Toward industrial production of isoprenoids in <i xmlns='http://www.wiley.com/namespaces/wiley'>Escherichia coliEscherichia coli : Lessons learned from CRISPR‐Cas9 based optimization of a chromosomally integrated mevalonate pathway

Alonso‐Gutierrez Jorge; Koma Daisuke; Hu Qijun; Yang Yuchen; Chan Leanne J. G.; Petzold Christopher J.; Adams Paul D.; Vickers Claudia E.; Nielsen Lars K.; Keasling Jay D.; Lee Taek S.

摘要

Abstract <section xml:id="bit26530-sec-0001" numbered="no"> > Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress‐responsive control. Production levels dropped dramatically relative to plasmid‐mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR‐Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid‐optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering. </section> </abstract> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> <div class="translation abstracttxt"> <span class="zhankaihshouqi fivelineshidden" id="abstract"> <span>机译：</span><Abstract XMLNS =“http://www.wiley.com/namespaces/wiley”type =“main”xml：lang =“en”> <title type =“main”>抽象</ title> <section XML：ID =“Bit26530-SEC-0001”编号=“否”> > 大肠杆菌是通过工程本地和异源途径产生不同化学品的选择的生物体。在本研究中，我们同时解决与 e相关的一些主要问题。 Coli </ i>作为异戊二烯的工业平台，包括无法在蔗糖上生长，缺乏对有毒甲戊酸（MVA）途径中间体的内源性控制，以及染色体中的有限途径工程。作为概念证明，我们生成了一个 e。 Coli </ i> DH1应变能够通过将 Cscakb </ i>操纵子与染色体中整合到染色体中，并通过在应力响应控制下表达异源MVA途径来产生异戊二烯酸二球果。当整个途径集成到染色体中时，产生水平相对于质粒介导的表达显着降低。为了优化染色体整合的MVA途径，我们建立了一种CRISPR-CAS9系统，以快速和系统地替换启动子序列。该策略导致了途径较高的途径表达和二元生产的五倍改善。更有趣的是，我们分析了蛋白质组学数据集，以了解和解决与染色体综合通路的代谢工程相关的一些挑战。本报告表明，将质粒优化的操纵子集成到基因组中并使它们最佳地工作不是直截了当的任务，并且染色体上的任何差的工程选择可能导致细胞死亡，而不是导致低滴度。基于这些结果，我们还提出了染色体代谢工程的方向。 </ p> </ section> </ abstract> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> </div> <div class="record"> <h2 class="all_title" id="enpatent33" > 著录项</h2> <ul> <li> <span class="lefttit">来源</span> <div style="width: 86%;vertical-align: text-top;display: inline-block;"> <a href='/journal-foreign-15017/'>《Biotechnology and Bioengineering 》</a> <b style="margin: 0 2px;">|</b><span>2018年第4期</span><b style="margin: 0 2px;">|</b> <span>共14页</span> </div> </li> <li> <div class="author"> <span class="lefttit">作者</span> <p id="fAuthorthree" class="threelineshidden zhankaihshouqi"> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Alonso‐Gutierrez Jorge&option=202" target="_blank" rel="nofollow">Alonso‐Gutierrez Jorge;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Koma Daisuke&option=202" target="_blank" rel="nofollow">Koma Daisuke;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Hu Qijun&option=202" target="_blank" rel="nofollow">Hu Qijun;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Yang Yuchen&option=202" target="_blank" rel="nofollow">Yang Yuchen;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Chan Leanne J. G.&option=202" target="_blank" rel="nofollow">Chan Leanne J. G.;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Petzold Christopher J.&option=202" target="_blank" rel="nofollow">Petzold Christopher J.;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Adams Paul D.&option=202" target="_blank" rel="nofollow">Adams Paul D.;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Vickers Claudia E.&option=202" target="_blank" rel="nofollow">Vickers Claudia E.;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Nielsen Lars K.&option=202" target="_blank" rel="nofollow">Nielsen Lars K.;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Keasling Jay D.&option=202" target="_blank" rel="nofollow">Keasling Jay D.;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Lee Taek S.&option=202" target="_blank" rel="nofollow">Lee Taek S.;</a> </p> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zkzz" style="display: none;">展开▼</span> </div> </li> <li> <div style="display: flex;"> <span class="lefttit">作者单位</span> <div style="position: relative;margin-left: 3px;max-width: 639px;"> <div class="threelineshidden zhankaihshouqi" id="fOrgthree"> <p>Joint BioEnergy Institute (JBEI)Emeryville California;</p> <p>Joint BioEnergy Institute (JBEI)Emeryville California;</p> <p>Joint BioEnergy Institute (JBEI)Emeryville California;</p> <p>Joint BioEnergy Institute (JBEI)Emeryville California;</p> <p>Joint BioEnergy Institute (JBEI)Emeryville California;</p> <p>Joint BioEnergy Institute (JBEI)Emeryville California;</p> <p>Joint BioEnergy Institute (JBEI)Emeryville California;</p> <p>Australian Institute for Bioengineering and NanotechnologyThe University of QueenslandSt. Lucia Queensland Australia;</p> <p>Australian Institute for Bioengineering and NanotechnologyThe University of QueenslandSt. Lucia Queensland Australia;</p> <p>Joint BioEnergy Institute (JBEI)Emeryville California;</p> <p>Joint BioEnergy Institute (JBEI)Emeryville California;</p> </div> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zhdw" style="display: none;">展开▼</span> </div> </div> </li> <li> <span class="lefttit">收录信息</span> <span style="width: 86%;vertical-align: text-top;display: inline-block;"></span> </li> <li> <span class="lefttit">原文格式</span> <span>PDF</span> </li> <li> <span class="lefttit">正文语种</span> <span>eng</span> </li> <li> <span class="lefttit">中图分类</span> <span> <a href="https://www.zhangqiaokeyan.com/clc/180.html" title="生物工程学（生物技术）">生物工程学（生物技术） ;</a></span> </li> <li class="antistop"> <span class="lefttit">关键词</span> <p style="width: 86%;vertical-align: text-top;"> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=biofuel&option=203" rel="nofollow">biofuel;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=chromosomal engineering&option=203" rel="nofollow">chromosomal engineering;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=CRISPR‐Cas9&option=203" rel="nofollow">CRISPR‐Cas9;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=E. coli DH1&option=203" rel="nofollow">E. coli DH1;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=mevalonate pathway&option=203" rel="nofollow">mevalonate pathway;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=sucrose utilization&option=203" rel="nofollow">sucrose utilization;</a> </p> <div class="translation"> 机译：生物燃料;染色体工程;CRISPR-CAS9;e。 COLI DH1;甲戊二醛途径;蔗糖利用; </div> </li> </ul> </div> </div> <div class="literature cardcommon"> <div class="similarity "> <h3 class="all_title" id="enpatent66">相似文献</h3> <div class="similaritytab clearfix"> <ul> <li class="active" >外文文献</li> <li >中文文献</li> <li >专利</li> </ul> </div> <div class="similarity_details"> <ul > <li> <div> <b>1. </b><a class="enjiyixqcontent" 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<span> <a href="/journal-cn-11277/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 高校化学工程学报 </a> </span> <span> . 2014</span><span> ,第001期</span> </span> </div> </li> <li> <div> <b>6. </b><a class="enjiyixqcontent" href="/academic-conference-cn_meeting-6674_thesis/020221733108.html">无卤阻燃剂季戊四醇二磷酸酯蜜胺盐生产工艺优化设计</a> <b>[C]</b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=LUO Wen&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . LUO Wen</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=罗文&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,罗文</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=WANG Ying-xi&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,WANG Ying-xi</a> <span> <a href="/conference-cn-6674/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 湖北省第五届大学生化学（化工）学术创新成果报告会 </a> <span> <span> . 2012</span> </span> </div> </li> <li> <div> <b>7. </b><a class="enjiyixqcontent" href="/academic-degree-domestic_mphd_thesis/020313533642.html">均匀设计法分析与优化连续硝化合成二甲戊乐灵过程</a> <b>[A] </b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=刘明月&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 刘明月</a> <span> . 2018</span> </span> </div> </li> </ul> <ul style="display: none;"> <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/patent-detail/06130400497341.html">GENE-THERAPEUTIC DNA-VECTOR BASED ON GENE-THERAPEUTIC DNA-VECTOR VTVAF17, CARRYING TARGET GENE CAS9 FOR HETEROLOGOUS EXPRESSION OF THIS TARGET GENE IN HUMAN AND ANIMAL CELLS IN IMPLEMENTING VARIOUS METHODS OF GENOMIC EDITING, METHOD OF PRODUCING AND USING GENE-THERAPEUTIC DNA VECTOR, ESCHERICHIA COLI SCS110-AF/VTVAF17-CAS9 STRAIN, CARRYING GENE-THERAPEUTIC DNA VECTOR, METHOD FOR PREPARING THEREOF, METHOD FOR PRODUCTION ON INDUSTRIAL SCALE OF GENE-THERAPEUTIC DNA VECTOR</a> <b>[P]</b> . <span> 外国专利：  RU2730663C2 </span> <span> . 2020-08-24</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：基于基因-治疗性DNA-载体VTVAF17的基因治疗性DNA-载体，携带目标基因CAS9在人和动物细胞中异源表达该靶基因，以实现各种基因编辑方法，制备方法和制备方法，大肠杆菌（Escherichia）COLI SCS110-AF / VTVAF17-CAS9菌株，携带基因-治疗性DNA矢量的载体，其制备方法，基因-治疗性DNA矢量的工业规模生产方法 </span> </p> </li> <li> <div> <b>2. </b><a class="enjiyixqcontent" href="/patent-detail/06130400512650.html">VTvaf17-Act1-Cas9 GENE-THERAPEUTIC DNA VECTOR BASED ON VTvaf17 GENE-THERAPEUTIC DNA VECTOR CARRYING TARGET Cas9 GENE FOR HETEROLOGOUS EXPRESSION OF THIS TARGET GENE IN PLANT CELLS DURING GENOME EDITING OF PLANTS, METHOD OF PRODUCING AND USING GENE-THERAPEUTIC DNA VECTOR, STRAIN OF ESCHERICHIA COLI SCS110-AF/VTvaf17-Act1-Cas9 CARRYING GENE-THERAPEUTIC DNA VECTOR, METHOD FOR PREPARING THEREOF, METHOD FOR INDUSTRIAL PRODUCTION OF GENE-THERAPEUTIC DNA VECTOR</a> <b>[P]</b> . <span> 外国专利：  RU2715314C1 </span> <span> . 2020-02-26</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：基于携带靶标Cas9基因的VTvaf17基因-治疗性DNA矢量的VTvaf17-Act1-Cas9基因-治疗性DNA矢量，用于在植物的基因编辑，基因编码，生产方法和制备过程中，在植物细胞中该靶基因的异源表达携带基因-治疗性DNA矢量的埃希氏大肠杆菌CSS110-AF / VTvaf17-Act1-Cas9的合成，制备方法，工业生产基因-治疗性DNA矢量的方法 </span> </p> </li> <li> <div> <b>3. </b><a class="enjiyixqcontent" href="/patent-detail/06130400278063.html">Gene therapy DNA vector based on vtvaf17 gene therapy DNA vector, Under different genome editing methods, the target gene of cas9 vector is applied to the heterogeneous expression of this specific gene in human and animal cells,Methods for obtaining and using DNA vectors for gene therapy,Escherichia coli scs110-af / vtvaf17-cas9, carrying gene therapy DNA vector,The way to get it,Gene therapy DNA vector industrial scale production method</a> <b>[P]</b> . <span> 外国专利：  RU2018147083A </span> <span> . 2020.06.29</span> </div> </li> </ul> </div> </div> </div> <div class="theme cardcommon" style="overflow: auto;display:none"> <h3 class="all_title" id="enpatent55">相关主题</h3> <ul id="subject"> </ul> </div> </div> </div> </div> <div class="right rightcon"> <div class="details_img cardcommon clearfix" style="margin-bottom: 10px;display:none;" > </div> </div> </div> <div id="thesis_get_original1" class="downloadBth" style="bottom: 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Toward industrial production of isoprenoids in Escherichia coliEscherichia coli : Lessons learned from CRISPR‐Cas9 based optimization of a chromosomally integrated mevalonate pathway

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