BioVector? CBS系列 經(jīng)典模式真菌與酵母菌標(biāo)準(zhǔn)菌株BioVector? CBS-Series Classic Model Fungal and Yeast Reference Strains Datasheet

第一部分：中文說(shuō)明

一、 產(chǎn)品基本信息、物種校正與詳細(xì)特征描述

本說(shuō)明書(shū)涵蓋了源自荷蘭中心菌種庫(kù) (Centraalbureau voor Schimmelcultures, 現(xiàn)名 WI-Oryx/Westerdijk Fungal Biodiversity Institute, 簡(jiǎn)稱 CBS) 的 7 株在遺傳學(xué)、天然產(chǎn)物化學(xué)、分類(lèi)學(xué)及病理學(xué)研究中極具里程碑意義的經(jīng)典模式真菌與酵母標(biāo)準(zhǔn)菌株。

二、 細(xì)胞培養(yǎng)環(huán)境、底盤(pán)培養(yǎng)基配方與理化參數(shù)

1. 酵母類(lèi)菌株 (CBS 289.85, 288.85, 789.73, 788.73, 449.87) 專用配方：

   • 常規(guī)擴(kuò)增培養(yǎng)基：BioVector? YPD 培養(yǎng)基（1% 酵母膏 Yeast Extract，2% 蛋白胨 Peptone，2% 葡萄糖 Glucose）。固體平板添加 1.5% - 2.0% 瓊脂粉。

   • 理化參數(shù)：培養(yǎng)溫度設(shè)定為 25°C 至 28°C（注：長(zhǎng)孢洛德酵母在 30°C 亦能迅速增殖，但為了保持孢子鏈完整性，建議 28°C 培養(yǎng)）。需氧培養(yǎng)。液體轉(zhuǎn)速：180 - 200 RPM。pH 控制在 6.0 - 6.5。

2. 絲狀真菌與大型真菌類(lèi) (CBS 410.71, 573.69) 專用配方：

   • 常規(guī)擴(kuò)增與產(chǎn)孢培養(yǎng)基：BioVector? 馬鈴薯葡萄糖瓊脂/肉湯 (PDA/PDB) 或 麥芽浸膏瓊脂 (MEA)。

   • 理化參數(shù)：培養(yǎng)溫度 25°C 至 26°C。黑曲霉在 28°C 生長(zhǎng)極快，3-4 天即可布滿黑色分生孢子堆。秀美裂褶菌（CBS 573.69）作為擔(dān)子菌，需保持濕度 $>80\%$ 以促進(jìn)菌絲平鋪。專性需氧，液體培養(yǎng)需使用帶擋板搖瓶（Baffled Flasks），轉(zhuǎn)速 150 - 180 RPM 以防菌絲體結(jié)成巨大過(guò)密團(tuán)塊。

三、 菌株復(fù)蘇、傳代與遺傳操作標(biāo)準(zhǔn)步驟

1. 凍干管/冷凍管無(wú)菌復(fù)蘇：

   • 在二級(jí)無(wú)菌超凈臺(tái)內(nèi)，使用酒精燈對(duì)安瓿瓶外壁消毒，敲碎頂端。

   • 依據(jù)菌株類(lèi)別，吸取 0.8 mL 預(yù)熱的 YPD 肉湯（針對(duì)酵母）或 PDB 肉湯（針對(duì)真菌）注入管底，輕輕吹吸使干燥菌絲/孢子餅徹底重懸。

   • 將重懸液分別接種至對(duì)應(yīng)的固體平板（YPD/PDA）上，置于 25°C - 28°C 培養(yǎng)箱中避光孵育。酵母類(lèi)通常在 24 - 48 小時(shí)內(nèi)長(zhǎng)出乳白色、濕潤(rùn)的單菌落；絲狀真菌（如黑曲霉）通常在第 3 天開(kāi)始從中心向外噴射分生孢子。

2. 常規(guī)傳代與菌絲/孢子純化：

   • 絲狀真菌（黑曲霉、裂褶菌）嚴(yán)禁用移液槍當(dāng)做細(xì)胞吹打。傳代時(shí)須使用無(wú)菌接種針/環(huán)，挑取邊緣剛長(zhǎng)出的新嫩氣生菌絲（營(yíng)養(yǎng)旺盛期）或者成熟孢子，轉(zhuǎn)接至新平板中央。

3. 針對(duì)釀酒酵母與洛德酵母的常規(guī)高效率醋酸鋰（LiAc）轉(zhuǎn)化法：

   • 收集對(duì)數(shù)生長(zhǎng)中期（$OD_{600} \approx 0.6 - 0.8$）的酵母細(xì)胞。

   • 利用 100 mM 醋酸鋰溶液洗滌細(xì)胞沉淀 2 次，使其處于高感受態(tài)狀態(tài)。

   • 在無(wú)菌離心管中順序加入：PEG 3350 (50% w/v) + 100 mM LiAc + 單鏈載體 DNA（如經(jīng)煮沸變性的鮭魚(yú)精 DNA 作為載體）+ 目標(biāo)重組質(zhì)粒（如 pYES2 表達(dá)質(zhì)粒）。

   • 混勻后于 30°C 孵育 30 分鐘，隨后立即投入 42°C 熱激 15 - 20 分鐘。低速離心棄去 PEG，用無(wú)菌水重懸，涂布于特定氨基酸缺陷型選擇性平板（SD-Minus 培養(yǎng)基）上進(jìn)行抗性克隆篩選。

四、 菌株長(zhǎng)期保藏與冷凍技術(shù)

• 深凍冷凍保護(hù)劑配方：

  • 酵母類(lèi)：使用基于 YPD 肉湯配制的復(fù)合凍存液，內(nèi)含最終體積百分比為 20% - 25% 優(yōu)質(zhì)醫(yī)用甘油。

  • 絲狀真菌孢子類(lèi)（黑曲霉）：使用 15% 甘油 + 0.05% Tween-80 無(wú)菌水溶液。Tween-80 作為表面活性劑能使極度疏水的真菌分生孢子均勻分散。

• 冷凍保存程序：對(duì)于酵母，直接吸取高濃度液體純培養(yǎng)物與等體積 50% 甘油混合；對(duì)于絲狀真菌，倒入保護(hù)液后用涂布棒輕輕刮取平板表面的孢子層，通過(guò)無(wú)菌擦鏡紙過(guò)濾菌絲，收集純孢子懸液。分裝入凍存管，置于程序降溫盒內(nèi)降溫至 -80°C，隨后長(zhǎng)久轉(zhuǎn)入液氮罐（-196°C）的氣相中保藏。

五、 質(zhì)量控制標(biāo)準(zhǔn)與核心科研應(yīng)用方向

• 質(zhì)量控制標(biāo)準(zhǔn)：BioVector? 提供的各批次 CBS 衍生標(biāo)準(zhǔn)菌株均通過(guò)了最嚴(yán)苛的表型形態(tài)學(xué)與分子生物學(xué)多重審核。經(jīng)真菌通用引物（ITS1/ITS4）PCR 擴(kuò)增及測(cè)序，基因組序列與原廠 CBS 數(shù)據(jù)庫(kù)完全呈現(xiàn) 100% 互補(bǔ)匹配；確保無(wú)任何外源細(xì)菌或外源雜菌污染；各菌株對(duì)特定碳源（如木糖、半乳糖）的選擇性發(fā)酵能力和產(chǎn)酶活性保持代際高度穩(wěn)定。

• 核心科研應(yīng)用方向：

  • CBS 289.85 / 288.85 / 449.87：作為非常規(guī)酵母與經(jīng)典發(fā)酵動(dòng)力學(xué)模型，用于解析非釀酒酵母在新能源發(fā)酵、極端高鹽/高糖逆境應(yīng)激下的糖代謝網(wǎng)絡(luò)重構(gòu)。

  • CBS 789.73 / 788.73：臨床近緣鑒定與真菌形態(tài)分化模型。用于篩查和研究抗真菌藥物（如氟康唑、兩性霉素B）對(duì)偽菌絲形成的特異性阻斷通路。

  • CBS 410.71 (黑曲霉)：絲狀真菌高產(chǎn)酶發(fā)酵工程。廣泛用于工業(yè)級(jí)酸性蛋白酶、糖化酶的工業(yè)發(fā)酵放大，或作為 CRISPR-Cas9 介導(dǎo)的多基因簇（BGCs）異源表達(dá)底盤(pán)細(xì)胞。

  • CBS 573.69 (秀美裂褶菌)：高等擔(dān)子菌遺傳分化與生物降解。用于剖析木質(zhì)素過(guò)氧化物酶、纖維素酶系在降解秸稈等生物質(zhì)能中的空間催化動(dòng)力學(xué)，以及解析真菌多性別（萬(wàn)余種交配型）識(shí)別的非編碼 RNA 調(diào)控機(jī)制。

PART 2: ENGLISH SECTION

I. General Information, Species Correction, and Detailed Characterization

This technical datasheet encompasses 7 classic model fungal and yeast reference strains originating from the renowned Centraalbureau voor Schimmelcultures (CBS, currently reorganized as the WI-Oryx/Westerdijk Fungal Biodiversity Institute). These strains serve as international touchstones across genetics, natural product chemistry, taxonomy, and phytopathology.

II. Cultivation Environments, Basal Medium Formulations, and Physical Parameters

1. Yeast-Form Lineages (CBS 289.85, 288.85, 789.73, 788.73, 449.87):

   • Standard Expansion Matrix: BioVector? YPD Medium (1% Yeast Extract, 2% Peptone, 2% Glucose). For solid plates, supplement with 1.5% - 2.0% bacteriological agar.

   • Physical Parameters: Incubation temperature configured at 25°C to 28°C. Note: While L. elongisporus scales rapidly up to 30°C, maintaining the thermostat at 28°C is strictly recommended to preserve stable ascospore chain geometry. Fully aerobic dynamics. Liquid agitation: 180 - 200 RPM. Calibrate pH to stay between 6.0 and 6.5.

2. Filamentous & Macro-Fungal Lineages (CBS 410.71, 573.69):

   • Standard Expansion Matrix: BioVector? Potato Dextrose Agar/Broth (PDA/PDB) or Malt Extract Agar (MEA).

   • Physical Parameters: Constantly regulated at 25°C to 26°C. A. niger undergoes aggressive vegetative expansion at 28°C, blanketing the agar with charcoal-black conidiophore heaps within 72–96 hours. For the basidiomycete S. commune (CBS 573.69), maintain an atmospheric relative humidity $>80\%$ to optimize flat mycelial extension. Strictly aerobic; liquid fermentation mandates baffled Erlenmeyer flasks run at 150 - 180 RPM to break up thick hyphal aggregates and avoid localized anoxic mycelial cores.

III. Thawing, Subculturing, and Genetic Transformation Protocols

1. Lyophilized / Cryopreserved Aliquot Rehydration:

   • Within a certified laminar flow hood, sanitize the glass ampoule exterior with ethanol and aseptically crack open the apex.

   • Utilizing a sterile pipette, dispense 0.8 mL of pre-warmed YPD broth (for yeasts) or PDB broth (for filamentous fungi) directly onto the dried matrix pellet. Gently aspirate until the cells are completely resuspended.

   • Inoculate the rehydrated slurry across matching solid TSA/PDA plates and incubate at 25°C - 28°C in darkness. Yeast colonies typically emerge as moist, creamy-white single discs within 24–48 hours; Aspergillus colonies will project radical aerial hyphae followed by dark pigmentation by Day 3.

2. Routine Stock Maintenance Loop:

   • Filamentous lineages must never be mechanically sheared with pipettes. Subculturing requires an aseptic inoculating needle or loop to delicately excise a small wedge of active, juvenile mycelium from the colony edge (exponential growth front) or mature conidia, transferring it directly to the center of a fresh agar plate.

3. High-Yield Lithium Acetate (LiAc) Yeast Transformation (For S. cerevisiae & Lodderomyces):

   • Harvest middle-logarithmic phase yeast cultures yielding an $OD_{600}$ density spanning 0.6 - 0.8.

   • Wash the cell sediment twice with sterile 100 mM LiAc solution to induce a high state of chemical competence.

   • Sequentially assemble the following transformation matrix inside a sterile microtube: PEG 3350 (50% w/v) + 100 mM LiAc + single-stranded carrier DNA (e.g., boiled, highly denatured salmon sperm DNA) + target recombinant plasmid vector (e.g., pYES2 expression system).

   • Vortex gently and incubate at 30°C for 30 minutes, immediately followed by a precise 42°C heat-shock window for 15 - 20 minutes. Centrifuge at low velocity to evacuate the viscous PEG supernatant, resuspend the transformed cell pellet in sterile water, and spread onto synthetic dropout amino acid selection agar (SD-Minus media).

IV. Strain Cryopreservation and Long-Term Archiving

• Cryoprotective Matrix Architecture:

  • Yeast Subsets: Formulate complete YPD broth enriched to a definitive final concentration of 20% - 25% analytical-grade medical Glycerol.

  • Filamentous Conidia Subsets (Aspergillus): Formulate a solution containing 15% Glycerol + 0.05% Tween-80 in sterile water. Tween-80 functions as an essential surfactant to wet and evenly disperse highly hydrophobic fungal conidial envelopes.

• Cryopreservation Workflow: For yeasts, blend late-log liquid cultures directly with an equal volume of 50% glycerol protection matrix. For filamentous fungi, pour the pre-chilled cryoprotective matrix over a mature sporulating plate, gently scrape the conidial lawn with a sterile loop, and pass the suspension through sterile lens paper to trap large hyphal strands. Collect the pure spore filtrate inside sterile cryovials. Cool the vials at a rate of 1°C per minute inside a controlled freezing container down to -80°C before transferring them to the vapor phase of a liquid nitrogen storage tank (-196°C) for indefinite preservation.

V. Quality Control Standards and Strategic Research Applications

• Quality Control Standards: Every production lot of BioVector? CBS-derived reference strains undergoes exhaustive phenotypic, morphological, and molecular verification. Full-length PCR sequencing utilizing fungal-specific universal primers (ITS1/ITS4) confirms a 100% identity match with the authentic genomic master records of the CBS database. All stocks are certified completely free from adventitious bacterial or fungal contaminants. Multi-generational profiling confirms that carbon-source fermentation kinetics and enzyme yields remain stable across extended passage windows.

• Core Experimental Applications:

  • CBS 289.85 / 288.85 / 449.87: Serving as premiere non-conventional yeast models to dissect carbohydrate pathway remodeling during biofuel optimization or metabolic adaptation to high salt and sugar stress.

  • CBS 789.73 / 788.73: Functioning as indispensable clinical diagnostic mimics and developmental models to evaluate how novel antifungal compounds (such as fluconazole or amphotericin B) selectively block pseudomycelial morphogenesis pathways.

  • CBS 410.71 (Aspergillus niger): High-yield fungal biomanufacturing. Widely utilized for scaling up industrial acid proteases and glucoamylases, or serving as a specialized high-capacity chassis for CRISPR-Cas9-mediated heterologous expression of complex biosynthetic gene clusters (BGCs).

  • CBS 573.69 (Schizophyllum commune): Basidiomycete developmental genetics and bioremediation. Deployed to trace the structural spatial kinetics of lignin peroxidases and cellulase complexes during biomass breakdown, and to dissect non-coding RNA networks that regulate multi-mating compatibility across thousands of distinct fungal sexes.

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