BioVector? pYLXP' Yarrowia lipolytica High-Efficiency Expression Vector / pYLXP' 解脂耶氏酵母高水平表達(dá)載體

一 產(chǎn)品基本信息與分子生物學(xué)背景

• 載體名稱：pYLXP'（亦常寫作 pYLXP' 或 pYLXP-prime）。

• 載體分類：非傳統(tǒng)工業(yè)酵母表達(dá)質(zhì)粒 / 解脂耶氏酵母（Yarrowia lipolytica）專用高拷貝表達(dá)載體。

• 質(zhì)粒大小：約 7.5 - 8.2 kb（依具體配置的選擇標(biāo)記和克隆多克隆位點(diǎn)微調(diào)）。

• 骨架源起與設(shè)計(jì)背景（合成生物學(xué)明星底盤）：

  pYLXP' 是一款在解脂耶氏酵母（Yarrowia lipolytica）合成生物學(xué)和代謝工程研究中極具統(tǒng)治地位的經(jīng)典高水平表達(dá)質(zhì)粒。解脂耶氏酵母作為一種著名的“富油酵母（Oleaginous yeast）”，具有極強(qiáng)的脂質(zhì)積累能力、廣闊的底物利用譜和卓越的蛋白質(zhì)分泌胞外能力，被廣泛用于生產(chǎn)脂肪酸、生物燃料、類胡蘿卜素及工業(yè)酶制劑。傳統(tǒng)的解脂酵母載體多為低拷貝整合型（Integration）質(zhì)粒，雖然穩(wěn)定但表達(dá)量有限。pYLXP' 及其系列衍生載體，通過配置特殊的自復(fù)制序列（Autonomously Replicating Sequence, ARS）和百倍級(jí)強(qiáng)啟動(dòng)子工程改造，實(shí)現(xiàn)了外源基因在解脂耶氏酵母體內(nèi)的高拷貝游離（Episomal）維持與爆發(fā)式轉(zhuǎn)錄表達(dá)，是目前國(guó)際上組裝各種復(fù)雜多基因代謝通路（Multigene metabolic pathways）的標(biāo)配骨架。

• 核心順式作用元件與圖譜特征：

  • $TEF_{in}$ 雜合超強(qiáng)啟動(dòng)子（Hybrid Promoter）：這是該載體高表達(dá)的靈魂元件。它由翻譯伸長(zhǎng)因子-1$\alpha$（TEF1）啟動(dòng)子與其內(nèi)含子（Intron）序列融合雜合而成。研究表明，該內(nèi)含子的存在能通過“內(nèi)含子介導(dǎo)的表達(dá)增強(qiáng)機(jī)制（IME）”使轉(zhuǎn)錄水平相較于傳統(tǒng) TEF 啟動(dòng)子激增數(shù)倍至數(shù)十倍，且屬于強(qiáng)烈的結(jié)構(gòu)性（Constitutive）驅(qū)動(dòng)，無(wú)需添加昂貴的誘導(dǎo)劑。

  • 解脂酵母特殊自復(fù)制元件（$ARS18$ / $CEN$ 盒）：賦予質(zhì)粒在解脂耶氏酵母胞內(nèi)不依賴基因組整合、即可進(jìn)行獨(dú)立自主復(fù)制的能力，每細(xì)胞拷貝數(shù)通常可維持在 10 - 20 個(gè)以上。

  • 解脂酵母篩選標(biāo)記（通常為 URA3 或 LEU2 營(yíng)養(yǎng)缺陷型標(biāo)記）：用于在相應(yīng)的解脂酵母營(yíng)養(yǎng)缺陷型宿主菌株（如 Po1f、Po1g、W29 衍生株）中進(jìn)行嚴(yán)格的非抗生素選擇性壓力篩選。

  • 大腸桿菌元件：含有 pUC ori 和 氨芐青霉素抗性基因（$Amp^R$），便于前期在 E. coli 中進(jìn)行極其高效的質(zhì)?？寺?、重組 DNA 組裝與大抽提。

二 核心科研價(jià)值與工業(yè)生物制造轉(zhuǎn)化應(yīng)用

pYLXP' 質(zhì)粒在現(xiàn)代現(xiàn)代白生物技術(shù)、油脂化學(xué)品發(fā)酵及合成生物學(xué)中扮演著核心角色：

1. 高附加值脂質(zhì)及類萜化合物的高產(chǎn)調(diào)控（脂質(zhì)工廠）：

   在解脂耶氏酵母中利用 pYLXP' 載體過表達(dá)關(guān)鍵限速酶（如 ACC1, DGA1 或 HMG1），可將酵母胞內(nèi)的油脂（TAG）積累量提升至細(xì)胞干重的 60% 以上，或用于高效合成諸如類胡蘿卜素（Carotenoids）、角鯊烯（Squalene）、EPA/DHA  omega-3 脂肪酸等高價(jià)值非天然產(chǎn)物。

2. 多基因發(fā)酵代謝通路的模塊化快速組裝（Modular Assembly）：

   由于 pYLXP' 具有良好的游離復(fù)制特性和極強(qiáng)的表達(dá)均一性，科研人員常通過重疊延伸 PCR（OE-PCR）或 Gibson 組裝技術(shù)，將多個(gè)基因分別置于獨(dú)立的 $TEF_{in}$ 驅(qū)動(dòng)夾層中，并排組裝進(jìn) pYLXP' 骨架。這使得在一顆質(zhì)粒上同時(shí)實(shí)現(xiàn)一條完整外源異源代謝途徑的高效運(yùn)轉(zhuǎn)成為現(xiàn)實(shí)。

3. 復(fù)雜工業(yè)酶制劑的高效胞外分泌表達(dá)：

   利用解脂耶氏酵母完美的蛋白質(zhì)折疊和強(qiáng)大的翻譯后修飾體系，在 pYLXP' 的超強(qiáng)啟動(dòng)子下游融合外源酶（如高級(jí)脂肪酶、纖維素酶、蛋白酶）及耶氏酵母內(nèi)源的分泌信號(hào)肽（如 XPR2 prepro 序列），可直接將大量具有完全生物活性的重組工業(yè)酶高濃度分泌到發(fā)酵液中，極大地簡(jiǎn)化了下游純化（Downstream processing）工序。

三 實(shí)驗(yàn)室大腸克隆、耶氏酵母電轉(zhuǎn)化/化學(xué)轉(zhuǎn)化與高產(chǎn)篩選標(biāo)準(zhǔn)步驟

1. 質(zhì)粒在大腸桿菌（E. coli）中的分子克隆與高質(zhì)純化

• 重組組裝：將目的基因（帶有解脂酵母密碼子優(yōu)化 Optimized codons 更佳）克隆至 pYLXP' 的多克隆位點(diǎn)（通常位于 $TEF_{in}$ 啟動(dòng)子與 $XPR2$ 終止子之間）。

• 轉(zhuǎn)化與擴(kuò)增：通過常規(guī)熱擊法轉(zhuǎn)化大腸桿菌常規(guī)感受態(tài)（如 DH5$\alpha$），涂布于含有 100 $\mu$g/mL 氨芐青霉素的 LB 固體平板上，37 ℃ 培養(yǎng)過夜。

• 高質(zhì)純化：挑取單菌落進(jìn)行液體 LB（100 $\mu$g/mL Amp）擴(kuò)增，使用優(yōu)質(zhì)高純度質(zhì)粒提取試劑盒提質(zhì)粒。用于解脂酵母轉(zhuǎn)化的質(zhì)粒濃度建議 $\ge 400\text{ ng/}\mu\text{L}$，純度 $OD_{260}/OD_{280} = 1.80 - 1.85$。若該載體設(shè)計(jì)為游離表達(dá)，則可直接使用環(huán)狀質(zhì)粒轉(zhuǎn)化；若需提高長(zhǎng)期工業(yè)發(fā)酵穩(wěn)定性，可選用特定限制性內(nèi)切酶將載體線性化（Linearization）后再進(jìn)行基因組同源整合轉(zhuǎn)化。

2. 解脂耶氏酵母的轉(zhuǎn)化操作（高效醋酸鋰/PEG 轉(zhuǎn)化法）

解脂耶氏酵母的細(xì)胞壁富含幾丁質(zhì)和甘露聚糖，通常使用改進(jìn)型的醋酸鋰/聚乙二醇（LiAc/PEG）化學(xué)轉(zhuǎn)化法或電擊法。以下為實(shí)驗(yàn)室最常用且高重復(fù)性的 LiAc/PEG 轉(zhuǎn)化步序：

1. 活化與菌體收集：

   • 挑取解脂耶氏酵母宿主菌（如缺失 URA3 的 Po1f 菌株）單菌落接種于 YPD 液體培養(yǎng)基中，28 ℃ - 30 ℃、200 rpm 劇烈振蕩培養(yǎng)過夜。

   • 次日轉(zhuǎn)接擴(kuò)大培養(yǎng)，待菌液密度達(dá)到對(duì)數(shù)生長(zhǎng)中期（$OD_{600} \approx 1.0 - 2.0$）時(shí)，4 ℃ 下 4000 rpm 離心 5 分鐘收集菌體。

2. 清洗與感受態(tài)激活：

   • 使用無(wú)菌去離子水洗滌菌體沉淀 1 次。

   • 使用 1 mL 新鮮配制的 100 mM 醋酸鋰（LiAc）溶液 重懸菌體，室溫下靜置或微速振蕩 10 - 15 分鐘，使酵母細(xì)胞壁充分溶脹變松散。

   • 再次離心棄去上清，沉淀即為待轉(zhuǎn)化的耶氏酵母感受態(tài)細(xì)胞。

3. 轉(zhuǎn)化混合物配制（核心加樣順序）：

   向收集好的感受態(tài)菌體沉淀中，嚴(yán)格按照以下順序依次疊加滴加轉(zhuǎn)化試劑：

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

   • 36 $\mu$L 1 M 醋酸鋰（LiAc）

   • 25 $\mu$L 提前進(jìn)行過煮沸熱變性并迅速冰激的單鏈載體 DNA（如 10 mg/mL Salmon Sperm DNA 鮭魚精 DNA，作為載體阻斷劑）

   • 5 - 10 $\mu$L 高純度的 pYLXP' 重組質(zhì)粒 DNA（總量約 1 - 3 $\mu$g）。

4. 懸浮與熱休克（Heat Shock）：

   • 使用移液槍非常徹底、輕柔地吹打，使粘稠的 PEG 混合物與酵母細(xì)胞完全混勻成均勻懸液。

   • 置于 30 ℃ 恒溫水浴或孵箱中靜置孵育 30 分鐘。

   • 隨后，立刻轉(zhuǎn)移至 39 ℃ - 42 ℃ 水浴箱中進(jìn)行精確熱休克 15 - 20 分鐘（解脂耶氏酵母的最適熱休克溫度低于傳統(tǒng)釀酒酵母，切勿超過 43 ℃，否則會(huì)導(dǎo)致細(xì)胞大面積熱致死）。

5. 離心與洗滌重懸：

   • 熱休克結(jié)束后，在室溫下 6000 rpm 離心 2 分鐘，極其小心地抽干或倒凈粘稠的 PEG 上清液。

   • 加入 1 mL 無(wú)菌水或無(wú)菌 PBS 緩沖液，極為輕柔地吹打重懸洗滌酵母沉淀（注意：剛經(jīng)受熱休克的酵母細(xì)胞膜極其脆弱，嚴(yán)禁劇烈振蕩振蕩或高速拍打）。

3. 營(yíng)養(yǎng)缺陷型最小培養(yǎng)基平板篩選與發(fā)酵驗(yàn)證

1. 固體缺陷平板初篩：

   吸取 100 - 200 $\mu$L 洗滌重懸后的酵母菌液，均勻涂布于未添加尿嘧啶的 YNB 選擇性最小固體平板（SD-Ura 培養(yǎng)基平板：由 0.67% 酵母無(wú)氨基氮源基底 YNB、2% 葡萄糖及缺 Ura 的完全混合氨基酸單體組成）。

   將平板倒置放入恒溫生化培養(yǎng)箱中，在 28 攝氏度 - 30 攝氏度（解脂耶氏酵母的標(biāo)準(zhǔn)推薦生長(zhǎng)溫度，絕對(duì)禁止放入 37 ℃ 孵箱）下，遮光暗培養(yǎng) 3 - 5 天。成功導(dǎo)入 pYLXP' 質(zhì)粒的轉(zhuǎn)化子因恢復(fù)了 URA3 基因表達(dá)，能合成自身所需的尿嘧啶，從而在缺陷平板上長(zhǎng)出飽滿、圓潤(rùn)、呈乳白色的陽(yáng)性單菌落。

2. 搖瓶液體發(fā)酵與產(chǎn)物表達(dá)定量分析：

   • 挑取選定好的陽(yáng)性單菌落，接種于 SD-Ura 液體選擇性培養(yǎng)基中，30 ℃ 預(yù)培養(yǎng) 24 小時(shí)以維持質(zhì)粒拷貝數(shù)。

   • 按照 1% - 5% 的接種量轉(zhuǎn)接至富含碳源的工業(yè)發(fā)酵培養(yǎng)基（如高濃度葡萄糖 YPD 培養(yǎng)基，或以甘油、廢棄油脂為底物的富脂發(fā)酵液）中進(jìn)行擴(kuò)大搖瓶發(fā)酵。由于 pYLXP' 游離質(zhì)粒在完全培養(yǎng)基（YPD）中長(zhǎng)期傳代可能會(huì)發(fā)生微量質(zhì)粒丟失，在工業(yè)放大時(shí)可選擇在發(fā)酵第 48 小時(shí)補(bǔ)加適量選擇性壓力。

   • 在發(fā)酵的關(guān)鍵時(shí)間節(jié)點(diǎn)（如 24h, 48h, 72h, 96h）抽取發(fā)酵粗提液：

     • 若目的產(chǎn)物為胞外分泌蛋白/酶：離心收集上清液，直接進(jìn)行 SDS-PAGE 膠電泳分析或特異性酶活顯色測(cè)定。

     • 若目的產(chǎn)物為胞內(nèi)積累的脂質(zhì)、胡蘿卜素或萜類化合物：離心收集酵母生物量細(xì)胞泥，利用尼羅紅（Nile Red）熒光染色法進(jìn)行胞內(nèi)脂滴可視化測(cè)定，或通過有機(jī)溶劑（如氯仿-甲醇法）破壁抽提胞內(nèi)總脂，利用氣相色譜-質(zhì)譜聯(lián)用儀（GC-MS）或液相色譜（HPLC）進(jìn)行精確的化學(xué)定量譜系分析，評(píng)估該超強(qiáng)表達(dá)系統(tǒng)下的真實(shí)代謝流向。

4. 菌株長(zhǎng)期保存標(biāo)準(zhǔn)

• 冷凍保存液配方：常規(guī)富集 YPD 液體培養(yǎng)基 或 SD-Ura 缺陷液體培養(yǎng)基 混合 30% 滅菌純甘油（Glycerol），最終使甘油總體積豐度維持在 15% - 20%。

• 冷凍存放規(guī)范：

  1. 收集處于對(duì)數(shù)生長(zhǎng)旺盛期、鏡檢未見任何細(xì)菌雜菌污染的健康 pYLXP' 重組解脂耶氏酵母液體菌液。

  2. 在無(wú)菌凍存管中，將 700 $\mu$L 菌液與 300 $\mu$L 無(wú)菌 50% 甘油水溶液徹底顛倒混勻。

  3. 降溫與儲(chǔ)存：可直接或裝入降溫盒放入 -80 ℃ 超低溫冰箱中鎖死長(zhǎng)期保存。解脂耶氏酵母在 -80 ℃ 甘油懸液狀態(tài)下具有極高的結(jié)構(gòu)穩(wěn)態(tài)，可穩(wěn)定存放數(shù)年而不發(fā)生質(zhì)粒丟失或自發(fā)降解，日常使用時(shí)嚴(yán)禁反復(fù)凍融，必須實(shí)行單管單次使用。

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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