BioVector? 敲除 KU70 的單倍體解脂耶氏酵母菌株 / BioVector? KU70-Knockout Haploid Yarrowia lipolytica Strain

通用定義 / General Definition:BioVector? 敲除 KU70 (ku70$\Delta$) 的解脂耶氏酵母菌株是一種專門為高效基因編輯而工程化的單倍體底盤細胞。在野生型解脂耶氏酵母中，非同源末端連接 (NHEJ) 修復機制占據(jù)主導地位，導致通過同源重組 (HDR) 進行精準基因整合的效率極低。通過敲除 KU70（NHEJ 途徑的關(guān)鍵蛋白），該菌株的 DNA 修復機制被強制轉(zhuǎn)向同源重組。這顯著提高了外源 DNA 片段在基因組特定位點的定點整合效率，是進行大規(guī)模代謝工程、多基因通路構(gòu)建以及蛋白質(zhì)表達優(yōu)化的理想遺傳背景。

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 技術(shù)說明書 (Technical Datasheet)

中文版說明書 (Chinese Datasheet)

1. 產(chǎn)品基本信息

• 產(chǎn)品名稱： BioVector? 敲除 KU70 解脂耶氏酵母 (單倍體)

• 親本菌株： 通?；?Po1f, W29 或 W29 衍生的單倍體背景。

• 基因型特征：ku70::loxP 或 ku70::Marker；通常帶有多種營養(yǎng)缺陷型標志（如 ura3-, leu2-, ade2-），便于多輪篩選。

• 主要優(yōu)勢： 同源重組效率比野生型提高 10-50 倍；適合使用長同源臂進行大片段整合。

2. 培養(yǎng)條件

• 基礎(chǔ)培養(yǎng)基： BioVector? YPD 培養(yǎng)基（完全培養(yǎng)基）或 SC/SD 最小培養(yǎng)基（用于缺陷型篩選）。

• 培養(yǎng)溫度： 28 - 30 攝氏度（超過 32°C 生長受阻）。

• 培養(yǎng)環(huán)境： 好氧培養(yǎng)，搖床轉(zhuǎn)速 200-250 rpm。

• 生長特性： 具有典型的二形性特征（酵母態(tài)與絲狀態(tài)轉(zhuǎn)化），在特定脅迫下可積累大量油脂。

3. 菌株應(yīng)用

• 代謝工程： 高效整合脂肪酸、類胡蘿卜素或有機酸合成通路。

• CRISPR 輔助編輯： 配合 CRISPR/Cas9 系統(tǒng)實現(xiàn)超高效率的基因組定點編輯。

• 蛋白質(zhì)工程： 在特定強啟動子（如 GPD, pTEF）驅(qū)動下，定點插入外源蛋白表達框。

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

4. 注意事項

• 表型穩(wěn)定性： KU70 缺失可能導致菌株對電離輻射或特定 DNA 損傷劑的敏感性增加，操作中應(yīng)盡量減少誘變因素。

• 轉(zhuǎn)化方法： 建議使用醋酸鋰 (LiAc) 轉(zhuǎn)化法或電轉(zhuǎn)化法。由于 NHEJ 被抑制，轉(zhuǎn)化所需的同源臂長度建議在 500bp - 1kb 以上以獲得最佳效果。

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學研究中的“明星底盤”。BioVector? 提醒您，雖然該菌株極大地方便了定點整合，但在完成多基因構(gòu)建后，有時研究者會選擇通過 Cre/loxP 系統(tǒng)回補 KU70 以恢復菌株的基因組穩(wěn)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.

BioVector NTCC質(zhì)粒載體菌株細胞蛋白抗體基因保藏中心

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