BioVector? pINA1312 解脂耶氏酵母表達/整合質(zhì)粒載體

BioVector? pINA1312 Yarrowia lipolytica Expression and Integration Vector

第一部分 中文說明

一 載體基本信息與科研用途

• 載體名稱 BioVector? pINA1312

• 載體類型 非常規(guī)酵母表達質(zhì)粒載體 / 基因組整合型載體。

• 核心用途 專門用于常規(guī)工業(yè)底盤及高產(chǎn)脂常規(guī)菌株——解脂耶氏酵母（Yarrowia lipolytica）中的外源基因高效表達、分泌克隆與基因組定向整合。

• 載體大小 約 5.5 kb到6.2 kb（根據(jù)內(nèi)部插入的特定啟動子及信號肽元件排列有所不同）。

• 抗性標記

  • 大腸桿菌篩選 卡那霉素抗性（Kanamycin，$Km^R$）或特定衍生骨架的氨芐青霉素抗性。

  • 酵母篩選 帶有修飾或弱化的尿嘧啶營養(yǎng)缺陷型標志基因（$URA3$ 核心選擇標記，如 $ura3d1$ 等位基因）。

• 常用宿主菌 大腸桿菌 DH5a、Top10 以及解脂耶氏酵母缺陷型工業(yè)底盤株（如 Po1f、Po1g、po1fe-3）。

二 關鍵結(jié)構(gòu)域與元件配置

• 啟動子系統(tǒng)（Promoter Systems）：根據(jù)具體衍生型號，該骨架通常搭載以下兩種高強度解脂耶氏酵母專用啟動子之一：

  • hp4d 強啟動子：一種經(jīng)典的非誘導型高強度雜合啟動子，包含4個串聯(lián)的 XPR2 啟動子上游激活序列（UAS）。其最大優(yōu)勢在于其轉(zhuǎn)錄活性幾乎不受培養(yǎng)基組分、碳源類型或 pH 值的波動干擾，在細胞生長的對數(shù)末期及穩(wěn)定期能夠?qū)崿F(xiàn)自發(fā)性的高效轉(zhuǎn)錄。

  • XPR2 或 POX2 啟動子：用于高精度環(huán)境條件誘導轉(zhuǎn)錄的調(diào)控機制。

• 終止子（Terminator）：下游搭載解脂耶氏酵母固有的 堿性細胞外蛋白酶終止子（XPR2 Terminator），提供極其高效和精確的 mRNA 3端加工、聚腺苷酸化及轉(zhuǎn)錄終止。

• 選擇標記與整合策略：內(nèi)置的 $URA3$ 營養(yǎng)缺陷型標志（多采用帶有特定啟動子或片段刪減的 $ura3d1$ 等位基因）。

  • 核心功能：該等位基因?qū)ｉT設計用于介導單拷貝定向同源重組整合（Single-copy integration）。由于該選擇標記的自主轉(zhuǎn)錄能力被故意弱化，在轉(zhuǎn)化對應的尿嘧啶缺陷型酵母株后，只有當整個質(zhì)粒重組轉(zhuǎn)錄盒在基因組特定平臺位點（如 $Zeta$ 序列或相應的宿主對接底盤）發(fā)生穩(wěn)定的染色體整合時，轉(zhuǎn)化子才能在缺乏尿嘧啶的合成選擇平板上存活。這種設計極大地保證了工程菌株的遺傳穩(wěn)定性，完全杜絕了多拷貝質(zhì)粒串聯(lián)丟失或表達量大幅波動的風險。

三 標準分子克隆與轉(zhuǎn)化操作步驟

1. 目的基因克隆 選用多克隆位點中的合適酶切位點，將經(jīng)密碼子優(yōu)化的目的基因（或融合了 LIP2 信號肽的分泌盒）插入到 BioVector? pINA1312 載體中，通過大腸桿菌進行擴增和測序鑒定。

2. 質(zhì)粒線性化制備 提取高純度的重組質(zhì)粒，使用特定限制性內(nèi)切酶（如 NotI）對質(zhì)粒進行徹底消化，將包含 $URA3$ 選擇標記、表達元件盒及兩端整合序列的 DNA 片段整體釋放線性化。

3. 耶氏酵母電轉(zhuǎn)化 采用醋酸鋰（LiAc）/聚乙二醇（PEG）法或高效電轉(zhuǎn)化法，將線性化片段導入處于對數(shù)生長期的解脂耶氏酵母（如 Po1f 缺陷株）中。

4. 陽性克隆篩選與驗證 將轉(zhuǎn)化后的酵母細胞涂布于缺乏尿嘧啶的 BioVector? 酵母合成完全選擇固體培養(yǎng)基（SD-Ura 培養(yǎng)基） 平板上，30攝氏度培養(yǎng) 3到5天。挑取長出的陽性轉(zhuǎn)化子，通過基因組 PCR 和染色體印跡分析驗證外源基因是否成功同源整合入基因組。

四 核心科研應用方向

1. 常規(guī)與復雜脂質(zhì)代謝調(diào)控網(wǎng)絡工程：由于解脂耶氏酵母天生具備龐大的乙酰輔酶A（Acetyl-CoA）和丙二酸單酰輔酶A（Malonyl-CoA）前體供應池，BioVector? pINA1312 經(jīng)常被用來過表達各類關鍵限速酶（如 DGA1、ACC1），用于創(chuàng)制高產(chǎn)類胡蘿卜素（Astaxanthin、蝦青素）、長鏈多不飽和脂肪酸或新型黃酮類化合物（如 icaritin）的微生物高效細胞工廠。

2. 重組異源復雜工業(yè)蛋白的分泌表達：利用該載體搭配高效的分泌信號肽，可實現(xiàn)各種外源工業(yè)酶（如脂肪酶、纖維素酶、淀粉酶）在解脂耶氏酵母中數(shù)十克每升級別的高純度周質(zhì)或胞外分泌，方便直接回收。

3. CRISPR/Cas9 多靶點編輯系統(tǒng)的穩(wěn)定集成：常作為 Cas9 基因或精準引導編輯工具（如 eSpCas9 表達盒）的穩(wěn)定載體骨架，通過 NotI 線性化將其永久集成整合入酵母基因組中，從而建立具備高效率、高穩(wěn)定編輯能力的工具菌株系。

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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