Pleiotropic functions of the yeast Greatwall-family protein kinase Rim15p: a novel target for the control of alcoholic fermentation

Rim15p, a Greatwall-family protein kinase in yeast Saccharomyces cerevisiae, is required for cellular nutrient responses, such as the entry into quiescence and the induction of meiosis and sporulation. In higher eukaryotes, the orthologous gene products are commonly involved in the cell cycle G₂/M transition. How are these pleiotropic functions generated from a single family of protein kinases? Recent advances in both research fields have identified the conserved Greatwall-mediated signaling pathway and a variety of downstream target molecules. In addition, our studies of S. cerevisiae sake yeast strains revealed that Rim15p also plays a significant role in the control of alcoholic fermentation. Despite an extensive history of research on glycolysis and alcoholic fermentation, there has been no critical clue to artificial modification of fermentation performance of yeast cells. Our finding of an in vivo metabolic regulatory mechanism is expected to provide a major breakthrough in yeast breeding technologies for fermentation applications.     

A loss-of-function mutation in the PAS kinase Rim15p is related to defective quiescence entry and high fermentation rates of Saccharomyces cerevisiae sake yeast strains

Sake yeast cells have defective entry into the quiescent state, allowing them to sustain high fermentation rates. To reveal the underlying mechanism, we investigated the PAS kinase Rim15p, which orchestrates initiation of the quiescence program in Saccharomyces cerevisiae. We found that Rim15p is truncated at the carboxyl terminus in modern sake yeast strains as a result of a frameshift mutation. Introduction of this mutation or deletion of the full-length RIM15 gene in a laboratory strain led to a defective stress response, decreased synthesis of the storage carbohydrates trehalose and glycogen, and impaired G(1) arrest, which together closely resemble the characteristic phenotypes of sake yeast. Notably, expression of a functional RIM15 gene in a modern sake strain suppressed all of these phenotypes, demonstrating that dysfunction of Rim15p prevents sake yeast cells from entering quiescence. Moreover, loss of Rim15p or its downstream targets Igo1p and Igo2p remarkably improved the fermentation rate in a laboratory strain. This finding verified that Rim15p-mediated entry into quiescence plays pivotal roles in the inhibition of ethanol fermentation. Taken together, our results suggest that the loss-of-function mutation in the RIM15 gene may be the key genetic determinant of the increased ethanol production rates in modern sake yeast strains.     

Phenolic composition and radical scavenging activity of sweetpotato-derived shochu distillery by-products treated with koji

Phenolic composition and radical scavenging activity in the shochu distillery by-products of sweetpotato (Ipomoea batatas L.) treated with koji (Aspergillus awamori mut.) and cellulase (Cellulosin T2) were investigated to develop new uses. Koji and Cellulosin T2 treatment of shochu distillery by-products from sweetpotatoes, rice, and barley increased phenolic content. Caffeic acid was identified as a dominant phenolic component in the shochu distillery by-products of the sweetpotato. Adding koji and/or Cellulosin T2 to the shochu distillery by-product indicated that koji was involved in caffeic acid production. Caffeic acid was not detected in raw or steamed roots of "Koganesengan", the material of sweetpotato for shochu production, suggesting that it is produced during shochu fermentation. The phenolic content and radical scavenging activity the shochu distillery by-product treated with koji and Cellulosin T2 were superior to those of commercial vinegar. These results suggest that koji treatment of sweetpotato-derived shochu distillery by-products has potential for food materials with physiological functions. Further koji treatment of sweetpotato shochu-distillery by-products may be applicable to mass production of caffeic acid.     

商业酵母在不同品种葡萄酒工业化生产中的定殖差异

[背景]酵母菌在葡萄酒酿造中起到重要的作用,接种商业活性干酵母(active dry yeast,ADY)进行葡萄酒酿造在国内较为普遍,然而商业酿酒酵母(Saccharomyces cerevisiae)对我国本土酵母菌资源的影响及二者竞争关系的相关报道不多.[目的]比较商业酿酒酵母在不同品种葡萄酒工业化生产中的定殖差...     

接种发酵和自然发酵中酿酒酵母菌株多样性比较

【目的】探究自然发酵和接种发酵两种发酵方式,对霞多丽葡萄发酵中酵母菌种多样性和酿酒酵母菌株遗传多样性的影响。【方法】以霞多丽葡萄为原料,分别进行自然发酵和接种不同酿酒酵母菌株(NXU17-26、UCD522和UCD2610)的发酵,利用26S rDNA D1/D2区序列分析和Interdelta指纹图谱技术分别进行酵母菌的种间及种内水平的区分,通过聚类分析及多样性指数对不同发酵方式下酿酒酵母菌株的多样性进行分析和比较。【结果】自然发酵的发酵曲线较平缓,接种发酵的发酵速度显著快于自然发酵。26S rDNA D1/D2区序列分析将4个发酵中分离到的酵母菌鉴定为6属11种,自然发酵中分离的酵母有5属6种,均为非酿酒酵母(non-Saccharomyces);而接种发酵中的酵母多样性远低于自然发酵,均由酿酒酵母和两种非酿酒酵母组成。Interdelta指纹图谱分析表明,接种UCD2610的发酵中,发酵后UCD2610是优势菌株,其基因型占比为48.78%;接种NXU17-26和UCD522的发酵中,未发现与NXU17-26和UCD522相同的基因型。聚类分析表明,分离自接种UCD522发酵中的酿酒酵母菌株间的遗传差异性较小;而分离自NXU17-26和UCD2610发酵中的酿酒酵母菌株间遗传差异性较大。多样性指数结果表明,接种UCD2610发酵中的优势菌株(UCD2610)在发酵过程中占据更加突出的地位;接种UCD522发酵中分离的酿酒酵母具有更高的多样性,影响其菌株多样性的未知因素较多,且不同基因型酿酒酵母的集中度较高。【结论】发酵方式对霞多丽葡萄发酵中酵母菌种多样性、以及酿酒酵母菌株遗传多样性的影响显著,研究结果对葡萄酒发酵中的微生物控制具有指导意义。     

Yeast mannan increases Bacteroides thetaiotaomicron abundance and suppresses putrefactive compound production in in vitro fecal microbiota fermentation

ABSTRACT

Yeast mannan is a part of yeast cell wall and can potentially affect gut microflora as a soluble dietary fiber. We demonstrated that yeast mannan suppressed putrefactive production and increased the relative abundance of Bacteroides thetaiotaomicron in in vitro fecal fermentation. These results suggest that yeast mannan can be used as a novel prebiotic food ingredient.     

Acquired resistance to severe ethanol stress-induced inhibition of proteasomal proteolysis in Saccharomyces cerevisiae

BackgroundAlthough the budding yeast, Saccharomyces cerevisiae, produces ethanol via alcoholic fermentation, high-concentration ethanol is harmful to yeast cells. Severe ethanol stress (> 9% v/v) inhibits protein synthesis and increases the level of intracellular protein aggregates. However, its effect on proteolysis in yeast cells remains largely unknown.MethodsWe examined the effects of ethanol on proteasomal proteolysis in yeast cells through the cycloheximide-chase analysis of short-lived proteins. We also assayed protein degradation in the auxin-inducible degron system and the ubiquitin-independent degradation of Spe1 under ethanol stress conditions.ResultsWe demonstrated that severe ethanol stress strongly inhibited the degradation of the short-lived proteins Rim101 and Gic2. Severe ethanol stress also inhibited protein degradation in the auxin-inducible degron system (Paf1-AID*-6FLAG) and the ubiquitin-independent degradation of Spe1. Proteasomal degradation of these proteins, which was inhibited by severe ethanol stress, resumed rapidly once the ethanol was removed. These results suggested that proteasomal proteolysis in yeast cells is reversibly inhibited by severe ethanol stress. Furthermore, yeast cells pretreated with mild ethanol stress (6% v/v) showed proteasomal proteolysis even with 10% (v/v) ethanol, indicating that yeast cells acquired resistance to proteasome inhibition caused by severe ethanol stress. However, yeast cells failed to acquire sufficient resistance to severe ethanol stress-induced proteasome inhibition when new protein synthesis was blocked with cycloheximide during pretreatment, or when Rpn4 was lost.Conclusions and general significanceOur results provide novel insights into the adverse effects of severe ethanol stress on proteasomal proteolysis and ethanol adaptability in yeast.     

刺梨自然发酵过程中非酿酒酵母多样性分析

【目的】分析刺梨果实自然发酵过程中非酿酒酵母菌群特征,为筛选优质刺梨非酿酒酵母提供参考。【方法】基于Illumina MiSeq高通量测序技术和WL营养琼脂鉴定培养基纯种分离技术,分析刺梨果实自然发酵1 d (F1)、3 d (F3)、5 d (F5)和15 d (F15) 4个阶段及YPD培养基富集培养样本中非酿酒酵母种群组成和多样性。【结果】高通量测序分析结果共获得182个OTUs (operational taxonomic units,OTUs),归属于81个属107个种;物种多样性分析结果表明,刺梨果实自然发酵前期,优势非酿酒酵母为汉逊酵母(Hanseniasporasp.)和伯顿丝孢毕赤酵母(Hyphopichiaburtonii),二者在样本F1中分别占42.59%和26.85%;随着自然发酵的不断进行,二者的比例逐渐降低,在第15天(F15),Hanseniaspora sp.和H. burtonii比例降低至7.73%和0.52%。相反,Pichia sporocuriosa和未培养的酵母,随着自然发酵不断进行所占比例逐渐增大,分别由F1中的0.23%和0.33%增至F15中的37.26%和32.62%。此外,采用WL营养琼脂鉴定培养基纯种分离和鉴定技术,从刺梨上分离到Hanseniasporasp.、H.burtonii、克鲁维毕赤酵母(Pichia kluyveri)、P. sporocuriosa和异常威克汉姆酵母(Wickerhamomyces anomalus) 5种类型的可培养非酿酒酵母。【结论】刺梨果实上存在着丰富的非酿酒酵母菌资源,研究刺梨自然发酵过程中非酿酒酵母多样性,为酵母资源开发和利用奠定基础。     

The Rim15-Endosulfine-PP2ACdc55 Signalling Module Regulates Entry into Gametogenesis and Quiescence via Distinct Mechanisms in Budding Yeast

Quiescence and gametogenesis represent two distinct survival strategies in response to nutrient starvation in budding yeast. Precisely how environmental signals are sensed by yeast cells to trigger quiescence and gametogenesis is not fully understood. A conserved signalling module consisting of Greatwall kinase, Endosulfine and Protein Phosphatase PP2A^Cdc55 proteins regulates entry into mitosis in Xenopus egg extracts and meiotic maturation in flies. We report here that an analogous signalling module consisting of the serine-threonine kinase Rim15, the Endosulfines Igo1 and Igo2 and the Protein Phosphatase PP2A^Cdc55, regulates entry into both quiescence and gametogenesis in budding yeast. PP2A^Cdc55 inhibits entry into gametogenesis and quiescence. Rim15 promotes entry into gametogenesis and quiescence by converting Igo1 into an inhibitor of PP2A^Cdc55 by phosphorylating at a conserved serine residue. Moreover, we show that the Rim15-Endosulfine-PP2A^Cdc55 pathway regulates entry into quiescence and gametogenesis by distinct mechanisms. In addition, we show that Igo1 and Igo2 are required for pre-meiotic autophagy but the lack of pre-meiotic autophagy is insufficient to explain the sporulation defect of igo1Δ igo2Δ cells. We propose that the Rim15-Endosulfine-PP2A^Cdc55 signalling module triggers entry into quiescence and gametogenesis by regulating dephosphorylation of distinct substrates.     

一株高产洛伐他汀红曲霉的筛选与液态发酵条件优化

【背景】洛伐他汀(lovastatin)是红曲霉的次生代谢产物,是重要的临床用降血脂药物。在液态发酵条件下,红曲霉的洛伐他汀产量较低,难以满足工业化生产的要求。【目的】筛选获得一株高产洛伐他汀的红曲霉株,并通过优化液态发酵条件提高洛伐他汀的产量。【方法】从红曲米中筛选获得一株高产洛伐他汀的红曲霉株,依据形态学特征、生理生化特性及18S rRNA基因序列分析对分离菌株进行鉴定;通过响应面法对其产洛伐他汀的液态发酵条件进行优化。【结果】获得一株产洛伐他汀的紫红曲霉(Monascus purpureus M4),该菌在甘油57.80g/L、酵母浸粉5.52 g/L、接种量为6.90%条件下,洛伐他汀产量(173.60 mg/L)较优化前提高了4.8倍。【结论】菌株M4产洛伐他汀最优液态发酵条件的建立,为洛伐他汀的大规模生产及该菌株的工业化应用提供了技术支撑。     

贺兰山东麓优良本土酿酒酵母筛选及发酵特性分析

【背景】商业酵母的使用造成葡萄酒同质化问题严重,发掘优良本土酿酒酵母具有十分重要的意义。【目的】从168株宁夏本土酿酒酵母菌株中筛选出性能优良、具有出色葡萄酒发酵能力的菌株。【方法】基于杜氏管发酵试验和乙醇、高糖等耐受性试验分析产H2S能力及生长曲线测定的方法,筛选出发酵力好、耐受性强、低产H2S的本土酿酒酵母进行赤霞珠葡萄酒发酵试验,测定葡萄酒样基础理化指标、酚类物质和挥发性成分,探究筛选出的酿酒酵母发酵特性。【结果】初步筛选出发酵快速,能适应13%乙醇、350 g/L葡萄糖、250 mg/L SO2、pH 1.0的生存环境且低产H2S的4株本土酿酒酵母YC-E8、QTX-D17、QTX-D7、YQY-E18。菌株YC-E8产甘油能力强,所发酵酒样香气与商业酵母XR、F33最为接近,适用于赤霞珠葡萄酒的发酵。菌株QTX-D17发酵酒样中酒精、单宁、总酚和花色苷含量最高,表现出本土酿酒酵母优良的发酵特性。菌株QTX-D7所发酵酒样香气中乙酸乙酯、辛酸乙酯、1-壬醇等物质含量较高,赋予了葡萄酒香蕉味、苹果味、菠萝味、椰子味等愉悦花果香。【结论】最终筛选出3株优良本土酿酒酵母QTX-D17...     

发酵产泡性能降低的解脂耶氏酵母菌株的获得及其评价

微生物发酵过程中泡沫的产生是发酵领域遇到的共性问题。在不影响发酵性能的前提下抑制菌株的产泡,对简化操作以及降低发酵成本具有较为重要的意义。解脂耶氏酵母(Yarrowia lipolytica,之前称为Candida lipolytica)是一种常用的合成生物学底盘,也是合成赤藓糖醇等功能糖醇的生产菌株。但在发酵合成赤藓糖醇的过程中会产生大量的泡沫,需要添加消泡剂以消除泡沫。【目的】本研究旨在开发一种产泡能力显著降低的解脂耶氏酵母新菌株,以减少赤藓糖醇发酵过程中消泡剂的添加。【方法】本研究利用解脂耶氏酵母中非同源靶向重组占支配地位的原理,采用一段外源DNA随机插入基因组的手段,随机突变基因组,改变菌株的发酵产泡性能,使突变株在发酵过程中不产泡或者降低其产泡的能力。【结果】通过筛选,获得一株在发酵过程中产泡性能显著降低的工程菌株,该菌株在保留高效合成赤藓糖醇性能的同时,显著降低了泡沫的产生。【结论】所获得的菌株对工业发酵合成赤藓糖醇具有较为重要的意义,也为控制其他微生物发酵过程中泡沫的生成提供了思路。     

An innovative consecutive batch fermentation process for very high gravity ethanol fermentation with self-flocculating yeast

An innovative consecutive batch fermentation process was developed for very high gravity (VHG) ethanol fermentation with the self-flocculating yeast under high biomass concentration conditions. On the one hand, the high biomass concentration significantly shortened the time required to complete the VHG fermentation and the duration of yeast cells suffering from strong ethanol inhibition, preventing them from losing viability and making them suitable for being repeatedly used in the process. On the other hand, the separation of yeast cells from the fermentation broth by sedimentation instead of centrifugation, making the process economically more competitive. The VHG medium composed of 255 g L⁻¹ glucose and 6.75 g L⁻¹ each of yeast extract and peptone was fed into the fermentation system for nine consecutive batch fermentations, which were completed within 8–14 h with an average ethanol concentration of 15% (v/v) and ethanol yield of 0.464, 90.8% of its theoretical value of 0.511. The average ethanol productivity that was calculated with the inclusion of the downstream time for the yeast flocs to settle from the fermentation broth and the supernatant to be removed from the fermentation system was 8.2 g L⁻¹ h⁻¹, much higher than those previously reported for VHG ethanol fermentation and regular ethanol fermentation with ethanol concentration around 12% (v/v) as well.     

四川甘孜州高山葡萄酒产区酵母菌多样性分析

【背景】西南高山葡萄酒产区的甘孜州产区,具有生产优质葡萄酒的自然禀赋。【目的】研究四川甘孜州葡萄酒产区真核微生物种类多样性、本土酿酒酵母遗传多样性,以及商业酵母对本土酵母多样性的影响。【方法】利用ITS高通量测序技术对赤霞珠接种发酵和自然发酵过程中的微生物进行多样性分析,并利用Interdelta指纹图谱分析法,对经过26S rRNA基因鉴定的野生酿酒酵母基因型进行分类。【结果】ITS测序结果显示,接种发酵和自然发酵各时期均注释到7个科7个属的酵母,通过Interdelta指纹图谱分析发现甘孜州产区的酿酒酵母共有5种基因型。该产区酿酒酵母的6株代表菌株与我国其他产区109株酿酒酵母的进化树分析结果显示,均与来自北京产区的酿酒酵母菌株亲缘关系更近。【结论】甘孜州葡萄酒子产区酵母资源丰富,表现出较高的微生物多样性和中等程度的本土酿酒酵母基因型多样性,为后续优良本土酵母菌株的筛选奠定基础。     

History,lineage and phenotypic differentiation of sake yeast

ABSTRACT

Sake yeast was first isolated as a single yeast strain, Saccharomyces sake, during the Meiji era. Yeast strains suitable for sake fermentation were subsequently isolated from sake brewers and distributed as Kyokai yeast strains. Sake yeast strains that produce characteristic flavors have been bred in response to various market demands and individual preferences. Interestingly, both genetic and morphological studies have indicated that sake yeast used during the Meiji era differs from new sake yeasts derived from Kyokai Strain No. 7 lineage. Here, we discuss the history of sake yeast breeding, from the discovery of sake yeast to the present day, to highlight the achievements of great Japanese scientists and engineers.     

Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002