CN108841771B - 一种产软骨素的重组谷氨酸棒杆菌及其应用 - Google Patents

一种产软骨素的重组谷氨酸棒杆菌及其应用 Download PDF

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CN108841771B
CN108841771B CN201810756333.5A CN201810756333A CN108841771B CN 108841771 B CN108841771 B CN 108841771B CN 201810756333 A CN201810756333 A CN 201810756333A CN 108841771 B CN108841771 B CN 108841771B
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康振
王阳
胡立涛
陈坚
堵国成
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Abstract

本发明公开了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。本发明首先在谷氨酸棒杆菌异源表达UDP‑N‑乙酰葡萄糖胺C4异构酶编码基因kfoA和软骨素合成酶编码基因kfoC，以及过表达glmU、galU和ugd以及glmS和glmM，初步构建了软骨素的合成途径；随后通过对软骨素合成酶(KfoC)进行随机突变，发现KfoC(H357G N363S)突变体可以将软骨素产量提高200％。利用蛋白质融合技术将kfoA,kfoC(突变体)和ugd基因(编码UDP‑葡萄糖脱氢酶)融合，使表达的UDP‑N‑乙酰葡萄糖胺C4异构酶，软骨素合成酶和UDP‑葡萄糖脱氢酶形成人工复合体，将软骨素的产量进一步提高。

Description

一种产软骨素的重组谷氨酸棒杆菌及其应用
技术领域
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。
发明内容
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(Escherichia coli O5:K4:H4(L):H4，Escherichia coli K4)。KfoA的氨基酸序列如SEQID NO.1所示，KfoC的氨基酸序列如SEQ ID NO.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CorynebactriumglutamicumATCC 13032。
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SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5，GalU的氨基酸序列包括SEQ ID NO.6和SEQ ID NO.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SEQ ID NO.9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(Generally Recognized as Safe,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GS linker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.glutamicum Chon1：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.glutamicum Chon2: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.glutamicum Chon3：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.glutamicum ATCC 13032，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PrimerSTAR Max DNA Polymerase(Takara)；限制性内切酶为FastDigest(Thermo Scientific)；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、50μg mL-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(μg mL-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glgA up-F和glgA down-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glgA up-F与glgAup-R、glgA down-F与glgA down-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.glutamicum Chon1的构建
Corynebacterium glutamicumATCC 13032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.glutamicumATCC 13032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.glutamicumATCC 13032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.glutamicum Chon1构建成功。
表1 Ptrc-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
Figure GDA0002691366150000041
Figure GDA0002691366150000051
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DiversifyTM PCR RandomMutagenesis Kit PCR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.glutamicum Chon2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.glutamicum Chon2菌株。
实施例5：C.glutamicum Chon3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GGSGGSGGGGS linker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GGSGGSGGGGS linker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GGSGGSGGGGS linker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.glutamicum Chon3。
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.glutamicum Chon1,C.glutamicum Chon1和C.glutamicum Chon3(三种菌株基因型见图3说明)的单克隆接于5ml BHI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1.5mM IPTG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.glutamicum Chon3的软骨素产量可以达到6.8g/L。
实施例7：C.glutamicum Chon3发酵罐发酵实验
第一步，种子液的制备：准备C.glutamicum Chon3种子液。将C.glutamicum Chon3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。
SEQUENCE LISTING
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Ala Gln Leu Lys Asp Ala Ile Lys Gln Ile Leu Ala Glu His Glu Ala
290 295 300
Ala Glu Arg Ile Ala Asp Asp Ser Gln Val Lys
305 310 315
<210> 7
<211> 315
<212> PRT
<213> Corynebacterium glutamicum ATCC 13032
<400> 7
Met Ser Leu Pro Ile Asp Glu His Val Asn Ala Val Lys Thr Val Val
1 5 10 15
Val Pro Ala Ala Gly Leu Gly Thr Arg Phe Leu Pro Ala Thr Lys Thr
20 25 30
Val Pro Lys Glu Leu Leu Pro Val Val Asp Thr Pro Gly Ile Glu Leu
35 40 45
Ile Ala Ala Glu Ala Ala Glu Leu Gly Ala Thr Arg Leu Ala Ile Ile
50 55 60
Thr Ala Pro Asn Lys Ala Gly Val Leu Ala His Phe Glu Arg Ser Ser
65 70 75 80
Glu Leu Glu Glu Thr Leu Met Glu Arg Gly Lys Thr Asp Gln Val Glu
85 90 95
Ile Ile Arg Arg Ala Ala Asp Leu Ile Lys Ala Val Pro Val Thr Gln
100 105 110
Asp Lys Pro Leu Gly Leu Gly His Ala Val Gly Leu Ala Glu Ser Val
115 120 125
Leu Asp Asp Asp Glu Asp Val Val Ala Val Met Leu Pro Asp Asp Leu
130 135 140
Val Leu Pro Thr Gly Val Met Glu Arg Met Ala Gln Val Arg Ala Glu
145 150 155 160
Phe Gly Gly Ser Val Leu Cys Ala Val Glu Val Ser Glu Ala Asp Val
165 170 175
Ser Lys Tyr Gly Ile Phe Glu Ile Glu Ala Asp Thr Lys Asp Ser Asp
180 185 190
Val Lys Lys Val Lys Gly Met Val Glu Lys Pro Ala Ile Glu Asp Ala
195 200 205
Pro Ser Arg Leu Ala Ala Thr Gly Arg Tyr Leu Leu Asp Arg Lys Ile
210 215 220
Phe Asp Ala Leu Arg Arg Ile Thr Pro Gly Ala Gly Gly Glu Leu Gln
225 230 235 240
Leu Thr Asp Ala Ile Asp Leu Leu Ile Asp Glu Gly His Pro Val His
245 250 255
Ile Val Ile His Gln Gly Lys Arg His Asp Leu Gly Asn Pro Gly Gly
260 265 270
Tyr Ile Pro Ala Cys Val Asp Phe Gly Leu Ser His Pro Val Tyr Gly
275 280 285
Ala Gln Leu Lys Asp Ala Ile Lys Gln Ile Leu Ala Glu His Glu Ala
290 295 300
Ala Glu Arg Ile Ala Asp Asp Ser Gln Val Lys
305 310 315
<210> 8
<211> 387
<212> PRT
<213> Corynebacterium glutamicum ATCC 13032
<400> 8
Met Lys Ile Ala Val Ala Gly Leu Gly Tyr Val Gly Leu Ser Asn Ala
1 5 10 15
Ala Leu Leu Ser Lys Asn His Lys Val Val Ala Val Asp Ile Asp Glu
20 25 30
Glu Arg Val Lys Leu Val Gln Glu Phe Arg Ser Pro Ile Val Asp Ser
35 40 45
Asp Leu Glu Glu Tyr Leu Ser Thr Lys Pro Gln Asn Leu Thr Ala Thr
50 55 60
Thr Asp Ala Glu Ala Ala Tyr Lys Gly Ala Asp Phe Ile Val Ile Ala
65 70 75 80
Thr Pro Thr Asn Tyr Asp Pro Glu Ser Asn Phe Phe Asp Thr Ser Ser
85 90 95
Val Glu Ser Val Ile Glu Ile Val Leu Lys Val Ser Pro Gly Ser Thr
100 105 110
Ile Val Ile Lys Ser Thr Ile Pro Val Gly Phe Thr Ser Glu Leu Arg
115 120 125
Ile Lys His Pro Glu Ala Ser Ile Ile Phe Ser Pro Glu Phe Leu Arg
130 135 140
Glu Gly Arg Ala Phe Tyr Asp Asn Leu Tyr Pro Ser Arg Val Val Val
145 150 155 160
Gly Asp Arg Ser Pro Leu Gly Glu Glu Phe Ala Thr Leu Leu Ala Glu
165 170 175
Gly Ala Lys Glu Lys Pro Pro Ile Leu Leu Thr Asp Ser Thr Glu Ala
180 185 190
Glu Ala Ile Lys Leu Phe Ser Asn Thr Tyr Leu Ala Leu Arg Val Ala
195 200 205
Phe Phe Asn Glu Leu Asp Thr Tyr Ala Ser Val Arg Ser Leu Asp Thr
210 215 220
Lys Gln Ile Ile Glu Gly Val Gly Leu Asp Pro Arg Ile Gly Ser His
225 230 235 240
Tyr Asn Asn Pro Ser Phe Gly Tyr Gly Gly Tyr Cys Leu Pro Lys Asp
245 250 255
Thr Lys Gln Leu Leu Ala Asn Tyr Lys Asp Val Pro Gln Asn Leu Ile
260 265 270
Ser Ala Val Val Gln Ala Asn Lys Thr Arg Lys Asp Phe Ile Ala Glu
275 280 285
Asp Ile Leu Ser Lys Ser Pro Thr Val Val Gly Ile Tyr Arg Leu Val
290 295 300
Met Lys Ser Gly Ser Asp Asn Phe Arg Ser Ser Ser Ile Gln Gly Val
305 310 315 320
Met Lys Arg Ile Lys Ala Lys Gly Ile Glu Ile Val Val Phe Glu Pro
325 330 335
Asn Leu Gly Glu Glu Thr Phe Tyr Asn Ser Lys Ile Leu Asn Asp Ile
340 345 350
Glu Glu Phe Lys Asp Tyr Cys Asp Ile Ile Ile Ala Asn Arg Pro Thr
355 360 365
Asp Glu Leu Ser Asp Val Pro Glu Lys Val Tyr Thr Arg Asp Ile Phe
370 375 380
Gln Arg Asp
385
<210> 9
<211> 439
<212> PRT
<213> Corynebacterium glutamicum ATCC 13032
<400> 9
Met Arg Met Thr Val Ile Gly Thr Gly Tyr Leu Gly Ala Thr His Ala
1 5 10 15
Ala Cys Met Ala Glu Leu Ser His Glu Val Leu Gly Val Asp Val Asp
20 25 30
Glu Ala Lys Ile Ala Ser Leu Lys Asp Ser Lys Val Pro Phe Phe Glu
35 40 45
Pro Gly Leu Pro Glu Val Leu Glu Arg Asn Leu Glu Asn Gly Arg Leu
50 55 60
Asn Phe Thr Thr Asp Tyr Ala Glu Ala Ala Ala Phe Ala Gln Val His
65 70 75 80
Phe Leu Gly Val Gly Thr Pro Gln Gln Lys Gly Thr Tyr Ala Ala Asp
85 90 95
Leu Thr Tyr Val Arg Gln Val Val Glu Asp Leu Val Pro Leu Leu Glu
100 105 110
Gly Glu His Ile Ile Phe Gly Lys Ser Thr Val Pro Val Gly Thr Ala
115 120 125
Glu Gln Leu Gln Glu Leu Ala Asp Ser Leu Val Lys Pro Gly Ser His
130 135 140
Val Glu Ile Ala Trp Asn Pro Glu Phe Leu Arg Glu Gly Tyr Ala Val
145 150 155 160
Lys Asp Thr Ile Thr Pro Asp Arg Ile Val Val Gly Val Arg Glu Gly
165 170 175
Ala Thr Ala Glu Ala Ile Ala Arg Glu Val Tyr Ala Thr Ala Ile Ala
180 185 190
Ala Asp Thr Pro Phe Leu Val Thr Asp Leu Ala Thr Ala Glu Leu Val
195 200 205
Lys Val Ser Ala Asn Ala Phe Leu Ala Thr Lys Ile Ser Phe Ile Asn
210 215 220
Ala Val Ala Glu Ile Cys Glu Gln Thr Gly Ala Asp Val Val Ala Leu
225 230 235 240
Ala Asp Ala Ile Gly His Asp Asp Arg Ile Gly Arg Lys Phe Leu Gly
245 250 255
Ala Gly Leu Gly Phe Gly Gly Gly Cys Leu Pro Lys Asp Ile Arg Ala
260 265 270
Phe Met Ala Arg Ala Gly Glu Leu Gly Ala Asp Gln Ala Leu Thr Phe
275 280 285
Leu Arg Glu Val Asp Ser Ile Asn Met Arg Arg Arg Asp Arg Val Val
290 295 300
Gln Leu Ala Lys Glu Met Cys Gly Gly Ser Leu Leu Gly Lys Arg Val
305 310 315 320
Thr Val Leu Gly Ala Ala Phe Lys Pro Asn Ser Asp Asp Val Arg Asp
325 330 335
Ser Pro Ala Leu Ser Val Ala Gly Ser Leu Ser Leu Gln Gly Ala Ala
340 345 350
Val Ser Val Tyr Asp Pro Glu Ala Met Asp Asn Ala Arg Arg Val Phe
355 360 365
Pro Thr Leu Ser Tyr Ala Ser Ser Thr Lys Glu Ala Leu Ile Asp Ala
370 375 380
His Leu Val Val Leu Ala Thr Glu Trp Gln Glu Phe Arg Asp Leu Asp
385 390 395 400
Pro Glu Val Ala Gly Gly Val Val Glu Lys Arg Ala Ile Ile Asp Gly
405 410 415
Arg Asn Val Leu Asp Val Ala Lys Trp Lys Ala Ala Gly Trp Glu Met
420 425 430
Glu Ala Leu Gly Arg Asn Leu
435
<210> 10
<211> 29
<212> DNA
<213> 人工序列
<400> 10
cgactgcacg gtgcaccaat gcttctggc 29
<210> 11
<211> 33
<212> DNA
<213> 人工序列
<400> 11
catggtctgt ttcctgtgtg aaattgttat ccg 33
<210> 12
<211> 47
<212> DNA
<213> 人工序列
<400> 12
tttcacacag gaaacagacc atggttttca agggtgaaaa atgaata 47
<210> 13
<211> 29
<212> DNA
<213> 人工序列
<400> 13
ttaaatataa ccatttgggt ttttcattt 29
<210> 14
<211> 51
<212> DNA
<213> 人工序列
<400> 14
aaaacccaaa tggttatatt taaagattag gttgaaataa tatgagtatt c 51
<210> 15
<211> 46
<212> DNA
<213> 人工序列
<400> 15
agaggttccc cggctttcgg ttttataaat cattctctat tttttc 46
<210> 16
<211> 46
<212> DNA
<213> 人工序列
<400> 16
cgaattcgag ctcggtaccc ggggtgctca tctagcatct ggcttg 46
<210> 17
<211> 24
<212> DNA
<213> 人工序列
<400> 17
taaaaggggc tatcattcgg accc 24
<210> 18
<211> 22
<212> DNA
<213> 人工序列
<400> 18
aaccgaaagc cggggaacct ct 22
<210> 19
<211> 46
<212> DNA
<213> 人工序列
<400> 19
aacgacggcc agtgccaagc ttagctcgtc ctcattgatc agttcg 46
<210> 20
<211> 25
<212> DNA
<213> 人工序列
<400> 20
ggcactcgta gaacgcatca atggt 25
<210> 21
<211> 27
<212> DNA
<213> 人工序列
<400> 21
caatcgttgc ccaggagaaa tcattga 27
<210> 22
<211> 25
<212> DNA
<213> 人工序列
<400> 22
ggataacaat ttcacacagg aaaca 25
<210> 23
<211> 25
<212> DNA
<213> 人工序列
<400> 23
gtgaaaagaa aaaccaccct ggcgc 25
<210> 24
<211> 47
<212> DNA
<213> 人工序列
<400> 24
gcgccagggt ggtttttctt ttcacctgag tcttagattt tgagaaa 47
<210> 25
<211> 21
<212> DNA
<213> 人工序列
<400> 25
aacaagagac cgccaagggt g 21
<210> 26
<211> 44
<212> DNA
<213> 人工序列
<400> 26
cacccttggc ggtctcttgt tgtactcttg gttccatgag tttg 44
<210> 27
<211> 43
<212> DNA
<213> 人工序列
<400> 27
gcaaactcat ggaaccaaga gtaccttagc cttcctggtt gtg 43
<210> 28
<211> 25
<212> DNA
<213> 人工序列
<400> 28
ggtactcttg gttccatgag tttgc 25
<210> 29
<211> 48
<212> DNA
<213> 人工序列
<400> 29
tgtttcctgt gtgaaattgt tatccctatt ttacttgaga atcgtctg 48
<210> 30
<211> 54
<212> DNA
<213> 人工序列
<400> 30
gaccatggaa ttcgagctcg gtaccaagtt gttgtaaagt catgcgcatg tgtg 54
<210> 31
<211> 26
<212> DNA
<213> 人工序列
<400> 31
ttattcgacg gtgacagact ttgcca 26
<210> 32
<211> 58
<212> DNA
<213> 人工序列
<400> 32
tggcaaagtc tgtcaccgtc gaataaggcc gtgcataatt aatcgcatga ctcgacta 58
<210> 33
<211> 47
<212> DNA
<213> 人工序列
<400> 33
caagcttgca tgcctgcagg tcgacttaga cttctgcaac cactgca 47
<210> 34
<211> 11
<212> PRT
<213> 人工序列
<400> 34
Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 35
<211> 33
<212> DNA
<213> 人工序列
<400> 35
ggtggaagtg gaggtagtgg aggtggaggt agt 33
<210> 36
<211> 33
<212> DNA
<213> 人工序列
<400> 36
ggaggtagcg gtggaagcgg tggaggtgga agc 33

Claims (7)

1.一种重组谷氨酸棒杆菌，其特征在于，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，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；所述UDP-N-乙酰葡萄糖胺C4异构酶KfoA的氨基酸序列如SEQID NO.1所示；所述软骨素合成酶KfoC为氨基酸序列如SEQ ID NO.2所示的野生型，或为将氨基酸序列如SEQ ID NO.2所示的野生型第357位突变为甘氨酸和第363位突变为丝氨酸的双突变体；所述谷氨酰胺-果糖-6-磷酸氨基转移酶glmS氨基酸序列如SEQ ID NO.3所示；所述磷酸葡萄糖变位酶glmM氨基酸序列如SEQ ID NO.4所示；所述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氨基酸序列如SEQ ID NO.5所示；所述葡萄糖-6-磷酸尿酰胺转移酶galU氨基酸序列包括SEQ ID NO.6所示。
2.根据权利要求1所述的一种重组谷氨酸棒杆菌，其特征在于，还将UDP-N-乙酰葡萄糖胺C4异构酶KfoA、软骨素合成酶KfoC和UDP-葡萄糖脱氢酶Ugd进行融合，形成人工酶复合体；所述UDP-葡萄糖脱氢酶Ugd核苷酸序列如SEQ ID NO.9所示。
3.根据权利要求2所述的一种重组谷氨酸棒杆菌，其特征在于，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。
4.根据权利要求1～3任一所述的一种重组谷氨酸棒杆菌，其特征在于，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。
5.权利要求1～4任一所述重组谷氨酸棒杆菌在生产软骨素中的应用。
6.根据权利要求5所述的应用，其特征在于，包括以下步骤：
第一步，种子液的制备：将重组谷氨酸棒杆菌的单克隆接入种子培养基，培养至对数中后期；
第二步，补料发酵：在发酵罐中装入发酵培养基，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35～40g/L；控制发酵温度为28～30℃，搅拌转速为480～500rpm；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，当葡萄糖消耗速率下降到低于2g/h时，停止补料，待残留葡萄糖耗尽后结束发酵。
7.根据权利要求6所述的应用，其特征在于，
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，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到低于2g/h时，停止补料，待残留葡萄糖耗尽后结束发酵。
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