Name: genetic engineering of an unconventional yeast for renewable biofuel and biochemical production
Uploaded: 2016-09-20T08:00:00.000+00:00
Duration: 10 min 10 s
Description: Watch this Scientific Journal Video about Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production at JoVE.com

我们非常感谢来自新加坡（ETRP 1201102）的国家环境局的资金支持，新加坡国立研究基金会（NRF-CRP5-2009-03），该机构对新加坡科学技术研究的竞争研究计划（ 1324004108），全球R＆D项目计划，知识经济部，韩国（N0000677），国防威胁降低局（DTRA，HDTRA1-13-1-0037）和合成生物学新加坡国立大学倡议（ DPRT / 943/09/14）。

1.新一代的Y的解脂 KU70删除应变<ol><li>中断盒建设 注:请参阅表1为聚合酶链反应（PCR）扩增使用的所有引物。 <ol><li>设计的引物20进行PCR扩增LEU2表达盒（ 见表1）从一个Y.和一个长的反向引物（＃2; 表1）分别; 解脂表达载体并引入LoxP位点到5&#39;和3&#39;LEU2启动盒与长正向引物（ 表1＃1）结束。引入用于克隆过程的后续步骤的额外限制性位点，加在引物＃1的巴姆 HI限制性位点。 </li><li>使用高保真DNA聚合酶如表2中详述的进行PCR。 </li><li>使用PCR purificatio纯化PCR产物loxP的启动子 - LEU2 - 终止-LoxP位盒根据制造商的说明Ñ套件。根据制造商的说明21 ...添加3&#39;-A突出的纯化的PCR产物。使用T4 DNA连接酶结扎A-尾PCR产物入TA克隆载体，产生质粒T-LEU2按表3。 </li><li>用PCR引物＃3/4＃从Y.扩增基因KU70的1 kb的5&#39;上游序列解脂 Po1g按表2的基因组DNA 22。纯化使用根据制造商的说明一个PCR纯化试剂盒将PCR产物。 <ol><li>双重消化两个纯化的PCR产物和T-LEU2质粒用Sac II和BamHⅠ酶按表4。使用PCR纯化试剂盒按照制造商的说明纯化消化混合物。使用T4 DNA连接酶，以结扎纯化和消化的PCR产物的囊 II / T-LEU2的巴姆 HI位点，得到质粒T-LEU2-5E按表3。 </li></ol></li><li>使用引物＃5 /＃6从Y.扩增KU70基因的1kb的3&#39;下游序列解脂 Po1g按表2的基因组DNA 22。纯化使用根据制造商的说明一个PCR纯化试剂盒将PCR产物。双重消化两个纯化的PCR产物和用Not I和的Nde I酶按表4的T-LEU2-5E质粒。 <ol><li>纯化使用PCR纯化试剂盒按照制造商的说明将消化混合物。然后，结扎纯化和消化的PCR产物的未 I / 的Nde I位点的T-LEU2-5E的，得到使用T4DNA连接酶按表3质粒的T-KO。 </li></ol></li><li>消化质粒T-KO用Sac II和NDE我酶按表4，产生中断盒（ 图1 </strong&gt;的）。纯化使用PCR纯化试剂盒按照制造商的说明将消化的DNA片段。 </li></ol></li><li>感受态细胞制备和Y改造解脂 Po1g应变<ol><li>感受态细胞制备<ol><li>接种Y.的殖民地从在10ml YPD培养基（1％酵母提取物，2％蛋白胨，2％葡萄糖和50mM柠檬酸盐缓冲液pH 4.0）中的100毫升烧瓶中新鲜酵母提取物-蛋白胨-葡萄糖（YPD）板解脂 Po1g菌株。孵育在30℃振荡培养箱中于225rpm下20小时，直到饱和度（的OD为约15 600，使用分光光度计测量）。 </li><li>通过在室温下以5000 xg离心离心5分钟沉淀细胞。清洗细胞用20ml的Tris-EDTA（TE）缓冲液（10mM的Tris，1mM EDTA中，pH值7.5），并如在步骤1.2.1.2所述细胞沉淀。重悬在1ml的0.1M乙酸锂（pH值6.0，用乙酸调节）的细胞中，并孵育在室温坦佩10分钟叉涂抹。 </li><li>分装感受态细胞（100微升）进入无菌1.5毫升管。进行到转化步骤下面马上或在-80℃下长期贮存添加甘油至25％的终浓度（体积/体积），并存储。 </li></ol></li><li>转型<ol><li>轻轻10微升变性的鲑精子DNA（10毫克/毫升）和1-5微克纯化的破坏盒的与100微升的感受态细胞混合在一起，并在30℃下孵育15分钟。 </li><li>加入700微升40％聚乙二醇（PEG）-4000（溶于0.1M醋酸锂pH值为6.0）中，拌匀，并在225转速在30℃摇床孵育60分钟。 注意:使用的PEG 4000平均分子量，代替PEG-3350（典型地在常规的酵母的转化中使用）是重要的。 </li><li>热火通过将管在39℃水浴60分钟震荡转化混合物。 </li><li>加入1 ml YPD中，恢复为在30℃下2小时，225转。离心机以9000rpm的XG在室温下1分钟，除去上清液和重悬沉淀在1ml TE缓冲液中。 </li><li> Repellet通过离心将细胞以9000rpm的XG的在室温下1分钟，并弃去上清液。重悬在100μlTE缓冲液中的沉淀和板到选择平板（ 例如 ，亮氨酸缺陷型板），并在30°C孵育2-3天。 </li><li>选择4至10个单菌落，并分别接种入2ml YPD培养基。在225rpm下在30℃振荡培养箱中孵育过夜。从转化22识别由基因组DNA的PCR分析阳性菌落按使用两组引物＃55 /＃56和＃56 /＃57 表5中 ，分别。 注意: 未 I线性pYLEX1载体转化到Y.解脂 Po1g感受态细胞，以便通过计数的数目来确定转化效率每微克质粒的菌落形成单位（cfu）的DNA用于23。 </li></ol></li></ol></li></ol> 2.标记救援<ol><li>华创表达载体的构建<ol><li> pYLEX1-CRE和pYLEX1-HPH建设<ol><li>设计的引物20进行PCR扩增CRE和HPH的开放阅读框。添加额外的腺嘌呤核苷酸的起始密码子上游的正向引物＃82和＃84，和插入在反向引物＃83和＃85的终止密码子的下游的PCR扩增CRE的开放读框的KpnI限制性位点和从pSH69（登录号HQ412578）24 HPH按表2。 </li><li>净化既CRE和使用根据制造商的说明一个PCR纯化试剂盒HPH片段。既消化酶我按照TABL与KPN纯化的综合招聘考试及HPH片段E 4。双酶切与PML和KpnI酶按表4中的pYLEX1质粒。用PCR纯化试剂盒按照制造商的说明纯化消化混合物。 </li><li>使用T4 DNA连接酶按表3结扎纯化和消化CRE和HPH片段至Y的PML的I / 的KpnI位点解脂表达载体，产生pYLEX1-CRE和pYLEX1-HPH分别。 </li></ol></li><li> pSL16-CRE-HPH建设<ol><li>使用引物＃86放大来自pYLEX1-CRE CRE的启动子-基因-终止子盒/＃87侧翼萨尔的I /PstⅠ限制位点按表2中 。根据制造商的说明纯化使用PCR纯化试剂盒将PCR产物。 <ol><li>双重消化两个纯化的PCR产物和pSL16-CEN1-1（227）的质粒25用Sal I和的PstⅠ酶按表4。纯化使用PCR纯化试剂盒按照制造商的说明将消化混合物。然后，在相应的位点结扎纯化和消化的PCR产品放入pSL16-CEN1-1（227），使用T4 DNA连接酶按表3来创建pSL16-CRE。 </li></ol></li><li> PCR使用引物扩增来自pYLEX1-HPH HPH的启动子-基因-终止子盒＃88 /＃89侧翼Xho I 位 /BglⅡ位限制性位点按表2中 。使用PCR纯化试剂盒根据制造商的说明纯化PCR产物。 <ol><li>双重消化两个纯化的PCR产物和用Xho I和BglⅡ酶切酶按表4 pSL16-CRE质粒。用PCR纯化试剂盒按照制造商的说明纯化消化混合物。然后，结扎纯化和消化的PCR产品放入pSL16-CRE在相应​​的网站使用T4DNA连接酶按表3，以产生pSL16-CRE-HPH。 注意:将所得的Cre表达的质粒，pSL16-CRE-HPH，怀有可选择潮霉素B的标记，是着丝粒和附加型复制载体（ 图2）。 </li></ol></li><li>验证所有通过消化适当的限制性内切酶（ 例如 ， 萨尔我/ PST我，切除从载体插入）按表4的结构。 </li></ol></li></ol></li><li> Cre重组酶介导的标记救援<ol><li>变换pSL16-CRE-HPH进入淘汰赛KU70菌株的感受态细胞按照上述第1.2节所述的改造方案。板转化细胞到YPD加潮霉素B（YPDH）板（含有400微克/毫升潮霉素B），并孵育在30℃下2-3天。 </li><li>进行菌落PCR按表5中使用引物＃84/85＃识别正面含改造后pSL16-CRE-HPH质粒的菌落。 注意:正向引物＃84和反向引物＃85位于潮霉素基因（ 表1）的编码区内部。只阳性克隆产生在PCR反应的特定PCR产物。 </li><li>挑选阳性克隆，接种于2ml YPDH培养基中，并在225rpm下在30℃振荡培养箱中孵育过夜饱和。测量用分光光度计细胞OD 600。收获细胞以约15的OD 600。离心机在5000 xg离心在室温下5分钟，并用2毫升无菌水中洗一次。 </li><li>再接种细胞于2ml YPD培养基具有初始OD为0.1 600和允许细胞在30℃振荡培养箱中于225rpm下生长过夜。条纹过夜细胞培养到YPD平板分离单菌落23。复制品板菌落到YPD，YPDH和亮氨酸缺陷型板23 </s了&gt;。 注:已经失去了LEU2标记，pSL16-CRE-HPH质粒只能生长在YPD平板（ 图3）菌落。 </li></ol></li></ol> 3.乙醇脱氢酶和醇氧化酶基因的缺失<ol><li>执行对Y.搜索使用基本局部比对搜索工具（BLAST）来识别要删除的26基因的候选解脂基因组数据库。输入S的蛋白质序列酵母的乙醇脱氢酶我到BLAST搜索工具（http://www.genome.jp/tools-bin/search_sequence?prog=blast&amp;db=yli&amp;seqid=aa）。 注:BLAST搜索工具将返回最相似的蛋白质序列中的Y.解脂基因组数据库。 </li><li>构建通过PCR缺失盒用引物＃7至＃56（ 表1），并执行酶切，并使用上文第1.1节描述的相同的方法连接反应。 </li><li>预备是KU70敲除菌株的感受态细胞，进行转化和进行PCR通过以下在上述1.2节中描述的协议用 ​​引物＃55，＃58〜＃81（ 表1），以确定阳性菌落。 注意:作为一个例子，对于YALI0E17787g基因缺失的方法在图4和5进行说明。报告的1 kb和2 kb的同源区域同源重组率的影响。 </li></ol>

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(63), e3064 (2012).</li><li>Hegemann, J. H., Heick, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delete+and+repeat:+a+comprehensive+toolkit+for+sequential+gene+knockout+in+the+budding+yeast+Saccharomyces+cerevisiae.">Delete and repeat: a comprehensive toolkit for sequential gene knockout in the budding yeast Saccharomyces cerevisiae.</a> Methods Mol. Biol. 765, 189-206 (2011).</li><li>Yamane, T., Sakai, H., Nagahama, K., Ogawa, T., Matsuoka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+centromeric+DNA+from+yeast+Yarrowia+lipolytica+and+identification+of+protein-binding+site+required+for+plasmid+transmission.">Dissection of centromeric DNA from yeast Yarrowia lipolytica and identification of protein-binding site required for plasmid transmission.</a> J. Biosci. Bioeng. 105 (6), 571-578 (2008).</li><li>McGinnis, S., Madden, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLAST:+at+the+core+of+a+powerful+and+diverse+set+of+sequence+analysis+tools.">BLAST: at the core of a powerful and diverse set of sequence analysis tools.</a> Nucleic Acids Res. 32 ((Web Server issue)), W20-W25 (2004).</li><li>Madzak, C., Tr&#233;ton, B., Blanchin-Roland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strong+hybrid+promoters+and+integrative+expression/secretion+vectors+for+quasi-constitutive+expression+of+heterologous+proteins+in+the+yeast+Yarrowia+lipolytica.">Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica.</a> J. Mol. Microbiol. Biotechnol. 2 (2), 207-216 (2000).</li><li>Nicaud, J. M., Le Clainche, A., Le Dall, M. T., Wang, H., Gaillardin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yarrowia+lipolytica,+a+yeast+model+for+the+genetic+studies+of+hydroxy+fatty+acids+biotransformation+into+lactones.">Yarrowia lipolytica, a yeast model for the genetic studies of hydroxy fatty acids biotransformation into lactones.</a> J. Mol. Catal. B-Enzym. 5 (1), 175-181 (1998).</li></ol>

The authors have nothing to disclose.

我们对这项研究的目的是来实现快速，高效的新一代靶向基因敲除在Y.解脂 Po1g应变。有好几个因素需要加以解决，以实现这一目标。首先，需要一个高转化效率。因此，高效，便捷的化学转化协议为Y.解脂 Po1g菌株在本研究中所描述。使用的PEG-4000的是该菌株的成功转化的关键因素。用PEG-3350时（为酿酒酵母的转化）代替PEG-4000质粒转化获得没有转化体。此外，需要使用?...

不像酿酒酵母 ， 解脂耶氏，一个非常规酵母，可以响应于环境条件1,2-变化在酵母或菌丝的形式生长。因此，这种二形酵母可用作真菌分化，形态发生和分类​​3,4,5的研究的良好模型。它通常被认为是安全的（GRAS）的酵母物种，它被广泛用于生产各种食品添加剂如有机酸，多元醇，芳香化合物，乳化剂和表面活性剂6,7,8,9这样。它是专性需氧菌和能够以高含量自然积累脂质的公知的含油酵母， 即高达细胞干重10的70％。它也可以利用碳源的广泛增长，其中包括浪费资源的养分11,12,13不同种类的残留物。所有这些独特的功能使Y.为解脂非常有吸引力各种生物技术应用。 虽然Y的全基因组序列解脂已发表​​14,15，这种非常规酵母的基因操作比其它酵母种更加复杂。第一，该酵母物种的转化是有效的要少得多，由于不存在稳定高效的遗传转化系统16,17的。第二，线性表达盒的费力的基因组整合通常用于感兴趣基因的表达，因为没有天然游离质粒系统已经在该酵母18被发现。第三，代遗传敲奏和敲造访受到限制，因为该基因通过准确同源重组在该酵母靶向效率是低的，并通过非同源末端连接（NHEJ）19最积分事件发生。 在这项研究中，我们报道了Y.优化改造协议解脂 </青霉&gt; Po1g应变，这是很容易，快速，高效和可重复的。以提高精确同源重组的频率，我们删除KU70基因，其编码在NHEJ途径的关键酶。通过使用优化的改造方案和改造含有侧翼的1 kb的同源地区，Y的KU70基因的敲除线性磁带解脂 Po1g被成功删除。这种基因缺失的方法的稳健性然后通过在Po1g KU70Δ应变靶向乙醇脱氢酶和醇氧化酶基因的证明。据观察，在KU70缺失菌株表现出同源重组介导的基因的一个相当高的效率比野生型Po1g应变的定位。此外，携带潮霉素B抗性标记的复制的Cre表达质粒构建以执行标记救援。标记救援促进多轮基因在针对第Ë获得的基因缺失突变体。除了 ​​基因缺失，我们对这里描述的遗传转化和基因缺失协议可应用到插入基因的具体位点，并引入位点特异性突变引入Y.解脂基因组。

<table><tbody><tr><td>&lt;strong&gt;Reagent/Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Oligonucleotide primers</td><td>Integrated DNA Technologies</td><td></td><td>25 nmole DNA oligos</td></tr><tr><td>&lt;em&gt;Y. lipolytica&lt;/em&gt; strain Po1g</td><td>Yeastern Biotech</td><td></td><td>leucine auxotrophic derivative of the wild-type strain W29 (ATCC 20460)</td></tr><tr><td>Vector pYLEX1</td><td>Yeastern Biotech</td><td>FYY203-5MG</td><td>&lt;em&gt;Y. lipolytica&lt;/em&gt; expression vector</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; TOP10</td><td>Invitrogen</td><td></td><td>For cloning and propagation of plasmids</td></tr><tr><td>pGEM-T vector&amp;nbsp;</td><td>Promega</td><td>A3600</td><td>TA cloning vector</td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27106</td><td>For plasmid isolation</td></tr><tr><td>Wizard SV Gel and&lt;br/&gt; PCR Clean-Up System</td><td>Promega</td><td>A9282</td><td>Extract DNA fragments from agarose gels&lt;br/&gt; and purify PCR products from an amplification reaction</td></tr><tr><td>The iProof high-fidelity&lt;br/&gt; DNA polymerase</td><td>Bio-Rad</td><td>172-5302</td><td>High-fidelity DNA polymerase</td></tr><tr><td>BamHI&amp;nbsp;</td><td>New England Biolabs</td><td>R0136S</td><td>Restriction enzyme</td></tr><tr><td>BglII&amp;nbsp;</td><td>New England Biolabs</td><td>R0144L</td><td>Restriction enzyme</td></tr><tr><td>KpnI</td><td>New England Biolabs</td><td>R0142S</td><td>Restriction enzyme</td></tr><tr><td>NdeI&amp;nbsp;</td><td>New England Biolabs</td><td>R0111S</td><td>Restriction enzyme</td></tr><tr><td>NotI</td><td>New England Biolabs</td><td>R0189L</td><td>Restriction enzyme</td></tr><tr><td>PmlI</td><td>New England Biolabs</td><td>R0532S</td><td>Restriction enzyme</td></tr><tr><td>PstI&amp;nbsp;</td><td>New England Biolabs</td><td>R0140S</td><td>Restriction enzyme</td></tr><tr><td>SacII</td><td>New England Biolabs</td><td>R0157S</td><td>Restriction enzyme</td></tr><tr><td>SalI</td><td>New England Biolabs</td><td>R0138S</td><td>Restriction enzyme</td></tr><tr><td>XhoI</td><td>New England Biolabs</td><td>R0146L</td><td>Restriction enzyme</td></tr><tr><td>T4 DNA ligase</td><td>New England Biolabs</td><td>M0202L</td><td></td></tr><tr><td>Taq DNA polymerase&amp;nbsp;</td><td>Bio-Rad</td><td>M0267L</td><td></td></tr><tr><td>Ampicillin</td><td>Gibco-Life Technologies</td><td>11593-027</td><td>Antibiotics</td></tr><tr><td>Hygromycin B</td><td>PAA</td><td>P21-014</td><td>Antibiotics</td></tr><tr><td>GeneRuler 1 kb DNA ladder</td><td>Thermo Scientific</td><td>SM0312</td><td>1 kb DNA ladder</td></tr><tr><td>PEG4000</td><td>Sigma</td><td>95904-F</td><td></td></tr><tr><td>Tris</td><td>Promega</td><td>H5135</td><td></td></tr><tr><td>EDTA</td><td>Bio-Rad</td><td>161-0729</td><td></td></tr><tr><td>Salmon Sperm DNA</td><td>Invitrogen</td><td>15-632-011</td><td></td></tr><tr><td>Lithium Acetate</td><td>Sigma</td><td></td><td></td></tr><tr><td>Acetic acid</td><td>Sigma</td><td></td><td></td></tr><tr><td>Glass beads (425-600 &amp;micro;m)</td><td>Sigma</td><td>G8772</td><td></td></tr><tr><td>RNAse A</td><td>Thermo Scientific</td><td>EN0531</td><td></td></tr><tr><td>DNA Loading Dye</td><td>Thermo Scientific</td><td>R0611</td><td></td></tr><tr><td>Bacto Yeast Extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>Bacto Peptone</td><td>BD</td><td>211677</td><td></td></tr><tr><td>D-Glucose</td><td>1st Base</td><td>BIO-1101</td><td></td></tr><tr><td>YNB without amino acids</td><td>Sigma</td><td>Y0626</td><td></td></tr><tr><td>DO Supplement-Leu</td><td>Clontech</td><td>630414</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>Difco LB Broth</td><td>BD</td><td>244620</td><td></td></tr><tr><td>Difco LB Agar</td><td>BD</td><td>244520</td><td></td></tr><tr><td>Bacto Agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PCR machine</td><td>Biorad</td><td>T100 Thermal Cycler</td><td></td></tr><tr><td>Water bath</td><td>Memmert</td><td>WNB 14</td><td></td></tr><tr><td>Stationary/Shaking Incubator</td><td>Yihder</td><td>LM-570RD</td><td></td></tr><tr><td>Thermo-shaker</td><td>Allsheng</td><td>MS-100</td><td></td></tr><tr><td>Micro centrifuge</td><td>Eppendorf</td><td>5424R</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Spectrophotometer</td><td>Eppendorf&amp;nbsp;</td><td>BioPhotometer plus</td><td></td></tr><tr><td>Gel imager</td><td>GE</td><td>Amersham Imager 600</td><td></td></tr></tbody></table>

genetic engineering of an unconventional yeast for renewable biofuel and biochemical production

解脂耶氏是一种非致病的，二相性和严格的有氧酵母物种。由于其独特的生理功能和代谢特征，这非常规酵母不仅对真菌分化的基本性质的研究的良好模型，但也用于生化生产和各种生物技术应用，其需要大量的基因操作一个有希望的微生物的平台。不过，Y的遗传操作解脂一直有限由于缺少一种有效的和稳定的遗传转化系统以及可主要归因于KU70基因的非同源重组率很高。在这里，我们报告一个简单而快速的协议为高效遗传转化并为Y.基因缺失解脂 Po1g。首先，外源DNA的高效转化为Y.协议解脂 Po1g成立。山高次，以达到增强的双交换同源重组速率为靶基因的进一步缺失，所述KU70基因通过转化中断箱运送1kb的同源臂删除。第三，以展示KU70基因的缺失后增强的基因缺失的效率，我们单独删除11编码上使用KU70淘汰赛平台菌株的相同程序乙醇脱氢酶和醇氧化酶的靶基因。据观察，精确的同源重组的速率从低于0.5％大幅增加为删除 在Po1g的KU70基因到33％-71％，为单基因缺失Po1g KU70Δ11靶基因。复制型质粒携带潮霉素B抗性标记和酶Cre / loxP系统构建，并最终被Cre重组酶表达去除酵母基因敲除菌株的选择标记基因便于多轮圆盾的特德遗传操作。所得单基因缺失突变体具有在生物燃料和生化生产的应用潜力。

线性Y.解脂表达载体插入到PBR对接平台在Y的基因组解脂 Po1g应变通过执行一个交叉重组27。通过使用本研究建立的快速化学转化过程中，线性Y.解脂表达载体在&gt; 100个/微克DNA的转化效率被成功转化到野生型Po1g菌株。由1kb的同源序列侧接敲除盒转化到Po1g菌株和KU70基因被成功删除（ 图1）。这里，正向引物＃56退火到位?...

Watch this Scientific Journal Video about Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production at JoVE.com

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

可再生生物燃料及生化生产的非常规酵母基因工程

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

genetic-engineering-an-unconventional-yeast-for-renewable-biofuel

Research

JoVE Journal

Genetics

13.6K 次观看.  National University of Singapore. We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

视频: Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

体内研究 使用酵母中的生物发光报告基因监测代谢转变过程中的转录活性

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

生物工程

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

设计和自动照明，培养和取样系统实现微生物光遗传学的应用

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

免疫与感染

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

高通量酵母菌株，带基于滴滴的RNA测序

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

绿色怪物承载多基因缺失的酵母菌株的产生过程

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

生物学

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

用于微生物化学和蛋白质生产的光控发酵

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

定向进化法<em&gt;酿酒酵母</em&gt;:突变体库的创建和筛选

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Biofuels: Producing Ethanol from Cellulosic Material

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
In this experiment, cellulosic material (such as corn stalks, leaves, grasses, etc.) will be used as a feedstock for the production of ethanol.&#160;The cellulosic material is first pretreated (ground and heated), digested with enzymes, and then fermented with yeast.&#160;Ethanol production is monitored using an ethanol probe.&#160;The experiment can be extended to optimize ethanol production by varying the feedstock used, pretreatment conditions, enzyme variation, yeast variation, etc.&#160;An alternative method of monitoring the reaction is to measure the carbon dioxide produced (using a gas sensor) instead of the ethanol.&#160;As a low-tech alternative, glucose meters (found in any drug store) can be used to monitor the glucose during the process, if an ethanol probe or carbon dioxide gas sensor is not available.
With an increased emphasis on &#8216;inquiry-based learning&#8221;, scientific probes are becoming more popular.&#160;Handheld devices like the Vernier Lab Quest used in conjunction with a variety of probes (such as those for conductivity, dissolved oxygen, voltage, and more) allow for less focus on collecting data and/or making graphs and more on analyzing the data and making predictions.&#160;Another advantage is that these are small and lightweight and can be taken into the field for measurements.

生物燃料: 生产乙醇从纤维素材料

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
In this experiment, cellulosic material (such as corn stalks, leaves, ...

可再生生物燃料及生化生产的非常规酵母基因工程

摘要

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此视频中的章节

机器人平台的高通量原生质体分离和转型

体内研究使用酵母中的生物发光报告基因监测代谢转变过程中的转录活性

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

设计和自动照明，培养和取样系统实现微生物光遗传学的应用

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

高通量酵母菌株，带基于滴滴的RNA测序

绿色怪物承载多基因缺失的酵母菌株的产生过程

用于微生物化学和蛋白质生产的光控发酵

定向进化法

生物燃料: 生产乙醇从纤维素材料

可再生生物燃料及生化生产的非常规酵母基因工程

摘要

探索更多视频

此视频中的章节

机器人平台的高通量原生质体分离和转型

体内研究 使用酵母中的生物发光报告基因监测代谢转变过程中的转录活性

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

设计和自动照明，培养和取样系统实现微生物光遗传学的应用

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

高通量酵母菌株，带基于滴滴的RNA测序

绿色怪物承载多基因缺失的酵母菌株的产生过程

用于微生物化学和蛋白质生产的光控发酵

定向进化法

生物燃料: 生产乙醇从纤维素材料

体内研究使用酵母中的生物发光报告基因监测代谢转变过程中的转录活性