BioVector® pYLSC1-POX2 表达质粒载体（解脂耶氏酵母表达质粒） / BioVector® pYLSC1-POX2 Expression Vector (Yarrowia lipolytica Expression Vector)

一、 背景与来源说明 / Background & Origin

BioVector® pYLSC1-POX2 是一种专用于解脂耶氏酵母（Yarrowia lipolytica）高效重组蛋白表达与分泌的整合型酵母表达载体（Yeast Expression Vector）。

解脂耶氏酵母是一种非常优良的非常规产油酵母（Oleaginous Yeast），具备强劲的脂质合成能力、极高密度的发酵生长特性以及强效的异源蛋白分泌表达能力。BioVector® pYLSC1-POX2 载体驱动表达的核心为酰基辅酶A氧化酶 2 启动子（$P_{POX2}$）。$P_{POX2}$ 是一种极强的脂肪酸/油酸（Oleic Acid）强诱导型启动子，在疏水性碳源（如甲基/乙基油酸酯、橄榄油、油酸等）存在下表达水平极高，而在葡萄糖存在时受到严格抑制。结合载体内置的分泌信号肽，该载体广泛应用于工业酶制剂、油脂代谢调控酶类及高附加值重组蛋白的高密度发酵生产。

二、 质粒载体生物学特征 / Vector Characteristics

• 载体类型 / Vector Type：解脂耶氏酵母分泌表达/基因整合型载体（Yeast Secretion & Expression Integration Vector）

• 表达宿主 / Expression Host：解脂耶氏酵母（Yarrowia lipolytica，如 Po1g、Po1h、Po1d 等营养缺陷型菌株）

• 克隆宿主 / Cloning Host：大肠杆菌（E. coli，如 BioVector® DH5$\alpha$ 或 BioVector® TOP10）

• 启动子 / Promoter：强诱导型 $P_{POX2}$ 启动子（受油酸/脂肪酸强效诱导，葡萄糖抑制）

• 分泌信号肽 / Secretion Signal：杂合/天然分泌信号肽（如 XPR2 信号肽或 LIP2 信号肽），介导蛋白质高效分泌至培养上清中

• 筛选标记 / Selection Markers：

  • 酵母筛选标记：URA3 或 LEU2（补足 Y. lipolytica 营养缺陷型宿主）

  • 原核筛选抗性：Ampicillin（氨苄青霉素，100 $\mu\text{g/mL}$）

• 主要应用 / Key Applications：解脂耶氏酵母工业级重组蛋白/脂肪酶/工业酶高密度发酵、油脂代谢基因工程研究

三、 培养条件与操作指南 / Handling & Protocols

1. 大肠杆菌克隆与扩增 / E. coli Cloning & Propagation

• 培养基 / Medium：BioVector® LB Broth 或 BioVector® LB Agar

• 抗性浓度 / Antibiotics：100 $\mu\text{g/mL}$ Ampicillin

• 培养条件 / Incubation：37 摄氏度，振荡培养（220–250 rpm）

2. 解脂耶氏酵母转化与发酵表达 / Yeast Transformation & Expression

1. 载体线性化（建议）：使用适当的 restriction enzyme（如 Not I）切割限制位点，将表达框线性化，以大幅提升同源重组整合至 Y. lipolytica 基因组的效率。

2. 酵母转化：制备 Y. lipolytica 感受态细胞（通常采用 LiAc/PEG 化学转化或电穿孔法），加入线性化或环状 DNA。

3. 筛选与诱导发酵：

   • 涂布于 BioVector® YNB（Yeast Nitrogen Base） 无氨基酸/核苷酸缺陷选择平板（如 YNB-Ura 或 YNB-Leu）筛选阳性重组菌株。

   • 菌体先在含葡萄糖/甘油的培养基中生长时间以积累生物量；随后换入以 油酸（Oleic Acid，0.5%–1.0%） 或 橄榄油 为唯一/诱导碳源的 BioVector® YP Medium 中诱导 $P_{POX2}$ 启动子高效表达与分泌蛋白。

四、 技术指标简表 / Technical Data Summary

BioVector® pYLSC1-POX2 Expression Vector (Yarrowia lipolytica Expression Vector)

1. Background and Origin

BioVector® pYLSC1-POX2 is an integrative yeast expression plasmid designed for high-efficiency expression and extracellular secretion of recombinant proteins in Yarrowia lipolytica.

Yarrowia lipolytica is a non-conventional oleaginous yeast known for its high cell-density fermentation capabilities, vast secretory capacity, and specialized lipid metabolic pathways. The BioVector® pYLSC1-POX2 vector utilizes the acyl-CoA oxidase 2 promoter ($P_{POX2}$) to drive gene expression. $P_{POX2}$ is a strong inducible promoter triggered by hydrophobic carbon sources such as fatty acids or oleic acid, while being tightly repressed by glucose. Coupled with an integrated signal peptide, this vector is widely used for industrial enzyme production and metabolic engineering.

2. Vector Characteristics

• Vector Type: Yeast Secretion, Expression, & Integration Vector

• Expression Host: Yarrowia lipolytica (e.g., Po1g, Po1h, Po1d auxotrophic strains)

• Cloning Host: E. coli (e.g., BioVector® DH5$\alpha$ or BioVector® TOP10)

• Promoter: Strong inducible $P_{POX2}$ promoter (induced by oleic acid/fatty acids, repressed by glucose)

• Secretion Signal: Hybrid or native signal sequence (e.g., XPR2 or LIP2 secretion signal) directing proteins to the extracellular space

• Selection Markers:

  • Yeast Marker: URA3 or LEU2 auxotrophic selection marker

  • Bacterial Marker: Ampicillin (100 $\mu\text{g/mL}$)

• Primary Applications: High-yield secretion of recombinant proteins, industrial enzymes, and lipases in Y. lipolytica

3. Handling Protocols

E. coli Propagation

• Growth Medium: BioVector® LB Broth / BioVector® LB Agar

• Antibiotics: Ampicillin (100 $\mu\text{g/mL}$)

• Incubation: 37 degrees Celsius, shaking at 220–250 rpm

Yeast Transformation & Expression Protocol

1. Linearization: Digest BioVector® pYLSC1-POX2 with a unique restriction enzyme (e.g., Not I) to generate a linear expression cassette, promoting targeted genomic integration in Y. lipolytica.

2. Transformation: Transform competent Y. lipolytica cells using lithium acetate/PEG or electroporation methods.

3. Selection & Induction: Plate transformants onto BioVector® YNB Minimal Media lacking uracil or leucine. Grow cells in glucose/glycerol medium to accumulate biomass, then transfer to media supplemented with 0.5–1.0% oleic acid or olive oil to induce $P_{POX2}$-driven expression.

4. Technical Data Summary

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