一种产软骨素的重组谷氨酸棒杆菌及其应用的制作方法

本发明涉及一种产软骨素的重组谷氨酸棒杆菌及其应用，属于生物工程技术领域。

背景技术

硫酸软骨素是一种重要的糖胺聚糖，由葡萄糖醛酸和n-乙酰半乳糖胺构成的二糖单位通过β1-3和β1-4糖苷键交替连接形成。硫酸软骨素广泛应用于医疗保健、化妆品和食品添加剂等领域。目前硫酸软骨素主要是以动物组织为原料通过化学提取法获得。由于不同动物组织中的硫酸软骨素硫酸化程度相差较大，很难获得纯度高且质量均一的产品。最近兴起的生物酶法催化代表了硫酸软骨素和生产方法的重要发展方向。该方法是分别以软骨素作为底物经过软骨素硫酸转移酶的硫酸化等修饰，获得高纯度，硫酸化程度均一的产品。但受限于软骨素生产量不足，质量不高，目前很难通过此方法工业化生产硫酸软骨素。因此获得高质量、高产量的软骨素是促进硫酸软骨素生产方法革新，提高硫酸软骨素产量和质量的重要节点。

在自然条件下，大肠杆菌k4能够分别产生果糖化修饰的软骨素构成其荚膜的重要成分。因此人们希望通过不断优化大肠杆菌k4代谢途径以提高果糖化的软骨素的产量。但是大肠杆菌k4对人和动物有致病性，会引起肠道感染，败血症等疾病，带有较大的安全隐患。此外，大肠杆菌k4生产是发生了果糖化修饰的软骨素，而不是软骨素，因此需要额外的脱果糖处理。第三，果糖化软骨素合成过程受到复杂的调控，造成获得的产量偏低。这些缺点不但会造成生产成本偏高，还会增加下游产物的加工、纯化难度。而在其他微生物细胞中如大肠杆菌bl21和枯草芽孢杆菌中重组构建的软骨素合成侧重对代谢途径进行优化，缺乏对软骨素合成关键酶的改造，造成途径效率不高，造成软骨素产量低。

因此有必要探索、构建安全高效的微生物细胞工厂，提高软骨素和前体的产量与质量。

技术实现要素：

为了解决上述问题，本发明对工业安全菌株谷氨酸棒杆菌(corynebacteriumglutamicum)进行改造，大幅提高了软骨素的产量。

本发明提供了一种重组谷氨酸棒杆菌，所述谷氨酸棒杆菌异源整合表达了udp-n-乙酰葡萄糖胺c4异构酶kfoa和软骨素合成酶kfoc，并通过以质粒表达谷氨酰胺-果糖-6-磷酸氨基转移酶glms、磷酸葡萄糖变位酶glmm、udp-n-乙酰葡萄糖胺焦磷酸化酶/葡萄糖-1-磷酸乙酰转移酶双功能酶glmu和葡萄糖-6-磷酸尿酰胺转移酶galu基因，以加强软骨素合成底物udp-n-乙酰葡萄糖胺和udp-葡萄糖醛酸的积累，在谷氨酸棒杆菌中构建了软骨素的合成通路。

所述软骨素合成酶是野生型或者h357g/n363s双突变体。选择软骨素合成酶h357g/n363s双突变体，可以大幅提高谷氨酸棒杆菌生产软骨素的能力。

进一步的，将单个软骨素合成关键酶kfoc、kfoa和udp-葡萄糖脱氢酶ugd进行融合改造，形成人工酶复合体，增加了软骨素合成关键酶之间的关联性，进一步促进软骨素的积累。

所述的udp-n-乙酰葡萄糖胺c4异构酶kfoa和软骨素合成酶kfoc来源于大肠杆菌k4(escherichiacolio5:k4:h4(l):h4，escherichiacolik4)。kfoa的氨基酸序列如seqidno.1所示，kfoc的氨基酸序列如seqidno.2所示。所述的谷氨酰胺-果糖-6-磷酸氨基转移酶glms，磷酸葡萄糖变位酶glmm，udp-n-乙酰葡萄糖胺焦磷酸化酶/葡萄糖-1-磷酸乙酰转移酶双功能酶glmu，葡萄糖-6-磷酸尿酰胺转移酶galu，udp-葡萄糖脱氢酶ugd来源于谷氨酸棒杆菌corynebactriumglutamicumatcc13032。

在本发明的一种实施方式中，glmu、galu以pxmj19质粒为表达载体，以ppyc为启动子，形成组成型表达框，在谷氨酸棒杆菌中持续表达。glms和glmm以pec-xk99e质粒为表达载体，以ptrc启动子，形成诱导型表达框，在谷氨酸棒杆菌中iptg诱导表达。glms、glmm、glmu氨基酸序列分别如seqidno.3、seqidno.4、seqidno.5，galu的氨基酸序列包括seqidno.6和seqidno.7所示。

在本发明的一种实施方式中，采用ggsggsggggs蛋白linker融合将kfoc,kfoa和ugd串联融合形成人工酶复合体。复合体中kfoc,kfoa和ugd的摩尔比例为1:1:1。ugd的氨基酸序列包括seqidno.9、seqidno.10所示。

本发明的有益效果：本发明利用谷氨酸棒杆菌产软骨素，与其它软骨素生产菌相比，该发明具有非常大的优势。首先，本发明过程中使用的谷氨酸棒杆菌为安全菌株(generallyrecognizedassafe,gras)，完全满足医疗卫生和食品安全生产的要求；与枯草芽孢杆菌、大肠杆菌相比谷氨酸棒杆菌培养成本低，发酵过程易于控制，且发酵菌体密度高，菌体无裂解,适应高密度发酵。本发明在打通软骨素在谷氨酸棒杆菌中合成通路的基础上，侧重对软骨素合成关键酶进行深入改造：通过对软骨素合成关键酶kfoc进行随机突变，提高酶活性，以及通过对kfoc，kfoa和ugd进行人工复合体构建提高多步代谢步骤之间的关联性，最终重组谷氨酸棒杆菌生产的软骨素的产量达到13.4g/l。本发明不但解决了大肠杆菌k4菌株生产软骨素携带的安全隐患，避免了软骨素的果糖化修饰，而且大幅提高了软骨素的产量，为通过重组谷氨酸棒杆菌工业化生产软骨素打下基础。

附图说明

图1：重组谷氨酸棒杆菌生产软骨素和前体的代谢途径及相关的酶类。

图2：kfoc的突变位点和kfoa、kfoc、ugd酶复合体的构建示意图；其中gslinker的氨基酸序列为ggsggsggggs，g为甘氨酸，s为丝氨酸。

图3:重组谷氨酸棒杆菌软骨素产量图；其中，c.glutamicumchon1：kfoa和kfoc野生型基因组整合表达，glmu和galu以pxmj19为载体表达，glms和glmm以pec-xk99e表达；c.glutamicumchon2:kfoa野生型和kfoc(h357g/n363s)突变体基因组整合表达，glmu和galu以pxmj19为载体表达，glms和glmm以pec-xk99e表达；c.glutamicumchon3：kfoa野生型和kfoc(h357g/n363s)突变体以及ugd串联形成人工酶复合体，并在基因组整合表达，glmu和galu以pxmj19为载体表达，glms和glmm以pec-xk99e表达。

具体实施方式

菌株：谷氨酸棒杆菌c.glutamicumatcc13032，质粒：pxmj19,pec-xk99e,pk18mobsacb。菌株构建培养基：bhis：脑心浸液肉汤37g/l，山梨糖醇90g/l；软骨素发酵培养基：葡萄糖：40g/l，玉米浆干粉：20g/l，(nh4)2so4：30g/l,kh2po4：1g/l，k2hpo4：1g/l，mgso4：25g/l，mops(3-吗啉丙磺酸)：42g/l，谷氨酸棒杆菌培养温度为30℃。分子实验工具酶：pcr实验所用酶为primerstarmaxdnapolymerase(takara)；限制性内切酶为fastdigest(thermoscientific)；一步克隆实验所用酶为clonexpress(诺唯赞)。软骨素的产量测定：向装有1ml硼砂硫酸(500ml浓h2so4中溶解4.77g硼砂)的玻璃管中加入适当稀释的200μl样品，混匀后在沸水浴中煮15min，冷却至室温。加入50μl咔唑试剂(100ml无水乙醇中溶解0.125g咔唑)，混匀后再煮沸15min。测定波长530nm处的吸光值。用不同浓度(l0、20、30、40、50μgml^-1)d-葡萄糖醛酸标准品进行相同反应，绘制标准曲线。标准曲线方程：y＝0.0149x-0.0239,r²＝0.9991(x,样品中葡萄糖醛酸含量(μgml^-1)；y,吸光度a530)。软骨素产量计算公式：软骨素含量(g/l)＝(标准曲线测出的浓度*稀释倍数*2.067)/1000。

实施例1：将ptrc-kfoa-kfoc整合到谷氨酸棒杆菌基因组

以ptrc-f与ptrc-r为引物，以pec-xk99e为模板扩增ptrc,以kfoa-f与kfoa-r为引物，以大肠杆菌k4基因组为模板扩增kfoa；以kfoc-f与kfoc-r为引物，以大肠杆菌k4基因组为模板扩增kfoc；将获得的ptrc、kfoa和kfoc以ptrc-f和kfoc-r为引物进行重叠延伸pcr，获得ptrc-kfoa-kfoc表达框。以glgaup-f和glgadown-r为引物，以ptrc-kfoa-kfoc和谷氨酸棒杆菌glga基因上、下游500bp同源臂(两个500bp同源臂分别通过引物glgaup-f与glgaup-r、glgadown-f与glgadown-r以谷氨酸棒杆菌atcc13032基因组为模板进行pcr获得)为模板再次进行重叠延伸pcr，获得h1-ptrc-kfoa-kfoc-h2。

将h1-ptrc-kfoa-kfoc-h2以一步克隆的方式连接入bamhi和hindiii双酶切的pk18mobsacb质粒，获得质粒pk18mobsacb-h1-ptrc-kfoa-kfoc-h2。将质粒pk18mobsacb-h1-ptrc-kfoa-kfoc-h2通过电穿孔(12.5kv/cm,5ms)方式转化谷氨酸棒杆菌。并在含有25μg/ml的bhis平板上筛选第一步重组的转化子。第一步重组的阳性转化子在bhis液体培养基中过夜培养后，涂布于含有20％蔗糖的bhis平板上进行蔗糖致死反选，获得第二步重组的转化子。以glga-check-f和glga-check-r为引物，通过菌落pcr验证成功整合ptrc-kfoa-kfoc的单克隆。

实施例2:重组菌c.glutamicumchon1的构建

corynebacteriumglutamicumatcc13032接种于5mlbhis液体培养基，在30℃200rpm培养24h，收集菌体，采用细胞基因组提取试剂盒提取基因组dna。第一步：pxmj19-glmu-galu的构建。设计引物ppyc-f/ppyc-r、glmu-f/glmu-r、galu-f/galu-r，以提取的c.glutamicumatcc13032基因组dna为模板，采用标准的pcr扩增体系和程序，扩增获取ppyc、glmu、galu基因。以扩增的glmu、galu为模板，以glmu-f和galu-r为引物进行重叠延伸pcr获得相连的glmu-galu片段。以pxmj19-f/pxmj19-r为引物扩增缺失ptac和laciq区域的线性pxmj19质粒。将glmu-galu片段和线性质粒pxmj19分别进行一步克隆拼接反应。拼接反应体系转化jm109感受态细胞。阳性转化子进行质粒测序，与序列比对，重组质粒pxmj19-glmu-galu构建成功。第二步：pec-xk99e-glms-glmm的构建。设计glms-f/glms-r、glmm-f/glmm-r，以提取的c.glutamicumatcc13032基因组dna为模板，采用标准的pcr扩增体系和程序，扩增获取glms、glmm基因。以扩增的glmu、galu为模板，以glmu-f和galu-r为引物进行重叠延伸pcr获得相连的glms-galm片段。将glmu-galu片段和由kpni和sali双酶切线性质粒pec-xk99e分别进行一步克隆拼接反应。拼接反应体系转化jm109感受态细胞。阳性转化子进行质粒测序，与序列比对，重组质粒pec-xk99e-glms-glmm构建成功。第三步：电转化质粒pxmj19-glmu-galu和pec-xk99e-glms-glmm进入谷氨酸棒杆菌，构建c.glutamicumchon1。质粒pxmj19-glmu-galu和pec-xk99e-glms-glmm通过电穿孔(12.5kv/cm,5ms)方式转化实施例1中构建的整合了ptrc-kfoa-kfoc的谷氨酸棒杆菌，在含有卡那霉素和氯霉素的bhis平板上筛选阳性转化子，c.glutamicumchon1构建成功。

表1ptrc-kfoa-kfoc整合到谷氨酸帮杆菌基因组以及重组质粒pxmj19-glmu-galu构建所用引物

实施例3：kfoc(h357g/n363s)突变体的构建与筛选

按照试剂盒的操作手册，以kfoc-f与kfoc-r为引物，利用diversify^tmpcrrandommutagenesiskitpcr随机突变试剂盒对kfoc基因进行随机突变。将随机突变的pcr片段按照实施例1中的描述将ptrc启动子以及和kfoa基因组装形成ptrc-kfoa-kfoc(随机突变体)表达框，并按照实施例1中的描述，将ptrc-kfoa-kfoc(随机突变体)表达框整合到谷氨酸棒杆菌基因组。含有ptrc-kfoa-kfoc(随机突变体)的重组谷氨酸棒杆菌单菌落库在96孔板中进行发酵培养。待葡萄糖耗尽后(40小时后)用96孔板离心收集菌体，并用去离子水洗涤菌体2次。菌体进行适当倍数的稀释后，用硫酸咔唑方法测定软骨素的含量，并筛选出产量最高的突变体。产量最高的突变体经过kfoc基因测序，确定产软骨素产量最高的重组谷氨酸棒杆菌中kfoc发生突变的类型为h357g/n363s。

实施例4：c.glutamicumchon2的构建

按照实施例1、2和3中的表述，将pxmj19-glmu-galu和pec-xk99e-glms-glmm质粒分别电转化进入整合了kfoa野生型和kfoc(h357g/n363s)突变体的谷氨酸棒杆菌中，获得c.glutamicumchon2菌株。

实施例5：c.glutamicumchon3的构建：

第一步构建：ptrc-kfoa-linker-kfoc。该表达框的构建方式与实施例1中所述基本相同。不同之处是ptrc-kfoa-linker-kfoc中kfoa的终止密码子taa和kfoc的核糖体结合位点(rbs序列aggagg)被编码ggsggsggggslinker的核苷酸序列ggtggaagtggaggtagtggaggtggaggtagt所替代。替代方法为重叠延伸pcr。第二步构建：ptrc-kfoa-linker-kfoc-linker-ugd。在第一步构建的基础上，将谷氨酸棒杆菌udp-葡萄糖脱氢酶的编码基因ugd通过编码ggsggsggggslinker的核苷酸序列ggaggtagcggtggaagcggtggaggtggaagc与ptrc-kfoa-linker-kfoc融合形成ptrc-kfoa-linker-kfoc-linker-ugd。构建方法同样为重叠延伸pcr。为避免dna出现正向重复，连接kfoc和ugd的ggsggsggggslinker的核苷酸序列与第一步中连接kfoa与kfoc的核苷酸序列有所不同，但两个linker的氨基酸序列保持一致。将ptrc-kfoa-linker-kfoc-linker-ugd转化谷氨酸棒杆菌。第三步：电转化质粒pxmj19-glmu-galu和pec-xk99e-glms-glmm进入步骤(2)中已经转化了ptrc-kfoa-linker-kfoc-linker-ugd的谷氨酸棒杆菌，构建c.glutamicumchon3。

实施例6:重组谷氨酸棒杆菌摇瓶发酵生产软骨素实验

将构建的重组谷氨酸棒杆菌菌株：c.glutamicumchon1,c.glutamicumchon1和c.glutamicumchon3(三种菌株基因型见图3说明)的单克隆接于5mlbhi培养基，置于200rpm，30℃过夜培养。培养12-16小时后,按1％的接种量转接于250ml三角摇瓶(装液量25ml发酵培养基)中。置于200rpm，28℃培养，并添加1.5mmiptg进行诱导基因表达。待葡萄糖耗尽后终止发酵实验，并通过硫酸咔唑法测定软骨素含量。结果如图3所示，c.glutamicumchon3的软骨素产量可以达到6.8g/l。

实施例7：c.glutamicumchon3发酵罐发酵实验

第一步，种子液的制备：准备c.glutamicumchon3种子液。将c.glutamicumchon3的单克隆接入100ml发酵培养基(葡萄糖10g/l)，置于200rpm，28℃过夜培养。第二步，补料发酵：在3l发酵罐中装入900ml发酵培养基，灭菌后接入第一步的种子液。发酵培养基中初始葡萄糖浓度为40g/l。控制发酵温度为28℃，搅拌转速为500rpm。之后每4小时左右取一次样品，测定菌体浓度和葡萄糖含量。ph用氨水控制在6.5-7.0之间，当葡萄糖浓度降低至5g/l以下时将葡萄糖浓度补至15g/l.当葡萄糖消耗速率明显下降时(低于2g/h)，停止补料。待残留葡萄糖耗尽后结束发酵。

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<110>江南大学

<120>一种产软骨素的重组谷氨酸棒杆菌及其应用

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210215220

lysaspglythrglyvalargasptyrilehisvalmetaspleuala

225230235240

gluglyhisvalalaalaleusertyrleupheargaspasnasnthr

245250255

asntyrhisvalpheasnleuglythrglylysglytyrservalleu

260265270

gluleuvalserthrpheglulysileserglyvalargileprotyr

275280285

gluilevalserargargaspglyaspilealaglusertrpserser

290295300

proglulysalaasnlystyrleuasntrplysalalysarggluleu

305310315320

gluthrmetleugluaspalatrpargtrpglnmetlysasnproasn

325330335

glytyrile

<210>2

<211>686

<212>prt

<213>escherichiacolik4

<400>2

metserileleuasnglnalaileasnleutyrlysasnlysasntyr

151015

argglnalaleuserleupheglulysvalalagluiletyraspval

202530

sertrpvalglualaasnilelysleucysglnthralaleuasnleu

354045

serglugluvalasplysleuasnarglysalavalileaspileasp

505560

alaalathrlysilemetcysserasnalalysalaileserleuasn

65707580

gluvalglulysasngluileileserlystyrarggluilethrala

859095

lyslyssergluargalagluleulysgluvalgluproileproleu

100105110

asptrpproseraspleuthrleuproproleuprogluserthrasn

115120125

asptyrvaltrpalaglylysarglysgluleuaspasptyrproarg

130135140

lysglnleuileileaspglyleuserilevalileprothrtyrasn

145150155160

argalalysileleualailethrleualacysleucysasnglnlys

165170175

thriletyrasptyrgluvalilevalalaaspaspglyserlysglu

180185190

asnileglugluilevalarggluphegluserleuleuasnilelys

195200205

tyrvalargglnlysasptyrglytyrglnleucysalavalargasn

210215220

leuglyleuargalaalalystyrasntyrvalalaileleuaspcys

225230235240

aspmetalaproasnproleutrpvalglnsertyrmetgluleuleu

245250255

alavalaspaspasnvalalaleuileglyproarglystyrileasp

260265270

thrserlyshisthrtyrleuasppheleuserglnlysserleuile

275280285

asngluileprogluileilethrasnasnglnvalalaglylysval

290295300

gluglnasnlysservalasptrpargilegluhisphelysasnthr

305310315320

aspasnleuargleucysasnthrpropheargphepheserglygly

325330335

asnvalalaphealalyslystrpleupheargalaglytrppheasp

340345350

glugluphethrhistrpglyglygluaspasnglupheglytyrarg

355360365

leutyrarggluglycystyrpheargservalgluglyalametala

370375380

tyrhisglngluproproglylysgluasngluthraspargalaala

385390395400

glylysasnilethrvalglnleuleuglnglnlysvalprotyrphe

405410415

tyrarglyslysglulysilegluseralathrleulysargvalpro

420425430

leuvalseriletyrileproalatyrasncysserlystyrileval

435440445

argcysvalgluseralaleuasnglnthrilethraspleugluval

450455460

cysilecysaspaspglyserthraspaspthrleuargileleugln

465470475480

gluhistyralaasnhisproargvalargpheileserglnlysasn

485490495

lysglyileglyseralaserasnthralavalargleucysarggly

500505510

phetyrileglyglnleuaspseraspasppheleugluproaspala

515520525

valgluleucysleuaspgluphearglysaspleuserleualacys

530535540

valtyrthrthrasnargasnileasparggluglyasnleuileser

545550555560

asnglytyrasntrpproiletyrserargglulysleuthrserala

565570575

metilecyshishispheargmetphethralaargalatrpasnleu

580585590

thrgluglypheasngluserileserasnalavalasptyraspmet

595600605

tyrleulysleusergluvalglyprophelyshisileasnlysile

610615620

cystyrasnargvalleuhisglygluasnthrserilelyslysleu

625630635640

aspileglnlysgluasnhisphelysvalvalasngluserleuser

645650655

argleuglyilelyslystyrlystyrserproleuthrasnleuasn

660665670

glucysarglystyrthrtrpglulysilegluasnaspleu

675680685

<210>3

<211>564

<212>prt

<213>corynebacteriumglutamicumatcc13032

<400>3

leuaspalagluilealalysalaproleuproaspserileleugly

151015

ileglyhisthrargtrpalathrhisglyglyprothraspvalasn

202530

alahisprohisvalvalserasnglylysleualavalvalhisasn

354045

glyileilegluasnphealagluleuargsergluleuseralalys

505560

glytyrasnphevalseraspthraspthrgluvalalaalaserleu

65707580

leualagluiletyrasnthrglnalaasnglyaspleuthrleuala

859095

metglnleuthrglyglnargleugluglyalaphethrleuleuala

100105110

ilehisalaasphisaspaspargilevalalaalaargargasnser

115120125

proleuvalileglyvalglygluglygluasnpheleuglyserasp

130135140

valserglypheileasptyrthrarglysalavalgluleualaasn

145150155160

aspglnvalvalthrilethralaaspasptyralailethrasnphe

165170175

aspglyserglualavalglylyspropheaspvalglutrpaspala

180185190

alaalaalaglulysglyglypheglyserphemetglulysgluile

195200205

hisaspglnproalaalavalargaspthrleumetglyargleuasp

210215220

gluaspglylysleuvalleuaspgluleuargileaspglualaile

225230235240

leuargservalasplysilevalilevalalacysglythralaala

245250255

tyralaglyglnvalalaargtyralailegluhistrpcysargile

260265270

prothrgluvalgluleualahisglupheargtyrargaspproile

275280285

leuasnglulysthrleuvalvalalaleuserglnserglygluthr

290295300

metaspthrleumetalavalarghisalaarggluglnglyalalys

305310315320

valvalalailecysasnthrvalglyserthrleuproarggluala

325330335

aspalaserleutyrthrtyralaglyprogluilealavalalaser

340345350

thrlysalapheleualaglnilethralasertyrleuleuglyleu

355360365

tyrleualaglnleuargglyasnlysphealaaspgluvalserser

370375380

ileleuaspserleuargglumetproglulysileglnglnvalile

385390395400

aspalaglugluglnilelyslysleuglyglnaspmetalaaspala

405410415

lysservalleupheleuglyarghisvalglypheprovalalaleu

420425430

gluglyalaleulysleulysgluilealatyrleuhisalaglugly

435440445

phealaalaglygluleulyshisglyproilealaleuvalgluglu

450455460

glyglnproilephevalilevalproserproargglyargaspser

465470475480

leuhisserlysvalvalserasnileglngluileargalaarggly

485490495

alavalthrilevalilealaglugluglyaspglualavalasnasp

500505510

tyralaasnpheileileargileproglnalaprothrleumetgln

515520525

proleuleuserthrvalproleuglnilephealacysalavalala

530535540

thralalysglytyrasnvalaspglnproargasnleualalysser

545550555560

valthrvalglu

<210>4

<211>447

<212>prt

<213>corynebacteriumglutamicumatcc13032

<400>4

metthrargleupheglythraspglyvalargglyleualaasnglu

151015

valleuthralaproleualaleulysleuglyalaalaalaalahis

202530

valleuthralaglulysargvalaspglyargargprovalalaile

354045

valglyargaspproargvalserglyglumetleualaalaalaleu

505560

seralaglymetalaserglnglyvalaspvalileargvalglyval

65707580

ileprothrproalavalalapheleuthraspasptyrglyalaasp

859095

metglyvalmetileseralaserhisasnprometproaspasngly

100105110

ilelysphepheseralaglyglyhislysleuproasphisvalglu

115120125

aspgluilegluargvalmetaspserleuproalagluglyprothr

130135140

glyhisglyvalglyargvalilegluglualathraspalaglnasp

145150155160

argtyrleugluhisleulysglualavalprothrserleuglugly

165170175

ilelysilevalvalaspalaalaasnglyalaalaservalvalala

180185190

prothralatyrglualaalaglyalathrvalilealailehisasn

195200205

lysproaspsertyrasnileasnmetaspcysglyserthrhisile

210215220

aspglnvalglnalaalavalleulyshisglyalaaspleuglyleu

225230235240

alahisaspglyaspalaaspargcysleualavalasplysaspgly

245250255

asnleuvalaspglyaspglnilemetalaleuleualailealamet

260265270

lysgluasnglygluleuarglysasnthrleuvalglythrvalmet

275280285

serasnleuglyleulysilealametaspglualaglyilethrleu

290295300

argthrthrlysvalglyaspargtyrvalleugluaspleuasnala

305310315320

glyglypheserleuglyglygluglnserglyhisilevalleupro

325330335

asphisglythrthrglyaspglythrleuthrglyleuserilemet

340345350

alaargmetalagluthrglylysserleuglygluleualaglnala

355360365

metthrvalleuproglnvalleuileasnvalprovalserasplys

370375380

serthrilevalserhisproservalvalalaalailealagluala

385390395400

glualagluleuglyalathrglyargvalleuleuargalasergly

405410415

thrglugluleupheargvalmetvalglualaglyasplysglugln

420425430

alaargargilealaglyargleualaalavalvalalagluval

435440445

<210>5

<211>485

<212>prt

<213>corynebacteriumglutamicumatcc13032

<400>5

metseralaserasppheserseralavalvalvalleualaalagly

151015

alaglythrargmetlysseraspleuglnlysthrleuhisserile

202530

glyglyargserleuileserhisserleuhisalaalaalaglyleu

354045

asnprogluhisilevalalavalileglyhisglyargaspglnval

505560

glyproalavalalaglnvalalaglugluleuasparggluvalleu

65707580

ilealaileglnglugluglnasnglythrglyhisalavalglncys

859095

alametaspglnleugluglyphegluglythrileilevalthrasn

100105110

glyaspvalproleuleuthrasphisthrleuseralaleuleuasp

115120125

alahisvalgluvalprothralavalthrvalleuthrmetargleu

130135140

aspaspprothrglytyrglyargilevalargasnglugluglyglu

145150155160

valthralailevalgluglnlysaspalaseralagluvalglnala

165170175

ileaspgluvalasnserglyvalphealapheaspalaalaileleu

180185190

argseralaleualagluleulysseraspasnalaglnglygluleu

195200205

tyrleuthraspvalleuglyilealaargglygluglyhisproval

210215220

argalahisthralaalaaspalaarggluleualaglyvalasnasp

225230235240

argvalglnleualaglualaglyalagluleuasnargargthrval

245250255

ilealaalametargglyglyalathrilevalaspproalathrthr

260265270

trpileaspvalgluvalserileglyargaspvalileilehispro

275280285

glythrglnleulysglygluthrvalileglyaspargvalgluval

290295300

glyproaspthrthrleuthrasnmetthrileglyaspglyalaser

305310315320

valileargthrhisglypheaspserthrileglygluasnalathr

325330335

valglyprophethrtyrileargproglythrthrleuglyproglu

340345350

glylysleuglyglyphevalgluthrlyslysalathrileglyarg

355360365

glyserlysvalprohisleuthrtyrvalglyaspalathrilegly

370375380

glugluserasnileglyalaserservalphevalasntyraspgly

385390395400

gluasnlyshishisthrthrileglyserhisvalargthrglyser

405410415

aspthrmetpheilealaprovalthrvalglyaspglyalatyrser

420425430

glyalaglythrvalilelysaspaspvalproproglyalaleuala

435440445

valserglyglyargglnargasnilegluglytrpvalglnlyslys

450455460

argproglythralaalaalaglnalaalaglualaalaglnasnval

465470475480

hisasnglnglugly

485

<210>6

<211>315

<212>prt

<213>corynebacteriumglutamicumatcc13032

<400>6

metserleuproileaspgluhisvalasnalavallysthrvalval

151015

valproalaalaglyleuglythrargpheleuproalathrlysthr

202530

valprolysgluleuleuprovalvalaspthrproglyilegluleu

354045

ilealaalaglualaalagluleuglyalathrargleualaileile

505560

thralaproasnlysalaglyvalleualahisphegluargserser

65707580

gluleuglugluthrleumetgluargglylysthraspglnvalglu

859095

ileileargargalaalaaspleuilelysalavalprovalthrgln

100105110

asplysproleuglyleuglyhisalavalglyleualagluserval

115120125

leuaspaspaspgluaspvalvalalavalmetleuproaspaspleu

130135140

valleuprothrglyvalmetgluargmetalaglnvalargalaglu

145150155160

pheglyglyservalleucysalavalgluvalserglualaaspval

165170175

serlystyrglyilephegluileglualaaspthrlysaspserasp

180185190

vallyslysvallysglymetvalglulysproalailegluaspala

195200205

proserargleualaalathrglyargtyrleuleuasparglysile

210215220

pheaspalaleuargargilethrproglyalaglyglygluleugln

225230235240

leuthraspalaileaspleuleuileaspgluglyhisprovalhis

245250255

ilevalilehisglnglylysarghisaspleuglyasnproglygly

260265270

tyrileproalacysvalasppheglyleuserhisprovaltyrgly

275280285

alaglnleulysaspalailelysglnileleualagluhisgluala

290295300

alagluargilealaaspaspserglnvallys

305310315

<210>7

<211>315

<212>prt

<213>corynebacteriumglutamicumatcc13032

<400>7

metserleuproileaspgluhisvalasnalavallysthrvalval

151015

valproalaalaglyleuglythrargpheleuproalathrlysthr

202530

valprolysgluleuleuprovalvalaspthrproglyilegluleu

354045

ilealaalaglualaalagluleuglyalathrargleualaileile

505560

thralaproasnlysalaglyvalleualahisphegluargserser

65707580

gluleuglugluthrleumetgluargglylysthraspglnvalglu

859095

ileileargargalaalaaspleuilelysalavalprovalthrgln

100105110

asplysproleuglyleuglyhisalavalglyleualagluserval

115120125

leuaspaspaspgluaspvalvalalavalmetleuproaspaspleu

130135140

valleuprothrglyvalmetgluargmetalaglnvalargalaglu

145150155160

pheglyglyservalleucysalavalgluvalserglualaaspval

165170175

serlystyrglyilephegluileglualaaspthrlysaspserasp

180185190

vallyslysvallysglymetvalglulysproalailegluaspala

195200205

proserargleualaalathrglyargtyrleuleuasparglysile

210215220

pheaspalaleuargargilethrproglyalaglyglygluleugln

225230235240

leuthraspalaileaspleuleuileaspgluglyhisprovalhis

245250255

ilevalilehisglnglylysarghisaspleuglyasnproglygly

260265270

tyrileproalacysvalasppheglyleuserhisprovaltyrgly

275280285

alaglnleulysaspalailelysglnileleualagluhisgluala

290295300

alagluargilealaaspaspserglnvallys

305310315

<210>8

<211>387

<212>prt

<213>corynebacteriumglutamicumatcc13032

<400>8

metlysilealavalalaglyleuglytyrvalglyleuserasnala

151015

alaleuleuserlysasnhislysvalvalalavalaspileaspglu

202530

gluargvallysleuvalglnglupheargserproilevalaspser

354045

aspleugluglutyrleuserthrlysproglnasnleuthralathr

505560

thraspalaglualaalatyrlysglyalaasppheilevalileala

65707580

thrprothrasntyraspprogluserasnphepheaspthrserser

859095

valgluservalilegluilevalleulysvalserproglyserthr

100105110

ilevalilelysserthrileprovalglyphethrsergluleuarg

115120125

ilelyshisproglualaserileilepheserproglupheleuarg

130135140

gluglyargalaphetyraspasnleutyrproserargvalvalval

145150155160

glyaspargserproleuglyglugluphealathrleuleualaglu

165170175

glyalalysglulysproproileleuleuthraspserthrgluala

180185190

glualailelysleupheserasnthrtyrleualaleuargvalala

195200205

phepheasngluleuaspthrtyralaservalargserleuaspthr

210215220

lysglnileilegluglyvalglyleuaspproargileglyserhis

225230235240

tyrasnasnproserpheglytyrglyglytyrcysleuprolysasp

245250255

thrlysglnleuleualaasntyrlysaspvalproglnasnleuile

260265270

seralavalvalglnalaasnlysthrarglysasppheilealaglu

275280285

aspileleuserlysserprothrvalvalglyiletyrargleuval

290295300

metlysserglyseraspasnpheargserserserileglnglyval

305310315320

metlysargilelysalalysglyilegluilevalvalpheglupro

325330335

asnleuglyglugluthrphetyrasnserlysileleuasnaspile

340345350

glugluphelysasptyrcysaspileileilealaasnargprothr

355360365

aspgluleuseraspvalproglulysvaltyrthrargaspilephe

370375380

glnargasp

385

<210>9

<211>439

<212>prt

<213>corynebacteriumglutamicumatcc13032

<400>9

metargmetthrvalileglythrglytyrleuglyalathrhisala

151015

alacysmetalagluleuserhisgluvalleuglyvalaspvalasp

202530

glualalysilealaserleulysaspserlysvalprophepheglu

354045

proglyleuprogluvalleugluargasnleugluasnglyargleu

505560

asnphethrthrasptyralaglualaalaalaphealaglnvalhis

65707580

pheleuglyvalglythrproglnglnlysglythrtyralaalaasp

859095

leuthrtyrvalargglnvalvalgluaspleuvalproleuleuglu

100105110

glygluhisileilepheglylysserthrvalprovalglythrala

115120125

gluglnleuglngluleualaaspserleuvallysproglyserhis

130135140

valgluilealatrpasnproglupheleuarggluglytyralaval

145150155160

lysaspthrilethrproaspargilevalvalglyvalargglugly

165170175

alathralaglualailealaarggluvaltyralathralaileala

180185190

alaaspthrpropheleuvalthraspleualathralagluleuval

195200205

lysvalseralaasnalapheleualathrlysileserpheileasn

210215220

alavalalagluilecysgluglnthrglyalaaspvalvalalaleu

225230235240

alaaspalaileglyhisaspaspargileglyarglyspheleugly

245250255

alaglyleuglypheglyglyglycysleuprolysaspileargala

260265270

phemetalaargalaglygluleuglyalaaspglnalaleuthrphe

275280285

leuarggluvalaspserileasnmetargargargaspargvalval

290295300

glnleualalysglumetcysglyglyserleuleuglylysargval

305310315320

thrvalleuglyalaalaphelysproasnseraspaspvalargasp

325330335

serproalaleuservalalaglyserleuserleuglnglyalaala

340345350

valservaltyraspproglualametaspasnalaargargvalphe

355360365

prothrleusertyralaserserthrlysglualaleuileaspala

370375380

hisleuvalvalleualathrglutrpglnglupheargaspleuasp

385390395400

progluvalalaglyglyvalvalglulysargalaileileaspgly

405410415

argasnvalleuaspvalalalystrplysalaalaglytrpglumet

420425430

glualaleuglyargasnleu

435

<210>10

<211>29

<212>dna

<213>人工序列

<400>10

cgactgcacggtgcaccaatgcttctggc29

<210>11

<211>33

<212>dna

<213>人工序列

<400>11

catggtctgtttcctgtgtgaaattgttatccg33

<210>12

<211>47

<212>dna

<213>人工序列

<400>12

tttcacacaggaaacagaccatggttttcaagggtgaaaaatgaata47

<210>13

<211>29

<212>dna

<213>人工序列

<400>13

ttaaatataaccatttgggtttttcattt29

<210>14

<211>51

<212>dna

<213>人工序列

<400>14

aaaacccaaatggttatatttaaagattaggttgaaataatatgagtattc51

<210>15

<211>46

<212>dna

<213>人工序列

<400>15

agaggttccccggctttcggttttataaatcattctctattttttc46

<210>16

<211>46

<212>dna

<213>人工序列

<400>16

cgaattcgagctcggtacccggggtgctcatctagcatctggcttg46

<210>17

<211>24

<212>dna

<213>人工序列

<400>17

taaaaggggctatcattcggaccc24

<210>18

<211>22

<212>dna

<213>人工序列

<400>18

aaccgaaagccggggaacctct22

<210>19

<211>46

<212>dna

<213>人工序列

<400>19

aacgacggccagtgccaagcttagctcgtcctcattgatcagttcg46

<210>20

<211>25

<212>dna

<213>人工序列

<400>20

ggcactcgtagaacgcatcaatggt25

<210>21

<211>27

<212>dna

<213>人工序列

<400>21

caatcgttgcccaggagaaatcattga27

<210>22

<211>25

<212>dna

<213>人工序列

<400>22

ggataacaatttcacacaggaaaca25

<210>23

<211>25

<212>dna

<213>人工序列

<400>23

gtgaaaagaaaaaccaccctggcgc25

<210>24

<211>47

<212>dna

<213>人工序列

<400>24

gcgccagggtggtttttcttttcacctgagtcttagattttgagaaa47

<210>25

<211>21

<212>dna

<213>人工序列

<400>25

aacaagagaccgccaagggtg21

<210>26

<211>44

<212>dna

<213>人工序列

<400>26

cacccttggcggtctcttgttgtactcttggttccatgagtttg44

<210>27

<211>43

<212>dna

<213>人工序列

<400>27

gcaaactcatggaaccaagagtaccttagccttcctggttgtg43

<210>28

<211>25

<212>dna

<213>人工序列

<400>28

ggtactcttggttccatgagtttgc25

<210>29

<211>48

<212>dna

<213>人工序列

<400>29

tgtttcctgtgtgaaattgttatccctattttacttgagaatcgtctg48

<210>30

<211>54

<212>dna

<213>人工序列

<400>30

gaccatggaattcgagctcggtaccaagttgttgtaaagtcatgcgcatgtgtg54

<210>31

<211>26

<212>dna

<213>人工序列

<400>31

ttattcgacggtgacagactttgcca26

<210>32

<211>58

<212>dna

<213>人工序列

<400>32

tggcaaagtctgtcaccgtcgaataaggccgtgcataattaatcgcatgactcgacta58

<210>33

<211>47

<212>dna

<213>人工序列

<400>33

caagcttgcatgcctgcaggtcgacttagacttctgcaaccactgca47

<210>34

<211>11

<212>prt

<213>人工序列

<400>34

glyglyserglyglyserglyglyglyglyser

1510

<210>35

<211>33

<212>dna

<213>人工序列

<400>35

ggtggaagtggaggtagtggaggtggaggtagt33

<210>36

<211>33

<212>dna

<213>人工序列

<400>36

ggaggtagcggtggaagcggtggaggtggaagc33