BioVector® pYLXP' Yarrowia lipolytica High-Efficiency Expression Vector / pYLXP' 解脂耶氏酵母高水平表达载体

一 产品基本信息与分子生物学背景

• 载体名称：pYLXP'（亦常写作 pYLXP' 或 pYLXP-prime）。

• 载体分类：非传统工业酵母表达质粒 / 解脂耶氏酵母（Yarrowia lipolytica）专用高拷贝表达载体。

• 质粒大小：约 7.5 - 8.2 kb（依具体配置的选择标记和克隆多克隆位点微调）。

• 骨架源起与设计背景（合成生物学明星底盘）：

  pYLXP' 是一款在解脂耶氏酵母（Yarrowia lipolytica）合成生物学和代谢工程研究中极具统治地位的经典高水平表达质粒。解脂耶氏酵母作为一种著名的“富油酵母（Oleaginous yeast）”，具有极强的脂质积累能力、广阔的底物利用谱和卓越的蛋白质分泌胞外能力，被广泛用于生产脂肪酸、生物燃料、类胡萝卜素及工业酶制剂。传统的解脂酵母载体多为低拷贝整合型（Integration）质粒，虽然稳定但表达量有限。pYLXP' 及其系列衍生载体，通过配置特殊的自复制序列（Autonomously Replicating Sequence, ARS）和百倍级强启动子工程改造，实现了外源基因在解脂耶氏酵母体内的高拷贝游离（Episomal）维持与爆发式转录表达，是目前国际上组装各种复杂多基因代谢通路（Multigene metabolic pathways）的标配骨架。

• 核心顺式作用元件与图谱特征：

  • $TEF_{in}$ 杂合超强启动子（Hybrid Promoter）：这是该载体高表达的灵魂元件。它由翻译伸长因子-1$\alpha$（TEF1）启动子与其内含子（Intron）序列融合杂合而成。研究表明，该内含子的存在能通过“内含子介导的表达增强机制（IME）”使转录水平相较于传统 TEF 启动子激增数倍至数十倍，且属于强烈的结构性（Constitutive）驱动，无需添加昂贵的诱导剂。

  • 解脂酵母特殊自复制元件（$ARS18$ / $CEN$ 盒）：赋予质粒在解脂耶氏酵母胞内不依赖基因组整合、即可进行独立自主复制的能力，每细胞拷贝数通常可维持在 10 - 20 个以上。

  • 解脂酵母筛选标记（通常为 URA3 或 LEU2 营养缺陷型标记）：用于在相应的解脂酵母营养缺陷型宿主菌株（如 Po1f、Po1g、W29 衍生株）中进行严格的非抗生素选择性压力筛选。

  • 大肠杆菌元件：含有 pUC ori 和 氨苄青霉素抗性基因（$Amp^R$），便于前期在 E. coli 中进行极其高效的质粒克隆、重组 DNA 组装与大抽提。

二 核心科研价值与工业生物制造转化应用

pYLXP' 质粒在现代现代白生物技术、油脂化学品发酵及合成生物学中扮演着核心角色：

1. 高附加值脂质及类萜化合物的高产调控（脂质工厂）：

   在解脂耶氏酵母中利用 pYLXP' 载体过表达关键限速酶（如 ACC1, DGA1 或 HMG1），可将酵母胞内的油脂（TAG）积累量提升至细胞干重的 60% 以上，或用于高效合成诸如类胡萝卜素（Carotenoids）、角鲨烯（Squalene）、EPA/DHA  omega-3 脂肪酸等高价值非天然产物。

2. 多基因发酵代谢通路的模块化快速组装（Modular Assembly）：

   由于 pYLXP' 具有良好的游离复制特性和极强的表达均一性，科研人员常通过重叠延伸 PCR（OE-PCR）或 Gibson 组装技术，将多个基因分别置于独立的 $TEF_{in}$ 驱动夹层中，并排组装进 pYLXP' 骨架。这使得在一颗质粒上同时实现一条完整外源异源代谢途径的高效运转成为现实。

3. 复杂工业酶制剂的高效胞外分泌表达：

   利用解脂耶氏酵母完美的蛋白质折叠和强大的翻译后修饰体系，在 pYLXP' 的超强启动子下游融合外源酶（如高级脂肪酶、纤维素酶、蛋白酶）及耶氏酵母内源的分泌信号肽（如 XPR2 prepro 序列），可直接将大量具有完全生物活性的重组工业酶高浓度分泌到发酵液中，极大地简化了下游纯化（Downstream processing）工序。

三 实验室大肠克隆、耶氏酵母电转化/化学转化与高产筛选标准步骤

1. 质粒在大肠杆菌（E. coli）中的分子克隆与高质纯化

• 重组组装：将目的基因（带有解脂酵母密码子优化 Optimized codons 更佳）克隆至 pYLXP' 的多克隆位点（通常位于 $TEF_{in}$ 启动子与 $XPR2$ 终止子之间）。

• 转化与扩增：通过常规热击法转化大肠杆菌常规感受态（如 DH5$\alpha$），涂布于含有 100 $\mu$g/mL 氨苄青霉素的 LB 固体平板上，37 ℃ 培养过夜。

• 高质纯化：挑取单菌落进行液体 LB（100 $\mu$g/mL Amp）扩增，使用优质高纯度质粒提取试剂盒提质粒。用于解脂酵母转化的质粒浓度建议 $\ge 400\text{ ng/}\mu\text{L}$，纯度 $OD_{260}/OD_{280} = 1.80 - 1.85$。若该载体设计为游离表达，则可直接使用环状质粒转化；若需提高长期工业发酵稳定性，可选用特定限制性内切酶将载体线性化（Linearization）后再进行基因组同源整合转化。

2. 解脂耶氏酵母的转化操作（高效醋酸锂/PEG 转化法）

解脂耶氏酵母的细胞壁富含几丁质和甘露聚糖，通常使用改进型的醋酸锂/聚乙二醇（LiAc/PEG）化学转化法或电击法。以下为实验室最常用且高重复性的 LiAc/PEG 转化步序：

1. 活化与菌体收集：

   • 挑取解脂耶氏酵母宿主菌（如缺失 URA3 的 Po1f 菌株）单菌落接种于 YPD 液体培养基中，28 ℃ - 30 ℃、200 rpm 剧烈振荡培养过夜。

   • 次日转接扩大培养，待菌液密度达到对数生长中期（$OD_{600} \approx 1.0 - 2.0$）时，4 ℃ 下 4000 rpm 离心 5 分钟收集菌体。

2. 清洗与感受态激活：

   • 使用无菌去离子水洗涤菌体沉淀 1 次。

   • 使用 1 mL 新鲜配制的 100 mM 醋酸锂（LiAc）溶液 重悬菌体，室温下静置或微速振荡 10 - 15 分钟，使酵母细胞壁充分溶胀变松散。

   • 再次离心弃去上清，沉淀即为待转化的耶氏酵母感受态细胞。

3. 转化混合物配制（核心加样顺序）：

   向收集好的感受态菌体沉淀中，严格按照以下顺序依次叠加滴加转化试剂：

   • 240 $\mu$L 50% 浓度聚乙二醇（PEG 3350 或 PEG 4000）

   • 36 $\mu$L 1 M 醋酸锂（LiAc）

   • 25 $\mu$L 提前进行过煮沸热变性并迅速冰激的单链载体 DNA（如 10 mg/mL Salmon Sperm DNA 鲑鱼精 DNA，作为载体阻断剂）

   • 5 - 10 $\mu$L 高纯度的 pYLXP' 重组质粒 DNA（总量约 1 - 3 $\mu$g）。

4. 悬浮与热休克（Heat Shock）：

   • 使用移液枪非常彻底、轻柔地吹打，使粘稠的 PEG 混合物与酵母细胞完全混匀成均匀悬液。

   • 置于 30 ℃ 恒温水浴或孵箱中静置孵育 30 分钟。

   • 随后，立刻转移至 39 ℃ - 42 ℃ 水浴箱中进行精确热休克 15 - 20 分钟（解脂耶氏酵母的最适热休克温度低于传统酿酒酵母，切勿超过 43 ℃，否则会导致细胞大面积热致死）。

5. 离心与洗涤重悬：

   • 热休克结束后，在室温下 6000 rpm 离心 2 分钟，极其小心地抽干或倒净粘稠的 PEG 上清液。

   • 加入 1 mL 无菌水或无菌 PBS 缓冲液，极为轻柔地吹打重悬洗涤酵母沉淀（注意：刚经受热休克的酵母细胞膜极其脆弱，严禁剧烈振荡振荡或高速拍打）。

3. 营养缺陷型最小培养基平板筛选与发酵验证

1. 固体缺陷平板初筛：

   吸取 100 - 200 $\mu$L 洗涤重悬后的酵母菌液，均匀涂布于未添加尿嘧啶的 YNB 选择性最小固体平板（SD-Ura 培养基平板：由 0.67% 酵母无氨基氮源基底 YNB、2% 葡萄糖及缺 Ura 的完全混合氨基酸单体组成）。

   将平板倒置放入恒温生化培养箱中，在 28 摄氏度 - 30 摄氏度（解脂耶氏酵母的标准推荐生长温度，绝对禁止放入 37 ℃ 孵箱）下，遮光暗培养 3 - 5 天。成功导入 pYLXP' 质粒的转化子因恢复了 URA3 基因表达，能合成自身所需的尿嘧啶，从而在缺陷平板上长出饱满、圆润、呈乳白色的阳性单菌落。

2. 摇瓶液体发酵与产物表达定量分析：

   • 挑取选定好的阳性单菌落，接种于 SD-Ura 液体选择性培养基中，30 ℃ 预培养 24 小时以维持质粒拷贝数。

   • 按照 1% - 5% 的接种量转接至富含碳源的工业发酵培养基（如高浓度葡萄糖 YPD 培养基，或以甘油、废弃油脂为底物的富脂发酵液）中进行扩大摇瓶发酵。由于 pYLXP' 游离质粒在完全培养基（YPD）中长期传代可能会发生微量质粒丢失，在工业放大时可选择在发酵第 48 小时补加适量选择性压力。

   • 在发酵的关键时间节点（如 24h, 48h, 72h, 96h）抽取发酵粗提液：

     • 若目的产物为胞外分泌蛋白/酶：离心收集上清液，直接进行 SDS-PAGE 胶电泳分析或特异性酶活显色测定。

     • 若目的产物为胞内积累的脂质、胡萝卜素或萜类化合物：离心收集酵母生物量细胞泥，利用尼罗红（Nile Red）荧光染色法进行胞内脂滴可视化测定，或通过有机溶剂（如氯仿-甲醇法）破壁抽提胞内总脂，利用气相色谱-质谱联用仪（GC-MS）或液相色谱（HPLC）进行精确的化学定量谱系分析，评估该超强表达系统下的真实代谢流向。

4. 菌株长期保存标准

• 冷冻保存液配方：常规富集 YPD 液体培养基 或 SD-Ura 缺陷液体培养基 混合 30% 灭菌纯甘油（Glycerol），最终使甘油总体积丰度维持在 15% - 20%。

• 冷冻存放规范：

  1. 收集处于对数生长旺盛期、镜检未见任何细菌杂菌污染的健康 pYLXP' 重组解脂耶氏酵母液体菌液。

  2. 在无菌冻存管中，将 700 $\mu$L 菌液与 300 $\mu$L 无菌 50% 甘油水溶液彻底颠倒混匀。

  3. 降温与储存：可直接或装入降温盒放入 -80 ℃ 超低温冰箱中锁死长期保存。解脂耶氏酵母在 -80 ℃ 甘油悬液状态下具有极高的结构稳态，可稳定存放数年而不发生质粒丢失或自发降解，日常使用时严禁反复冻融，必须实行单管单次使用。

Part 2 English Section

I General Information and Molecular Biological Background

• Vector Name: pYLXP' (also standardly cataloged across dynamic repositories as pYLXP' or pYLXP-prime).

• Vector Classification: Non-conventional industrial yeast expression vehicle / Episomal high-copy expression framework optimized exclusively for Yarrowia lipolytica.

• Plasmid Size Scale: Approximately 7.5 - 8.2 kb (subject to minor size tuning depending on the targeted selection auxotrophic markers and customized multi-cloning sites).

• Backbone Origin and Synthetic Biology Background:

  The pYLXP' expression vector represents a foundational molecular tool utilizing a highly robust architecture widely standardly deployed in Yarrowia lipolytica metabolic engineering and synthetic biology. Yarrowia lipolytica is a non-conventional, oleaginous yeast model globally prized for its phenomenal intracellular lipid accumulation framework, broad substrate utilization kinetics, and high-capacity protein secretion pathways.

  Legacy Y. lipolytica engineering relied on integration-style vectors which, though chromosomes-stable, restricted gene expression to a single-copy low yield parameter. The pYLXP' episomal vector circumvents this design barrier by pairing a highly autonomous replication sequence (ARS) alongside an engineered hybrid ultra-strong promoter cassette, enabling high-copy non-integrative episomal replication combined with high transcriptional outputs. This configuration is widely implemented for assembling complex multigene metabolic arrays inside oleaginous chassis cells.

• Core Cis-Acting Elements and Structural Features:

  • Engineered $TEF_{in}$ Hybrid Ultra-Strong Promoter: The metabolic core driving the massive expression velocity of this vector. It consists of the translation elongation factor-1$\alpha$ (TEF1) promoter core fused to its native intron element. The localized retention of this structural intron activates Intron-Mediated Enhancement (IME) mechanisms, forcing a multi-fold transcriptional surge compared to standard baseline TEF promoters. It acts constitutively, completely eliminating the need for expensive chemical induction agents (e.g., galactose, methanol) during high-density cultivation.

  • Endogenous Autonomous Replication Assembly ($ARS18$ / $CEN$ Box): Grants pYLXP' the genetic infrastructure required to persist, replicate, and segregate as a self-sustaining episomal unit inside the Y. lipolytica nucleoplasm without chromosome integration, maintaining a stable copy number parameter of approximately 10 to 20 copies per cell.

  • Yarrowia Auxotrophic Selection Marker (typically URA3 or LEU2): Enables tight non-antibiotic selection and screening parameters when deployed inside matched nutrient-deficient host strains (e.g., Po1f, Po1g, W29 base variants).

  • E. coli Propagation Engine: Outfitted with a high-copy pUC ori and a functional Ampicillin resistance gene ($Amp^R$) to allow investigators to perform standard plasmid scaling, multi-fragment Gibson assemblies, and high-purity extractions inside Escherichia coli intermediate hosts.

II Strategic Research Value and Industrial Biomanufacturing Applications

The pYLXP' vector matrix serves as a vital genetic engine for high-yield industrial biotechnology and cell-factory optimization:

1. Hyper-Accumulation of Tailored Lipids and Terpenoid Metabolites:

   By overexpressing critical rate-limiting metabolic checkpoints (such as ACC1, DGA1, or HMG1) via pYLXP' inside Y. lipolytica, investigators can force cellular lipid accumulation to exceed 60% of total cell dry weight. This setup is widely standardly implemented to engineer specialized lipid factories producing premium omega-3 fatty acids (EPA/DHA), carotenoids, squalene, or tailored biofuels.

2. Modular Multigene Pathway Coordination:

   The stable episomal persistence and high transcriptional output of pYLXP' facilitate the rapid, modular synchronization of complex heterologous pathways. Investigators can stack multiple gene modules—each independently driven by its own $TEF_{in}$ promoter—onto a single pYLXP' vector via Gibson assembly, ensuring coordinated expression of multi-step enzyme cascades.

3. High-Density Secretion of Recombinant Industrial Enzymes:

   Leveraging the superior post-translational processing machinery of Yarrowia, fusing targeted industrial enzymes (e.g., high-performance lipases, cellulases, or proteases) downstream of the $TEF_{in}$ promoter and pairing them with native signal peptides (such as the XPR2 prepro peptide) enables direct secretion of mature, active proteins into the culture broth, facilitating simplified downstream isolation and purification protocols.

III Laboratory E. coli Cloning, LiAc/PEG Transfection, and Fermentation Analytics

1. Vector Manipulation and High-Yield Preparation inside E. coli

• Recombinant Assembly: Clone the optimized coding sequence of interest into the Multiple Cloning Site (MCS) situated precisely between the hybrid $TEF_{in}$ promoter and the $XPR2$ transcription terminator. Codon optimization tailored to Y. lipolytica translational preferences is strongly recommended to optimize output yields.

• E. coli Amplification: Introduce the assembled pYLXP' vector into competent E. coli cells (e.g., DH5$\alpha$) via classic heat-shock processing. Spread onto standard LB agar plates containing 100 $\mu$g/mL Ampicillin and incubate at 37 °C overnight.

• Plasmid Harvesting: Isolate a single colony for liquid LB expansion. Extract the plasmid matrix using a high-purity miniprep or midiprep kit, ensuring final elution metrics reach $\ge 400\text{ ng/}\mu\text{L}$ with an uncompromised purity profile ($OD_{260}/OD_{280} = 1.80 - 1.85$). Linearize the plasmid via targeted restriction digest if the experimental blueprint demands permanent chromosomal integration rather than episomal maintenance.

2. High-Efficiency Lithium Acetate/PEG Transformation Protocol

Because the cell wall of Yarrowia lipolytica is heavily reinforced with chitin and mannan polymers, a specialized Lithium Acetate / Polyethylene Glycol (LiAc/PEG) chemical permeabilization protocol is standardly utilized:

1. Biomass Activation and Collection:

   • Streak out the targeted auxotrophic host strain (e.g., URA3-deficient strain Po1f) onto a YPD agar plate. Inoculate a single colony into liquid YPD broth and cultivate at 28 °C - 30 °C with vigorous shaking at 200 rpm overnight.

   • Transfect an aliquot into fresh YPD medium to scale up the culture until the optical density reaches mid-log phase ($OD_{600} \approx 1.0 - 2.0$). Spin down the cells at 4000 rpm for 5 minutes at 4 °C to collect the biomass.

2. Permeabilization and Competence Induction:

   • Decant the spent media supernatant and wash the cell pellet once with sterile deionized water.

   • Resuspend the washed cell mass in 1 mL of freshly prepared 100 mM Lithium Acetate (LiAc) solution. Incubate at room temperature for 10 - 15 minutes with gentle agitation to permeabilize the yeast cell wall structure.

   • Spin down the cells and discard the LiAc solution supernatant; the remaining cellular pellet constitutes the transformation-ready competent cell matrix.

3. Formulating the Transformation Cocktail (Strict Multi-Component Sequence):

   Layer the following transformation components directly onto the prepared competent cell pellet in the precise order specified below:

   • 240 $\mu$L Polyethylene Glycol (50% w/v PEG 3350 or PEG 4000)

   • 36 $\mu$L Lithium Acetate (1 M LiAc)

   • 25 $\mu$L Carrier DNA (10 mg/mL Single-Stranded Salmon Sperm DNA, pre-boiled at 95 °C for 5 minutes and instantly chilled on ice to serve as a cellular transport shield)

   • 5 - 10 $\mu$L High-Purity pYLXP' Recombinant Plasmid DNA (Containing roughly 1 - 3 $\mu$g of total vector input).

4. Resuspension and Thermal Shock Processing:

   • Pipette the highly viscous mixture gently but thoroughly to transform the yeast pellet into a homogenous suspension.

   • Incubate the mixture statically inside a 30 °C incubator or water bath for 30 minutes.

   • Following incubation, transfer the tubes directly into a water bath calibrated to 39 °C - 42 °C to execute a precise heat shock for 15 - 20 minutes. Monitor the thermal threshold closely; Yarrowia cells are sensitive to high-temperature stress—exceeding 43 °C can cause severe cell mortality.

5. Recovery Washing Sequence:

   • Pellet the heat-shocked cells via centrifugation at 6000 rpm for 2 minutes at room temperature, then carefully remove the viscous PEG supernatant layer.

   • Dispense 1 mL of sterile deionized water or PBS buffer and resuspend the cells using gentle pipetting. Avoid vortexing or harsh mechanical agitation at this stage, as the cell membranes remain fragile post-heat shock.

3. Selection and Biomanufacturing Fermentation Analytics

1. Auxotrophic Solid-Plate Selection:

   Aspirate a 100 - 200 $\mu$L aliquot of the washed yeast suspension and spread it uniformly across Synthetic Defined Uracil-deficient agar plates (SD-Ura plates) (comprising 0.67% Yeast Nitrogen Base without amino acids, 2% D-Glucose, 1.5% agar, and an optimized drop-out amino acid mixture lacking Uracil).

   Invert the plates and place them inside a constant incubator calibrated strictly to 28 °C - 30 °C for 3 - 5 days in complete darkness. Never incubate Yarrowia cultures at 37 °C. Transformed single colonies harboring the pYLXP' vector will synthesize endogenous Uracil via the rescued URA3 gene marker, emerging as prominent, creamy-white circular colonies.

2. Liquid Scaling and High-Yield Fermentation Assays:

   • Inoculate a confirmed single colony into liquid SD-Ura drop-out media and grow at 30 °C for 24 hours to stabilize episomal vector copy numbers.

   • Inoculate this pre-culture at a 1% - 5% ratio into carbon-rich production media (such as high-glucose YPD broth or specialized industrial matrices supplemented with glycerol or crude lipids). While pYLXP' exhibits high replication stability, a slight drop-out selection pressure can be maintained if prolonged fermentation phases are required.

   • Collect samples at specific time points (e.g., 24h, 48h, 72h, 96h) to analyze metabolic yields:

     • For Secreted Recombinant Proteins: Centrifuge the fermentation broth, harvest the cell-free supernatant, and evaluate via SDS-PAGE or specific enzymatic colorimetric assays to quantify production metrics.

     • For Intracellular Lipids or Terpenoid Compounds: Collect the yeast biomass pellet. Intracellular lipid droplet formation can be monitored via Nile Red fluorescent imaging. To quantify compound yields, disrupt the cell wall and extract the total lipid fraction using organic solvent matrices (e.g., the chloroform-methanol method), followed by precise quantitative profiling via Gas Chromatography-Mass Spectrometry (GC-MS) or High-Performance Liquid Chromatography (HPLC).

4. Long-Term Strain Preservation Standards

• Cryoprotectant Preservation Formula: Combine active, mid-log phase YPD or SD-Ura liquid cultures with sterile 30% v/v Glycerol in a 7:3 ratio, yielding a final cryoprotectant mixture containing approximately 15% - 20% glycerol.

• Storage Protocol:

  1. Verify that the liquid culture shows optimal cell density and is entirely clear of external bacterial or fungal contamination.

  2. Combine 700 $\mu$L of active culture with 300 $\mu$L of the sterile 50% glycerol stock inside a sterile cryovial and mix thoroughly by inversion.

  3. Freezing Matrix: Transfer the prepared cryovials directly into an ultra-low freezer calibrated to -80 °C for long-term storage. Yarrowia lipolytica remains viable for several years when stored under these conditions without displaying significant plasmid loss or genetic drift. To maintain viability, avoid repeated freeze-thaw cycles; thaw each cryovial only once for direct experimental activation.

  

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