酿酒酵母乙醇脱氢酶Ⅱ基因反义干扰及对乙醇发酵的影响

通过构建酿酒酵母沉默表达载体PURH-ADH2,使ADH2基因在PGK强启动子、CYC1终止子在特定区域内进行干扰和表达。采用Bam HI和Xmal I限制性内切酶对SADH2基因和PURH质粒进行双酶切,构建反义重组表达质粒PURH-SADH2,通过高效酵母转化法和电转法将重组质粒组件转化至酿酒酵母SY01细胞中,获得阳性克隆菌株JY01。酿酒酵母JY01突变菌株与出发菌株SY01和Y01发酵试验结果相比,JY01甘油脱氢酶酶活比出发菌株Y01与SY01分别下降了16.31%和13.5%;当酿酒酵母Y01、SY01和JY01菌株发酵36~60 h时,JY01菌株甘油含量相比Y01分别下降了16.34%、14.25%、14.89%;当酿酒酵母突变菌株发酵48 h时,Y01、SY01和JY01的乙醇浓度分别为6.243 g/100 m L、7.145 g/100 m L和7.288 g/100 m L,酿酒酵母JY01发酵液乙醇量比比原始菌株Y01乙醇含量提高了14.33%。结果表明通过反义干扰ADH2基因5’UTR序列,能有效干扰酵母工程菌株ADH2转录与表达,削弱甘油积累途径,促进乙醇代谢途径。     

不同发酵条件对酿酒酵母甘油产量的影响

为提高酿酒酵母的甘油产量,分别考察不同初始葡萄糖和果糖质量浓度、pH值、发酵温度及SO2添加量对酿酒酵母D254甘油产量的影响。对酿酒酵母D254不同发酵初始条件进行单因素试验,其他因素固定条件下,葡萄糖质量浓度180g/L时酵母菌体生长平稳、生长量最高;果糖质量浓度108g/L时酵母甘油产量最高;pH值为3.5更适宜酵母菌体生长和合成甘油;在发酵温度和SO2添加量的单因素试验中也分别得出适宜发酵温度为28℃和适宜SO2添加量为 20mg/L。通过单因素试验,筛选出最利于酿酒酵母D254生长和产甘油的各因素的最佳质量浓度,进行Plackett-Burman发酵条件组合试验,得到发酵条件最佳组合为：初始葡萄糖质量浓度216g/L、果糖质量浓度144g/L、发酵温度32℃、pH 3.0、SO2添加量40mg/L,此条件下,酿酒酵母D254获得最高甘油产量达655.64μmol/L。     

GUT2, a gene for mitochondrial glycerol 3-phosphate dehydrogenase of Saccharomyces cerevisiae

A gut2 mutant of Saccharomyces cerevisiae is deficient in the mitochondrial glycerol 3-phosphate dehydrogenase and hence cannot utilize glycerol. Upon transformation of a gut2 mutant strain with a low-copy yeast genomic library, hybrid plasmids were isolated which complemented the gut2 mutation. The nucleotide sequence of a 3·2 kb PstI-XhoI fragment complementing a gut2 mutant strain is presented. The fragment reveals an open reading frame (ORF) encoding a polypeptide with a predicted molecular weight of 68·8 kDa. Disruption of the ORF leads to a glycerol non-utilizing phenotype. A putative flavin-binding domain, located at the amino terminus, was identified by comparison with the amino acid sequences of other flavoproteins. The cloned gene has been mapped both physically and genetically to the left arm of chromosome IX, where the original gut2 mutation also maps. We conclude that the presented ORF is the GUT2 gene and propose that it is the structural gene for the mitochondrial glycerol 3-phosphate dehydrogenase.     

Fermentation properties of a wine yeast over-expressing the Saccharomyces cerevisiae glycerol 3-phosphate dehydrogenase gene (GPD2)

Wine yeasts efficiently convert sugar into ethanol. The possibility of diverting some of the sugar into compounds other than ethanol by using molecular genetic methods was tested. Over-expression of the yeast glycerol 3-phosphate dehydrogenase gene ( GPD2 ) in a laboratory strain of Saccharomyces cerevisiae led to an approximate two-fold increase in the extracellular glycerol concentration. In the medium fermented with the modified strain, acetic acid concentration also increased approximately two-fold when respiration was blocked. A strain deleted for the GPD2 gene had the opposite phenotype, producing lower amounts of glycerol and acetic acid, with the latter compound only reduced during non-respiratory growth. A commercial wine yeast over-expressing GPD2 produced 16.5 g/L glycerol in a wine fermentation, compared to 7.9 g/L obtained with the parent strain. As seen for the laboratory strain, acetic acid concentrations were also increased when using the genetically modified wine yeast. A panel of wine judges confirmed the increase in volatile acidity of these wines. The altered glycerol biosynthetic pathway sequestered carbon from glycolysis and reduced the production of ethanol by 6 g/L.     

Reduced pyruvate decarboxylase and increased glycerol-3-phosphate dehydrogenase [NAD+] levels enhance glycerol production in Saccharomyces cerevisiae

This investigation deals with factors affecting the production of glycerol in Saccharomyces cerevisiae. In particular, the impact of reduced pyruvate-decarboxylase (PDC) and increased NAD-dependent glycerol-3-phosphate dehydrogenase (GPD) levels was studied. The glycerol yield was 4·7 times (a pdc mutant exhibiting 19% of normal PDC activity) and 6·5 times (a strain exhibiting 20-fold increased GPD activity resulting from overexpression of GPD1 gene) that of the wild type. In the strain carrying both enzyme activity alterations, the glycerol yield was 8·1 times higher than that of the wild type. In all cases, the substantial increase in glycerol yield was associated with a reduction in ethanol yield and a higher by-product formation. The rate of glycerol formation in the pdc mutant was, due to a slower rate of glucose catabolism, only twice that of the wild type, and was increased by GPD1 overexpression to three times that of the wild-type level. Overexpression of GPD1 in the wild-type background, however, led to a six- to seven-fold increase in the rate of glycerol formation. The experimental work clearly demonstrates the rate-limiting role of GPD in glycerol formation in S. cerevisiae.     

CRISPR/Cas9介导的酿酒酵母ADH2基因中断及反义RNA干扰GPD1的表达

CRISPR/Cas9是一个简单、高效的用于靶向目的基因和无标记的基因组工程的工具。本文通过构建酿酒酵母沉默组件PGK-SGPD1-CYC1,使甘油-3-磷酸脱氢酶I(Glycerol-3-phosphate dehydrogenase,GPD1)基因在PGK强启动子、CYC1终止子在特定区域内进行干扰和表达。应用CRISPR/Cas9基因编辑技术,在中断乙醇脱氢酶Ⅱ(alcohol dehydrogenase Ⅱ,ADH2)基因的同时,定点敲入GPD1基因的反义干扰组件,从而特定地干扰GPD1的表达。采用高效的酵母化学转化法将反应组件敲入酿酒酵母Y1H中,CRISPR/Cas9介导的同源重组效率达43.48%,由此获得了ADH2基因中断和GPD1反义干扰的酿酒酵母突变株。发酵实验结果表明,酿酒酵母突变菌株SG1-1与出发菌株Y1H相比,乙醇产率提高了9.07%,甘油产率下降了12.05%,乙酸产率下降了12.30%,结果表明通过中断ADH2基因及插入GPD1反义干扰组件,既能够中断ADH2基因的功能,减少乙醇转化为乙醛,同时也能在一定程度上干扰GPD1基因的表达,提高乙醇产率。     

高乙醇转化率酿酒酵母工程菌株构建研究进展

酿酒酵母(Saccharomyces cerevisiae)发酵产生乙醇的过程中,甘油的生成所消耗的碳源约占总碳源的4%~10%。减少甘油合成量可提高乙醇产率与碳源利用率。其主要策略是修饰或切除一步或多步代谢反应,或引入外源相关基因以改变碳流方向与碳流量,从而使反应向有利于生成更多乙醇而少生成甘油的方向进行。文中主要综述了近年来通过代谢工程手段阻断酿酒酵母甘油的合成或降低甘油的合成量,以提高乙醇发酵糖醇转化率的研究进展。     

Fermentation of Soybean Meal Hydrolyzates with Saccharomyces cerevisiae and Zymomonas mobilis for Ethanol Production

Most of the ethanol currently produced by fermentation is derived from sugar cane, corn, or beets. However, it makes good ecological and economic sense to use the carbohydrates contained in by‐products and coproducts of the food processing industry for ethanol production. Soybean meal, a co‐product of the production of soybean oil, has a relatively high carbohydrate content that could be a reasonable substrate for ethanol production after fermentable sugars are released via hydrolysis. In this research, the capability of Saccharomyces cerevisiae NRRL Y‐2233 and Zymomonas mobilis subsp. mobilis NRRL B‐4286 to produce ethanol was evaluated using soybean meal hydrolyzates as substrates for the fermentation. These substrates were produced from the dilute‐acid hydrolysis of soybean meal at 135 °C for 45 min with 0, 0.5%, 1.25%, and 2% H₂SO₄ and at 120 °C for 30 min with 1.25% H₂SO₄. Kinetic parameters of the fermentation were estimated using the logistic model. Ethanol production using S. cerevisiae was highest with the substrates obtained at 135 °C, 45 min, and 0.5% H₂SO₄ and fermented for 8 h, 8 g/L (4 g ethanol/100 g fresh SBM), while Z. mobilis reached its maximum ethanol production, 9.2 g/L (4.6 g ethanol/100 g fresh SBM) in the first 20 h of fermentation with the same hydrolyzate.     

扁平云假丝酵母与酿酒酵母混合发酵对葡萄酒乙醇含量及香气的影响

为降低葡萄酒中的乙醇含量,采用不同接种方式（同时接种和顺序接种）在两个温度下（13?℃和23?℃）进行扁平云假丝酵母（Candida humilis）与酿酒酵母（Saccharomyces cerevisiae）的发酵实验。结果表明：23 ℃时混合发酵除高产甘油外,还有效降低了乙醇含量,其中与S. cerevisiae单独发酵相比,顺序接种可降低乙醇体积分数约2.59%,降醇幅度达到17.56%。在香气方面,混合发酵能产生较高含量的酯类物质,其中顺序接种最为明显,如乙酯类物质总量和乙酸乙酯含量分别增加了18.30%和16.03%,丁酸乙酯含量提高了2.77?倍,明显增加了花香和果香;13?℃时,混合发酵显著提高了β-大马士酮的含量,其中同时接种发酵增加26.19%。结果表明C. humilis与S. cerevisiae进行混合发酵具有较好的降醇效果,可以为葡萄酒的降醇研究提供一种新的解决方法。     

面包酵母高糖发酵力与蔗糖酶活力关系的研究

本文研究了面包酵母高糖耐性与蔗糖酶活性的关系。通过对八株酵母菌株的蔗糖酶活性和高糖面团发酵力比较分析,其中ADY2蔗糖酶酶活最大,BY-6最小,分别为128.70 U/g干酵母和30.55 U/g干酵母,而在高糖面团中发酵力却是BY-6最大,ADY2最小,CO2的产生量分别为850 ccm和225 ccm,证实了较低蔗糖酶活性的面包酵母菌株具有在高糖面团中发酵力较高的特性。通过测定蔗糖酶酶活相差较大的株菌BY-6和ADY2在蔗糖模拟面团中的蔗糖消耗和葡萄糖积累曲线,结果表明ADY2不仅蔗糖消耗速度比BY-6快,且其积累葡萄糖的速度比BY-6快,同时所积累的最高葡萄糖量也比BY-6高,分别为5.89?10-2和4.50?10-2 g/mL。此外,即便是蔗糖酶酶活低且高糖面团发酵力大菌株BY-6在蔗糖模拟面团培养基中仍有较多葡萄糖积累,因此选育蔗糖酶水解生成葡萄糖速度与其利用葡萄糖速度一致或相差不大的菌株是我们选育耐高糖面包酵母菌株的一个控制靶点。     

GPD1过表达调控葡萄酒酵母酒精和风味物质的生成量

本研究以葡萄酒酵母WY1为出发菌株,探究GPD1基因过表达对葡萄酒酵母菌产酒精能力及风味物质的影响。利用分子生物学技术,采用PCR技术扩增葡萄酒酵母WY1中甘油-3磷酸脱氢酶基因GPD1,构建了单拷贝GPD1,两拷贝GPD1和三拷贝GPD1重组菌株。利用Real-TimePCR技术对GPD1基因表达量进行检测,与出发菌株WY1相比,单拷贝GPD1,两拷贝GPD1和三拷贝GPD1重组菌株中GPD1基因的表达水平分别提高了1.75倍、3.02倍和3.42倍。葡萄酒发酵实验数据显示,GPD1基因的过表达对乙醇的降低有显著效果,单拷贝GPD1,两拷贝GPD1和三拷贝GPD1重组菌株分别比WY1降低了8.07%,14.36%和15.34%;总高级醇含量分别减少了15.20%、17.11%和16.55%;乙酸乙酯的含量分别下降了17.63%、23.81%和27.42%,乙酸异戊酯含量分别下降了11.78%、15.87%、17.79%;甘油生成量分别提高了1.02倍、1.80倍和1.83倍。本研究表明过表达GPD1基因可以影响葡萄酒酵母产酒精的能力,同时对改善葡萄酒风味有重要意义。     

酿酒酵母发酵过程中褪黑素及其同分异构体变化规律的研究

本文研究了A、B、F三株酿酒酵母在模拟葡萄汁发酵过程中褪黑素及同分异构体产生及变化的规律,并初步探讨了与酵母酒精发酵进程的关系,最后用草莓、蓝莓、桑葚、树莓四种浆果发酵进行了验证。结果发现褪黑素含量在发酵的第1 d达到最大值,A、B、F菌含量分别为0.773、0.647、0.825 ng/m L,随后迅速下降。褪黑素同分异构体含量在发酵的第3 d达到最大值,A、B、F菌含量分别为34.89、19.24、26.79 ng/m L,后期逐渐下降,且同分异构体含量远高于褪黑素(p0.01)。草莓、蓝莓、桑葚、树莓发酵前后褪黑素含量无显著性差异,同分异构体含量分别达到64.4、7.37、81.9、55.5 ng/m L,均显著高于褪黑素的含量。总之,褪黑素及其同分异构体在酒精发酵初期产生,中后期逐渐下降,推测与酵母发酵初期逆境代谢密切相关。     

Glycerol formation during wine fermentation is mainly linked to Gpd1p and is only partially controlled by the HOG pathway

Glycerol 3-phosphate dehydrogenase, a key enzyme in the production of glycerol, is encoded by GPD1 and GPD2. The isoforms encoded by these genes have different functions, in osmoregulation and redox balance, respectively. We investigated the roles of GPD1, GPD2 and HOG1-the kinase involved in the response to osmotic stress-in glycerol production during wine fermentation. We found that the deletion of GPD2 in a wine yeast-derived strain did not affect growth or fermentation performance and reduced glycerol production by only 20%. In contrast, a gpd1delta mutant displayed a prolonged lag phase, and produced 40% less glycerol than the wild-type strain. The deletion of HOG1 resulted in a slight decrease in growth rate and a 20% decrease in glycerol production, indicating that the HOG pathway operates under wine fermentation conditions. However, the hog1delta mutant was not as severely affected as the gpd1delta mutant during the first few hours of fermentation, and continued to express GPD1 strongly. The hog1delta mutant was able to increase glycerol production in response to high sugar concentration (15-28% glucose), to almost the same extent as the wild-type, whereas this response was totally abolished in the gpd1delta mutant. These data show that Gpd1p plays a major role in glycerol formation, particularly during the first few hours of exposure to high sugar concentration, and that GPD2 is only of little significance in anaerobic fermentation by wine yeast. The results also demonstrate that the HOG pathway exerts only limited control over GPD1 expression and glycerol production during wine fermentation.     

桓仁产区优良冰酒酵母菌株的筛选及酿造特性评价（英文）

为获得可用于东北桓仁地区威代尔冰酒生产的酿酒酵母菌株,采用一种快速的酵母菌株筛选方法,通过测定菌株乙醇和二氧化硫耐受性,从威代尔冰酒自然发酵过程中筛选到9株酿酒酵母菌株。进一步酿造实验结果显示,所筛选的酿酒酵母可以顺利完成冰酒发酵,产生的香气轮廓与商业酵母DV10相比也不同(主成分分析结果),最终获得了2株具有高发酵活力且香气特征与商业酵母差异显著的酵母菌株SC42和SC45,其中SC42能够高产高级醇和酯类物质,并且低产乙酸,而SC45能够产生高含量的甘油、酯类物质以及反式玫瑰醚和β-大马士酮。结果表明,采用本研究的筛选方法能够快速有效地筛选到具有应用潜力的冰酒生产菌株,同时也证明了使用本土野生酵母菌株能够有效地改善冰酒香气品质,生产出与接种商业酵母不同风格的冰酒产品。     

The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains

Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wild-type level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD(+) into NADP(+) and NADH, and TH-overproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization. Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulose-derived inhibitors. Low G6PDH-activity strains were also more sensitive to H(2)O(2) than the control strain TMB3001.     

内蒙古乌海地区沙漠葡萄发酵醪液中酿酒酵母菌的筛选与鉴定

以内蒙古乌海地区沙漠葡萄自然发酵醪液为筛选来源,共分离纯化出22株酵母菌,通过复筛得到1株优良酵母菌KT3,经26S r DNA D1/D2区测序,鉴定为酵母菌属(Saccharomyces)的酿酒酵母菌(Saccharomyces cerevisiae);该酵母菌具有良好的耐受能力,耐酒精度为16%,耐SO2浓度为450 mg/L,耐高糖浓度为50%,耐pH值为2.5;并且具备高产酒精和低产硫化氢的能力,发酵产品风味协调,典型性强,可以用于葡萄酒的生产。     

Study of Two Pools of Glycogen in Saccharomyces cerevisiae and their Role in Fermentation Performance

Glycogen in Saccharomyces cerevisiae is present in two pools as cell wall bound and intracellular glycogen. The content of the cell wall bound glycogen was found to be almost three times higher than the intracellular glycogen. The content varied with the sugar concentration in the medium and an optimum value of 22.11 mg glycogen/g yeast was observed for the cell wall bound glycogen, while it was 7 mg/g yeast for the intracellular glycogen at a 12% medium sugar content. The two pools also varied with fermentation time reaching an optimal value at 36 h of fermentation. The cell wall bound glycogen was reduced by 85% during the first three hours after pitching, when sugar uptake was minimal and started to accumulate when almost 50% of the medium sugar was utilized. It was the cell wall bound glycogen that correlated with fermentation performance. Cells grown in 8% sugar content and rich in cell wall bound glycogen, when pitched into a 1% sugar medium showed an enhancement in ethanol content by 21%. The depletion of glycogen also affected fermentation performance.     

异常威克汉姆酵母与酿酒酵母在混合发酵中的相互作用机制

为研究异常威克汉姆酵母(Wickerhamomyces anomalus,Wa)K-008在混菌发酵中的应用潜力以及与酿酒酵母(Saccharomyces cerevisiae,Sc)ZM-005共发酵中的相互作用机制,使用保藏的两株菌进行混合发酵,以纯接种Sc和Wa作为对照,通过监测发酵体系中不同菌种的生物量、葡萄糖、乙醇、发酵时间以及CO₂质量损失变化探究其相互作用方式。研究发现,在混合发酵24 h后,Wa菌株出现生长停滞和死亡的现象,但Sc菌株生长未受到明显影响,初步排除Wa早期死亡现象是Wa自溶和凋亡,再通过发酵上清液实验和乙醇耐受实验,发现Wa菌株在发酵前期出现的死亡现象不是营养物质缺乏以及Sc产生有毒代谢物的作用。分别通过添加高浓度Sc细胞、破碎Sc细胞和死Sc细胞进行混合发酵,发现Wa在前期出现死亡现象是由于体系中存在高浓度的活Sc细胞,而与死亡和破碎Sc细胞无关。进一步通过透析实验探究其作用机制,透析袋将Sc与Wa细胞分离,避免两者之间出现接触,实验发现Sc和Wa都能在体系中正常生长。结果表明,在本实验设计的混合发酵体系中,Wa在混合发酵前期出现生...