L、pH6.5、转化时间6h。     

提高酵母抽提物得率的工艺条件

以啤酒废酵母为原料 ,研究了影响酵母抽提物得率的因素和提高酵母抽提物得率的方法。结果表明 ,机械破碎可缩短自溶时间 ,提高酵母抽提物氨基酸态氮含量和得率 ,且以高压均质法破碎效果为好 ;均质条件是 1 0 %酵母悬浮液于 3 5MPa二次均质。均质后在 5 5℃、pH5 5下自溶4h ,添加 0 8%木瓜蛋白酶 (以酵母干重计 )自溶 2 8h ,酵母抽提液中氨基酸态氮含量达 7 1 4g/L ,得率 69 47% ,风味醇厚 ,与以NaCl为自溶促进剂的酵母抽提液相比 ,氨基酸态氮含量增加42 97% ,得率净增 2 1 97%。