BACKGROUND & AIMS: :Exposure to low temperatures reduces protein folding rates and induces the cold denaturation of proteins. Considering the roles played by chaperones in facilitating protein folding and preventing protein aggregation, chaperones must exist that confer tolerance to cold stress. Here, yeast strains lacking individual chaperones were screened for reduced freezing tolerance. In total, 19 of 82 chaperone-deleted strains tested were more sensitive to freeze-thaw treatment than wild-type cells. The reintroduction of the respective chaperone genes into the deletion mutants recovered the freeze tolerance. The freeze sensitivity of the chaperone-knockout strains was also retained in the presence of 20% glycerol.

背景与目标： ：暴露于低温下会降低蛋白质的折叠速率，并引起蛋白质的冷变性。考虑到伴侣在促进蛋白质折叠和防止蛋白质聚集中所起的作用，必须存在赋予对冷胁迫的耐受性的伴侣。在此，筛选了缺乏单个伴侣的酵母菌株以降低冷冻耐受性。总共测试了82个缺失伴侣的菌株中的19个比野生型细胞对冻融处理更敏感。将各自的伴侣基因重新引入缺失突变体中恢复了冷冻耐受性。在存在20％甘油的情况下，伴侣伴侣敲除菌株的冷冻敏感性也得以保持。

BACKGROUND & AIMS: :Set2-mediated histone methylation at H3K36 regulates diverse activities, including DNA repair, mRNA splicing, and suppression of inappropriate (cryptic) transcription. Although failure of Set2 to suppress cryptic transcription has been linked to decreased lifespan, the extent to which cryptic transcription influences other cellular functions is poorly understood. Here, we uncover a role for H3K36 methylation in the regulation of the nutrient stress response pathway. We found that the transcriptional response to nutrient stress was dysregulated in SET2-deleted (set2Δ) cells and was correlated with genome-wide bi-directional cryptic transcription that originated from within gene bodies. Antisense transcripts arising from these cryptic events extended into the promoters of the genes from which they arose and were associated with decreased sense transcription under nutrient stress conditions. These results suggest that Set2-enforced transcriptional fidelity is critical to the proper regulation of inducible and highly regulated transcription programs.

背景与目标： ：Set2介导的H3K36的组蛋白甲基化调节多种活性，包括DNA修复，mRNA剪接和抑制不适当的（隐式）转录。尽管Set2未能抑制隐性转录与寿命缩短有关，但对隐性转录影响其他细胞功能的程度了解甚少。在这里，我们揭示了H3K36甲基化在营养胁迫响应途径调控中的作用。我们发现，对营养胁迫的转录反应在SET2缺失（set2Δ）细胞中失调，并且与源自基因体内的全基因组双向隐秘转录相关。由这些隐性事件引起的反义转录物延伸到它们产生的基因的启动子中，并与在营养胁迫条件下的有义转录物减少有关。这些结果表明，Set2增强的转录保真度对于诱导性和高度调控的转录程序的正确调控至关重要。

BACKGROUND & AIMS: While protein synthesis is rapidly inactivated in Saccharomyces cerevisiae, cells shifted from log growth at 30 degrees C to 43 degrees C, a 1-h 37 degrees C treatment given to cells just prior to the shift to 43 degrees C partially blocks this inactivation. By contrast, such a pre-heat shock treatment has no protective effect on translational inactivation at 45 degrees C or higher. Cells allowed to approach stationary phase not only develop an enhanced thermotolerance relative to log cells but also exhibit a pronounced resistance to inactivation of protein synthesis at 43 degrees C as well as at 45 degrees C. We have found that this 'translational thermotolerance' can also be induced in S. cerevisiae by briefly treating log phase cells at 30 degrees C with cycloheximide. Using such a procedure to induce stabilization of protein synthesis at 43 degrees C, we have been able to show that heat shock-induced proteins are not responsible for the establishment of this protective effect. This work shows that enhanced thermotolerance can be induced in log cells even after a shift to 43 degrees C, as long as a prior translational thermotolerance has been established. Furthermore, we show that the capacity of plateau cells to maintain translation at 43 degrees C contributes significantly to their state of enhanced thermotolerance.

背景与目标： 尽管在酿酒酵母中蛋白质的合成迅速失活，但细胞从30℃的对数生长转移到43℃，而在转移到43℃之前对细胞进行1小时37℃处理可以部分阻止这种失活。相反，这种预热激处理对45℃或更高温度下的翻译失活没有保护作用。允许进入固定相的细胞不仅相对于对数细胞发展出增强的耐热性，而且在43摄氏度和45摄氏度下均表现出显着的抗蛋白质合成失活的能力。我们发现，这种“翻译耐热性”还可以通过用环己酰亚胺在30摄氏度下短暂处理对数期细胞，可在酿酒酵母中诱导其生长。使用这种程序在43摄氏度下诱导蛋白质合成的稳定化，我们已经能够证明热激诱导的蛋白质与这种保护作用的建立无关。这项工作表明，即使已经建立了先前的翻译耐热性，即使转移到43摄氏度之后，也可以在对数单元中诱导增强的耐热性。此外，我们表明高原细胞在43摄氏度下保持翻译的能力显着促进了其耐热性的提高。

BACKGROUND & AIMS: :The osmotolerant yeast Zygosaccharomyces rouxii is sensitive to the toxic L-proline analogue, L-azetidine-2-carboxylate (AZC). The possibility of use of the Saccharomyces cerevisiae MPR1 gene (ScMPR1) encoding the AZC-detoxifying enzyme as a dominant selection marker in Z. rouxii was examined. The heterologous expression of ScMPR1 in two Z. rouxii strains resulted in AZC-resistant colonies, but that of ScMPR1 as a dominant marker gene in vectors was affected by a high frequency of spontaneously resistant colonies. The same was found for an AZC-sensitive S. cerevisiae strain in which the ScMPR1 was expressed. In both yeasts, ScMPR1 can be used only as an auxiliary marker gene.

背景与目标： 耐渗透酵母酵母Zygosaccharomyces rouxii对有毒的L-脯氨酸类似物L-氮杂环丁烷-2-羧酸盐（AZC）敏感。考察了将编码AZC解毒酶的啤酒酵母MPR1基因（ScMPR1）用作鲁氏沼虾的主要选择标记的可能性。 ScMPR1在两个鲁氏弧菌菌株中的异源表达导致了AZC抗性菌落，但作为载体中显性标记基因的ScMPR1的表达受到了高频率的自发抗性菌落的影响。对于其中表达ScMPR1的AZC敏感啤酒酵母菌株也发现了相同的情况。在两种酵母中，ScMPR1只能用作辅助标记基因。

BACKGROUND & AIMS: The effects of medium composition, nutrient limitation and dilution rate on the loss of the recombinant plasmid pLG669-z and plasmid-borne beta-galactosidase expression were studied in batch and chemostat cultures of Saccharomyces cerevisiae strain CGpLG. The difference in growth rates between plasmid-free and plasmid-containing cells (delta mu) and the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were identified. Glucose-limited chemostat cultures of CGpLG grown on defined medium were more stable at higher dilution rates and exhibited delta mu-dominated plasmid loss kinetics. Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-dominated plasmid loss kinetics. Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was least stable in magnesium-limited cultures grown on defined medium. delta mu decreased and R increased with increased dilution rate, irrespective of medium composition. Increased plasmid loss rates at high or low dilution rates would appear to be characteristic of loss kinetics dominated by R or delta mu, respectively. Growth of glucose-limited chemostat cultures on complex medium decreased delta mu values but increased R values, in comparison to those cultures grown on defined medium. Any increased stability that a complex medium-induced reduction of delta mu may have conferred was counteracted by an increased R value. Increased beta-galactosidase productivity was correlated with increased plasmid stability only in glucose-limited chemostat cultures grown on defined medium and not in those grown on complex medium. Previous studies have yielded contrasting responses with regard to the effect of dilution rate on recombinant plasmid loss from S. cerevisiae. Our findings can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs. This information would facilitate the design of bioprocesses, where recombinant plasmid instability results in reduced culture productivity.

背景与目标： 在酿酒酵母菌株CGpLG的分批培养和恒化培养中，研究了培养基组成，养分限制和稀释率对重组质粒pLG669-z丢失和质粒携带的β-半乳糖苷酶表达的影响。确定了无质粒和含有质粒的细胞之间的生长速率差异（δ亩）和分离速率（R），并鉴定了培养基组成对质粒损失的影响所导致的一些共同因素。在限定的培养基上生长的CGpLG的葡萄糖有限的恒化器培养物在更高的稀释率下更稳定，并表现出delta占主导的质粒损失动力学。在复杂的培养基上生长的相似培养物在较低的稀释率下更稳定，并表现出R占主导地位的质粒损失动力学。总体质粒稳定性在限定培养基上生长的磷酸盐限制的恒化器培养物中最大，而在限定培养基上生长的镁限制的培养物中最不稳定。不管培养基组成如何，随着稀释率的增加，Δmu减小而R增加。在高或低稀释率下增加的质粒损失率似乎是分别由R或δmu决定的损失动力学的特征。与在限定的培养基上生长的那些培养物相比，在复合培养基上的葡萄糖有限的恒化器培养物的生长降低了Δmu值，但是增加了R值。 R值的增加抵消了复合介质诱导的delta减少可能带来的任何增加的稳定性。仅在限定培养基上生长的葡萄糖有限的恒化器培养物中而不是在复杂培养基上生长的那些中，β-半乳糖苷酶生产率的提高与质粒稳定性的提高相关。先前的研究在稀释率对啤酒酵母中重组质粒丢失的影响方面产生了不同的响应。我们的发现可以解释这些差异，并且对于相似的酵母质粒构建体的稳定性通常可能是有效的。此信息将有助于设计生物过程，其中重组质粒的不稳定性会导致培养效率的降低。

BACKGROUND & AIMS: :Carbon feedstocks from fossilized sources are being rapidly depleted due to rising demand for industrial and commercial applications. Many petroleum-derived chemicals can be directly or functionally substituted with chemicals derived from renewable feedstocks. Several short chain organic acids may fulfill this role using their functional groups as a target for chemical catalysis. Saccharomyces cerevisiae was engineered to produce short chain carboxylic acids (C6 to C10 ) from glucose using the heterologous Homo sapiens type I fatty acid synthase (hFAS). This synthase was activated by phosphopantetheine transfereases AcpS and Sfp from Escherichia coli and Bacillus subtilis, respectively, both in vitro and in vivo. hFAS was produced in the holo-form and produced carboxylic acids in vitro, confirmed by NADPH and ADIFAB assays. Overexpression of hFAS in a yeast FAS2 knockout strain, deficient in de novo fatty acid synthesis, demonstrated the full functional replacement of the native fungal FAS by hFAS. Two active heterologous short chain thioesterases (TEs) from Cuphea palustris (CpFatB1) and Rattus norvegicus (TEII) were evaluated for short chain fatty acid (SCFA) synthesis in vitro and in vivo. Three hFAS mutants were constructed: a mutant deficient in the native TE domain, a mutant with a linked CpFatB1 TE and a mutant with a linked TEII TE. Using the native yeast fatty acid synthase for growth, the overexpression of the hFAS mutants and the short-chain TEs (linked or plasmid-based) increased in vivo caprylic acid and total SCFA production up to 64-fold (63 mg/L) and 52-fold (68 mg/L), respectively, over the native yeast levels. Combined over-expression of the phosphopantetheine transferase with the hFAS mutant resulted in C8 titers of up to 82 mg/L and total SCFA titers of up to 111 mg/L.

背景与目标： ：由于对工业和商业应用需求的增长，来自化石资源的碳原料正在迅速消耗。许多石油衍生的化学品可以直接或功能上替换为可再生原料衍生的化学品。几种短链有机酸可以利用其官能团作为化学催化的目标来实现这一功能。利用异源的智人I型脂肪酸合酶（hFAS），将酿酒酵母（Saccharomyces cerevisiae）工程化以从葡萄糖产生短链羧酸（C6至C10）。在体外和体内，该合酶分别被大肠杆菌和枯草芽孢杆菌的磷酸泛酸转移酶AcpS和Sfp激活。 NFAPH和ADIFAB分析证实，hFAS呈完整形式，并在体外产生羧酸。酵母FAS2敲除菌株中hFAS的过表达，从头脂肪酸合成不足，证明了hFAS可以完全替代天然真菌FAS。评价了来自古铜杯（CupatB1）和褐家鼠（TEII）的两种活性异源短链硫酯酶（TEs）的体内和体外短链脂肪酸（SCFA）合成。构建了三个hFAS突变体：天然TE结构域缺失的突变体，具有连接的CpFatB1 TE的突变体和具有连接的TEII TE的突变体。使用天然酵母脂肪酸合酶进行生长，hFAS突变体和短链TE（连接或基于质粒的）的过表达将体内辛酸和总SCFA产量提高到64倍（63μmg/ L），并且分别是天然酵母水平的52倍（68µmg / L）。磷酸泛酸转移酶与hFAS突变体的过表达相结合，导致C8效价高达82 mg / L，总SCFA效价高达111 mg / L。

BACKGROUND & AIMS: :High productivity processes are critical for commercial production of cellulosic ethanol. One high productivity process-continuous hydrolysis and fermentation-has been applied in corn ethanol industry. However, little research related to this process has been conducted on cellulosic ethanol production. Here, we report and compare the kinetics of both batch SHF (separate hydrolysis and co-fermentation) and SSCF (simultaneous saccharification and co-fermentation) of AFEX™ (Ammonia Fiber Expansion) pretreated corn stover (AFEX™-CS). Subsequently, we designed a SSCF process to evaluate continuous hydrolysis and fermentation performance on AFEX™-CS in a series of continuous stirred tank reactors (CSTRs). Based on similar sugar to ethanol conversions (around 80% glucose-to-ethanol conversion and 47% xylose-to-ethanol conversion), the overall process ethanol productivity for continuous SSCF was 2.3- and 1.8-fold higher than batch SHF and SSCF, respectively. Slow xylose fermentation and high concentrations of xylose oligomers were the major factors limiting further enhancement of productivity.

背景与目标： ：高生产率工艺对于纤维素乙醇的商业化生产至关重要。一种高生产率的方法-连续水解和发酵-已应用于玉米乙醇工业。但是，有关纤维素乙醇生产的研究很少与此过程有关。在这里，我们报告并比较了AFEX™（氨纤维膨胀）预处理玉米秸秆（AFEX™-CS）的批次SHF（单独的水解和共同发酵）和SSCF（同时糖化和共同发酵）的动力学。随后，我们设计了一种SSCF工艺，以评估一系列连续搅拌釜反应器（CSTR）在AFEX™-CS上的连续水解和发酵性能。基于相似的糖到乙醇转化率（大约80％的葡萄糖到乙醇转化率和47％的木糖到乙醇转化率），连续SSCF的总工艺乙醇生产率比批次SHF和SSCF分别高2.3倍和1.8倍，分别。木糖缓慢发酵和高浓度的木糖低聚物是限制生产率进一步提高的主要因素。

BACKGROUND & AIMS: :A phospholipid flippase activity from the endoplasmic reticulum (ER) of the model organism Saccharomyces cerevisiae has been characterized and functionally reconstituted into proteoliposomes. Analysis of the transbilayer movement of acyl-7-nitrobenz-2-oxa-1,3-diazol-4-yl (acyl-NBD)-labeled phosphatidylcholine in yeast microsomes using a fluorescence stopped-flow back exchange assay revealed a rapid, ATP-independent flip-flop (half-time, <2 min). Proteoliposomes prepared from a Triton X-100 extract of yeast microsomal membranes were also capable of flipping NBD-labeled phospholipid analogues rapidly in an ATP-independent fashion. Flippase activity was sensitive to the protein modification reagents N-ethylmaleimide and diethylpyrocarbonate. Resolution of the Triton X-100 extract by velocity gradient centrifugation resulted in the identification of a approximately 4S protein fraction enriched in flippase activity as well as of other fractions where flippase activity was depleted or undetectable. We estimate that flippase activity is due to a protein(s) representing approximately 2% (wt/wt) of proteins in the Triton X-100 extract. These results indicate that specific proteins are required to facilitate ATP-independent phospholipid flip-flop in the ER and that their identification is feasible. The architecture of the ER protein translocon suggests that it could account for the flippase activity in the ER. We tested this hypothesis using microsomes prepared from a temperature-sensitive yeast mutant in which the major translocon component, Sec61p, was quantitatively depleted. We found that the protein translocon is not required for transbilayer movement of phospholipids across the ER. Our work defines yeast as a promising model system for future attempts to identify the ER phospholipid flippase and to test and purify candidate flippases.

背景与目标： ：已鉴定出来自模型生物酿酒酵母的内质网（ER）的磷脂翻转酶活性，并在功能上重构为蛋白脂质体。使用荧光停止流反向交换测定法分析酰基-N-硝基苯-2-氧杂-1,3-二氮杂-4-基（酰基-NBD）标记的磷脂酰胆碱在酵母微粒体中的跨双分子运动，显示了快速的ATP独立的触发器（半时，<2分钟）。由酵母微粒体膜的Triton X-100提取物制备的蛋白脂质体也能够以不依赖ATP的方式快速翻转NBD标记的磷脂类似物。 Flippase的活性对蛋白质修饰试剂N-乙基马来酰亚胺和焦碳酸二乙酯敏感。通过速度梯度离心分离Triton X-100提取物可鉴定出富含脂肪酶活性的约4S蛋白级分，以及脂肪酶活性被耗尽或无法检测到的其他组分。我们估计，flippase的活性是由于蛋白质代表了Triton X-100提取物中蛋白质的大约2％（wt / wt）。这些结果表明，需要特定的蛋白质来促进ER中不依赖ATP的磷脂触发器，并且它们的鉴定是可行的。 ER蛋白转运蛋白的结构表明它可以解释ER中的flippase活性。我们使用由温度敏感的酵母突变体制备的微粒体测试了这一假设，其中主要的转座子成分Sec61p被定量耗尽。我们发现，蛋白质的跨膜转运不是跨ER的磷脂的跨双层运动所必需的。我们的工作将酵母定义为一种有前途的模型系统，可用于未来鉴定ER磷脂翻转酶以及测试和纯化候选翻转酶的尝试。

BACKGROUND & AIMS: :Ethanol fermentation from sweet sorghum juice containing 240 g/l of total sugar by Saccharomyces cerevisiae TISTR 5048 and S. cerevisiae NP 01 immobilized on low-cost support materials, corncob pieces, was investigated. In batch fermentation, S. cerevisiae TISTR 5048 immobilized on 6 × 6 × 6 mm(3) corncobs gave higher ethanol production than those immobilized on 12 × 12 × 12 mm(3) corncobs in terms of ethanol concentration (P), yield (Y ( p/s )) and productivity (Q ( p )) with the values of 102.39 ± 1.11 g/l, 0.48 ± 0.01 and 2.13 ± 0.02 g/l h, respectively. In repeated-batch fermentation, the yeasts immobilized on the 6 × 6 × 6 mm(3) corncobs could be used at least eight successive cycles with the average P, Y ( p/s ) and Q ( p ) of 97.19 ± 5.02 g/l, 0.48 ± 0.02 and 2.02 ± 0.11 g/l h, respectively. Under the same immobilization and repeated-batch fermentation conditions, P (90.75 ± 3.05 g/l) and Q ( p ) (1.89 ± 0.06 g/l h) obtained from S. cerevisiae NP 01 were significantly lower than those from S. cerevisiae TISTR 5048 (P < 0.05), while Y ( p/s ) from both strains were not different. S. cerevisiae TISTR 5048 immobilized on the corncobs also gave significantly higher P, Y ( p/s ) and Q ( p ) than those immobilized on calcium alginate beads (P < 0.05).

背景与目标： ：研究了固定在低成本支撑材料玉米芯上的酿酒酵母TISTR 5048和酿酒酵母NP 01从甜高粱汁中所含的总糖为240 g / l的乙醇发酵。在分批发酵中，固定在6×6×6mm（3）玉米芯上的酿酒酵母TISTR 5048与固定在12×12×12mm（3）玉米芯上的酿酒酵母相比，乙醇浓度（P），产量（ Y（p / s））和生产率（Q（p））的值分别为102.39±1.11 g / l，0.48±0.01和2.13±0.02 g / l h。在重复分批发酵中，固定在6×6×6mm（3）玉米芯上的酵母至少可以连续使用八个循环，平均P，Y（p / s）和Q（p）为97.19±5.02 g / l，分别为0.48±0.02和2.02±0.11 g / l h。在相同的固定化和重复分批发酵条件下，从酿酒酵母NP 01获得的P（90.75±3.05 g / l）和Q（p）（1.89±0.06 g / l h）显着低于酿酒酵母NP。 TISTR 5048（P <0.05），两种菌株的Y（p / s）均无差异。固定在玉米芯上的酿酒酵母TISTR 5048也比固定在藻酸钙珠上的P，Y（p / s）和Q（p）高得多（P <0.05）。

BACKGROUND & AIMS: :Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h-1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h-1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h-1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h-1 The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast.IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are insufficient to sustain fast growth and product formation. Consequently, this expensive vitamin is routinely added to baker's yeast cultures. In this study, laboratory evolution in biotin-free growth medium yielded new strains that grew as fast in the absence of biotin as in its presence. By analyzing the DNA sequences of evolved biotin-independent strains, mutations were identified that contributed to this ability. This work demonstrates full biotin independence of an industrially relevant yeast and identifies mutations whose introduction into other yeast strains may reduce or eliminate their biotin requirements.

背景与目标： ：生物素原养是酿酒酵母菌株的一种罕见，尚未完全理解并且在工业上具有相关性的特征。单倍体实验室菌株CEN.PK113-7D的基因组包含完整的生物素生物合成基因，但在不含生物素的合成培养基中的生长极为缓慢（比生长率[μ]≈0.01 h-1）。在重复的分批培养和加速器中进行的四个独立进化实验得出的菌株在无生物素和补充生物素的培养基中的生长速率（μ≤0.36 h-1）相似。这些进化菌株的全基因组重测序揭示了BIO1的40倍扩增，该BIO1编码庚二酰辅酶A（CoA）合成酶。在不同的染色体上发现了BIO1的其他副本，其扩增与大量的染色体重排相符。 BIO1在菌株CEN.PK113-7D中的过表达证实了该基因扩增的关键作用，该菌株能够在不含生物素的培养基中生长（μ= 0.15 h-1）。在一些进化的菌株中发现了膜转运蛋白基因TPO1和/或PDR12的突变。在过表达BIO1的菌株中TPO1和PDR12的缺失将其比生长速率提高到0.25 h-1。这些基因中无效突变的作用是非可累加的，这些突变以前未与生物素代谢相关。这项研究表明，具有生物素合成基本遗传信息的酿酒酵母菌株可以进化为完整的生物素原营养型，并为将生物素原营养型工程化到该酵母的实验室和工业菌株中确定了新的目标。在所有生物体中发挥作用，并非所有生物体都能合成这种维生素。面包酵母的许多菌株是工业生物技术中的重要微生物，至少含有一些生物素合成所需的基因。然而，这些菌株中的大多数不能完全合成生物素，或者不能以足以维持快速生长和产物形成的速率合成生物素。因此，通常将这种昂贵的维生素添加到面包师的酵母培养物中。在这项研究中，实验室在无生物素生长培养基中的进化产生了新菌株，该菌株在不存在生物素的情况下的生长速度与在存在生物素的情况下一样快。通过分析进化的不依赖生物素的菌株的DNA序列，鉴定出有助于此能力的突变。这项工作证明了与工业相关的酵母具有完全的生物素独立性，并鉴定了突变，将其引入其他酵母菌株可以降低或消除其对生物素的需求。

BACKGROUND & AIMS: :Although microorganisms account for the largest fraction of Earth's biodiversity, we know little about how their reproductive barriers evolve. Sexual microorganisms such as Saccharomyces yeasts rapidly develop strong intrinsic post-zygotic isolation, but the role of extrinsic isolation in the early speciation process remains to be investigated. We measured the growth of F1 hybrids between two incipient species of Saccharomyces paradoxus to assess the presence of extrinsic post-zygotic isolation across 32 environments. More than 80% of hybrids showed either partial dominance of the best parent or over-dominance for growth, revealing no fitness defects in F1 hybrids. Extrinsic reproductive isolation therefore likely plays little role in limiting gene flow between incipient yeast species and is not a requirement for speciation.

背景与目标： ：尽管微生物是地球生物多样性的最大组成部分，但我们对其生殖屏障如何进化知之甚少。性微生物（例如酵母）迅速发展出强大的内在合子后分离，但是外源分离在早期物种形成过程中的作用仍有待研究。我们测量了两个Saccharomyces paradoxus初始物种之间的F1杂种的生长，以评估在32个环境中外部合子后分离的存在。超过80％的杂种显示出最佳亲本的部分优势或对生长的过度主导，表明F1杂种中没有适合性缺陷。因此，外源性生殖分离可能在限制初始酵母物种之间的基因流动方面几乎没有作用，并且不是物种形成的必要条件。

BACKGROUND & AIMS: :In Saccharomyces cerevisiae, Y family DNA polymerase Rev1 is involved in the repair of DNA damage by translesion DNA synthesis (TLS). In the current study, to elucidate the role of Rev1 in oxidative stress-induced DNA damage in S. cerevisiae, REV1 was deleted and overexpressed; transcriptome analysis of these mutants along with the wild-type strain was performed to screen potential genes that could be associated with REV1 during response to DNA damage. When the yeast cells were treated with 2 mM H2O2, the deletion of REV1 resulted in a 1.5- and 2.8-fold decrease in the survival rate and mutation frequency, respectively, whereas overexpression of REV1 increased the survival rate and mutation frequency by 1.1- and 2.9-fold, respectively, compared to the survival rate and mutation frequency of the wild-type strain. Transcriptome and phenotypic analyses identified that Sml1 aggravated oxidative stress in the yeast cells by inhibiting the activity of Rev1. This inhibition was due to the physical interaction between the BRCA1 C terminus (BRCT) domain of Rev1 and amino acid residues 36 to 70 of Sml1; the cell survival rate and mutation frequency increased by 1.8- and 3.1-fold, respectively, when this interaction was blocked. We also found that Sml1 inhibited Rev1 phosphorylation under oxidative stress and that deletion of SML1 increased the phosphorylation of Rev1 by 46%, whereas overexpression of SML1 reduced phosphorylation of Rev1. Overall, these findings demonstrate that Sml1 could be a novel regulator that mediates Rev1 dephosphorylation to inhibit its activity during oxidative stress.IMPORTANCE Rev1 was critical for cell growth in S. cerevisiae, and the deletion of REV1 caused a severe growth defect in cells exposed to oxidative stress (2 mM H2O2). Furthermore, we found that Sml1 physically interacted with Rev1 and inhibited Rev1 phosphorylation, thereby inhibiting Rev1 DNA antioxidant activity. These findings indicate that Sml1 could be a novel regulator for Rev1 in response to DNA damage by oxidative stress.

背景与目标： ：在酿酒酵母中，Y家族DNA聚合酶Rev1参与通过病灶DNA合成（TLS）修复DNA损伤。在当前的研究中，为阐明Rev1在酿酒酵母中氧化应激诱导的DNA损伤中的作用，将REV1删除并过表达。对这些突变体以及野生型菌株进行转录组分析，以筛选在对DNA损伤的反应过程中可能与REV1相关的潜在基因。当用2μmMH2O2处理酵母细胞时，REV1的缺失导致存活率和突变频率分别降低1.5倍和2.8倍，而REV1的过表达使存活率和突变频率提高1.1倍和2.8倍。分别是野生型菌株的存活率和突变频率的2.9倍。转录组和表型分析确定Sml1通过抑制Rev1的活性加剧了酵母细胞中的氧化应激。这种抑制是由于Rev1的BRCA1 C末端（BRCT）结构域与Sml1的36至70位氨基酸残基之间的物理相互作用所致。当这种相互作用被阻止时，细胞存活率和突变频率分别增加了1.8倍和3.1倍。我们还发现，Sml1在氧化应激下抑制Rev1的磷酸化，SML1的缺失使Rev1的磷酸化增加46％，而SML1的过表达减少了Rev1的磷酸化。总的来说，这些发现表明Sml1可能是介导Rev1去磷酸化以抑制其在氧化应激过程中的活性的新型调节剂。氧化应激（2 mM H2O2）。此外，我们发现Sml1与Rev1发生物理相互作用并抑制Rev1的磷酸化，从而抑制Rev1 DNA的抗氧化活性。这些发现表明Sml1可能是Rev1的新型调节剂，以响应氧化应激对DNA的损伤。

BACKGROUND & AIMS: :Rosmarinic acid is a hydroxycinnamic acid ester commonly found in the Boraginaceae and Lamiaceae plant families. It exhibits various biological activities, including antioxidant, anti-inflammatory, antibacterial, antiallergic, and antiviral properties. Rosmarinic acid is used as a food and cosmetic ingredient, and several pharmaceutical applications have been suggested as well. Rosmarinic acid is currently produced by extraction from plants or chemical synthesis; however, due to limited availability of the plant sources and the complexity of the chemical synthesis method, there is an increasing interest in producing this compound by microbial fermentation. In this study, we aimed to produce rosmarinic acid by engineered baker's yeast Saccharomyces cerevisiae. Multiple biosynthetic pathway variants, carrying only plant genes or a combination of plant and Escherichia coli genes, were implemented using a full factorial design of experiment. Through analysis of variances, the effect of each enzyme variant (factors), together with possible interactions between these factors, was assessed. The best pathway variant produced 2.95 ± 0.08 mg/L rosmarinic acid in mineral medium with glucose as the sole carbon source. Increasing the copy number of rosmarinic acid biosynthetic genes increased the titer to 5.93 ± 0.06 mg/L. The study shows the feasibility of producing rosmarinic acid by yeast fermentation.

背景与目标： ：迷迭香酸是常见于紫草科和唇形科植物家族中的羟基肉桂酸酯。它具有多种生物活性，包括抗氧化，抗炎，抗菌，抗过敏和抗病毒特性。迷迭香酸被用作食品和化妆品成分，并且已经提出了几种药物应用。迷迭香酸目前是通过从植物中提取或化学合成生产的。然而，由于植物来源的可获得性有限以及化学合成方法的复杂性，通过微生物发酵生产该化合物的兴趣日益浓厚。在这项研究中，我们旨在通过工程面包酵母酿酒酵母生产迷迭香酸。使用完整的因子设计实验，可以实现仅携带植物基因或植物和大肠杆菌基因组合的多种生物合成途径变体。通过方差分析，评估了每种酶变体（因子）的作用，以及这些因子之间可能的相互作用。最佳途径的变体在葡萄糖为唯一碳源的矿物培养基中产生了2.95±0.08 mg / L迷迭香酸。迷迭香酸生物合成基因的拷贝数增加将滴度提高到5.93±0.06 mg / L。研究表明通过酵母发酵生产迷迭香酸的可行性。

BACKGROUND & AIMS: :The monitoring of fermentation at low temperatures (12-15°C) is a current practice in the winery for retention and enhancement of the flavour volatile content of wines. Among Saccharomyces species, Saccharomyces uvarum and Saccharomyces kudriavzevii have revealed interesting industrial properties, including better adaptation at low temperatures. To gather deeper knowledge of the fermentative metabolism at a low temperature of these species together with S. cerevisiae, we performed a comparative metabolomic analysis using four representative strains. We used batch cultures to obtain an exhaustive and dynamic image of the metabolome of strains passing through the sequential stresses related to the winemaking environment. A great variety of intra- and extracellular metabolites (>500 compounds) were quantified across fermentation using distinct chromatographic methods. Besides a global decrease in the lipid composition of the four strains when they entered into the stationary phase, we reported some strain-specific high magnitude changes. Examples of these differences included divergent patterns of production of short-chain fatty acids and erythritol in the S. uvarum strain. Strains also differed in expression for aromatic amino acid biosynthesis and sulphur metabolism, including the glutathione pathway. These data will allow us to refine and obtain the most value of fermentations with this alternative Saccharomyces species.

背景与目标： ：在酿酒厂中，为了保持和提高葡萄酒的风味挥发物含量，目前在低温（12-15°C）下对发酵进行监控是目前的做法。在酿酒酵母种类中，葡萄酿酒酵母和库氏酿酒酵母显示出令人感兴趣的工业特性，包括在低温下更好的适应性。为了收集这些物种与酿酒酵母在低温下的发酵代谢的更深入的知识，我们使用四种代表性菌株进行了比较代谢组学分析。我们使用分批培养来获得菌株代谢组的详尽而动态的图像，这些菌株通过与酿酒环境有关的顺序应力传递。在整个发酵过程中，使用不同的色谱方法对各种各样的细胞内和细胞外代谢产物（> 500种化合物）进行了定量。除了当它们进入固定相时这四个菌株的脂质组成总体降低外，我们还报道了一些菌株特异性的高强度变化。这些差异的例子包括在葡萄链球菌菌株中短链脂肪酸和赤藓糖醇生产的不同模式。菌株在芳香族氨基酸生物合成和硫代谢（包括谷胱甘肽途径）的表达上也有所不同。这些数据将使我们能够精炼并获得这种替代酵母菌发酵的最大价值。

BACKGROUND & AIMS: :In this study we show that inactivation of Hsl1 or Hsl7, negative regulators of the Swe1 kinase, enhances the invasive behavior of haploid and diploid cells. The enhancement of filamentous growth caused by inactivation of both genes is mediated via the Swe1 protein kinase. Whereas Swe1 contributes noticeably to the effectiveness of haploid invasive growth under all conditions tested, its contribution to pseudohyphal growth is limited to the morphological response under standard assay conditions. However, Swe1 is essential for pseudohyphal differentiation under a number of nonstandard assay conditions including altered temperature and increased nitrogen. Swe1 is also required for pseudohyphal growth in the absence of Tec1 and for the induction of filamentation by butanol, a related phenomenon. Although inactivation of Hsl1 is sufficient to suppress the defect in filamentous growth caused by inactivation of Tec1 or Flo8, it is insufficient to promote filamentous growth in the absence of both factors. Moreover, inactivation of Hsl1 will not bypass the requirement for nitrogen starvation or growth on solid medium for pseudohyphal differentiation. We conclude that the Swe1 kinase modulates filamentous development under a broad spectrum of conditions and that its role is partially redundant with the Tec1 and Flo8 transcription factors.

背景与目标： ：在这项研究中，我们显示Hsl1或Hsl7（Swe1激酶的负调节剂）的失活增强了单倍体和二倍体细胞的侵袭行为。由两个基因的失活引起的丝状生长的增强是通过Swe1蛋白激酶介导的。尽管在所有测试条件下，Swe1均对单倍体侵袭性生长的有效性做出了显着贡献，但其对假菌丝生长的贡献仅限于标准测定条件下的形态学响应。但是，Swe1对于在许多非标准测定条件（包括温度变化和氮增加）下的假菌丝分化必不可少。在不存在Tec1的情况下，假菌丝的生长也需要Swe1，并且通过丁醇（相关现象）诱导丝化也需要Swe1。尽管Hsl1的失活足以抑制由Tec1或Flo8失活引起的丝状生长缺陷，但在没有这两种因素的情况下，不足以促进丝状生长。此外，Hsl1的灭活不会绕过氮饥饿或在假菌丝分化的固体培养基上生长的需求。我们得出的结论是，Swe1激酶在广泛的条件下调节丝状发育，并且其作用与Tec1和Flo8转录因子部分重叠。