Summary: The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial (“white”) biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate.

Progress in Metabolic Engineering of Saccharomyces cerevisiae

Glycogen and trehalose are the two glucose stores of yeast cells. The large variations in the cell content of these two compounds in response to different environmental changes indicate that their metabolism is controlled by complex regulatory systems. In this review we present information on the regulation of the activity of the enzymes implicated in the pathways of synthesis and degradation of glycogen and trehalose as well as on the transcriptional control of the genes encoding them. cAMP and the protein kinases Snf1 and Pho85 appear as major actors in this regulation. From a metabolic point of view, glucose-6-phosphate seems the major effector in the net synthesis of glycogen and trehalose. We discuss also the implication of the recently elucidated TOR-dependent nutrient signalling pathway in the control of the yeast glucose stores and its integration in growth and cell division. The unexpected roles of glycogen and trehalose found in the control of glycolytic flux, stress responses and energy stores for the budding process, demonstrate that their presence confers survival and reproductive advantages to the cell. The findings discussed provide for the first time a teleonomic value for the presence of two different glucose stores in the yeast cell.

Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae

The disaccharide trehalose is widely distributed in nature and can be found in many organisms, including bacteria, fungi, plants, invertebrates and mammals. Due to its particular physical features, trehalose is able to protect the integrity of the cell against a variety of environmental injuries and nutritional limitations. In addition, data available on several species of bacteria and yeast suggest specific functions for trehalose in these organisms. Bacteria can use exogenous trehalose as the sole source of carbon and energy as well as synthesize enormous amounts of the disaccharide as compatible solute. This ability to accumulate trehalose is the result of an elaborate genetic system, which is regulated by osmolarity. Some mycobacteria contain sterified trehalose as a structural component of the cell wall, whereas yeast cells are largely unable to grow on trehalose as carbon source. In these lower eukaryotes, trehalose appears to play a dual function: as a reserve compound, mainly stored in vegetative resting cells and reproductive structures, and as a stress metabolite. Recent findings also point to important biotechnological applications for trehalose.

Physiological roles of trehalose in bacteria and yeasts: a comparative analysis.

Bio-functional components in the processed pre-germinated brown rice by a twin-screw extruder

Fermentation predictability and wine quality are directly dependent on wine yeast attributes that assist in the rapid establishment of numerical dominance in the early phase of wine fermentation, and that determine the ability to conduct an even and efficient fermentation to obtain a desirable alcohol degree. It is therefore not surprising that the primary selection criteria applied to most wine yeast strain development programmes relate to the overall objective of achieving an efficient conversion of grape sugar to alcohol and carbon dioxide, at a controlled rate and without the development of off-flavours. Numerous factors influence the fermentation performance of wine yeast.  Following a successful inoculation of grape must with an appropriate starter culture strain, the ability of a wine yeast to adapt to and cope with the hostile environment and stress conditions prevailing in grape juice fermentation are of vital importance to fermentation performance. There is a direct correlation between fermentation efficiency and stress resistance, which refers to the ability of a yeast strain to adapt efficiently to a changing environment and unfavourable growth conditions. Successful yeast cellular adaptation to changes in extracellular parameters during wine fermentation requires the timely perception (sensing) of chemical or physical environmental parameters, followed by accurate transmission of the information to the relevant compartments of the cell.  Chemical parameters perceived during wine fermentation include the availability/concentration of certain nutrients (e.g., fermentable sugars, assimilable nitrogen, oxygen, vitamins, minerals, ergosterol and unsaturated fatty acids) and the presence of inhibitory substances (e.g., ethanol, acetic acid, fatty acids, sulfite, phenolic phytoalexins, mycotoxins, bacterial toxins and agrochemical residues). Signals of a physical nature include temperature, pH, agitation and osmotic pressure. The sensing of these environmental signals is carried out by specific receptor proteins, most of them situated on the cellular surface. Once perceived, the information is transmitted by a network of dedicated, interconnected signal transduction pathways to the relevant cellular compartments which implement the adaptive response, a process referred to as "stress response". Intensive research has focused on elucidating the molecular mechanisms involved in stress responses, which are evolutionarily well conserved. Besides furthering our understanding of the fundamental strategies for adaptation to hostile, industrial environments, and the biological resilience of Saccharomyces cerevisiae, the data are of key importance to the future improvement of wine yeast strains. This review describes the different types of stress experienced by wine yeast cells during their life cycles, summarises our current knowledge of some of the most important molecular processes required for the survival of the yeast cell, and highlights the potential benefits for future yeast strain development which can be derived from this research.

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Yeast Stress Response and Fermentation Efficiency: How to Survive the Making of Wine - A Review

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Δnth1), acid trehalase mutants (Δath1), and double mutants (Δnth1 ath1) by using commercial baker’s yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Δnth1 and Δath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Δnth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough.

Stress Tolerance in Doughs of Saccharomyces cerevisiae Trehalase Mutants Derived from Commercial Baker’s Yeast

The effect of intracellular charged amino acids on freeze tolerance in doughs was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.

Disruption of the CAR1 gene encoding arginase enhances freeze tolerance of the commercial baker's yeast Saccharomyces cerevisiae

パン生地発酵力試験の際にパン生地から発生するガス量を長時間にわたり自動計測する装置を開発した.基本的には発生ガス量を水量・液面の変化に置換して,ロータリエンコーダの動きに変え,その動きを光学センサにより検知・測定することであり,このデータの収集と処理をマイクロコンピュータ処理化したものである.この装置ではパン生地から発生するガス量と,生地内に内蔵されるガス量を流路の途中にアルカリ液を置くことで測定している. 1台のコンピュータで48チャンネルの装置を制御でき,チャンネル間の測定精度は, ±2%もしくは±0.2ml以内である. N 88-日本語BASIC (86)を用いたプログラムによりコンピュータが, 5分毎のCO2ガス発生量と生地からのガス漏洩時刻を算出する.現在装備しているデータ処理としては総ガス発生量,単位時間毎のガス発生量,グラフ化,単純移動平均,および内蔵ガス量の概算である.測定例としてチューブポンプによるデータの信頼性に関するものと,無糖パン生地,および低糖パン生地・高糖パン生地におけるシリンダー試験法との相関性について示した,それらのデータをもとに,装置の性能と問題点について検討した,この装置は現在Fermograph (ATTO Co., Ltd., Japan)として市販されている.終わりに本装置のソフトウェア開発にあたり御指導,御助言をいただいた北海道大学工学部魚住純博士,農林水産省食品総合研究所鍋谷浩志氏に深く感謝の意を表します.

https://www.jstage.jst.go.jp/article/nskkk1962/35/5/35_5_344/_pdf

Automatic Measuring System for Dough Testing; Design, Construction and Reproducibility

A diploid hybrid bakers' yeast belonging to Saccharomyces cerevisiae having at least strong fermentative ability of non-sugar bread dough and strong freeze-resistance and frozen bread dough containing the same are provided.

Novel Bakers' yeast

米粉へのα化の有無がライスブレッド製造時の発酵および発酵中の糖の消長に及ぼす影響について検討を行ない,以下の結果を得た。1. 米粉20%置換添加した時の無糖生地の発酵において,生米粉を用いると初期発酵が増大し, α化処理米粉を用いると後期発酵つまりマルトース発酵が増大する。2. 米粉単独で発酵中における炭酸ガス発生量を測定したところ, α化処理米粉では1.5時間以内に炭酸ガス発生は停止したが,生米粉では長時間炭酸ガス発生が持続された。3. 生米粉を135℃4時間加熱して小麦粉に加えた時には,初期発酵の増大は認められなかった。このことから生米粉を添加した際に認められる初期発酵の増大は,米粉中に存在する酵素によって発酵性糖が生成されたためと考えられる。4. 加水前および加水後に30℃1時間インキュベーションした時の小麦粉と米粉の糖組成の変化を調べた結果, α化処理米粉には糖組成に変化が認められなかった。生米粉ではインキュベーションによってマルトースが著しく増大し,またグルコースとフラクトースも増加したが,シュクロースは減少した。小麦粉の場合,インキュベーションによってマルトースの顕著な増大が認められた。5. 小麦粉に対して米粉を20%置換添加し,同様にして糖の消長を調べた結果, 30℃1時間のインキュベーションによって生米粉添加ではマルトースとグルコースが著しく増加した。一方α化処理米粉添加では,マルトースのみの増大が顕著であった。6. 米粉を20%置換添加した無糖生地の発酵中における全糖量の消長は,生米粉添加では小麦粉のみのコントロールとほとんど差が認められなかった。一方α化処理米粉添加では,全糖量は前者の2倍以上となり,長時間にわたって高いレベルが維持された。7. 発酵中における糖組成の消長を調べた結果,小麦粉のみの場合,初期発酵の段階でグルコース,フラクトース,シュクロースおよびラフィノースは減少した。マルトースは極めて高含量を示し,発酵後約1.5時間から減少し始めたが,これはマルトース発酵への移行が起ることによるものである。生米粉添加では初期発酵の段階からマルトースが減少したが,これはマルトース発酵によるものではなく, α-グルコシダーゼによる加水分解作用によるものであると考えられ,一方でグルコースの増大が認められた。またα化処理米粉添加では,マルトースが小麦粉のみに比べ2倍以上に増大し,マルトース発酵が長時間にわたって高く維持されたのは,このためであることがわかった。8. 以上の結果から,生米粉を添加した時に認められる初期発酵の増大は,生米粉中に存在するα-グルコシダーゼによってマルトースがグルコースに分解されるためであり,一方, α化処理米粉を添加した時に認められる後期発酵つまりマルトース発酵の増大は, α化した米粉中のでん粉が,小麦粉中に存在するβ-アミラーゼの基質となり,マルトースが多量に生成されたためであるといえる。

https://www.jstage.jst.go.jp/article/nskkk1962/27/10/27_10_522/_pdf

Hiroyuki Takano

Papers

Stress Tolerance in Doughs of Saccharomyces cerevisiae Trehalase Mutants Derived from Commercial Baker’s Yeast

Disruption of the CAR1 gene encoding arginase enhances freeze tolerance of the commercial baker's yeast Saccharomyces cerevisiae

Automatic Measuring System for Dough Testing; Design, Construction and Reproducibility

Novel Bakers' yeast

On the Fermentation Properties of Wheat Flour-yeast Dough Mixed with Rice Flour and its Change in Sugar Content during Fermentation