CN113881613B - 一种微生物发酵法生产制备超高分子量透明质酸的方法 - Google Patents

一种微生物发酵法生产制备超高分子量透明质酸的方法 Download PDF

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CN113881613B
CN113881613B CN202110396678.6A CN202110396678A CN113881613B CN 113881613 B CN113881613 B CN 113881613B CN 202110396678 A CN202110396678 A CN 202110396678A CN 113881613 B CN113881613 B CN 113881613B
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康振
王阳
堵国成
汪昊
陈坚
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Abstract

本发明公开了一种微生物发酵法生产制备超高分子量透明质酸的方法，属于生物工程技术领域。本发明在谷氨酸棒杆菌中游离表达透明质酸合酶编码基因pmhasA，构建了透明质酸的合成途径；采取弱化RBS强度的方法，弱化合酶pmHAS的表达；采取定点突变的策略理性改造透明质酸合成酶编码基因pmhasA；在谷氨酸棒杆菌中通过组合调控策略强化表达前体UDP‑葡萄糖醛酸和UDP‑N‑乙酰葡萄糖胺的合成途径酶编码基因；对发酵工艺进行优化，采取低温发酵的策略；重组谷氨酸棒杆菌生产的透明质酸分子量达到5‑9MDa。本发明构建的重组谷氨酸棒杆菌适合于工业化生产应用。

Description

一种微生物发酵法生产制备超高分子量透明质酸的方法
技术领域
本发明涉及一种微生物发酵法生产制备超高分子量透明质酸的方法，属于生物工程技术领域。
背景技术
透明质酸(Hyaluronan,HA)是一种广泛存在于微生物胞外的大分子酸性粘多糖，由(1-β-4)D-葡糖醛酸(1-β-3)N-乙酰基-D-氨基葡萄糖的双糖重复单位所组成的一种线性聚阴离子电解质。HA粘稠的物理特性使其在眼科手术、关节炎治疗和化妆品制造中扮演重要角色。细胞组织的构架、细胞外液的水分调节、润滑及治疗创伤时均需要透明质酸。
相比普通分子量透明质酸，超高分子量透明质酸(分子量高于6MDa)有着更为重要的生理功能。它具有更加优秀的保湿功能、促进皮肤营养吸收、增加皮肤弹性并且能够延缓皮肤衰老、促进创伤愈合、调节免疫能力和抗肿瘤。
目前，在工业领域还没有能够大规模生产超高分子量透明质酸的方法。现有制备超高分子量透明质酸的方法主要依赖组织提取法。部分动物的组织，如裸鼹鼠，富含大量的超高分子量透明质酸，其组织内透明质酸分子量可达到的6-12MDa，是超高分子量透明质酸的主要来源之一；但是动物组织材料来源十分有限，且提取过程繁琐、成本高昂、工艺复杂，透明质酸产物纯度和最终提取的透明质酸分子量极易受到提取过程的影响因而限制了超高分子量透明质酸的实际应用。
随着我国经济的迅速发展，医药行业对其需求量也越来越大。采用人工提取的方法得到的超高分子透明质酸远远不能满足社会的需求，提纯透明质酸的方法较为困难、成本较高。而兽疫链球菌可致各种炎症和败血症，在工业生产安全问题上具有一定风险。人们需要利用成本较低、安全的微生物发酵法来生产超高分子量透明质酸。
发明内容
[技术问题]
本发明要解决的技术问题是，通过生物合成，如发酵兽疫链球菌，生产透明质酸得到的透明质酸的分子量较低，且因为其致病性在工业生产上具有一定风险。
[技术方案]
本发明提供了一株能够合成超高分子量透明质酸的重组谷氨酸棒杆菌，以工业安全菌株谷氨酸棒杆菌(Corynebacterium glutamicum)异源表达透明质酸合酶pmHasA，并通过RBS弱化和定点突变的方法理性改造透明质酸合酶pmHasA，提高透明质酸合酶合成长链透明质酸的能力；通过构建表达框强化葡萄糖-6-磷酸尿酰胺转移酶编码基因galU、UDP-葡萄糖脱氢酶编码基因ugd、谷氨酰胺-果糖-6-磷酸氨基转移酶编码基因glmS、磷酸葡萄糖变位酶编码基因glmM和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编码基因glmU基因的表达，加强透明质酸的合成底物UDP-N-乙酰葡萄糖胺和UDP-葡萄糖醛酸的积累，提高了重组谷氨酸棒杆菌生产超高分子量透明质酸的能力；并在此基础上，对提纯超高分子量透明质酸的方法进行了优化。
所述超高分子量透明质酸是指分子量大于6MDa的透明质酸。
所述重组谷氨酸棒杆菌的宿主选自：Corynebacterium glutamicum ATCC 13032、Corynebacterium glutamicum K051、Corynebacterium glutamicum ATCC 14067、Corynebacterium glutamicum MB001、Corynebacterium glutamicum R、Corynebacteriumglutamicum S9114、Corynebacterium glutamicum SCgG1、Corynebacterium glutamicumSCgG2、Corynebacterium glutamicum Z188、Corynebacterium glutamicum ZL-2、Corynebacterium glutamicum ZL-5、Corynebacterium glutamicum ZL-6；优选Corynebacterium glutamicum ATCC 13032。
在本发明的一种实施方式中，弱化用于表达透明质酸合成酶pmHasA的编码基因的RBS序列时，采用的RBS序列为AAGGAGG、AAGGGCC、AAGGCTC、AAGGAAC、AAGGCAT、AAGGATC或AAGGTTG，其中优选AAGGAGG。
在本发明的一种实施方式中，所述透明质酸合酶pmHasA来源于多杀性巴斯德杆菌(Pasteurella multocida)，其氨基酸序列如SEQ ID NO.1所示。编码所述透明质酸合成酶pmHasA的基因是来源于多杀巴斯德杆菌的，或者人工合成的。或者，透明质酸合成酶pmHasA还可以是与SEQ ID NO.1有高于60％的氨基酸序列相似性的其他来源的透明质酸合成酶。或者，所述透明质酸合成酶pmHasA是经过定点突变改造的，例如，T104A、V59M、T104A/K106A/K107A，突变体的氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4所示的透明质酸合成酶。
在本发明的一种实施方式中，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Corynebactrium glutamicum。
在本发明的一种实施方式中，GlmU、GalU、Ugd、GlmS和GlmM的启动子为Ptrc、Ppyc、PglnA、PlysE、PlysG、PilvB、PleuA、PleuB、PthrB、PthrE、PgltB、PlpdA、Prep、Pper、PorfB、Porf3-aro、PsigA、PaceA、PaceB、Pamt、PctRNA、PbrnF、PbrnE、Plrp；优选Ptrc。
在本发明的一种实施方式中，GlmU、GalU、Ugd、GlmS和GlmM的表达载体选自：pXMJ19、pEC-XK99E、pEC-XC99E、pEC-XT99A、pEKEx1、pEKEx2、pVWEx1、pVWEx2、pZ8-1、pECTAC-K99、pAPE12。
在本发明的一种实施方式中，以pEC-XK99E为载体，以Ptrc为启动子，形成表达框在谷氨酸棒杆菌中利用IPTG诱导表达。GlmS、GlmM、GlmU、GalU和Ugd的氨基酸序列分别如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9所示。
本发明还提供了应用所述重组谷氨酸棒杆菌发酵生产超高分子量透明质酸的方法，采取低温发酵的策略，进一步提高了重组谷氨酸棒杆菌生产超高分子量透明质酸的能力。最终重组谷氨酸棒杆菌生产的超高分子量透明质酸分子量为5-9MDa。所述方法包括以下步骤：(1)制备重组谷氨酸棒杆菌种子培养液，(2)将种子液转接至发酵培养基中，使得发酵罐中菌体初始OD600为1-1.5，在28℃通风培养3h，待OD600涨到10-15，添加IPTG诱导，降温至20℃继续发酵69h。可选择的发酵培养基配方为：玉米浆干粉20g/L，(NH4)2SO4 20g/L，KH2PO4 1g/L，K2HPO4 1g/L，MgSO4 0.25g/L，MOPS(3-吗啉丙磺酸)4.2g/L，葡萄糖浓度40g/L。。可选地，发酵过程中，以50％氨水调节pH约为7.0，通过流加70％葡萄糖维持发酵培养基中的葡萄糖浓度约10g/L左右。
[有益效果]
本发明利用谷氨酸棒杆菌产超高分子量透明质酸，与其它菌相比，该发明具有非常大的应用优势。首先，它完全满足医疗卫生和食品安全生产的要求，其次，与枯草芽孢杆菌、大肠杆菌、兽疫链球菌相比，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。
本发明通过理性改造透明质酸合成关键酶pmHasA，调控透明质酸合成途径的两个前体物质UDP-GlcNAc和UDP-GlcUA合成途径，采取低温发酵策略，能够明显提高透明质酸的分子量。
本发明利用重组谷氨酸棒杆菌合成了分子量为5-9MDa的超高分子量透明质酸，而现有技术如酶法合成透明质酸分子量约为4.7MDa，利用兽疫链球菌发酵生产透明质酸分子量最高达到3.4MDa。因此，本发明大幅提高了透明质酸的分子量，在工业上用于安全高效的制备与提纯超高分子量透明质酸具有广泛的应用前景。
采用传统高温高压法提取发酵液中的透明质酸时，透明质酸链会发生断裂，提取得到的透明质酸分子量偏小；而本发明开发的碱裂提取法提取透明质酸，更有利于提取得到超高分子量透明质酸。
附图说明
图1：重组谷氨酸棒杆菌生产透明质酸的代谢途径及相关酶类
图2：重组谷氨酸棒杆菌的透明质酸的分子量液相图
具体实施方式
材料与方法
菌株：谷氨酸棒杆菌C.glutamicum ATCC 13032，质粒：pXMJ19,pEC-XK99E。
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。
超高分子量透明质酸的碱裂解提纯方法：取10mL发酵液，离心去除上清保留菌体，加入5mL PI溶液轻轻重悬菌体，放于37℃水浴1h；加入5mL PⅡ溶液裂解菌体溶液，上下轻轻颠倒至溶液澄清使细胞完全裂开；加入7.5mL PⅢ溶液使蛋白与基因组共沉淀；离心取上清，加入四倍体积无水乙醇，反复醇沉复溶两次；加入20mg/mL DNase和100μg/mL RNase，37℃水浴1h，去除溶液内残余DNA和RNA，醇沉复溶后得到高纯度透明质酸溶液。(PI：25mMTris-HCl、10mM EDTA、20mg/mL Lyzsome、pH 8.0；PⅡ:0.2M NaOH、1％SDS；PⅢ:3M KoAC、pH6.0；使用DNAse/RNAse时的缓冲体系：10mM Tris-HCl pH 7.5、2.5mM MgCl2、0.5mM CaCl2)。
透明质酸分子量测定：将发酵样品通过反复醇沉获得纯度较高的透明质酸，经过水系膜过滤出去颗粒杂质后，通过High Performance Size Exclusion Chromatography(HPSEC)测定透明质酸分子量。
实施例1:重组谷氨酸棒杆菌低温发酵生产超高分子量透明质酸
步骤1:构建重组质粒表达多杀巴斯德杆菌来源透明质酸合成酶pmHasA
根据多杀性巴斯德杆菌的基因组序列，由GENEWIZ(苏州)合成透明质酸合酶基因pmHasA，pmHasA的序列如Gene ID:11805012所示。以合成的pmHasA基因序列为模板，设计引物pmhasA-F和pmhasA-R进行PCR扩增，再通过T5 exonuclease-dependent assembly(TEDA)方法将pmHasA基因序列连接至质粒pXMJ19，构建得到重组表达质粒pXMJ19-pmhasA。
表1构建表达透明质酸合酶的重组质粒所用的引物
步骤2:构建RBS弱化表达透明质酸合酶pmHasA的重组质粒
设计引物pmhasA-RBS1-F/pmhasA-RBS1-R，以质粒pXMJ19-pmhasA为模板，采用环形PCR的体系和程序进行扩增，回收纯化PCR产物并直接转化JM109感受态细胞。提取阳性转化子的质粒进行测序，将测序结果进行比对，确定RBS弱化表达透明质酸合酶pmHasA重组质粒pXMJ19-RBS1-pmhasA构建成功。采用相同步骤，分别利用引物pmhasA-RBS2-F/pmhasA-RBS2-R、pmhasA-RBS3-F/pmhasA-RBS3-R、pmhasA-RBS4-F/pmhasA-RBS4-R、pmhasA-RBS5-F/pmhasA-RBS5-R，来获得弱化表达透明质酸合酶pmHasA的重组质粒pXMJ19-RBS2-pmhasA、pXMJ19-RBS3-pmhasA、pXMJ19-RBS4-pmhasA、pXMJ19-RBS5-pmhasA。
表2表达透明质酸合酶重组质粒构建所用引物
步骤3:定点突变理性改造多杀巴斯德杆菌来源透明质酸合成酶pmHasA
设计引物pmhasA-T104A-F/pmhasA-T104A-/R和pmhasA-V59M-F/pmhasA-V59M-/R，以质粒pXMJ19-RBS1-pmhasA为模板采用环形PCR的体系和程序进行扩增；设计引物pmhasA-T104A K106A K107A-F/pmhasA-T104A K106A K107A-/R，以质粒pXMJ19-RBS1-pmhasAT104A为模板采用环形PCR的体系和程序进行扩增，回收纯化PCR产物，并直接转化JM109感受态细胞。提取阳性转化子的质粒进行测序，将测序结果与透明质酸合成酶序列进行比对，确定透明质酸合酶突变体构建成功。
采用相同的思路，对步骤2构建得到的其他弱化表达透明质酸合酶pmHasA的重组质粒进行T104A、V59M、T104A/K106A/K107A的定点突变。
表3透明质酸合酶突变体构建所用引物
步骤4:重组质粒pEC-XK99E-ugd-glms-glmm构建
将Corynebacterium glutamicum ATCC 13032接种于5ml LBG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。设计引物ugd-F/ugd-R,glms-F/glms-R和glmm-F/glmm-R,以提取的基因组DNA为模板，采用标准的PCR扩增体系和程序，扩增获取ugd、glms、glmm基因。
以扩增的ugd、glms、glmm为模板，以ugd-F和glmm-R为引物进行重叠延伸PCR获得相连的ugd-glms-glmm片段。以EcoRI和BamHI为酶切位点，双酶切线性pEC-XK99E质粒。将ugd-glms-glmm片段和线性质粒pEC-XK99E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，比对确认重组质粒pEC-XK99E-ugd-glms-glmm构建成功。
同理构建：pEC-XK99E-ugd，pEC-XK99E-ugd-galu，pEC-XK99E-ugd-glmu，pEC-XK99E-ugd-glms，pEC-XK99E-ugd-glmm重组质粒。
表4重组质粒pEC-XK99E-ugd-glms-glmm构建所用引物
步骤5重组谷氨酸棒杆菌低温发酵生产超高分子量透明质酸
将步骤3构建得到的弱化表达透明质酸合酶pmHasA且发生定点突变的重组质粒与步骤4构建得到的用于加强透明质酸的合成底物UDP-N-乙酰葡萄糖胺和UDP-葡萄糖醛酸的积累的质粒转化进入C.glutamicum ATCC13032。例如：
将步骤3构建得到的pXMJ19-RBS1-pmhasA-T104A K106A K107A以及步骤4构建得到的pEC-XK99E-ugd转化进入C.glutamicum ATCC13032，得到C.glutamicum ATCC13032pXMJ19-RBS1-pmhasA-T104A K106A K107A/pEC-XK99E-ugd；
将步骤3构建得到的pXMJ19-RBS1-pmhasA-T104A以及步骤4构建得到的pEC-XK99E-ugd转化进入C.glutamicum ATCC13032，得到C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA-T104A/pEC-XK99E-ugd；
将步骤3构建得到的pXMJ19-pmhasA-V59M以及步骤4构建得到的pEC-XK99E-ugd转化进入C.glutamicum ATCC13032，得到C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA-V59M/pEC-XK99E-ugd；
将步骤3构建得到的突变质粒pXMJ19-pmhasA-T104A K106A K107A或pXMJ19-pmhasA-T104A或pXMJ19-pmhasA-V59M以及步骤4得到的pEC-XK99E-ugd-galu、pEC-XK99E-ugd-glmu、pEC-XK99E-ugd-glms或pEC-XK99E-ugd-glmm转化进入C.glutamicumATCC13032。
还构建得到一系列重组谷氨酸帮杆菌菌株，例如：C.glutamicum ATCC13032pXMJ19-RBS1-pmhasA-T104A K106A K107A/pEC-XK99E-ugd-galu、C.glutamicumATCC13032 pXMJ19-RBS1-pmhasA-T104A/pEC-XK99E-ugd-galu、C.glutamicum ATCC13032pXMJ19-RBS1-pmhasA-V59M/pEC-XK99E-ugd-galu、C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA-T104A K106A K107A/pEC-XK99E-ugd-glms、C.glutamicum ATCC13032pXMJ19-RBS1-pmhasA-T104A/pEC-XK99E-ugd-glms、C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA-V59M/pEC-XK99E-ugd-glms、C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA-T104A K106A K107A/pEC-XK99E-ugd-glmm、C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA-T104A/pEC-XK99E-ugd-glmm、C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA-V59M/pEC-XK99E-ugd-glmm、C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA-T104A K106A K107A/pEC-XK99E-ugd-glmu、C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA-T104A/pEC-XK99E-ugd-glmu、C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA-V59M/pEC-XK99E-ugd-glmu。
对照菌1：将步骤1的重组表达质粒pXMJ19-pmhasA转入C.glutamicum ATCC13032，得到C.glutamicum ATCC13032 pXMJ19-pmhasA。
对照菌2：将步骤2构建的pXMJ19-RBS1-pmhasA转入C.glutamicum ATCC13032，得到C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA。
将所得上述重组菌的单克隆分别接于5mL BHI培养基，置于200rpm，30℃过夜培养。12h后，按1％的接种量转接于250mL三角摇瓶(装25mL发酵培养基)中，置于200rpm、28℃培养，培养3h后，添加0.25mM L-1IPTG诱导基因表达，并将培养温度降至为20℃，继续诱导培养69h。同时设置不降温诱导表达的对照组，该对照组在加入IPTG后，于28℃诱导培养69h。
发酵结束后，取10mL发酵样品，于8000rpm、离心10min去除上清保留菌体，加入5mLPI溶液轻轻重悬菌体，放于37℃水浴1h；加入5mL PⅡ溶液裂解细胞，上下轻轻颠倒至溶液澄清使细胞完全裂开，释放胞内透明质酸；加入7.5mL PⅢ溶液使蛋白与基因组共沉淀；8000rpm、30min离心取上清，加入四倍体积无水乙醇，反复醇沉复溶两次；加入20mg/mLDNAse和100μg/mL RNAse，37℃水浴1h，去除溶液内残余DNA和RNA，醇沉复溶浓缩后得到高纯度透明质酸溶液。通过HPSEC测定透明质酸分子量。
(1)添加IPTG后常温28℃摇瓶水平诱导发酵时
表达透明质酸合酶的对照重组菌C.glutamicum ATCC13032 pXMJ19-pmhasA的透明质酸分子量为1.3MDa，产量为0.12g/L。
优化RBS序列后，对照重组菌C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA透明质酸分子量为1.5MDa，产量为0.14g/L。
在优化RBS序列的基础上，表达透明质酸合酶突变体的重组菌C.glutamicumATCC13032 pXMJ19-RBS1-pmhasA V59M的透明质酸分子量为2.8MDa，重组菌C.glutamicumATCC13032 pXMJ19-RBS1-pmhasA T104A的透明质酸分子量为3MDa，重组菌C.glutamicumATCC13032 pXMJ19-RBS1-pmhasA T104A K106A K107A的透明质酸分子量为3.2MDa。
同时表达RBS优化、透明质酸合酶突变体以及透明质酸前体途径基因的重组菌当中，C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA V59M/pEC-XK99E-ugd透明质酸分子量达到4MDa，产量为0.3g/L；C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA T104A/pEC-XK99E-ugd透明质酸分子量达到5MDa，产量提高至0.5g/L；C.glutamicum ATCC13032pXMJ19-RBS1-pmhasA T104A K106A K107A/pEC-XK99E-ugd透明质酸分子量达到5.5MDa，产量提高至0.5g/L。表达透明质酸前体途径基因时，单独表达ugd对透明质酸分子量及产量的提升效果最明显，组合表达其他基因时也能提升透明质酸产量及分子量。
(2)添加IPTG后低温20℃摇瓶水平诱导发酵时
重组菌C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA V59M/pEC-XK99E-ugd透明质酸分子量达到5MDa，产量为0.27g/L；C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasAT104A/pEC-XK99E-ugd透明质酸分子量达到6MDa，产量为0.43g/L，C.glutamicumATCC13032 pXMJ19-RBS1-pmhasA T104A K106A K107A/pEC-XK99E-ugd透明质酸分子量达到6.5-9MDa，产量为0.45g/L。
实施例2:重组谷氨酸棒杆菌发酵罐水平低温发酵生产超高分子量透明质酸
1.培养基
发酵培养基：玉米浆干粉20g/L，(NH4)2SO4 20g/L，KH2PO4 1g/L，K2HPO4 1g/L，MgSO4 0.25g/L，MOPS(3-吗啉丙磺酸)4.2g/L，葡萄糖浓度40g/L。
2.培养方法
挑取平板中的单克隆菌体，置于装有5mL BHIS培养基的摇菌管中，在28℃，220rpm条件下过夜培养。
以初始OD600为0.1的接种量取过夜培养的种子液，转移至装有25mL发酵培养基的挡板摇瓶中。在28℃，220rpm条件下培养约10-12h后，摇瓶中OD600涨到10-15后，以10％接种量转接至3L发酵罐中，使得发酵罐中菌体初始OD600为1-1.5。在28℃，220rpm条件下培养3h后，OD600涨到约10-15，添加0.25mM IPTG诱导，诱导后降温至20℃继续发酵69h。发酵过程中以50％氨水调节pH约为7.0，通过流加70％葡萄糖维持葡萄糖浓度约10g/L左右。
在3L发酵罐中分批补料发酵结果表明，C.glutamicum ATCC13032 pXMJ19-RBS1-pmhasA T104A K106A K107A/pEC-XK99E-ugd是最优重组菌，透明质酸分子量为6-9MDa，产量达到1.5g/L。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
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<120> 一种微生物发酵法生产制备超高分子量透明质酸的方法
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Val Thr Asp Asp Gly Ser Gln Glu Asp Leu Ser Pro Ile Ile Arg Gln
195 200 205
Tyr Glu Asn Lys Leu Asp Ile Arg Tyr Val Arg Gln Lys Asp Asn Gly
210 215 220
Phe Gln Ala Ser Ala Ala Arg Asn Met Gly Leu Arg Leu Ala Lys Tyr
225 230 235 240
Asp Phe Ile Gly Leu Leu Asp Cys Asp Met Ala Pro Asn Pro Leu Trp
245 250 255
Val His Ser Tyr Val Ala Glu Leu Leu Glu Asp Asp Asp Leu Thr Ile
260 265 270
Ile Gly Pro Arg Lys Tyr Ile Asp Thr Gln His Ile Asp Pro Lys Asp
275 280 285
Phe Leu Asn Asn Ala Ser Leu Leu Glu Ser Leu Pro Glu Val Lys Thr
290 295 300
Asn Asn Ser Val Ala Ala Lys Gly Glu Gly Thr Val Ser Leu Asp Trp
305 310 315 320
Arg Leu Glu Gln Phe Glu Lys Thr Glu Asn Leu Arg Leu Ser Asp Ser
325 330 335
Pro Phe Arg Phe Phe Ala Ala Gly Asn Val Ala Phe Ala Lys Lys Trp
340 345 350
Leu Asn Lys Ser Gly Phe Phe Asp Glu Glu Phe Asn His Trp Gly Gly
355 360 365
Glu Asp Val Glu Phe Gly Tyr Arg Leu Phe Arg Tyr Gly Ser Phe Phe
370 375 380
Lys Thr Ile Asp Gly Ile Met Ala Tyr His Gln Glu Pro Pro Gly Lys
385 390 395 400
Glu Asn Glu Thr Asp Arg Glu Ala Gly Lys Asn Ile Thr Leu Asp Ile
405 410 415
Met Arg Glu Lys Val Pro Tyr Ile Tyr Arg Lys Leu Leu Pro Ile Glu
420 425 430
Asp Ser His Ile Asn Arg Val Pro Leu Val Ser Ile Tyr Ile Pro Ala
435 440 445
Tyr Asn Cys Ala Asn Tyr Ile Gln Arg Cys Val Asp Ser Ala Leu Asn
450 455 460
Gln Thr Val Val Asp Leu Glu Val Cys Ile Cys Asn Asp Gly Ser Thr
465 470 475 480
Asp Asn Thr Leu Glu Val Ile Asn Lys Leu Tyr Gly Asn Asn Pro Arg
485 490 495
Val Arg Ile Met Ser Lys Pro Asn Gly Gly Ile Ala Ser Ala Ser Asn
500 505 510
Ala Ala Val Ser Phe Ala Lys Gly Tyr Tyr Ile Gly Gln Leu Asp Ser
515 520 525
Asp Asp Tyr Leu Glu Pro Asp Ala Val Glu Leu Cys Leu Lys Glu Phe
530 535 540
Leu Lys Asp Lys Thr Leu Ala Cys Val Tyr Thr Thr Asn Arg Asn Val
545 550 555 560
Asn Pro Asp Gly Ser Leu Ile Ala Asn Gly Tyr Asn Trp Pro Glu Phe
565 570 575
Ser Arg Glu Lys Leu Thr Thr Ala Met Ile Ala His His Phe Arg Met
580 585 590
Phe Thr Ile Arg Ala Trp His Leu Thr Asp Gly Phe Asn Glu Lys Ile
595 600 605
Glu Asn Ala Val Asp Tyr Asp Met Phe Leu Lys Leu Ser Glu Val Gly
610 615 620
Lys Phe Lys His Leu Asn Lys Ile Cys Tyr Asn Arg Val Leu His Gly
625 630 635 640
Asp Asn Thr Ser Ile Lys Lys Leu Gly Ile Gln Lys Lys Asn His Phe
645 650 655
Val Val Val Asn Gln Ser Leu Asn Arg Gln Gly Ile Thr Tyr Tyr Asn
660 665 670
Tyr Asp Glu Phe Asp Asp Leu Asp Glu Ser Arg Lys Tyr Ile Phe Asn
675 680 685
Lys Thr Ala Glu Tyr Gln Glu Glu Ile Asp Ile Leu Lys Asp Ile Lys
690 695 700
Ile Ile Gln Asn Lys Asp Ala Lys Ile Ala Val Ser Ile Phe Tyr Pro
705 710 715 720
Asn Thr Leu Asn Gly Leu Val Lys Lys Leu Asn Asn Ile Ile Glu Tyr
725 730 735
Asn Lys Asn Ile Phe Val Ile Val Leu His Val Asp Lys Asn His Leu
740 745 750
Thr Pro Asp Ile Lys Lys Glu Ile Leu Ala Phe Tyr His Lys His Gln
755 760 765
Val Asn Ile Leu Leu Asn Asn Asp Ile Ser Tyr Tyr Thr Ser Asn Arg
770 775 780
Leu Ile Lys Thr Glu Ala His Leu Ser Asn Ile Asn Lys Leu Ser Gln
785 790 795 800
Leu Asn Leu Asn Cys Glu Tyr Ile Ile Phe Asp Asn His Asp Ser Leu
805 810 815
Phe Val Lys Asn Asp Ser Tyr Ala Tyr Met Lys Lys Tyr Asp Val Gly
820 825 830
Met Asn Phe Ser Ala Leu Thr His Asp Trp Ile Glu Lys Ile Asn Ala
835 840 845
His Pro Pro Phe Lys Lys Leu Ile Lys Thr Tyr Phe Asn Asp Asn Asp
850 855 860
Leu Lys Ser Met Asn Val Lys Gly Ala Ser Gln Gly Met Phe Met Thr
865 870 875 880
Tyr Ala Leu Ala His Glu Leu Leu Thr Ile Ile Lys Glu Val Ile Thr
885 890 895
Ser Cys Gln Ser Ile Asp Ser Val Pro Glu Tyr Asn Thr Glu Asp Ile
900 905 910
Trp Phe Gln Phe Ala Leu Leu Ile Leu Glu Lys Lys Thr Gly His Val
915 920 925
Phe Asn Lys Thr Ser Thr Leu Thr Tyr Met Pro Trp Glu Arg Lys Leu
930 935 940
Gln Trp Thr Asn Glu Gln Ile Glu Ser Ala Lys Arg Gly Glu Asn Ile
945 950 955 960
Pro Val Asn Lys Phe Ile Ile Asn Ser Ile Thr Leu
965 970
<210> 3
<211> 972
<212> PRT
<213> Pasteurella multocida
<400> 3
Met Asn Thr Leu Ser Gln Ala Ile Lys Ala Tyr Asn Ser Asn Asp Tyr
1 5 10 15
Gln Leu Ala Leu Lys Leu Phe Glu Lys Ser Ala Glu Ile Tyr Gly Arg
20 25 30
Lys Ile Val Glu Phe Gln Ile Thr Lys Cys Lys Glu Lys Leu Ser Ala
35 40 45
His Pro Ser Val Asn Ser Ala His Leu Ser Met Asn Lys Glu Glu Lys
50 55 60
Val Asn Val Cys Asp Ser Pro Leu Asp Ile Ala Thr Gln Leu Leu Leu
65 70 75 80
Ser Asn Val Lys Lys Leu Val Leu Ser Asp Ser Glu Lys Asn Thr Leu
85 90 95
Lys Asn Lys Trp Lys Leu Leu Thr Glu Lys Lys Ser Glu Asn Ala Glu
100 105 110
Val Arg Ala Val Ala Leu Val Pro Lys Asp Phe Pro Lys Asp Leu Val
115 120 125
Leu Ala Pro Leu Pro Asp His Val Asn Asp Phe Thr Trp Tyr Lys Lys
130 135 140
Arg Lys Lys Arg Leu Gly Ile Lys Pro Glu His Gln His Val Gly Leu
145 150 155 160
Ser Ile Ile Val Thr Thr Phe Asn Arg Pro Ala Ile Leu Ser Ile Thr
165 170 175
Leu Ala Cys Leu Val Asn Gln Lys Thr His Tyr Pro Phe Glu Val Ile
180 185 190
Val Thr Asp Asp Gly Ser Gln Glu Asp Leu Ser Pro Ile Ile Arg Gln
195 200 205
Tyr Glu Asn Lys Leu Asp Ile Arg Tyr Val Arg Gln Lys Asp Asn Gly
210 215 220
Phe Gln Ala Ser Ala Ala Arg Asn Met Gly Leu Arg Leu Ala Lys Tyr
225 230 235 240
Asp Phe Ile Gly Leu Leu Asp Cys Asp Met Ala Pro Asn Pro Leu Trp
245 250 255
Val His Ser Tyr Val Ala Glu Leu Leu Glu Asp Asp Asp Leu Thr Ile
260 265 270
Ile Gly Pro Arg Lys Tyr Ile Asp Thr Gln His Ile Asp Pro Lys Asp
275 280 285
Phe Leu Asn Asn Ala Ser Leu Leu Glu Ser Leu Pro Glu Val Lys Thr
290 295 300
Asn Asn Ser Val Ala Ala Lys Gly Glu Gly Thr Val Ser Leu Asp Trp
305 310 315 320
Arg Leu Glu Gln Phe Glu Lys Thr Glu Asn Leu Arg Leu Ser Asp Ser
325 330 335
Pro Phe Arg Phe Phe Ala Ala Gly Asn Val Ala Phe Ala Lys Lys Trp
340 345 350
Leu Asn Lys Ser Gly Phe Phe Asp Glu Glu Phe Asn His Trp Gly Gly
355 360 365
Glu Asp Val Glu Phe Gly Tyr Arg Leu Phe Arg Tyr Gly Ser Phe Phe
370 375 380
Lys Thr Ile Asp Gly Ile Met Ala Tyr His Gln Glu Pro Pro Gly Lys
385 390 395 400
Glu Asn Glu Thr Asp Arg Glu Ala Gly Lys Asn Ile Thr Leu Asp Ile
405 410 415
Met Arg Glu Lys Val Pro Tyr Ile Tyr Arg Lys Leu Leu Pro Ile Glu
420 425 430
Asp Ser His Ile Asn Arg Val Pro Leu Val Ser Ile Tyr Ile Pro Ala
435 440 445
Tyr Asn Cys Ala Asn Tyr Ile Gln Arg Cys Val Asp Ser Ala Leu Asn
450 455 460
Gln Thr Val Val Asp Leu Glu Val Cys Ile Cys Asn Asp Gly Ser Thr
465 470 475 480
Asp Asn Thr Leu Glu Val Ile Asn Lys Leu Tyr Gly Asn Asn Pro Arg
485 490 495
Val Arg Ile Met Ser Lys Pro Asn Gly Gly Ile Ala Ser Ala Ser Asn
500 505 510
Ala Ala Val Ser Phe Ala Lys Gly Tyr Tyr Ile Gly Gln Leu Asp Ser
515 520 525
Asp Asp Tyr Leu Glu Pro Asp Ala Val Glu Leu Cys Leu Lys Glu Phe
530 535 540
Leu Lys Asp Lys Thr Leu Ala Cys Val Tyr Thr Thr Asn Arg Asn Val
545 550 555 560
Asn Pro Asp Gly Ser Leu Ile Ala Asn Gly Tyr Asn Trp Pro Glu Phe
565 570 575
Ser Arg Glu Lys Leu Thr Thr Ala Met Ile Ala His His Phe Arg Met
580 585 590
Phe Thr Ile Arg Ala Trp His Leu Thr Asp Gly Phe Asn Glu Lys Ile
595 600 605
Glu Asn Ala Val Asp Tyr Asp Met Phe Leu Lys Leu Ser Glu Val Gly
610 615 620
Lys Phe Lys His Leu Asn Lys Ile Cys Tyr Asn Arg Val Leu His Gly
625 630 635 640
Asp Asn Thr Ser Ile Lys Lys Leu Gly Ile Gln Lys Lys Asn His Phe
645 650 655
Val Val Val Asn Gln Ser Leu Asn Arg Gln Gly Ile Thr Tyr Tyr Asn
660 665 670
Tyr Asp Glu Phe Asp Asp Leu Asp Glu Ser Arg Lys Tyr Ile Phe Asn
675 680 685
Lys Thr Ala Glu Tyr Gln Glu Glu Ile Asp Ile Leu Lys Asp Ile Lys
690 695 700
Ile Ile Gln Asn Lys Asp Ala Lys Ile Ala Val Ser Ile Phe Tyr Pro
705 710 715 720
Asn Thr Leu Asn Gly Leu Val Lys Lys Leu Asn Asn Ile Ile Glu Tyr
725 730 735
Asn Lys Asn Ile Phe Val Ile Val Leu His Val Asp Lys Asn His Leu
740 745 750
Thr Pro Asp Ile Lys Lys Glu Ile Leu Ala Phe Tyr His Lys His Gln
755 760 765
Val Asn Ile Leu Leu Asn Asn Asp Ile Ser Tyr Tyr Thr Ser Asn Arg
770 775 780
Leu Ile Lys Thr Glu Ala His Leu Ser Asn Ile Asn Lys Leu Ser Gln
785 790 795 800
Leu Asn Leu Asn Cys Glu Tyr Ile Ile Phe Asp Asn His Asp Ser Leu
805 810 815
Phe Val Lys Asn Asp Ser Tyr Ala Tyr Met Lys Lys Tyr Asp Val Gly
820 825 830
Met Asn Phe Ser Ala Leu Thr His Asp Trp Ile Glu Lys Ile Asn Ala
835 840 845
His Pro Pro Phe Lys Lys Leu Ile Lys Thr Tyr Phe Asn Asp Asn Asp
850 855 860
Leu Lys Ser Met Asn Val Lys Gly Ala Ser Gln Gly Met Phe Met Thr
865 870 875 880
Tyr Ala Leu Ala His Glu Leu Leu Thr Ile Ile Lys Glu Val Ile Thr
885 890 895
Ser Cys Gln Ser Ile Asp Ser Val Pro Glu Tyr Asn Thr Glu Asp Ile
900 905 910
Trp Phe Gln Phe Ala Leu Leu Ile Leu Glu Lys Lys Thr Gly His Val
915 920 925
Phe Asn Lys Thr Ser Thr Leu Thr Tyr Met Pro Trp Glu Arg Lys Leu
930 935 940
Gln Trp Thr Asn Glu Gln Ile Glu Ser Ala Lys Arg Gly Glu Asn Ile
945 950 955 960
Pro Val Asn Lys Phe Ile Ile Asn Ser Ile Thr Leu
965 970
<210> 4
<211> 972
<212> PRT
<213> Pasteurella multocida
<400> 4
Met Asn Thr Leu Ser Gln Ala Ile Lys Ala Tyr Asn Ser Asn Asp Tyr
1 5 10 15
Gln Leu Ala Leu Lys Leu Phe Glu Lys Ser Ala Glu Ile Tyr Gly Arg
20 25 30
Lys Ile Val Glu Phe Gln Ile Thr Lys Cys Lys Glu Lys Leu Ser Ala
35 40 45
His Pro Ser Val Asn Ser Ala His Leu Ser Val Asn Lys Glu Glu Lys
50 55 60
Val Asn Val Cys Asp Ser Pro Leu Asp Ile Ala Thr Gln Leu Leu Leu
65 70 75 80
Ser Asn Val Lys Lys Leu Val Leu Ser Asp Ser Glu Lys Asn Thr Leu
85 90 95
Lys Asn Lys Trp Lys Leu Leu Ala Glu Ala Ala Ser Glu Asn Ala Glu
100 105 110
Val Arg Ala Val Ala Leu Val Pro Lys Asp Phe Pro Lys Asp Leu Val
115 120 125
Leu Ala Pro Leu Pro Asp His Val Asn Asp Phe Thr Trp Tyr Lys Lys
130 135 140
Arg Lys Lys Arg Leu Gly Ile Lys Pro Glu His Gln His Val Gly Leu
145 150 155 160
Ser Ile Ile Val Thr Thr Phe Asn Arg Pro Ala Ile Leu Ser Ile Thr
165 170 175
Leu Ala Cys Leu Val Asn Gln Lys Thr His Tyr Pro Phe Glu Val Ile
180 185 190
Val Thr Asp Asp Gly Ser Gln Glu Asp Leu Ser Pro Ile Ile Arg Gln
195 200 205
Tyr Glu Asn Lys Leu Asp Ile Arg Tyr Val Arg Gln Lys Asp Asn Gly
210 215 220
Phe Gln Ala Ser Ala Ala Arg Asn Met Gly Leu Arg Leu Ala Lys Tyr
225 230 235 240
Asp Phe Ile Gly Leu Leu Asp Cys Asp Met Ala Pro Asn Pro Leu Trp
245 250 255
Val His Ser Tyr Val Ala Glu Leu Leu Glu Asp Asp Asp Leu Thr Ile
260 265 270
Ile Gly Pro Arg Lys Tyr Ile Asp Thr Gln His Ile Asp Pro Lys Asp
275 280 285
Phe Leu Asn Asn Ala Ser Leu Leu Glu Ser Leu Pro Glu Val Lys Thr
290 295 300
Asn Asn Ser Val Ala Ala Lys Gly Glu Gly Thr Val Ser Leu Asp Trp
305 310 315 320
Arg Leu Glu Gln Phe Glu Lys Thr Glu Asn Leu Arg Leu Ser Asp Ser
325 330 335
Pro Phe Arg Phe Phe Ala Ala Gly Asn Val Ala Phe Ala Lys Lys Trp
340 345 350
Leu Asn Lys Ser Gly Phe Phe Asp Glu Glu Phe Asn His Trp Gly Gly
355 360 365
Glu Asp Val Glu Phe Gly Tyr Arg Leu Phe Arg Tyr Gly Ser Phe Phe
370 375 380
Lys Thr Ile Asp Gly Ile Met Ala Tyr His Gln Glu Pro Pro Gly Lys
385 390 395 400
Glu Asn Glu Thr Asp Arg Glu Ala Gly Lys Asn Ile Thr Leu Asp Ile
405 410 415
Met Arg Glu Lys Val Pro Tyr Ile Tyr Arg Lys Leu Leu Pro Ile Glu
420 425 430
Asp Ser His Ile Asn Arg Val Pro Leu Val Ser Ile Tyr Ile Pro Ala
435 440 445
Tyr Asn Cys Ala Asn Tyr Ile Gln Arg Cys Val Asp Ser Ala Leu Asn
450 455 460
Gln Thr Val Val Asp Leu Glu Val Cys Ile Cys Asn Asp Gly Ser Thr
465 470 475 480
Asp Asn Thr Leu Glu Val Ile Asn Lys Leu Tyr Gly Asn Asn Pro Arg
485 490 495
Val Arg Ile Met Ser Lys Pro Asn Gly Gly Ile Ala Ser Ala Ser Asn
500 505 510
Ala Ala Val Ser Phe Ala Lys Gly Tyr Tyr Ile Gly Gln Leu Asp Ser
515 520 525
Asp Asp Tyr Leu Glu Pro Asp Ala Val Glu Leu Cys Leu Lys Glu Phe
530 535 540
Leu Lys Asp Lys Thr Leu Ala Cys Val Tyr Thr Thr Asn Arg Asn Val
545 550 555 560
Asn Pro Asp Gly Ser Leu Ile Ala Asn Gly Tyr Asn Trp Pro Glu Phe
565 570 575
Ser Arg Glu Lys Leu Thr Thr Ala Met Ile Ala His His Phe Arg Met
580 585 590
Phe Thr Ile Arg Ala Trp His Leu Thr Asp Gly Phe Asn Glu Lys Ile
595 600 605
Glu Asn Ala Val Asp Tyr Asp Met Phe Leu Lys Leu Ser Glu Val Gly
610 615 620
Lys Phe Lys His Leu Asn Lys Ile Cys Tyr Asn Arg Val Leu His Gly
625 630 635 640
Asp Asn Thr Ser Ile Lys Lys Leu Gly Ile Gln Lys Lys Asn His Phe
645 650 655
Val Val Val Asn Gln Ser Leu Asn Arg Gln Gly Ile Thr Tyr Tyr Asn
660 665 670
Tyr Asp Glu Phe Asp Asp Leu Asp Glu Ser Arg Lys Tyr Ile Phe Asn
675 680 685
Lys Thr Ala Glu Tyr Gln Glu Glu Ile Asp Ile Leu Lys Asp Ile Lys
690 695 700
Ile Ile Gln Asn Lys Asp Ala Lys Ile Ala Val Ser Ile Phe Tyr Pro
705 710 715 720
Asn Thr Leu Asn Gly Leu Val Lys Lys Leu Asn Asn Ile Ile Glu Tyr
725 730 735
Asn Lys Asn Ile Phe Val Ile Val Leu His Val Asp Lys Asn His Leu
740 745 750
Thr Pro Asp Ile Lys Lys Glu Ile Leu Ala Phe Tyr His Lys His Gln
755 760 765
Val Asn Ile Leu Leu Asn Asn Asp Ile Ser Tyr Tyr Thr Ser Asn Arg
770 775 780
Leu Ile Lys Thr Glu Ala His Leu Ser Asn Ile Asn Lys Leu Ser Gln
785 790 795 800
Leu Asn Leu Asn Cys Glu Tyr Ile Ile Phe Asp Asn His Asp Ser Leu
805 810 815
Phe Val Lys Asn Asp Ser Tyr Ala Tyr Met Lys Lys Tyr Asp Val Gly
820 825 830
Met Asn Phe Ser Ala Leu Thr His Asp Trp Ile Glu Lys Ile Asn Ala
835 840 845
His Pro Pro Phe Lys Lys Leu Ile Lys Thr Tyr Phe Asn Asp Asn Asp
850 855 860
Leu Lys Ser Met Asn Val Lys Gly Ala Ser Gln Gly Met Phe Met Thr
865 870 875 880
Tyr Ala Leu Ala His Glu Leu Leu Thr Ile Ile Lys Glu Val Ile Thr
885 890 895
Ser Cys Gln Ser Ile Asp Ser Val Pro Glu Tyr Asn Thr Glu Asp Ile
900 905 910
Trp Phe Gln Phe Ala Leu Leu Ile Leu Glu Lys Lys Thr Gly His Val
915 920 925
Phe Asn Lys Thr Ser Thr Leu Thr Tyr Met Pro Trp Glu Arg Lys Leu
930 935 940
Gln Trp Thr Asn Glu Gln Ile Glu Ser Ala Lys Arg Gly Glu Asn Ile
945 950 955 960
Pro Val Asn Lys Phe Ile Ile Asn Ser Ile Thr Leu
965 970
<210> 5
<211> 564
<212> PRT
<213> Corynebacterium glutamicum
<400> 5
Leu Asp Ala Glu Ile Ala Lys Ala Pro Leu Pro Asp Ser Ile Leu Gly
1 5 10 15
Ile Gly His Thr Arg Trp Ala Thr His Gly Gly Pro Thr Asp Val Asn
20 25 30
Ala His Pro His Val Val Ser Asn Gly Lys Leu Ala Val Val His Asn
35 40 45
Gly Ile Ile Glu Asn Phe Ala Glu Leu Arg Ser Glu Leu Ser Ala Lys
50 55 60
Gly Tyr Asn Phe Val Ser Asp Thr Asp Thr Glu Val Ala Ala Ser Leu
65 70 75 80
Leu Ala Glu Ile Tyr Asn Thr Gln Ala Asn Gly Asp Leu Thr Leu Ala
85 90 95
Met Gln Leu Thr Gly Gln Arg Leu Glu Gly Ala Phe Thr Leu Leu Ala
100 105 110
Ile His Ala Asp His Asp Asp Arg Ile Val Ala Ala Arg Arg Asn Ser
115 120 125
Pro Leu Val Ile Gly Val Gly Glu Gly Glu Asn Phe Leu Gly Ser Asp
130 135 140
Val Ser Gly Phe Ile Asp Tyr Thr Arg Lys Ala Val Glu Leu Ala Asn
145 150 155 160
Asp Gln Val Val Thr Ile Thr Ala Asp Asp Tyr Ala Ile Thr Asn Phe
165 170 175
Asp Gly Ser Glu Ala Val Gly Lys Pro Phe Asp Val Glu Trp Asp Ala
180 185 190
Ala Ala Ala Glu Lys Gly Gly Phe Gly Ser Phe Met Glu Lys Glu Ile
195 200 205
His Asp Gln Pro Ala Ala Val Arg Asp Thr Leu Met Gly Arg Leu Asp
210 215 220
Glu Asp Gly Lys Leu Val Leu Asp Glu Leu Arg Ile Asp Glu Ala Ile
225 230 235 240
Leu Arg Ser Val Asp Lys Ile Val Ile Val Ala Cys Gly Thr Ala Ala
245 250 255
Tyr Ala Gly Gln Val Ala Arg Tyr Ala Ile Glu His Trp Cys Arg Ile
260 265 270
Pro Thr Glu Val Glu Leu Ala His Glu Phe Arg Tyr Arg Asp Pro Ile
275 280 285
Leu Asn Glu Lys Thr Leu Val Val Ala Leu Ser Gln Ser Gly Glu Thr
290 295 300
Met Asp Thr Leu Met Ala Val Arg His Ala Arg Glu Gln Gly Ala Lys
305 310 315 320
Val Val Ala Ile Cys Asn Thr Val Gly Ser Thr Leu Pro Arg Glu Ala
325 330 335
Asp Ala Ser Leu Tyr Thr Tyr Ala Gly Pro Glu Ile Ala Val Ala Ser
340 345 350
Thr Lys Ala Phe Leu Ala Gln Ile Thr Ala Ser Tyr Leu Leu Gly Leu
355 360 365
Tyr Leu Ala Gln Leu Arg Gly Asn Lys Phe Ala Asp Glu Val Ser Ser
370 375 380
Ile Leu Asp Ser Leu Arg Glu Met Pro Glu Lys Ile Gln Gln Val Ile
385 390 395 400
Asp Ala Glu Glu Gln Ile Lys Lys Leu Gly Gln Asp Met Ala Asp Ala
405 410 415
Lys Ser Val Leu Phe Leu Gly Arg His Val Gly Phe Pro Val Ala Leu
420 425 430
Glu Gly Ala Leu Lys Leu Lys Glu Ile Ala Tyr Leu His Ala Glu Gly
435 440 445
Phe Ala Ala Gly Glu Leu Lys His Gly Pro Ile Ala Leu Val Glu Glu
450 455 460
Gly Gln Pro Ile Phe Val Ile Val Pro Ser Pro Arg Gly Arg Asp Ser
465 470 475 480
Leu His Ser Lys Val Val Ser Asn Ile Gln Glu Ile Arg Ala Arg Gly
485 490 495
Ala Val Thr Ile Val Ile Ala Glu Glu Gly Asp Glu Ala Val Asn Asp
500 505 510
Tyr Ala Asn Phe Ile Ile Arg Ile Pro Gln Ala Pro Thr Leu Met Gln
515 520 525
Pro Leu Leu Ser Thr Val Pro Leu Gln Ile Phe Ala Cys Ala Val Ala
530 535 540
Thr Ala Lys Gly Tyr Asn Val Asp Gln Pro Arg Asn Leu Ala Lys Ser
545 550 555 560
Val Thr Val Glu
<210> 6
<211> 447
<212> PRT
<213> Corynebacterium glutamicum
<400> 6
Met Thr Arg Leu Phe Gly Thr Asp Gly Val Arg Gly Leu Ala Asn Glu
1 5 10 15
Val Leu Thr Ala Pro Leu Ala Leu Lys Leu Gly Ala Ala Ala Ala His
20 25 30
Val Leu Thr Ala Glu Lys Arg Val Asp Gly Arg Arg Pro Val Ala Ile
35 40 45
Val Gly Arg Asp Pro Arg Val Ser Gly Glu Met Leu Ala Ala Ala Leu
50 55 60
Ser Ala Gly Met Ala Ser Gln Gly Val Asp Val Ile Arg Val Gly Val
65 70 75 80
Ile Pro Thr Pro Ala Val Ala Phe Leu Thr Asp Asp Tyr Gly Ala Asp
85 90 95
Met Gly Val Met Ile Ser Ala Ser His Asn Pro Met Pro Asp Asn Gly
100 105 110
Ile Lys Phe Phe Ser Ala Gly Gly His Lys Leu Pro Asp His Val Glu
115 120 125
Asp Glu Ile Glu Arg Val Met Asp Ser Leu Pro Ala Glu Gly Pro Thr
130 135 140
Gly His Gly Val Gly Arg Val Ile Glu Glu Ala Thr Asp Ala Gln Asp
145 150 155 160
Arg Tyr Leu Glu His Leu Lys Glu Ala Val Pro Thr Ser Leu Glu Gly
165 170 175
Ile Lys Ile Val Val Asp Ala Ala Asn Gly Ala Ala Ser Val Val Ala
180 185 190
Pro Thr Ala Tyr Glu Ala Ala Gly Ala Thr Val Ile Ala Ile His Asn
195 200 205
Lys Pro Asp Ser Tyr Asn Ile Asn Met Asp Cys Gly Ser Thr His Ile
210 215 220
Asp Gln Val Gln Ala Ala Val Leu Lys His Gly Ala Asp Leu Gly Leu
225 230 235 240
Ala His Asp Gly Asp Ala Asp Arg Cys Leu Ala Val Asp Lys Asp Gly
245 250 255
Asn Leu Val Asp Gly Asp Gln Ile Met Ala Leu Leu Ala Ile Ala Met
260 265 270
Lys Glu Asn Gly Glu Leu Arg Lys Asn Thr Leu Val Gly Thr Val Met
275 280 285
Ser Asn Leu Gly Leu Lys Ile Ala Met Asp Glu Ala Gly Ile Thr Leu
290 295 300
Arg Thr Thr Lys Val Gly Asp Arg Tyr Val Leu Glu Asp Leu Asn Ala
305 310 315 320
Gly Gly Phe Ser Leu Gly Gly Glu Gln Ser Gly His Ile Val Leu Pro
325 330 335
Asp His Gly Thr Thr Gly Asp Gly Thr Leu Thr Gly Leu Ser Ile Met
340 345 350
Ala Arg Met Ala Glu Thr Gly Lys Ser Leu Gly Glu Leu Ala Gln Ala
355 360 365
Met Thr Val Leu Pro Gln Val Leu Ile Asn Val Pro Val Ser Asp Lys
370 375 380
Ser Thr Ile Val Ser His Pro Ser Val Val Ala Ala Ile Ala Glu Ala
385 390 395 400
Glu Ala Glu Leu Gly Ala Thr Gly Arg Val Leu Leu Arg Ala Ser Gly
405 410 415
Thr Glu Glu Leu Phe Arg Val Met Val Glu Ala Gly Asp Lys Glu Gln
420 425 430
Ala Arg Arg Ile Ala Gly Arg Leu Ala Ala Val Val Ala Glu Val
435 440 445
<210> 7
<211> 485
<212> PRT
<213> Corynebacterium glutamicum
<400> 7
Met Ser Ala Ser Asp Phe Ser Ser Ala Val Val Val Leu Ala Ala Gly
1 5 10 15
Ala Gly Thr Arg Met Lys Ser Asp Leu Gln Lys Thr Leu His Ser Ile
20 25 30
Gly Gly Arg Ser Leu Ile Ser His Ser Leu His Ala Ala Ala Gly Leu
35 40 45
Asn Pro Glu His Ile Val Ala Val Ile Gly His Gly Arg Asp Gln Val
50 55 60
Gly Pro Ala Val Ala Gln Val Ala Glu Glu Leu Asp Arg Glu Val Leu
65 70 75 80
Ile Ala Ile Gln Glu Glu Gln Asn Gly Thr Gly His Ala Val Gln Cys
85 90 95
Ala Met Asp Gln Leu Glu Gly Phe Glu Gly Thr Ile Ile Val Thr Asn
100 105 110
Gly Asp Val Pro Leu Leu Thr Asp His Thr Leu Ser Ala Leu Leu Asp
115 120 125
Ala His Val Glu Val Pro Thr Ala Val Thr Val Leu Thr Met Arg Leu
130 135 140
Asp Asp Pro Thr Gly Tyr Gly Arg Ile Val Arg Asn Glu Glu Gly Glu
145 150 155 160
Val Thr Ala Ile Val Glu Gln Lys Asp Ala Ser Ala Glu Val Gln Ala
165 170 175
Ile Asp Glu Val Asn Ser Gly Val Phe Ala Phe Asp Ala Ala Ile Leu
180 185 190
Arg Ser Ala Leu Ala Glu Leu Lys Ser Asp Asn Ala Gln Gly Glu Leu
195 200 205
Tyr Leu Thr Asp Val Leu Gly Ile Ala Arg Gly Glu Gly His Pro Val
210 215 220
Arg Ala His Thr Ala Ala Asp Ala Arg Glu Leu Ala Gly Val Asn Asp
225 230 235 240
Arg Val Gln Leu Ala Glu Ala Gly Ala Glu Leu Asn Arg Arg Thr Val
245 250 255
Ile Ala Ala Met Arg Gly Gly Ala Thr Ile Val Asp Pro Ala Thr Thr
260 265 270
Trp Ile Asp Val Glu Val Ser Ile Gly Arg Asp Val Ile Ile His Pro
275 280 285
Gly Thr Gln Leu Lys Gly Glu Thr Val Ile Gly Asp Arg Val Glu Val
290 295 300
Gly Pro Asp Thr Thr Leu Thr Asn Met Thr Ile Gly Asp Gly Ala Ser
305 310 315 320
Val Ile Arg Thr His Gly Phe Asp Ser Thr Ile Gly Glu Asn Ala Thr
325 330 335
Val Gly Pro Phe Thr Tyr Ile Arg Pro Gly Thr Thr Leu Gly Pro Glu
340 345 350
Gly Lys Leu Gly Gly Phe Val Glu Thr Lys Lys Ala Thr Ile Gly Arg
355 360 365
Gly Ser Lys Val Pro His Leu Thr Tyr Val Gly Asp Ala Thr Ile Gly
370 375 380
Glu Glu Ser Asn Ile Gly Ala Ser Ser Val Phe Val Asn Tyr Asp Gly
385 390 395 400
Glu Asn Lys His His Thr Thr Ile Gly Ser His Val Arg Thr Gly Ser
405 410 415
Asp Thr Met Phe Ile Ala Pro Val Thr Val Gly Asp Gly Ala Tyr Ser
420 425 430
Gly Ala Gly Thr Val Ile Lys Asp Asp Val Pro Pro Gly Ala Leu Ala
435 440 445
Val Ser Gly Gly Arg Gln Arg Asn Ile Glu Gly Trp Val Gln Lys Lys
450 455 460
Arg Pro Gly Thr Ala Ala Ala Gln Ala Ala Glu Ala Ala Gln Asn Val
465 470 475 480
His Asn Gln Glu Gly
485
<210> 8
<211> 315
<212> PRT
<213> Corynebacterium glutamicum
<400> 8
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> 9
<211> 439
<212> PRT
<213> Corynebacterium glutamicum
<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> 33
<212> DNA
<213> 人工序列
<400> 10
atgaatacat tatcacaagc aataaaagca tat 33
<210> 11
<211> 59
<212> DNA
<213> 人工序列
<400> 11
atgcttttat tgcttgtgat aatgtattca tctacaggcc cttctgcagg catgcaagc 59
<210> 12
<211> 56
<212> DNA
<213> 人工序列
<400> 12
aattaagctt gcatgcctgc agaaggaggc atttacatga atacattatc acaagc 56
<210> 13
<211> 41
<212> DNA
<213> 人工序列
<400> 13
gctggaattc gagactcgcg taccttatag agttatacta t 41
<210> 14
<211> 33
<212> DNA
<213> 人工序列
<400> 14
atgaatacat tatcacaagc aataaaagca tat 33
<210> 15
<211> 59
<212> DNA
<213> 人工序列
<400> 15
cttttattgc ttgtgataat gtattcatga caggagcctt ctgcaggcat gcaagctta 59
<210> 16
<211> 33
<212> DNA
<213> 人工序列
<400> 16
atgaatacat tatcacaagc aataaaagca tat 33
<210> 17
<211> 50
<212> DNA
<213> 人工序列
<400> 17
ttgcttgtga taatgtattc attagtcgtt ccttctgcag gcatgcaagc 50
<210> 18
<211> 33
<212> DNA
<213> 人工序列
<400> 18
atgaatacat tatcacaagc aataaaagca tat 33
<210> 19
<211> 56
<212> DNA
<213> 人工序列
<400> 19
cttttattgc ttgtgataat gtattcatca tgggatcctt ctgcaggcat gcaagc 56
<210> 20
<211> 33
<212> DNA
<213> 人工序列
<400> 20
atgaatacat tatcacaagc aataaaagca tat 33
<210> 21
<211> 56
<212> DNA
<213> 人工序列
<400> 21
cttttattgc ttgtgataat gtattcatct gaacaacctt ctgcaggcat gcaagc 56
<210> 22
<211> 59
<212> DNA
<213> 人工序列
<400> 22
aaaacacgtt aaaaaataaa tggaaattgc tcgcagagaa gaaatctgaa aatgcggag 59
<210> 23
<211> 30
<212> DNA
<213> 人工序列
<400> 23
gcaatttcca tttatttttt aacgtgtttt 30
<210> 24
<211> 59
<212> DNA
<213> 人工序列
<400> 24
ctgttaattc agcacatctt tctatgaata aagaagaaaa agtcaatgtt tgcgatagt 59
<210> 25
<211> 30
<212> DNA
<213> 人工序列
<400> 25
tattcataga aagatgtgct gaattaacag 30
<210> 26
<211> 59
<212> DNA
<213> 人工序列
<400> 26
taaaaaataa atggaaattg ctcgcagagg cagcatctga aaatgcggag gtaagagcg 59
<210> 27
<211> 32
<212> DNA
<213> 人工序列
<400> 27
ctctgcgagc aatttccatt tattttttaa cg 32

Claims (8)

1. 用于合成超高分子量透明质酸的重组谷氨酸棒杆菌，其特征在于，以谷氨酸棒杆菌（Corynebacterium glutamicum）异源表达透明质酸合酶pmHasA，并通过RBS弱化和定点突变改造透明质酸合酶pmHasA；同时，通过构建表达框强化UDP-葡萄糖脱氢酶编码基因ugd，加强透明质酸的合成底物UDP-N-乙酰葡萄糖胺和UDP-葡萄糖醛酸的积累；
所述超高分子量透明质酸是指分子量大于6 MDa的透明质酸；
所述定点突变是指透明质酸合成酶pmHasA发生T104A/K106A/K107A突变，突变体的氨基酸序列如SEQ ID NO .4所示；
弱化用于表达透明质酸合成酶pmHasA的编码基因的RBS序列时，用于替换原RBS序列的RBS序列为AAGGAGG。
2.根据权利要求1所述的用于合成超高分子量透明质酸的重组谷氨酸棒杆菌，其特征在于，进一步通过构建表达框强化葡萄糖-6-磷酸尿酰胺转移酶编码基因galU、谷氨酰胺-果糖-6-磷酸氨基转移酶编码基因glmS、磷酸葡萄糖变位酶编码基因glmM和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编码基因glmU中至少一种基因的表达。
3. 根据权利要求1所述的用于合成超高分子量透明质酸的重组谷氨酸棒杆菌，其特征在于，所述重组谷氨酸棒杆菌的宿主选自：Corynebacterium glutamicum ATCC 13032、Corynebacterium glutamicum K051、Corynebacterium glutamicum ATCC 14067、Corynebacterium glutamicum MB001、Corynebacterium glutamicum R、Corynebacterium glutamicum S9114、Corynebacterium glutamicum SCgG1、Corynebacterium glutamicumSCgG2、Corynebacterium glutamicum Z188、Corynebacterium glutamicum ZL-2、Corynebacterium glutamicum ZL-5或Corynebacterium glutamicum ZL-6。
4. 根据权利要求2所述的用于合成超高分子量透明质酸的重组谷氨酸棒杆菌，其特征在于，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Corynebactrium glutamicum。
5. 根据权利要求4所述的用于合成超高分子量透明质酸的重组谷氨酸棒杆菌，其特征在于，GlmU、GalU、Ugd、GlmS和GlmM的启动子为Ptrc、Ppyc、PglnA、PlysE、PlysG、PilvB、PleuA、PleuB、PthrB、PthrE、PgltB、PlpdA、Prep、Pper、PorfB、Porf3-aro、PsigA、PaceA、PaceB 、Pamt、PctRNA、PbrnF、PbrnE或Plrp。
6.根据权利要求4所述的用于合成超高分子量透明质酸的重组谷氨酸棒杆菌，其特征在于，GlmU、GalU、Ugd、GlmS和GlmM的表达载体选自：pXMJ19、pEC-XK99E、pEC-XC99E、pEC-XT99A、pEKEx1、pEKEx2、pVWEx1、pVWEx2、pZ8-1、pECTAC-K99、pAPE12。
7.根据权利要求4所述的用于合成超高分子量透明质酸的重组谷氨酸棒杆菌，其特征在于，以pEC-XK99E为载体，以Ptrc为启动子。
8. 应用权利要求1-7任一所述重组谷氨酸棒杆菌发酵生产超高分子量透明质酸的方法，其特征在于，包括以下步骤：（1）制备重组谷氨酸棒杆菌种子培养液，（2）将种子液转接至发酵培养基中，使得发酵罐中菌体初始OD600为1-1.5，在28℃通风培养3 h，待OD600涨到10-15，添加IPTG诱导，降温至20℃继续发酵69 h；发酵培养基配方为：玉米浆干粉 20 g/L，(NH4) 2SO4 20 g/L，KH2PO4 1 g/L，K2HPO4 1 g/L，MgSO4 0.25 g/L，3-吗啉丙磺酸 4.2 g/L，葡萄糖浓度40g/L。
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