BioVector® 敲除 KU70 的单倍体解脂耶氏酵母菌株 / BioVector® KU70-Knockout Haploid Yarrowia lipolytica Strain

通用定义 / General Definition:BioVector® 敲除 KU70 (ku70$\Delta$) 的解脂耶氏酵母菌株是一种专门为高效基因编辑而工程化的单倍体底盘细胞。在野生型解脂耶氏酵母中，非同源末端连接 (NHEJ) 修复机制占据主导地位，导致通过同源重组 (HDR) 进行精准基因整合的效率极低。通过敲除 KU70（NHEJ 途径的关键蛋白），该菌株的 DNA 修复机制被强制转向同源重组。这显著提高了外源 DNA 片段在基因组特定位点的定点整合效率，是进行大规模代谢工程、多基因通路构建以及蛋白质表达优化的理想遗传背景。

BioVector® KU70-knockout (ku70$\Delta$)Yarrowia lipolytica is a haploid chassis strain specifically engineered for high-efficiency genetic manipulation. In wild-type Y. lipolytica, the Non-Homologous End Joining (NHEJ) repair pathway is dominant, resulting in extremely low rates of precise gene integration via Homologous Recombination (HDR). By deleting KU70, a key protein in the NHEJ pathway, the DNA repair machinery is shifted toward HDR. This significantly enhances the efficiency of site-specific integration of exogenous DNA, making this strain an ideal genetic background for large-scale metabolic engineering, multi-gene pathway assembly, and protein expression optimization.

BioVector® ku70$\Delta$ Y. lipolytica 技术说明书 (Technical Datasheet)

中文版说明书 (Chinese Datasheet)

1. 产品基本信息

• 产品名称： BioVector® 敲除 KU70 解脂耶氏酵母 (单倍体)

• 亲本菌株： 通常基于 Po1f, W29 或 W29 衍生的单倍体背景。

• 基因型特征：ku70::loxP 或 ku70::Marker；通常带有多种营养缺陷型标志（如 ura3-, leu2-, ade2-），便于多轮筛选。

• 主要优势： 同源重组效率比野生型提高 10-50 倍；适合使用长同源臂进行大片段整合。

2. 培养条件

• 基础培养基： BioVector® YPD 培养基（完全培养基）或 SC/SD 最小培养基（用于缺陷型筛选）。

• 培养温度： 28 - 30 摄氏度（超过 32°C 生长受阻）。

• 培养环境： 好氧培养，摇床转速 200-250 rpm。

• 生长特性： 具有典型的二形性特征（酵母态与丝状态转化），在特定胁迫下可积累大量油脂。

3. 菌株应用

• 代谢工程： 高效整合脂肪酸、类胡萝卜素或有机酸合成通路。

• CRISPR 辅助编辑： 配合 CRISPR/Cas9 系统实现超高效率的基因组定点编辑。

• 蛋白质工程： 在特定强启动子（如 GPD, pTEF）驱动下，定点插入外源蛋白表达框。

[Image showing the mechanism of HDR vs NHEJ with KU70 deletion]

4. 注意事项

• 表型稳定性： KU70 缺失可能导致菌株对电离辐射或特定 DNA 损伤剂的敏感性增加，操作中应尽量减少诱变因素。

• 转化方法： 建议使用醋酸锂 (LiAc) 转化法或电转化法。由于 NHEJ 被抑制，转化所需的同源臂长度建议在 500bp - 1kb 以上以获得最佳效果。

English Datasheet

1. General Product Information

• Product Name: BioVector® KU70-Knockout Yarrowia lipolytica (Haploid)

• Parental Strain: Typically based on Po1f, W29, or other W29-derived haploid backgrounds.

• Genotypic Features:ku70::loxP or ku70::Marker; usually carries multiple auxotrophic markers (e.g., ura3-, leu2-, ade2-) for iterative selection.

• Key Advantage: Enhances homologous recombination efficiency by 10- to 50-fold compared to wild-type, facilitating large-fragment integration using long homology arms.

2. Culture Conditions

• Basal Media: BioVector® YPD (Complete) or SC/SD (Synthetic Defined) media for auxotrophic selection.

• Incubation Temperature: 28°C - 30°C (Growth is significantly inhibited above 32°C).

• Environment: Aerobic; shaking at 200-250 rpm.

• Growth Properties: Exhibits dimorphism (transition between yeast and hyphal forms) and high capacity for lipid accumulation under stress.

3. Applications

• Metabolic Engineering: Efficient integration of complex pathways for fatty acids, carotenoids, or organic acid production.

• CRISPR-Mediated Editing: Synergizing with CRISPR/Cas9 systems for ultra-high-efficiency genome-wide engineering.

• Protein Expression: Site-specific insertion of expression cassettes driven by strong promoters (e.g., GPD, pTEF).

4. Key Usage Notes

• Phenotypic Sensitivity: The absence of KU70 may increase sensitivity to ionizing radiation or DNA-damaging agents; minimize exposure to mutagenic factors during handling.

• Transformation Protocol: Lithium Acetate (LiAc) or Electroporation is recommended. Since NHEJ is suppressed, BioVector® advises using homology arms of 500bp to 1kb to maximize integration rates.

注意 / Note: 敲除 KU70 的解脂耶氏酵母菌株是合成生物学研究中的“明星底盘”。BioVector® 提醒您，虽然该菌株极大地方便了定点整合，但在完成多基因构建后，有时研究者会选择通过 Cre/loxP 系统回补 KU70 以恢复菌株的基因组稳定性。The ku70$\Delta$ Yarrowia lipolytica strain is a "star chassis" in synthetic biology. BioVector® notes that while it greatly facilitates site-specific integration, researchers may sometimes choose to restore KU70 using the Cre/loxP system after completing multi-gene assembly to recover genomic stability.

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E-mail: BioVector@163.com

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