BioVector® pINA1312 解脂耶氏酵母表达/整合质粒载体

BioVector® pINA1312 Yarrowia lipolytica Expression and Integration Vector

第一部分 中文说明

一 载体基本信息与科研用途

• 载体名称 BioVector® pINA1312

• 载体类型 非常规酵母表达质粒载体 / 基因组整合型载体。

• 核心用途 专门用于常规工业底盘及高产脂常规菌株——解脂耶氏酵母（Yarrowia lipolytica）中的外源基因高效表达、分泌克隆与基因组定向整合。

• 载体大小 约 5.5 kb到6.2 kb（根据内部插入的特定启动子及信号肽元件排列有所不同）。

• 抗性标记

  • 大肠杆菌筛选 卡那霉素抗性（Kanamycin，$Km^R$）或特定衍生骨架的氨苄青霉素抗性。

  • 酵母筛选 带有修饰或弱化的尿嘧啶营养缺陷型标志基因（$URA3$ 核心选择标记，如 $ura3d1$ 等位基因）。

• 常用宿主菌 大肠杆菌 DH5a、Top10 以及解脂耶氏酵母缺陷型工业底盘株（如 Po1f、Po1g、po1fe-3）。

二 关键结构域与元件配置

• 启动子系统（Promoter Systems）：根据具体衍生型号，该骨架通常搭载以下两种高强度解脂耶氏酵母专用启动子之一：

  • hp4d 强启动子：一种经典的非诱导型高强度杂合启动子，包含4个串联的 XPR2 启动子上游激活序列（UAS）。其最大优势在于其转录活性几乎不受培养基组分、碳源类型或 pH 值的波动干扰，在细胞生长的对数末期及稳定期能够实现自发性的高效转录。

  • XPR2 或 POX2 启动子：用于高精度环境条件诱导转录的调控机制。

• 终止子（Terminator）：下游搭载解脂耶氏酵母固有的 碱性细胞外蛋白酶终止子（XPR2 Terminator），提供极其高效和精确的 mRNA 3端加工、聚腺苷酸化及转录终止。

• 选择标记与整合策略：内置的 $URA3$ 营养缺陷型标志（多采用带有特定启动子或片段删减的 $ura3d1$ 等位基因）。

  • 核心功能：该等位基因专门设计用于介导单拷贝定向同源重组整合（Single-copy integration）。由于该选择标记的自主转录能力被故意弱化，在转化对应的尿嘧啶缺陷型酵母株后，只有当整个质粒重组转录盒在基因组特定平台位点（如 $Zeta$ 序列或相应的宿主对接底盘）发生稳定的染色体整合时，转化子才能在缺乏尿嘧啶的合成选择平板上存活。这种设计极大地保证了工程菌株的遗传稳定性，完全杜绝了多拷贝质粒串联丢失或表达量大幅波动的风险。

三 标准分子克隆与转化操作步骤

1. 目的基因克隆 选用多克隆位点中的合适酶切位点，将经密码子优化的目的基因（或融合了 LIP2 信号肽的分泌盒）插入到 BioVector® pINA1312 载体中，通过大肠杆菌进行扩增和测序鉴定。

2. 质粒线性化制备 提取高纯度的重组质粒，使用特定限制性内切酶（如 NotI）对质粒进行彻底消化，将包含 $URA3$ 选择标记、表达元件盒及两端整合序列的 DNA 片段整体释放线性化。

3. 耶氏酵母电转化 采用醋酸锂（LiAc）/聚乙二醇（PEG）法或高效电转化法，将线性化片段导入处于对数生长期的解脂耶氏酵母（如 Po1f 缺陷株）中。

4. 阳性克隆筛选与验证 将转化后的酵母细胞涂布于缺乏尿嘧啶的 BioVector® 酵母合成完全选择固体培养基（SD-Ura 培养基） 平板上，30摄氏度培养 3到5天。挑取长出的阳性转化子，通过基因组 PCR 和染色体印迹分析验证外源基因是否成功同源整合入基因组。

四 核心科研应用方向

1. 常规与复杂脂质代谢调控网络工程：由于解脂耶氏酵母天生具备庞大的乙酰辅酶A（Acetyl-CoA）和丙二酸单酰辅酶A（Malonyl-CoA）前体供应池，BioVector® pINA1312 经常被用来过表达各类关键限速酶（如 DGA1、ACC1），用于创制高产类胡萝卜素（Astaxanthin、虾青素）、长链多不饱和脂肪酸或新型黄酮类化合物（如 icaritin）的微生物高效细胞工厂。

2. 重组异源复杂工业蛋白的分泌表达：利用该载体搭配高效的分泌信号肽，可实现各种外源工业酶（如脂肪酶、纤维素酶、淀粉酶）在解脂耶氏酵母中数十克每升级别的高纯度周质或胞外分泌，方便直接回收。

3. CRISPR/Cas9 多靶点编辑系统的稳定集成：常作为 Cas9 基因或精准引导编辑工具（如 eSpCas9 表达盒）的稳定载体骨架，通过 NotI 线性化将其永久集成整合入酵母基因组中，从而建立具备高效率、高稳定编辑能力的工具菌株系。

PART 2 ENGLISH SECTION

I General Information and Applications

• Vector Name BioVector® pINA1312

• Vector Type Non-conventional Yeast Expression and Non-replicative Genomic Integration Vector.

• Primary Application Specifically tailored for high-level heterologous gene expression, protein secretion, and chromosomal targeted integration in the oleaginous industrial yeast host Yarrowia lipolytica.

• Vector Footprint Approximately 5.5 kb to 6.2 kb (subject to custom internal modifications containing varying signal sequences or promoter fragments).

• Selection Markers

  • Bacterial Selection Propagated inside Escherichia coli using Kanamycin resistance ($Km^R$) or Ampicillin modules depending on exact downstream MCS subclones.

  • Yeast Selection Outfitted with a specialized, defective uracil auxotrophic marker variant ($URA3$ expression allele, e.g., $ura3d1$).

• Common Host Strains E. coli DH5a, Top10, and auxotrophic Yarrowia lipolytica chasses (e.g., Po1f, Po1g, or genomic platforms like po1fe-3).

II Vector Anatomy and Component Configuration

• Promoter Infrastructure: Depending on the specific variant configuration, this platform typically deploys one of the two major standard Y. lipolytica trans-activation units:

  • hp4d Hybrid Promoter: A powerful, constitutive, and non-regulatable artificial promoter structure consisting of four tandem copies of the XPR2 upstream activating sequences (UAS) linked to a minimal core promoter. It functions independently of medium formulations, pH flux, or specific carbon feeds, maximizing its peak transcript activity from the late log phase seamlessly through the extended stationary cultivation window.

  • XPR2 or POX2 Inducible Promoters: Optioned for conditional, micro-environmentally triggered transcript controls.

• Terminator Configuration: Equipped downstream with the genuine Yarrowia alkaline extracellular protease terminator (XPR2 Terminator), ensuring proper polyadenylation processing, mRNA transcript stability, and exact elongation termination.

• Selection-Driven Integration Strategy: Features the disabled $ura3d1$ marker allele which strictly drives stable single-copy genomic homologous recombination. Because the marker lacks autonomous transcript driving elements, it fails to complement the host auxotrophy as a free floating episomal circular vector. Consequently, survival on synthetic uracil drop-out matrices requires a precise chromosomal integration event into target docking loci (such as $Zeta$ repetitive arrays or designated engineered host integration sites), providing ultimate genetic stability during long term fermentations.

III Subcloning and Yeast Transformation Protocols

1. Target Insertion Clone codon-optimized open reading frames or specialized fusion expression blocks (e.g., matching the LIP2 or XPR2 secretion prepro leaders) into the dense MCS of BioVector® pINA1312, propagating the construct inside E. coli.

2. Vector Linearization Extract high-purity recombinant plasmids and subject them to absolute restriction digestion via targeted endonucleases like NotI to fully release the linear integration fragment containing the functional $URA3$ cassette, the target open reading frame, and flanking genomic sequence arms.

3. Competent Cell Delivery Introduce the purified linear DNA cassette into mid-log phase competent Yarrowia lipolytica via optimized Lithium Acetate/Polyethylene Glycol (LiAc/PEG) or high-voltage electroporation methods.

4. Selection and Monoclonal Validation Plat the transformed yeast suspension onto BioVector® Synthetic Dextrose Uracil Dropout agar (SD-Ura plates) and incubate undisturbed at 30 degrees Celsius for 3 to 5 days. Confirm targeted insertion via colony PCR and Southern blot strategies to ensure single copy integration architecture.

IV Strategic Research Applications

1. Oleaginous Metabolic Engineering and Synthetic Biochemistry: Since Y. lipolytica maintains massive internal pools of Acetyl-CoA and Malonyl-CoA, BioVector® pINA1312 is widely deployed to drive rate-limiting lipogenic genes (e.g., DGA1, ACC1), generating high-titer microbial cell factories for carotenoids (Astaxanthin, beta-carotene), valuable polyunsaturated fatty acids, or premium plant flavonoids (like icaritin).

2. Heterologous Bio-Enzyme Secretion Production: Paired with the highly efficient prepro peptide signals, this backbone reliably directs robust processing and extracellular transport of commercial enzymes (e.g., lipases, cellulases, peptidases) yielding tens of grams per liter of pure protein directly out of cell-free fermentation broths.

3. CRISPR/Cas9 Tool Suite Stabilization: Extensively used as a robust delivery platform to integrate Cas9 variants or base editing machineries permanently into the host chromosome, establishing uniform, non-fluctuating gene editing baseline strains for parallel synthetic biology projects.

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