Saturation mutagenesis and self‐inducible expression of trehalose synthase in Bacillus subtilis

Liu Hongling; Wang Xihui; Yang Shaojie; Wang Ruiming; Wang Tengfei

摘要

Abstract > Trehalose is a nonreducing disaccharide synthesized by trehalose synthase (TreS), which catalyzes the reversible interconversion of maltose and trehalose. We aimed to enhance the catalytic conversion of maltose to trehalose by saturation mutagenesis, and constructed a self‐inducible TreS expression system by generating a robust Bacillus subtilis recombinant. We found that the conversion yield and enzymatic activity of TreS was enhanced by saturation mutations, especially by the combination of V407M and K490L mutations. At the same time, these saturation mutations were contributing to reducing by‐products in the reaction. Compared to WT TreS, the conversion yield of maltose to trehalose was increased by 11.9%, and the k <sub>cat</sub> / K <sub>m</sub> toward trehalose was 1.33 times higher in the reaction catalyzed by treS V407M‐K490L . treS V407M‐K490L expression was further observed in the recombinant <fc> <fi>B. subtilis</fi> </fc> W800N(Δσ F ) under the influence of P <sub>srfA</sub> , P <sub>cry3Aa</sub> , and P <sub>srfA‐cry3Aa</sub> promoters without an inducer. It was shown that P <sub>srfA‐cry3Aa</sub> was evidently a stronger promoter for treS V407M‐K490L expression, with the intracellular enzymatic activity of recombinant treS V407M‐K490L being over 5,800?U/g at 35?hr in TB medium. These results suggested the combination of two mutations, V407M and K490L, was conducive for the production of trehalose. In addition, the self‐inducible TreS V407M/K490L mutant in the <fc> <fi>B. subtilis</fi> </fc> host provides a low‐cost choice for the industrial production of endotoxin‐free trehalose with high yields. </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 Type =“Main”XML：Lang =“en”> <标题类型=“main”>抽象</ title> > 海藻糖是由海藻糖合成酶（TRE）合成的非加强二糖，其催化麦芽糖和海藻糖的可逆互联。我们旨在通过饱和诱变增强麦芽糖对海藻糖的催化转化，并通过产生稳健构建自诱导的TRE表达系统枯草芽孢杆菌</ i> 重组。我们发现，通过饱和突变增强了TRE的转化产率和酶活性，尤其是V407M和K490L突变的组合。同时，这些饱和突变是有助于减少反应中的副产物。与WT TRE相比，麦芽糖对海藻糖的转化产率增加了11.9％，而且 k </ i> <sub> cat </ sub> / k </ i> <sub> m </ sub> 致海藻糖催化的反应率较高1.33倍 tres </ i> v407m-k490l </ sup> 。 tres </ i> v407m-k490l </ sup> 在重组中进一步观察到表达 <FC> <fi> b。 subtilis </ fi> </ FC> W800N（Δσ. f </ sup> ）在p的影响下 <sub> srfa </ sub> ，P. <sub> cry3aa </ sub> 和p <sub> srfa-cry3aa </ sub> 没有诱导剂的启动子。表明p <sub> srfa-cry3aa </ sub> 显然是一个更强大的推动者 tres </ i> v407m-k490l </ sup> 表达，具有重组的细胞内酶活性 tres </ i> v407m-k490l </ sup> 在TB培养基中以超过5,800？U / g？HR。这些结果表明，两个突变，V407M和K490L的组合有利于产生海藻糖。此外，自诱导的TRE v407m / k490l </ sup> 突变体在 <FC> <fi> b。 subtilis </ fi> </ FC> 宿主为工业生产的内毒素的天藻糖产业生产提供了低产率。 </ p> </摘要> </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-15005/'>《Biotechnology Progress》</a> <b style="margin: 0 2px;">|</b><span>2019年第4期</span><b style="margin: 0 2px;">|</b><span>共12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=Liu Hongling&option=202" target="_blank" rel="nofollow">Liu Hongling;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Wang Xihui&option=202" target="_blank" rel="nofollow">Wang Xihui;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Yang Shaojie&option=202" target="_blank" rel="nofollow">Yang Shaojie;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Wang Ruiming&option=202" target="_blank" rel="nofollow">Wang Ruiming;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Wang Tengfei&option=202" target="_blank" rel="nofollow">Wang Tengfei;</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>State Key Laboratory of Biobased Material and Green Papermaking (LBMP)Qilu University of Technology (Shandong Academy of Sciences)Jinan Shandong People's Republic of China;</p> <p>State Key Laboratory of Biobased Material and Green Papermaking (LBMP)Qilu University of Technology (Shandong Academy of Sciences)Jinan Shandong People's Republic of China;</p> <p>State Key Laboratory of Biobased Material and Green Papermaking (LBMP)Qilu University of Technology (Shandong Academy of Sciences)Jinan Shandong People's Republic of China;</p> <p>State Key Laboratory of Biobased Material and Green Papermaking (LBMP)Qilu University of Technology (Shandong Academy of Sciences)Jinan Shandong People's Republic of China;</p> <p>State Key Laboratory of Biobased Material and Green Papermaking (LBMP)Qilu University of Technology (Shandong Academy of Sciences)Jinan Shandong People's Republic of China;</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/15.html" title="生物科学">生物科学;</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=Bacillus subtilis W800N&option=203" rel="nofollow">Bacillus subtilis W800N;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=PsrfA‐cry3Aa promoter&option=203" rel="nofollow">PsrfA‐cry3Aa promoter;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=saturation mutagenesis&option=203" rel="nofollow">saturation mutagenesis;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=self‐induction expression&option=203" rel="nofollow">self‐induction expression;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=trehalose synthase&option=203" rel="nofollow">trehalose synthase;</a> </p> <div class="translation"> 机译：枯草芽孢杆菌W800N;PSRFA-Cry3AA启动子;饱和诱变;自诱导表达;海藻糖合成酶; </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" href="/academic-journal-cn_agricultural-biotechnology_thesis/0201272471934.html">Self-induced Secretion Expression of Trehalose Synthase in Bacillus subtilis WB800n</a> <b>[J]</b> . <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Xihui WANG&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">Xihui WANG,</a> <a 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Nilson Nicolau Junior</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Mariana Sant’Anna Pereira Nicolau&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,Mariana Sant’Anna Pereira Nicolau</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Luana Jandhy Mantovanini&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,Luana Jandhy Mantovanini</a> <span> <a href="/journal-cn-56204/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 美国植物学期刊（英文） </a> </span> <span> . 2013</span><span>,第12期</span> </span> </div> </li> <li> <div> <b>4. </b><a class="enjiyixqcontent" href="/academic-conference-cn_meeting-21637_thesis/02022417442.html">Expression and Pathological Significance of Inducible Nitric Oxide Synthase Expression in Osteosarcoma of the Jaws</a> <b>[C]</b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Chen Wei-liang&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . Chen Wei-liang</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Feng Hong-jie&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,Feng Hong-jie</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=LiJin-song&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,LiJin-song</a> <span> <a href="/conference-cn-21637/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 第三次全国口腔颌面—头颈肿瘤综合治疗研讨会暨中华口腔医学会外科专委会口腔颌面—头颈肿瘤内科协作组成立大会 </a> <span> <span> . 2007</span> </span> </div> </li> <li> <div> <b>5. </b><a class="enjiyixqcontent" href="/academic-degree-domestic_mphd_thesis/020312375784.html">Electroacupuncture Attenuates Visceral Hypersensitivity by Suppressing Spinal Par-2 and Cgrp Expressions in Animals with Tnbs-Induced Ileitis</a> <b>[A] </b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=马诺基·库马尔·沙阿&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 马诺基·库马尔·沙阿</a> <span> . 2016</span> </span> </div> </li> </ul> <ul style="display: none;"> <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/patent-detail/06120114114054.html">一株枯草芽孢杆菌（Bacillus subtilis）及含有该菌株的土壤改良剂</a> <b>[P]</b> . <span> 中国专利： CN114196595A </span> <span> . 2022-03-18</span> </div> </li> <li> <div> <b>2. </b><a class="enjiyixqcontent" href="/patent-detail/061204403701.html">一种枯草芽孢杆菌Bacillus Subtilis CZ162菌株及其应用</a> <b>[P]</b> . <span> 中国专利： CN110607265B </span> <span> . 2021.05.14</span> </div> </li> <li> <div> <b>3. </b><a class="enjiyixqcontent" href="/patent-detail/06130464616852.html">Process for a thermo-inducible gene expression in Gram positive bacteria, Bacillus subtilis strains containing plasmids which induce such an expression and portable vector systems</a> <b>[P]</b> . <span> 外国专利：  EP0182562B1 </span> <span> . 1991-10-09</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：在革兰氏阳性细菌，枯草芽孢杆菌菌株中热诱导基因表达的方法，该菌株包含诱导这种表达的质粒和便携式载体系统 </span> </p> </li> <li> <div> <b>4. </b><a class="enjiyixqcontent" href="/patent-detail/06130467014271.html">PROCESS FOR A THERMO-INDUCIBLE GENE EXPRESSION IN GRAM POSITIVE BACTERIA AND BACILLUS SUBTILIS STRAINS CONTAINING PLASMIDS WHICH INDUCE SUCH AN EXPRESSION</a> <b>[P]</b> . <span> 外国专利：  GB2166743B </span> <span> . 1989-01-05</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：在包含这种表达的质粒的革兰氏阳性细菌和芽胞杆菌菌株中热诱导基因表达的过程 </span> </p> </li> <li> <div> <b>5. </b><a class="enjiyixqcontent" href="/patent-detail/06130471401500.html">Process for a thermo-inducible gene expression in Gram positive bacteria, Bacillus subtilis strains containing plasmids which induce such an expression and portable vector systems</a> <b>[P]</b> . <span> 外国专利：  EP0182562A2 </span> <span> . 1986-05-28</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：在革兰氏阳性细菌，枯草芽孢杆菌菌株中热诱导基因表达的方法，该菌株包含诱导这种表达的质粒和便携式载体系统 </span> </p> </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: 19px;z-index: 999;" onclick="ywcd('0704028287788','4',7,2,1,'',this,24)" class="delivery" prompt="010401" title="通过人工服务将文献原文发送至邮箱" >获取原文</div> <div class="journalsub-pop-up" style="display: none"> <div class="journal-sub"> <h2>期刊订阅</h2> <img src="https://cdn.zhangqiaokeyan.com/img/loginclose.png" 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