酿酒酵母PDEs基因缺失对蓝莓果酒抗氧化及挥发性物质的影响

比较野生型酿酒酵母(Saccharomyces cerevisiae)与环核苷酸磷酸二酯酶PDE1和PDE2基因敲除的突变菌株(PDE1/Δpde1、Δpde1/Δpde1、PDE2/Δpde2和Δpde2/Δpde2)对蓝莓果酒发酵特性及品质的影响。测定了蓝莓果酒发酵醪液总糖、酒精度、总酸、pH值、DPPH自由基清除能力、羟自由基清除能力、ABTS自由基清除能力、总酚、总黄酮和花青素等指标。采用顶空固相微萃取技术(HS-SPME)与气相色谱-质谱联用(GC-MS)相结合的方法对蓝莓果酒挥发性物质进行比较。探究了PDE1和PDE2基因对酿酒酵母的发酵特性的影响。结果表明:Δpde1/Δpde1菌株能有效提高蓝莓果酒发酵速度,酿造的蓝莓果酒活性物质含量最高,抗氧化能力最强;PDE1和PDE2基因的缺失会降低发酵蓝莓果酒的抗氧化能力,且敲除PDE2的酿酒酵母酿造的蓝莓果酒的抗氧化能力比敲除PDE1的更弱,而Δpde1/Δpde1突变菌株酿造的果酒中香气成分含量较多。     

酿酒酵母SOD1、SOD2基因缺失对胁迫耐受性的影响

以sod1Δ、sod2Δ、sod1Δsod2Δ酿酒酵母基因缺失菌株为遗传材料,采用休止细胞梯度生长法,分析SOD1和SOD2基因缺失对高温、乙醇毒性、高渗透压、高盐、乙酸毒性及营养饥饿胁迫条件耐受性的影响。结果显示,与野生型菌株相比,sod1Δ菌株对高温、高渗透压和乙酸胁迫的耐受性降低;sod2Δ菌株耐受性无明显变化;sod1Δsod2Δ双缺失菌株对高温、乙醇毒性、乙酸毒性、高渗透压和高盐的耐受性均下降,表明酵母超氧化物歧化酶基因与多种胁迫耐受性密切相关。     

BAT基因改造对酿酒酵母高级醇生成量的影响

酿酒酵母BAT基因编码支链氨基酸转氨酶,其中BAT1和BAT2基因分别编码线粒体和细胞质氨基酸转氨酶,位于细胞不同的位置导致二者的生理功能有所差异,BAT1基因在线粒体中倾向催化α-酮酸合成氨基酸,细胞质中的BAT2基因将氨基酸转化为α-酮酸,通过敲除BAT2以减少α-酮酸合成,过表达BAT1以增加α-酮酸消耗达到降低酿酒酵母高级醇的合成的目的。本研究以酿酒酵母AY15单倍体α5为出发菌株,结合融合PCR技术构建重组质粒p UC-BABPB1K,获得BA-PGK-BAT1-BB重组盒,并利用醋酸锂转化法和同源重组技术筛选出缺失BAT2基因同时过表达BAT1基因的突变株B-8,将其和亲本菌株α5、BAT2基因缺失菌株α5ΔBAT2进行酒精发酵实验,发酵结束后进行发酵性能和高级醇的测定。实验结果表明,与亲本菌株相比,异丁醇降低了25%,异戊醇降低了15%,活性戊醇降低了30%;与α5ΔBAT2菌株相比,异丁醇提高了0.5倍,异戊醇增加了0.1倍,活性戊醇增加了0.3倍。     

CRISPR/Cas9介导的果糖低消耗酿酒酵母工程菌的构建

利用酿酒酵母为宿主,以果糖为原料合成D-阿洛酮糖具有食品方面的先天优势。为了减少酿酒酵母宿主本身对果糖的消耗,对酿酒酵母的己糖激酶同工酶2(Hexokinaseisoenzyme2,hxk2)基因进行编辑。本研究采用CRISPR/Cas9技术,构建了Cas9和gRNA共表达质粒p YES2-CG-Δhxk2,以酿酒酵母BY4741为出发菌株,URA3为筛选标记,采用高效电击转化法和胞内同源重组技术,获得hxk2基因缺陷株BY4741-Δhxk2。在此基础上,进行果糖发酵实验以评估突变菌株的果糖消耗速率。实验结果显示,发酵培养14 h时,缺陷株BY4741-Δhxk2果糖消耗速率为3.35 mg/h;与野生型菌株相比其下降了6.42%。此外,发酵培养22 h,BY4741-Δhxk2的OD600nm值为8.65,相比于野生型提高了6.40%。研究表明,己糖激酶hxk2基因的缺陷编辑可以一定程度降低酿酒酵母对果糖的利用,同时缺陷株较野生型表现出一定的生长优势,这为后续以酿酒酵母为宿主生产D-阿洛酮糖奠定了初步基础。     

酿酒酵母ICT1基因敲除对其耐盐性的影响

该研究以酿酒酵母(Saccharomyces cerevisiae)BY4741为出发菌,采用同源重组技术构建一株ICT1基因缺失菌株,分析ICT1基因敲除对酿酒酵母耐盐性的影响。分别采用梯度点滴实验、碘化丙啶(PI)荧光染色法、实时荧光定量聚合酶链式反应(RT-QPCR)等技术分析ICT1基因在酿酒酵母耐盐机制中的作用。结果表明,成功获得ICT1基因缺失菌株BY4741-ΔICT1。与出发菌BY4741相比,菌株BY4741-ΔICT1的盐耐受性减弱。经1.0 mol/L Na Cl处理后,麦角固醇含量减少66.9%,细胞膜受损,细胞膜上的转运蛋白基因NHA1和ENA1的转录水平分别下降65.6%和90.5%。说明ICT1基因的敲除使菌株BY4741-ΔICT1的盐耐受性下降可能与胞内麦角固醇合成减少、膜受损、离子转运蛋白基因的转录水平下降有关。     

TPK3基因敲除对酿酒酵母耐受性的影响

酿酒酵母是乙醇发酵时常用的微生物,在发酵过程中会受到高温、乙醇、渗透压、乙酸等胁迫环境的影响。为增强酵母对胁迫环境的耐受,该试验以AY12a为出发菌株,利用胞内同源重组,敲除编码蛋白激酶A（protein kinase A,PKA）催化亚基的TPK3基因,构建TPK3基因敲除菌株AY12a-2。利用敲除TPK3基因的分子操作来调控PKA活性,以此来提升酵母耐受性。耐受性测定结果表明,该菌株的耐热击性能、耐乙酸性能及耐盐性明显优于出发菌株。发酵数据表明,AY12a-2的葡萄糖消耗量增加及CO2的失重量提升34%,乙醇产量增长了17.8%。     

扣囊复膜酵母菌高产突变株转化可溶性淀粉生产海藻糖的研究

以前的研究曾发现扣囊复膜酵母菌 (Saccharomycopsisfibuligerasdu)能利用可溶性淀粉发酵累积质量浓度为 180 g/L的海藻糖。然而 ,该菌株含有高活性的酸性和中性海藻糖酶 ,使发酵过程中海藻糖的累积下降。为了提高海藻糖的产量 ,去除海藻糖酶的活性是必要的。野生型菌株通过EMS的诱变作用 ,筛选出了 1株不能同化海藻糖 ,但生长速率不变的突变株。该突变株与野生型菌株相比能够利用淀粉发酵累积更高含量的海藻糖 ,与野生型菌株相比 ,突变株细胞的酸性海藻糖酶的活性低 3倍 ,中性海藻糖酶的活性低 3 7倍。这意味着酸性和中性海藻糖酶活性的降低对该突变株的海藻糖产量的提高起关键作用     

群体感应系统对单增李斯特菌生物被膜形成的影响

以单增李斯特菌（Listeria monocytogenes）为研究对象,分析Agr群体感应系统和LuxS/AI-2系统对其生物被膜形成的调控作用。通过同源重组对LMB33426菌株进行agrD基因以及luxS基因的无痕敲除,比较野生菌株与agrD、luxS基因缺失菌株的生物被膜特性差异。结果表明,与野生株相比,ΔagrD突变株和ΔluxS突变株的生物被膜形成能力下降;ΔagrD突变株的疏水性显著下降,在37 ℃下的泳动能力较野生株增强;群体感应系统基因敲除对菌株的耐药性没有产生较大影响。基因缺失株的构建为进一步研究群体感应系统对单增李斯特菌生物被膜形成的调控机制提供参考,同时为单增李斯特菌的预防控制奠定了基础。     

LEU1基因缺失对酿酒酵母高级醇生成量的影响

LEU1基因编码异丙基苹果酸合成酶,为了研究该基因对酿酒酵母高级醇生成量的影响,以酿酒酵母AY15单倍体A8为出发菌株,通过构建重组质粒p UC-LABK获得LA-Kan MX-LB重组盒,并利用醋酸锂转化法和同源重组技术,筛选出LEU1基因缺失的突变株A-L9。将突变株和亲本菌株分别进行酒精发酵实验,发酵结束后进行发酵性能和高级醇生成量的测定。两种发酵条件下的实验结果表明,与亲本菌株相比,突变株的正丙醇生成量分别提高了0.18倍和0.47倍,异丁醇的生成量分别提高了0.52倍和1.58倍,而异戊醇的生成量则分别降低了0.29倍和0.51倍。     

ATF1过表达和BAT2敲除酿酒酵母发酵性能的研究

以啤酒酵母工业菌株S5为对照,对过表达醇乙酰基转移酶编码基因ATF1同时敲除氨基酸转氨酶编码基因BAT2酿酒酵母工程菌株S5-1进行发酵性能的研究。结果表明,与出发菌株相比,突变株生长状况、发酵速度、酒精度等基本发酵性能没有明显变化,而突变株发酵后的乙酸酯总量有较大程度的提升,为85.44mg/L,提高了6.96倍,高级醇总量有较大程度的下降,为79.25mg/L,降低了27.28%。研究结果为啤酒风味的改善奠定了良好的基础。     

A knockout strain of CPR1 induced during fermentation of Saccharomyces cerevisiae KNU5377 is susceptible to various types of stress

To investigate the tolerance factor of Saccharomyces cerevisiae KNU5377 against various types of environmental stress during fermentation, we identified the protein that is upregulated at high temperatures. The highly upregulated protein was high-score-matched as a cytoplasmic peptidyl-prolyl cis-trans isomerase, cyclophilin (Cpr1p), by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). We constructed a CPR1-deleted KNU5377 strain (KNU5377Y cpr1Delta) to determine the roles of the protein under fermentative or stress condition. The growth of the S. cerevisiae KNU5377Y cpr1Delta strain was completely inhibited under the following conditions: heat (40 degrees C), hydrogen peroxide (20-30 mM), menadione (0.3 mM), ethanol (16%), sulfuric acid (5 mm), and lactic acid (0.4-0.8%). However, the wild-type and cpr1Delta mutant of S. cerevisiae BY4741 as a positive control did not show differences in sensitivity to stress. It is interesting to note that the wild-type KNU5377Y and KNU5377Y cpr1Delta mutant showed high sensitivity against various stresses, particularly, acid stress such as in the presence of sulfuric and lactic acid. Although the alcohol fermentation rate of the KNU5377Y cpr1Delta mutant markedly decreased with an increase in temperature up to 40 degrees C, we observed no decrease in that of the wild-type strain under the same conditions. These results suggest that CPR1 contributes to the stress tolerance of KNU5377 against various types of environmental stress caused during fermentation, thus leading to the physiological role of maintaining an alcohol fermentation yield, even at high temperatures such as 40 degrees C.     

TOR1基因缺失对酿酒酵母耐受性的影响

酿酒酵母是最常见且应用最为广泛的酵母菌种,是以糖质和淀粉质为原料的乙醇发酵最经典的菌株.在发酵过程中,有很多不可避免的胁迫环境如高温条件、高渗条件等出现,这些胁迫会阻碍细胞生长并降低细胞的发酵能力,给发酵行业带来一定的经济损失.因此,为改善菌种的耐受性,该研究主要以实验室现有菌株AY12a为亲本菌株,URA3基因作筛选...     

GIS1基因对酿酒酵母耐受性的研究

该研究主要通过过表达GIS1基因来提高酿酒酵母的耐受性。在GIS1基因的N端加入强启动子PGK1p来实现GIS1基因的过表达,然后通过稀释点板实验来对比其对热激、乙醇、渗透压以及乙酸的耐受性,同时通过高温生长曲线和高温浓醪发酵测定其高温耐受性。结果表明,在相同的稀释倍数下,55 ℃热击4 min之后菌株AY12a-gis1的生长能力明显优于出发菌株,在含有5%（V/V）乙酸的平板上改造菌和出发菌耐受性相差不大,同时通过38 ℃浓醪发酵实验发现菌株AY12a-gis1的酒精度提高了3.79%,残糖略有下降,且发酵时间与亲本菌株相一致。因此通过过表达GIS1基因得到菌株AY12a-gis1,是对工业乙醇发酵有一定应用价值的优良耐逆性菌株。     

Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene

Glycerol is a major fermentation product of Saccharomyces cerevisiae that contributes to the sensory character of wine. Diverting sugar to glycerol overproduction and away from ethanol production by overexpressing the glycerol 3-phosphate dehydrogenase gene,GPD2, caused S. cerevisiae to produce more than twice as much acetic acid as the wild-type strain (S288C background) in anaerobic cell culture. Deletion of the aldehyde dehydrogenase gene, ALD6, in wild-type and GPD2 overexpressing strains (GPD2-OP) decreased acetic acid production by three- and four-fold, respectively. In conjunction with reduced acetic acid production, the GPD2-OP ald6Delta strain produced more glycerol and less ethanol than the wild-type. The growth rate and fermentation rate were similar for the modified and wild-type strains, although the fermentation rate for the GPD2 ald6Delta strain was slightly less than that of the other strains from 24h onwards. Analysis of the metabolome of the mutants revealed that genetic modification affected the production of some secondary metabolites of fermentation, including acids, esters, aldehydes and higher alcohols, many of which are flavour-active in wine. Modification of GPD2 and ALD6 expression represents an effective strategy to increase the glycerol and decrease the ethanol concentration during fermentation, and alters the chemical composition of the medium such that, potentially, novel flavour diversity is possible. The implications for the use of these modifications in commercial wine production require further investigation in wine yeast strains.     

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...     

Expression of the AZR1 gene (ORF YGR224w), encoding a plasma membrane transporter of the major facilitator superfamily, is required for adaptation to acetic acid and resistance to azoles in Saccharomyces cerevisiae

In this work, we report results on the functional analysis of Saccharomyces cerevisiae ORF YGR224w, predicted to code for an integral membrane protein, with 14 potential transmembrane segments, belonging to the major facilitator superfamily (MFS) of transporters which are required for multiple-drug resistance (MDR). This MFS-MDR homologue is required for yeast adaptation to high stress imposed by low-chain organic acids, in particular by acetic acid, and for resistance to azoles, especially to ketoconazole and fluconazole; the encoding gene was thus named the AZR1 gene. These conclusions were based on the higher susceptibility to these compounds of an azr1Delta deletion mutant strain compared with the wild-type and on the increased resistance of both azr1Delta and wild-type strains upon increased expression of the AZR1 gene from a centromeric plasmid clone. AZR1 gene expression reduces the duration of acetic acid-induced latency, although the growth kinetics of adapted cells under acetic acid stress is apparently independent of AZR1 expression level. Fluorescence microscopy observation of the distribution of the Azr1-GFP fusion protein in yeast living cells indicated that Azr1 is a plasma membrane protein. Studies carried out to gain some understanding of how this plasma membrane putative transporter facilitates yeast adaptation to acetic acid did not implicate Azr1p in the alteration of acetic acid accumulation into the cell through the active efflux of acetate.     

利用同源重组技术调节啤酒中SO2含量研究

二氧化硫在啤酒中具有抗氧化的重要功能,亚硫酸盐还原酶(MET10编码)在啤酒酵母硫代谢过程中起重要作用。利用同源重组技术,采用醋酸锂转化法,将一段目的基因转入到酵母体内,从而获得一株亚硫酸盐还原酶基因突变的工业酿酒酵母。突变株通过驯养,对比发酵栓试验,结果表明在发酵结束时,突变菌株的SO2产量是出发菌株的1.5倍。     

常温常压等离子诱变结合玉米秸秆水解液驯化酿酒酵母生产生物乙醇

因玉米秸秆水解液抑制物中的糠醛和乙酸通常会抑制酿酒酵母的活力,造成乙醇产量下降。该实验为了获得抗水解抑制物并且提高乙醇产量的优良菌株,以酿酒酵母xp为出发菌株,通过常温常压等离子诱变(atmospheric and room-temperature plasma mutagenesis,ARTP)技术,得到突变体库,并以玉米秸秆水解液为筛选压力,经过25代驯化培养,筛选出优良菌株xp2。该菌体的生物量DCW最高为3. 71 g/L,较原菌的3. 10 g/L上升了19. 68%。生长对数期为6～22 h,稳定期为22～42 h,比原菌的对数期明显提前。稳定期持续时间延长4 h,生长性能优势明显。发酵上清液中的糠醛1. 43 g/L,乙酸1. 21 g/L,较出发菌株发酵上清液的糠醛3. 78 g/L,乙酸1. 65 g/L,分别降低了62. 17%和26. 67%,乙醇产量和平均得率分别为38. 7 g/L和0. 806 g/(L·h),较出发菌株的30. 3 g/L和0. 631 g/(L·h),提高了17. 12%和27. 73%。该实验表明改造菌性状优良,转化糠醛和乙酸的能力明显...     

己酸菌对高产酯酿酒酵母酒精发酵及酯醇代谢的影响

将己酸菌与高产酯酿酒酵母在高粱汁培养基中进行混合发酵,研究己酸菌对高产酯酿酒酵母酒精发酵及酯醇代谢的影响。结果表明,己酸菌菌悬液对高产酯酿酒酵母的生长及代谢没有直接影响;己酸菌的代谢产物对高产酯酿酒酵母的发酵速度及酒精产量有所促进,在培养基己酸浓度为220mg/L时,酒精产量提高了6.63%;在己酸浓度为45~220mg/L时,高产酯酿酒酵母的乙酸酯类和主要高级醇受到的抑制作用逐渐增强,乙酸乙酯最多降低65.32%,乙酸异丁酯最多降低91.32%,乙酸异戊酯最多降低82.14%,高级醇最多降低71.14%;在己酸浓度为220mg/L时,与己酸浓度为0mg/L时对比,高产酯酿酒酵母共有1032个基因转录水平上调,有1037个基因转录水平下调,这些转录水平变化的基因主要涉及到高产酯酿酒酵母的氮代谢、糖酵解、苯丙氨酸代谢、碳代谢等多条代谢途径。     

转录因子MSN2基因过表达对酿酒酵母耐受性的影响

cAMP信号通路在调控酵母细胞代谢、增殖、分化及压力抗性的获得过程中具有重要的作用。工业应用中对酵母的耐受性有很高的要求,在发酵过程中,胁迫环境如高温、高渗透压、营养饥饿和高浓度酒精毒性等不可避免,故而提高酵母菌种的耐受性,可以提高菌种的发酵性能,降低发酵过程中的能量消耗。本文构建的突变株在工业应用方面具有重要的意义。以实验室现有菌种AY12a为出发菌株,URA3基因作筛选标记,利用胞内重组,在MSN2基因的N端加上强启动子PGK1_p以实现基因的过表达,最终通过PCR验证,成功构建突变株AY12a-msn2。对酵母进行耐受性的测定,发现AY12a-msn2不具有一定的耐高温性能。同时将突变株与AY12a进行玉米高温浓醪发酵,并测定发酵完成后的酒度、残糖、48 h细胞存活率、CO_2失重及发酵时间。结果发现突变株AY12a-msn2酒度下降,残糖含量上升,48 h细胞存活率上升,发酵时间较长。