BioVector® CBS系列 经典模式真菌与酵母菌标准菌株BioVector® CBS-Series Classic Model Fungal and Yeast Reference Strains Datasheet

第一部分：中文说明

一、 产品基本信息、物种校正与详细特征描述

本说明书涵盖了源自荷兰中心菌种库 (Centraalbureau voor Schimmelcultures, 现名 WI-Oryx/Westerdijk Fungal Biodiversity Institute, 简称 CBS) 的 7 株在遗传学、天然产物化学、分类学及病理学研究中极具里程碑意义的经典模式真菌与酵母标准菌株。

二、 细胞培养环境、底盘培养基配方与理化参数

1. 酵母类菌株 (CBS 289.85, 288.85, 789.73, 788.73, 449.87) 专用配方：

   • 常规扩增培养基：BioVector® YPD 培养基（1% 酵母膏 Yeast Extract，2% 蛋白胨 Peptone，2% 葡萄糖 Glucose）。固体平板添加 1.5% - 2.0% 琼脂粉。

   • 理化参数：培养温度设定为 25°C 至 28°C（注：长孢洛德酵母在 30°C 亦能迅速增殖，但为了保持孢子链完整性，建议 28°C 培养）。需氧培养。液体转速：180 - 200 RPM。pH 控制在 6.0 - 6.5。

2. 丝状真菌与大型真菌类 (CBS 410.71, 573.69) 专用配方：

   • 常规扩增与产孢培养基：BioVector® 马铃薯葡萄糖琼脂/肉汤 (PDA/PDB) 或 麦芽浸膏琼脂 (MEA)。

   • 理化参数：培养温度 25°C 至 26°C。黑曲霉在 28°C 生长极快，3-4 天即可布满黑色分生孢子堆。秀美裂褶菌（CBS 573.69）作为担子菌，需保持湿度 $>80\%$ 以促进菌丝平铺。专性需氧，液体培养需使用带挡板摇瓶（Baffled Flasks），转速 150 - 180 RPM 以防菌丝体结成巨大过密团块。

三、 菌株复苏、传代与遗传操作标准步骤

1. 冻干管/冷冻管无菌复苏：

   • 在二级无菌超净台内，使用酒精灯对安瓿瓶外壁消毒，敲碎顶端。

   • 依据菌株类别，吸取 0.8 mL 预热的 YPD 肉汤（针对酵母）或 PDB 肉汤（针对真菌）注入管底，轻轻吹吸使干燥菌丝/孢子饼彻底重悬。

   • 将重悬液分别接种至对应的固体平板（YPD/PDA）上，置于 25°C - 28°C 培养箱中避光孵育。酵母类通常在 24 - 48 小时内长出乳白色、湿润的单菌落；丝状真菌（如黑曲霉）通常在第 3 天开始从中心向外喷射分生孢子。

2. 常规传代与菌丝/孢子纯化：

   • 丝状真菌（黑曲霉、裂褶菌）严禁用移液枪当做细胞吹打。传代时须使用无菌接种针/环，挑取边缘刚长出的新嫩气生菌丝（营养旺盛期）或者成熟孢子，转接至新平板中央。

3. 针对酿酒酵母与洛德酵母的常规高效率醋酸锂（LiAc）转化法：

   • 收集对数生长中期（$OD_{600} \approx 0.6 - 0.8$）的酵母细胞。

   • 利用 100 mM 醋酸锂溶液洗涤细胞沉淀 2 次，使其处于高感受态状态。

   • 在无菌离心管中顺序加入：PEG 3350 (50% w/v) + 100 mM LiAc + 单链载体 DNA（如经煮沸变性的鲑鱼精 DNA 作为载体）+ 目标重组质粒（如 pYES2 表达质粒）。

   • 混匀后于 30°C 孵育 30 分钟，随后立即投入 42°C 热激 15 - 20 分钟。低速离心弃去 PEG，用无菌水重悬，涂布于特定氨基酸缺陷型选择性平板（SD-Minus 培养基）上进行抗性克隆筛选。

四、 菌株长期保藏与冷冻技术

• 深冻冷冻保护剂配方：

  • 酵母类：使用基于 YPD 肉汤配制的复合冻存液，内含最终体积百分比为 20% - 25% 优质医用甘油。

  • 丝状真菌孢子类（黑曲霉）：使用 15% 甘油 + 0.05% Tween-80 无菌水溶液。Tween-80 作为表面活性剂能使极度疏水的真菌分生孢子均匀分散。

• 冷冻保存程序：对于酵母，直接吸取高浓度液体纯培养物与等体积 50% 甘油混合；对于丝状真菌，倒入保护液后用涂布棒轻轻刮取平板表面的孢子层，通过无菌擦镜纸过滤菌丝，收集纯孢子悬液。分装入冻存管，置于程序降温盒内降温至 -80°C，随后长久转入液氮罐（-196°C）的气相中保藏。

五、 质量控制标准与核心科研应用方向

• 质量控制标准：BioVector® 提供的各批次 CBS 衍生标准菌株均通过了最严苛的表型形态学与分子生物学多重审核。经真菌通用引物（ITS1/ITS4）PCR 扩增及测序，基因组序列与原厂 CBS 数据库完全呈现 100% 互补匹配；确保无任何外源细菌或外源杂菌污染；各菌株对特定碳源（如木糖、半乳糖）的选择性发酵能力和产酶活性保持代际高度稳定。

• 核心科研应用方向：

  • CBS 289.85 / 288.85 / 449.87：作为非常规酵母与经典发酵动力学模型，用于解析非酿酒酵母在新能源发酵、极端高盐/高糖逆境应激下的糖代谢网络重构。

  • CBS 789.73 / 788.73：临床近缘鉴定与真菌形态分化模型。用于筛查和研究抗真菌药物（如氟康唑、两性霉素B）对伪菌丝形成的特异性阻断通路。

  • CBS 410.71 (黑曲霉)：丝状真菌高产酶发酵工程。广泛用于工业级酸性蛋白酶、糖化酶的工业发酵放大，或作为 CRISPR-Cas9 介导的多基因簇（BGCs）异源表达底盘细胞。

  • CBS 573.69 (秀美裂褶菌)：高等担子菌遗传分化与生物降解。用于剖析木质素过氧化物酶、纤维素酶系在降解秸秆等生物质能中的空间催化动力学，以及解析真菌多性别（万余种交配型）识别的非编码 RNA 调控机制。

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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