INFLUENCE OF THE CONCENTRATION OF BRANCHED CHAIN AMINO ACIDS ON THE FORMATION OF FUSEL ALCOHOLS

The influence of concentrations of branched-chain amino acids on the total, anabolic and catabolic production of isobutyl, active amyl, and isoamyl alcohol by Saccharomyces cerevisiae X 2180-1A has been examined in media with high nitrogen levels. It has been found that the total and catabolic production of higher alcohols increased with increasing concentrations of the corresponding amino acids. The anabolic pathway can be suppressed by high levels of the corresponding amino acid. Lower concentrations up to 1 mm for isoleucine and 10mm for leucine stimulate the anabolic formation of active amyl alcohol and isoamyl alcohol respectively. From the data obtained, it is shown that, under the conditions used, 50% or more of the fusel alcohols may arise in a beer wort fermentation from carbohydrates.     

酿酒酵母高级醇合成路径及关键基因

高级醇是在酒精发酵过程中由酿酒酵母代谢产生,在酿酒酵母细胞质和线粒体中由基因编码相关的酶催化α-酮酸脱羧还原而成,是酒的重要香气成分,对酒的香气和口感有重要影响。该文综述了高级醇代谢的相关途径及部分关键基因及功能,以期深入分析了解高级醇并为通过分子生物学方式构建适用于工业生产的低产高级醇的酵母菌株提供理论参考。     

酒中高级醇调控的研究进展

酒作为大众喜爱的饮品,人们对其需求量和品质的要求在不断提高。高级醇是酒中最主要的呈味物质之一,适量的高级醇可赋予酒体醇香的口感、增加酒体协调性,而高级醇含量过高或过低均会影响酒体的口感,当高级醇含量超过一定限度时还会对饮用者的身体健康造成损害。因此,调控酒中高级醇的含量及比例对有效提高酒体品质、促进酒产业健康发展具有重要意义。该文系统阐述酒中高级醇的形成机制,总结发酵工艺、酿酒酵母菌株以及菌株选育对高级醇的影响,旨在为酒工业化生产中高级醇精细化调控提供参考。     

Regulation of cystathionine gamma-lyase in Saccharomyces cerevisiae.

Regulation of the two enzymes in reverse trans-sulfuration was investigated in Saccharomyces cerevisiae. In wild-type strains, cystathionine gamma-lyase, but not cystathionine beta-synthase, was depressed nearly 15-fold if cells were starved for both inorganic and organic sulfur compounds. In a met17 strain which is defective of O-acetylserine and O-acetylhomoserine sulfhydrylase, the same enzyme was derepressed if organic sulfur compounds were limited; the repressive effect was in the order of glutathione greater than methionine greater than cysteine. The repressive effect of methionine was not observed, however, in a cys2 cys4 strain which is deficient of serine O-acetyltransferase and cystathionine beta-synthase, indicating that methionine itself is not the effector. The weak repressive effect of cysteine was attributed to inefficient uptake of this amino acid. Our observations indicate that cystathionine gamma-lyase is the target of regulation in reverse trans-sulfuration and that cysteine is very likely to be the effector of this regulation.     

啤酒发酵中代谢工程的初步研究

将代谢工程理论应用于啤酒的发酵过程,通过构建啤酒发酵过程中酵母的代谢网络模型,对啤酒发酵过程进行代谢通量分析。研究不同压力下啤酒酵母代谢能力的变化,并对其进行了代谢通量分析,结果表明,高压可对酵母在啤酒发酵过程中的代谢产生一定的抑制作用。     

两株不同体积酵母酿酒过程中的代谢网络分析

构建了啤酒酵母的代谢网络,通过代谢通量分析的方法,对两株不同体积的酵母进行啤酒发酵过程的代谢网络分析。结果显示体积较小的酵母BB细胞合成能力较强,酿造乙醇的速率较快,并且双乙酰合成较少。     

Bicarbonate-mediated social communication stimulates meiosis and sporulation of Saccharomyces cerevisiae

Meiosis and sporulation in the yeast Saccharomyces cerevisiae requires social communication, mediated by an extracellular factor which is secreted from cells during sporulation and accumulates in a cell density-dependent manner. We show here genetic and biochemical analyses supporting our conclusion that the extracellular factor is bicarbonate acting as an alkali to elevate extracellular pH. Sporulation defects of mdh1 (mitochondrial malate dehydrogenase) mutants and of wild-type cells at low density were rescued extracellularly by addition of bicarbonate or other alkaline solutions to raise medium pH. Addition of bicarbonate (or alkalization of medium) raised steady-state levels of mRNA in respiration-deficient mdh1 mutants and inhibited proliferation of wild-type cells at low density. These results indicate that the two conditions (respiration competency and high cell density), required for meiosis and sporulation, are essential for extracellular accumulation of bicarbonate and resulting alkalization of medium. © 1998 John Wiley & Sons, Ltd.     

Overproduction of glycolytic enzymes in yeast

Eight different enzyymes for glycolysis and alcoholic fermentation were overproduced in a common Saccharomyces cerevisiae strain by placing their genes on multicopy vectors. The specific enzyme activities were increased between 3·7-and 13·9-fold above the wild-type level. The overproduction of the different glycolytic enzymes had no effect on the rate of ethanol formation, even with those enzymes that catalyse irreversible steps: hexokinase, phosphofructokinase and pyruvate kinase. Also the simultaneous increase in the activities of pairs of enzymes such as pyruvate kinase and phosphofructokinase or pyruvate decarboxylase and alcohol dehyrogenase, did not increase the rate of ethanol production. The levels of key glycolytic metabolites were also normal, compared to the reference strain.     

代谢工程改造酿酒酵母生产L-苹果酸

为了研究胞质还原路径对酿酒酵母积累L-苹果酸的影响,通过在酿酒酵母中过量表达源于黄曲霉的丙酮酸羧化酶(Afpyc)、苹果酸脱氢酶(Afmdh)及C4-二羧酸转运蛋白(Afmae),成功构建了L-苹果酸合成的胞质还原路径。结果表明:(1)当低水平表达Afpyc时,其丙酮酸浓度降低了42%,但不能积累L-苹果酸;(2)当共表达Afpyc和Afmdh时,菌株W005积累了1.93 g/L的L-苹果酸,与对照菌株W004相比细胞干重提高了350%,丙酮酸降低了65.9%;(3)当共表达Afpyc、Afmdh和Afmae时,菌株W006的L-苹果酸产量提高了21.2%,达到2.34 g/L;4)通过提高接种量至初始OD_(600)=2,L-苹果酸的产量提高到3.28 g/L。通过在酿酒酵母中过量表达黄曲霉胞质还原路径的关键基因,使得工程菌能够积累L-苹果酸,为目标产物的高效积累提供了一种可借鉴的思路。     

Synchronization affector of autonomous short-period-sustained oscillation of Saccharomyces cerevisiae

When the yeast Saccharomyces cerevisiae was grown under aerobic continuous culture, an autonomous shortperiod-sustained oscillation appeared. This oscillation was observed in concentrations of various extracellular and intracellular parameters, such as ethanol, acetate, glycogen, dissolved oxygen and intracellular pH. In this work the synchronization affecter of this oscillation was investigated. Ethanol was found not to be the synchronizer of the oscillation because a pulse of ethanol did not affect the phase or period of the oscillation. The oscillation was dependent on the aeration rate, i.e., the oscillation occurred only between 150 and 600 ml min⁻¹. However, the oxygen concentration did not influence synchronization as an upward shift in the oxygen concentration of the gas flow did not affect the sustainability of the oscillation. On the other hand, synchronization was stopped by an enhanced gas flow rate, keeping dissolved oxygen tension at the oscillatory condition, suggesting that synchronization was caused by a volatile compound in the culture. A stepwise increase in carbon dioxide concentration of the gas flow rate ceased synchronization, yet the oscillation seem to continue in each individual cell. Oscillatory behaviour of intracellular pH and carbon dioxide evolution rate showed a phase difference of 90 degrees. Based on these facts it is postulated that carbon dioxide, through the influence of its dissociation on intracellular pH, could be the synchronization affector of the autonomous short-period-sustained oscillation of S. cerevisiae.     

酿酒酵母异源合成柠檬烯的研究进展

植物萜类化合物是以异戊二烯为结构单位的一大类植物天然的次生代谢产物。柠檬烯属于单萜类化合物,具有抑菌、增香、抗癌、止咳、平喘等多种功能,因此在食品、药品、化妆品、医疗等领域具有广泛的应用前景。目前柠檬烯的生产主要是从植物中提取,受到季节性原材料、产物分离纯化复杂、产率低等因素的限制,而化学合成又存在能耗高、污染严重等缺点。随着合成生物学技术的兴起,诞生了以微生物生物合成法生产柠檬烯的新方法,该方法具有能耗低、绿色环保、可持续等优势。然而微生物法合成柠檬烯也存在低产量、低效率等问题,这就限制了其商业化,因此构建高效的异源合成柠檬烯微生物细胞工厂,实现微生物发酵法替换传统的植物提取法,具有重要的经济与社会效益。本文主要回顾了近几年利用代谢工程改造酿酒酵母异源合成柠檬烯取得的成就,阐述了以酿酒酵母作为底盘微生物,利用代谢工程和合成生物学的手段构建高产柠檬烯的合成策略,还讨论了如何减轻柠檬烯对宿主细胞的毒性和提高宿主对柠檬烯的耐受性。     

利用酿酒酵母工程菌株生产2,3-丁二醇的 研究进展

2,3-丁二醇在食品、化妆品、医药和运输燃料等行业具有广泛的应用,因而提高2,3-丁二醇的产量一直备受研究者们关注。目前,研究的热点主要集中于利用微生物发酵可再生资源生产2,3-丁二醇以取代传统的化学合成法,并取得了较大的进展。常见的2,3-丁二醇产生菌有克雷伯氏菌属和芽孢杆菌属,它们能有效利用可再生资源高效生产2,3-丁二醇。然而这些细菌被认为是潜在的病原菌,难以应用于大规模生产。因此,研究者们又将目光转向了酿酒酵母。就安全性和工业化生产要求而言,酿酒酵母是生产2,3-丁二醇的理想菌种。本文对国内外的相关研究进行了总结,介绍了酿酒酵母产2,3-丁二醇的优势和不足,2,3-丁二醇合成代谢途径及其基因工程菌株构建的方向,以及通过代谢工程将酿酒酵母改造成能高效、高质、高量产生2,3-丁二醇的理想菌株的研究关键。