酿酒酵母对酚类抑制物耐受性研究

通过短期驯化策略提高酿酒酵母对木质纤维素预处理产生的酚类物质的耐受性。考察酚酸对酵母的抑制作用,比较驯化菌株在酚酸中生理指标,通过流式细胞仪分析酵母细胞膜完整性。单一酚酸低浓度对酿酒酵母生长和发酵没有明显抑制作用,而高浓度抑制强烈。混合酚酸具有更强的抑制作用,特别对乙醇发酵影响显著。相比原始菌株,短期驯化菌株在混合酚酸胁迫下的生长发酵动力学参数明显提高,细胞膜保持良好的完整性。酚酸对酿酒酵母生长有直接抑制作用,短期驯化能提高酵母酚酸耐受性,这与细胞膜应激反应保持其完整性有关。     

酿酒酵母乙醇耐性的分子机制及基因工程改造

提高工业微生物对毒性代谢产物及高温等环境胁迫因素的耐受性对工业生产具有重要的意义。发酵过程中产生的乙醇对酵母细胞的生长和代谢都具有较强的抑制作用,是酿酒酵母的重要环境胁迫因素之一。对酿酒酵母乙醇耐性的分子机制的研究可为选育具有较强乙醇耐受性的酵母菌种提供理论基础。近年来,通过细胞全局基因转录分析和基因功能分析,对酿酒酵母乙醇耐性的分子机制有了更多新的认识,揭示了很多新的与乙醇耐性相关的基因,并在此基础上,通过对相关基因进行过量表达或敲除,成功提高了酵母菌的乙醇耐性。以下综述了近年来酵母菌乙醇耐性的生物化学与分子生物学机制的研究进展,以及构建具有较高乙醇耐性的酵母菌的基因工程操作。这些研究不仅加深了对酿酒酵母乙醇耐性的机理认识,也可为高效进行生物转化生产生物质能源奠定理论基础。     

一株耐受低pH、高浓度乳酸及琥珀酸菌株的筛选与鉴定

【目的】通过构建的人工耐酸系统,筛选耐受低pH值、乳酸及琥珀酸的菌株。【方法】构建人工耐酸系统长期驯化菌株,利用不同p H的酸性平板进行筛选,从环境中筛选出一株对低p H值、高浓度乳酸以及琥珀酸有很好耐受性的菌株。通过形态学特征、生理生化特征研究,并结合18S rDNA基因序列分析及分子系统发育树的构建结果,确定菌株的种类。【结果】经过酸性人工系统的长期驯化,筛选分离出一株耐受低pH值、高浓度乳酸以及琥珀酸的菌株WJ-2,经鉴定该菌株为酿酒酵母(Saccharomyces cerevisiae),其最适生长温度为30°C。酸性平板实验显示该菌株能够耐受pH2.5的酸性环境,同时对9%的乳酸及8%的琥珀酸也有很好的耐受性。另外,耐酸菌株WJ-2在pH 2.5、9%乳酸和8%琥珀酸的培养环境中仍能保持相对中性的细胞内pH值。【结论】通过构建人工酸性系统,成功筛选出一株对低pH值、高浓度乳酸以及琥珀酸具有耐受性的菌株——酿酒酵母菌WJ-2,该方法可为筛选具有特定耐受能力菌株提供一个新思路。     

紫外诱变结合驯化提高酿酒酵母对抑制物耐受性

[目的]提高工业酿酒酵母Sc4126对木质纤维素预处理过程中产生的发酵抑制物的耐受性。[方法]采用紫外诱变结合驯化对工业酿酒酵母Sc4126进行选育,对筛选出的三株菌株Sc4126-1、Sc4126-2、Sc4126-3在复合抑制剂和单一抑制剂存在下对比考查其发酵性能。[结果]相比原始菌株,3株菌株对抑制剂的耐受性均不同程度地提高。在复合抑制剂存在下,优势菌株Sc4126-1发酵时间为48 h,乙醇平均产率0.19 g/L·h,而原始菌株Sc4126发酵时间138 h,乙醇平均产率0.06 g/L·h,相比原始菌株,优势菌株发酵时间缩短65.22%,乙醇平均产率提高2.17倍。对于单一抑制剂,改造后的菌株对糠醛、香草醛的耐受性明显提高,而对乙酸的耐受性提高较少。[结论]通过紫外诱变结合驯化的方法,有效提高了工业酿酒酵母Sc4126对抑制剂的耐受性。     

酿酒酵母乙酸耐性分子机制的功能基因组进展

提高工业酿酒酵母对高浓度代谢产物及原料中的毒性底物等环境胁迫因素的耐受性,对提高工业生产效率具有重要的意义。乙酸是纤维素原料水解产生的主要毒性副产物之一,其对酵母细胞的生长和代谢都具有较强的抑制作用,因此,对酿酒酵母乙酸耐性分子机制的研究可为选育优良菌种提供理论依据。近年来,通过细胞全局基因表达分析和代谢组分析,以及对单基因敲除的所有突变体的表型组研究,对酿酒酵母乙酸耐性的分子机制有了更多新的认识,揭示了很多新的与乙酸毒性适应性反应和乙酸耐性提高相关的基因。综述了近年来酿酒酵母乙酸耐性的基因组规模的研究进展,以及在此基础上构建乙酸耐性提高的工业酵母菌的代谢工程操作。结合本课题组的研究,对金属离子锌在酿酒酵母乙酸耐性中的作用进行了深入分析。未来对酿酒酵母乙酸耐性分子机理的认识及改造将深入到翻译后修饰和合成生物学等新的水平,所获得的认知,将为选育可高效进行纤维素原料生物转化、高效生产生物燃料和生物基化学品的工业酿酒酵母的菌株奠定理论基础。     

生物质转化工程酿酒酵母的研究进展

石油、煤炭等非再生资源的供给日益紧张,人类迫切需要对可再生资源的利用进一步开发。以生物质为原料生产纤维素乙醇具有原料可再生,成本低廉的显著优点。酿酒酵母菌株因其易于进行基因操作,并具有较高的抑制剂耐受性,被广泛用于发酵生产纤维素乙醇。酿酒酵母作为生物质转化工程菌,在如何提升菌株对抑制剂的耐受性和木糖转化效率等方面仍具有很大的挑战。综述了在生物质转化利用上酿酒酵母工程化的研究进展。     

酿酒酵母芳樟醇耐受性的工程改造

【背景】芳樟醇具有特殊的香气和多种生物学活性,是食品、医药和化妆品行业的重要原料。随着合成生物学的高速发展,代谢改造微生物进行芳樟醇生物合成是当前研究的一大热点。然而在微生物的生物合成中,芳樟醇对底盘细胞的毒性是一大瓶颈问题,也是其他单萜物质生物合成的共性问题。【目的】建立合理的耐受性改造方法,以提高微生物宿主细胞对芳樟醇的耐受性。【方法】以酿酒酵母BY4741为研究对象,通过对ABC转运蛋白、活性氧调控相关酶及转录调控因子的过表达,考察它们对酿酒酵母芳樟醇耐受性的影响,并通过对酿酒酵母细胞进行定向驯化,筛选耐受性提高的酿酒酵母突变株。【结果】单独过表达ABC转运蛋白(Yor1、Snq2、Pdr5、Pdr15和Pdr18)、ROS调控相关酶(Gre2、Ctt1、Yhb1、Gpx2、Trr1、Trx2和Gsh2)及转录调控因子(Ino2、Yap1、Yap5和Stb5)并不能有效提高酿酒酵母的耐受性,但在传代适应性驯化过程中获得了两株耐受性提高的酿酒酵母突变株,将芳樟醇的致死浓度从430mg/L提高到了645mg/L以上。进一步通过基因组重测序分析揭示了驯化菌株突变位点。其中YBR074W...     

过表达MRP8提高酿酒酵母乙酸耐性及乙醇发酵效率

乙酸是木质纤维素水解液中含量较多的抑制物,因此提高酿酒酵母菌株对乙酸的耐受性有助于提高纤维素乙醇生产效率。本文中,笔者利用基于CRISPR/Cas9系统的基因组编辑技术过表达了酿酒酵母(Saccharomyces cerevisiae)S288c线粒体核糖体蛋白编码基因MRP8,并比较了过表达MRP8的菌株与对照菌株的生长和发酵特性。平板耐性检测发现,MRP8过表达明显提高了菌株的乙酸胁迫耐受性;乙醇发酵结果表明,在4.8 g/L乙酸胁迫条件下,过表达菌株MRP8-3在51 h消耗全部的葡萄糖,发酵时间缩短了25 h,显著优于相同时间的对照菌株。本研究结果为构建高效纤维素乙醇发酵的酿酒酵母菌株提供了新思路。     

利用人工锌指文库选育高乙醇耐受性工业酵母菌株

选育高乙醇耐性的酿酒酵母菌株对提高燃料乙醇的发酵效率具有重要意义.锌指蛋白广泛存在于多种生物中,对基因的转录和翻译起重要的调节作用.利用人工设计的锌指蛋白可定向设计锌指序列及其排列顺序,实现对细胞内多个基因的全局调控.由于与环境胁迫反应相关的基因很多,因此可利用人工锌指蛋白技术获得耐受性提高的微生物重组菌.文中将人工锌指文库转入到酿酒酵母模式菌株S288c,选育了具有高乙醇耐受性的重组菌株M01,并分离了与乙醇耐受性提高相关的人工锌指蛋白表达载体pRS316ZFP-M01,转入工业酿酒酵母Sc4126,在含有不同浓度乙醇的平板上,工业酵母Sc4126的重组菌株表现出显著的耐受性提高.在高糖培养基(250 g/L)条件下进行乙醇发酵,发现重组菌的乙醇发酵效率明显快于野生型,发酵时间提前24 h,且发酵终点乙醇浓度提高6.3％.结果表明人工锌指文库能够提高酵母的乙醇耐受性,为构建发酵性能优良的酵母菌种奠定了基础.     

酿酒酵母耐受单萜类化合物的机理研究进展

增强酿酒酵母对单萜的耐受性对于利用其生产单萜和利用含有单萜的生物质均具有重要意义.深入了解酿酒酵母应对单萜胁迫机理有助于构建一株较高单萜耐受性的酵母菌株,该菌株将有助于更高效率的单萜生产效率.研究表明,单萜会破坏酿酒酵母体内的氧化还原平衡,造成活性氧积累并进而导致菌体死亡.为了应对单萜诱发氧胁迫造成的损伤,酿酒酵母需要系统提升其抗氧化能力.本文归纳了酿酒酵母耐受多种典型单萜化合物胁迫机制的研究进展,并从酿酒酵母自身抗氧化机制方面,介绍了酿酒酵母应对氧胁迫的策略,并提出了进一步研究的方向.     

Involvement of manganese in conversion of phenylalanine to benzaldehyde by lactic acid bacteria

We examined the involvement of Mn(II) in the conversion of phenylalanine to benzaldehyde in cell extracts of lactic acid bacteria. Experiments performed with Lactobacillus plantarum demonstrated that Mn(II), present at high levels in this strain, is involved in benzaldehyde formation by catalyzing the conversion of phenylpyruvic acid. Experiments performed with various lactic acid bacterial strains belonging to different genera revealed that benzaldehyde formation in a strain was related to a high Mn(II) level.     

Involvement of Manganese in Conversion of Phenylalanine to Benzaldehyde by Lactic Acid Bacteria

We examined the involvement of Mn(II) in the conversion of phenylalanine to benzaldehyde in cell extracts of lactic acid bacteria. Experiments performed with Lactobacillus plantarum demonstrated that Mn(II), present at high levels in this strain, is involved in benzaldehyde formation by catalyzing the conversion of phenylpyruvic acid. Experiments performed with various lactic acid bacterial strains belonging to different genera revealed that benzaldehyde formation in a strain was related to a high Mn(II) level.     

Enhanced benzaldehyde tolerance in Zymomonas mobilis biofilms and the potential of biofilm applications in fine-chemical production

Biotransformation plays an increasingly important role in the industrial production of fine chemicals due to its high product specificity and low energy requirement. One challenge in biotransformation is the toxicity of substrates and/or products to biocatalytic microorganisms and enzymes. Biofilms are known for their enhanced tolerance of hostile environments compared to planktonic free-living cells. Zymomonas mobilis was used in this study as a model organism to examine the potential of surface-associated biofilms for biotransformation of chemicals into value-added products. Z. mobilis formed a biofilm with a complex three-dimensional architecture comprised of microcolonies with an average thickness of 20 microm, interspersed with water channels. Microscopic analysis and metabolic activity studies revealed that Z. mobilis biofilm cells were more tolerant to the toxic substrate benzaldehyde than planktonic cells were. When exposed to 50 mM benzaldehyde for 1 h, biofilm cells exhibited an average of 45% residual metabolic activity, while planktonic cells were completely inactivated. Three hours of exposure to 30 mM benzaldehyde resulted in sixfold-higher residual metabolic activity in biofilm cells than in planktonic cells. Cells inactivated by benzaldehyde were evenly distributed throughout the biofilm, indicating that the resistance mechanism was different from mass transfer limitation. We also found that enhanced tolerance to benzaldehyde was not due to the conversion of benzaldehyde into less toxic compounds. In the presence of glucose, Z. mobilis biofilms in continuous cultures transformed 10 mM benzaldehyde into benzyl alcohol at a steady rate of 8.11 g (g dry weight)(-1) day(-1) with a 90% molar yield over a 45-h production period.     

Enhanced Benzaldehyde Tolerance in Zymomonas mobilis Biofilms and the Potential of Biofilm Applications in Fine-Chemical Production

Biotransformation plays an increasingly important role in the industrial production of fine chemicals due to its high product specificity and low energy requirement. One challenge in biotransformation is the toxicity of substrates and/or products to biocatalytic microorganisms and enzymes. Biofilms are known for their enhanced tolerance of hostile environments compared to planktonic free-living cells. Zymomonas mobilis was used in this study as a model organism to examine the potential of surface-associated biofilms for biotransformation of chemicals into value-added products. Z. mobilis formed a biofilm with a complex three-dimensional architecture comprised of microcolonies with an average thickness of 20 μm, interspersed with water channels. Microscopic analysis and metabolic activity studies revealed that Z. mobilis biofilm cells were more tolerant to the toxic substrate benzaldehyde than planktonic cells were. When exposed to 50 mM benzaldehyde for 1 h, biofilm cells exhibited an average of 45% residual metabolic activity, while planktonic cells were completely inactivated. Three hours of exposure to 30 mM benzaldehyde resulted in sixfold-higher residual metabolic activity in biofilm cells than in planktonic cells. Cells inactivated by benzaldehyde were evenly distributed throughout the biofilm, indicating that the resistance mechanism was different from mass transfer limitation. We also found that enhanced tolerance to benzaldehyde was not due to the conversion of benzaldehyde into less toxic compounds. In the presence of glucose, Z. mobilis biofilms in continuous cultures transformed 10 mM benzaldehyde into benzyl alcohol at a steady rate of 8.11 g (g dry weight)⁻¹ day⁻¹ with a 90% molar yield over a 45-h production period.     

Genetic aspects of ethanol tolerance and production bySaccharomyces cerevisiae

We developed a method of hybrid selection between homothallic wild-type and heterothallic strains. The hybrids obtained were used to study the heredity of ethanol tolerance and production. Both characters segregated independently, but no ethanol-sensitive strains were able to produce high levels of ethanol. At least four genes are implicated in ethanol tolerance.     

模拟人体胃肠道环境筛选益生乳杆菌

【目的】筛选具有益生特性的乳杆菌作为保健型酸奶的候选菌株。【方法】从健康人肠道和奶豆腐中分离筛选出耐受人工胃液的乳杆菌,对其进行体外益生特性(人工胃肠液耐受性、胆盐耐受性、抑菌活性及胆固醇降解能力)研究。【结果】从在乳杆菌分离培养基上有溶钙圈的41株菌株中筛选出5株耐酸、耐人工胃液较强的菌株,经16S rR NA基因测序鉴定,其中3株为乳杆菌,分别命名为植物乳杆菌Lp MT-3、植物乳杆菌Lp MT-5和唾液乳杆菌LsA F-7。在人工胃液中3株菌的耐受力均强于商品化的对照菌株LGG(鼠李糖乳杆菌GG);转入肠液4 h后直至26 h,Lp MT-5存活率基本稳定在45%左右,仅次于LGG。胆盐浓度为0.10%时,3株乳杆菌的耐胆盐能力均强于LGG;胆盐浓度为0.20%时,Lp MT-3和LsA F-7仍能存活。3株乳杆菌均具有抑菌活性,对粪肠球菌的抑制最明显,其次是金黄色葡萄球菌,对大肠杆菌、沙门氏菌的抑制作用较差。3株乳杆菌对胆固醇的清除效力依次为Lp MT-3LpM T-5Ls AF-7;清除率依次为Ls AF-7Lp MT-3LpM T-5。【结论】筛选出3株适应人体胃肠液环境、耐胆盐、抑菌及降胆固醇活力强的乳杆菌,可作为进一步开发新的益生菌产品和保健型酸奶的菌株。     

Two benzaldehyde dehydrogenases in bacterium N.C.I.B. 8250. Distinguishing properties and regulation

Evidence is presented for the existence in bacterium N.C.I.B. 8250 of two inducible NAD⁺-linked benzaldehyde dehydrogenases. They may be distinguished in crude extracts by their different thermal stabilities at high pH values, benzaldehyde dehydrogenase I being much more heat-stable than benzaldehyde dehydrogenase II. Only benzaldehyde dehydrogenase I is activated by K⁺ and certain other univalent cations. Gel-filtration experiments indicate that both enzymes have molecular weights of about 180000. Both enzymes are induced by growth on l-mandelate or phenylglyoxylate; only benzaldehyde dehydrogenase I is gratuitously induced by thiophenoxyacetate and only benzaldehyde dehydrogenase II is induced by benzyl alcohol, by benzaldehyde, and by a number of heterocyclic compounds which do not support growth. Mutants have been isolated that lack either benzaldehyde dehydrogenase II or benzyl alcohol dehydrogenase, or both of the enzymes. Results obtained in induction experiments with the wild-type bacterium N.C.I.B. 8250 and with the mutants show that benzaldehyde dehydrogenase II and benzyl alcohol dehydrogenase are co-ordinately regulated. Overall, the results suggest that benzaldehyde dehydrogenase I is associated with the metabolism of l-mandelate whereas benzaldehyde dehydrogenase II is associated with the metabolism of benzyl alcohol.     

Yeast pyruvate decarboxylases: variation in biocatalytic characteristics for (R)-phenylacetylcarbinol production

Based on previous studies, Candida utilis pyruvate decarboxylase (PDC) proved to be a stable and high productivity enzyme for the production (R)-phenylacetylcarbinol (PAC), a pharmaceutical precursor. However, a portion of the substrate pyruvate was lost to by-product formation. To identify a source of PDC which might overcome this problem, strains of four yeasts -- C. utilis, Candida tropicalis, Saccharomyces cerevisiae and Kluyveromyces marxianus -- were investigated for their PDC biocatalytic properties. Biotransformations were conducted with benzaldehyde and pyruvate as substrates and three experimental systems were employed (in the order of increasing benzaldehyde concentrations): (I) aqueous (soluble benzaldehyde), (II) aqueous/benzaldehyde emulsion, and (III) aqueous/octanol-benzaldehyde emulsion. Although C. utilis PDC resulted in the highest concentrations of PAC and was the most stable enzyme, C. tropicalis PDC was associated with the lowest acetoin formation. For example, in system (III) the ratio of PAC over acetoin was 35 g g(-1) for C. tropicalis PDC and 9.2 g g(-1) for C. utilis PDC. The study thereby opens up the potential to design a PDC with both high productivity and high yield characteristics.     

Isolation and characterization of ethanol tolerant yeast strains

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains.