CN114107149B - 一种高产透明质酸的方法、重组菌构建及其应用 - Google Patents
一种高产透明质酸的方法、重组菌构建及其应用 Download PDFInfo
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Abstract
一种高产透明质酸的重组菌及其应用，其特征在于，是将心磷脂合成途径中的心磷脂合酶基因转入至原始菌株，并失活了转录调控因子IolR，和/或转入透明颤菌血红蛋白基因制备而成。本发明通过强化透明质酸合成酶的“激活因子”—心磷脂的合成从而提高透明质酸的产量，并进一步失活转录调控因子IolR和/或引入透明颤菌血红蛋白基因来增强效果。优选的重组菌与出发菌株相比，透明质酸摇瓶产量可提高2.15倍，在透明质酸的生产中具有良好的工业应用前景。
Description
技术领域
本发明属于生物化工、合成生物学、基因工程及代谢工程领域，具体涉及一种高产透明质酸的重组菌及其应用。
背景技术
透明质酸(Hyaluronic acid)是一种由N-乙酰-氨基葡萄糖和D-葡萄糖醛酸交替排列而成的线性高分子聚合物，是糖胺聚糖家族的重要成员。透明质酸主要存在于动物组织当中，如雄鸡的鸡冠。由于具有良好的生物相容性和保水性能，透明质酸在临床、化妆品、制药和食品工业中都有着广泛的应用。高分子量透明质酸(分子量大于106Da)粘弹性及润滑性优异，主要用于关节腔注射、修复软骨退变及用于整形手术。低分子量透明质酸(分子量介于1×104-1×106Da之间)主要用于化妆品和护肤品添加，可以起到增强皮肤弹性、减少皱纹的作用。透明质酸低聚糖(分子量低于104Da)则可以作为口服保健品，来增强细胞新陈代谢，预防衰老。
20世纪80年代之前，透明质酸主要通过动物组织提取法获得。但该方法工艺复杂、成本高昂、产量较低；此外，产品中可能残留动物源蛋白质，对过敏性人群具有一定的安全隐患。目前，工业透明质酸主要通过微生物发酵法进行生产。常用的生产菌株主要为兽疫链球菌等动物致病菌，发酵罐产量为6-12g/L。该种方法同样也具有一定的局限性：首先，由于链球菌对发酵培养基营养要求较高，培养基中需要添加血清/脑心浸出液等昂贵成分，使得工业生产成本增加；另一方面，这些菌株在生产过程中可能产生一些外毒素，具有一定的致病风险。因此，开发新型的安全经济的重组菌对透明质酸产业具有重要的意义。
构建生产透明质酸的重组菌需要引入透明质酸合成酶，迄今为止，透明质酸已经在大肠杆菌、乳酸菌、枯草芽孢杆菌、谷氨酸棒杆菌等多种微生物中实现了异源合成。目前，提高微生物透明质酸发酵水平的方法包括优化透明质酸合成操纵子结构、优化培养条件、敲除或者弱化透明质酸合成竞争途径等。在透明质酸合成操纵子优化方面，研究者报道了将不同来源的透明质酸合成酶引入到大肠杆菌中，并进行了透明质酸合成操纵子的优化，证明了大肠杆菌合成透明质酸的可行性(Yu H,Stephanopoulos G.Metabolicengineering of Escherichia coli for biosynthesis of hyaluronic acid[J].Metabolic Engineering,2008,10(1):24-32.)。在培养条件优化方面，研究人员公开了谷氨酸棒杆菌发酵生产透明质酸过程中培养基及培养条件的优化，提高了发酵液中透明质酸的产量(Yu H,Stephanopoulos G.Metabolic engineering of Escherichia coli forbiosynthesis of hyaluronic acid[J].Metabolic Engineering,2008,10(1):24-32.)。敲除或者弱化透明质酸合成的竞争途径方面，研究人员公开了通过成簇规则间隔回文序列干扰技术(CRISPRi)同时弱化磷酸戊糖途径和糖酵解途径，使枯草芽孢杆菌生产透明质酸的产量较出发菌株相比提升108％，产量达到2.26g/L，同时所生产的透明质酸分子量也有提高(Westbrook A W,Ren X,Oh J,et al.Metabolic engineering to enhanceheterologous production of hyaluronic acid in Bacillus subtilis[J].MetabolicEngineering,2018,47:401-413.)。还有研究人员公开了敲除透明质酸竞争途径中的Cg0420、Cg0424基因使谷氨酸棒杆菌生产透明质酸的产量提高14.9％，摇瓶产量达到6.4g/L(Wang Y,Hu L,Huang H,et al.Eliminating the capsule-like layer to promoteglucose uptake for hyaluronan production by engineered Corynebacteriumglutamicum[J].Nature Communications,2020,11(1):1-10.)。进一步的，该期刊文献还报道了通过外源添加透明质酸酶将透明质酸生产和降解偶联，发酵罐产量可达74.1g/L，此时其产品为超低分子量透明质酸(分子量区间：51-56kDa)。专利文献CN110305827A和研究论文则公开了通过强化透明质酸合成途径的同时弱化竞争途径构建重组谷氨酸棒杆菌，其发酵罐产量高达28.7g/L，分子量为300-500kDa(Cheng F,Yu H,StephanopoulosG.Engineering Corynebacterium glutamicum for high-titer biosynthesis ofhyaluronic acid[J].Metabolic Engineering,2019,55:276-289.)。
除了通过调控代谢路径来强化透明质酸的合成外，提高透明质酸合成酶的活力对提高透明质酸的产量也至关重要。为此，研究人员对透明质酸合成酶的催化机理及突变改造进行了一系列研究。研究人员通过对来源于Pasteurella multocida的透明质酸合成酶(pmHAS)进行定向进化，在无细胞体系中合成了分子量为4.7MDa的透明质酸(Mandawe J,Infanzon B,Eisele A,et al.Directed Evolution of Hyaluronic Acid Synthase fromPasteurella multocida towards High-Molecular-Weight Hyaluronic Acid[J].ChemBioChem,2018,19(13):1414-1423.)。Garima Agarwal等对来源于Streptococcal的透明质酸合成酶(seHAS)进行了同源建模，预测了活性口袋中重要氨基酸，并通过实验进行了验证(Agarwal G,Krishnan K V,Prasad S B,et al.Biosynthesis of Hyaluronicacid polymer:Dissecting the role of sub structural elements of hyaluronansynthase[J].Scientific reports,2019,9(1):1-12.)。此外，研究人员还通过突变seHASC末端414-417位氨基酸，确定了C末端在透明质酸分子聚合以及分子量调控中起到关键作用。本发明创造性地提出，从透明质酸合成酶这一膜蛋白在细胞膜上保持正常构象所需要的辅助因子——心磷脂分子的合成途径强化出发，进行透明质酸生产菌株的改造，并取得了显著的产量提升效果。到目前为止，通过强化心磷脂合成来提高透明质酸产量还未见报道。
发明内容
心磷脂分子是保障透明质酸合成酶在细胞膜上正确组装的辅助因子：每分子的透明质酸合成酶需结合约16分子的心磷脂分子才能保持生物学活性。因此，转入心磷脂合成相关基因能够促进谷氨酸棒杆菌合成心磷脂，从而维持透明质酸合成酶的活性。此外，谷氨酸棒杆菌中的转录调控因子IolR会抑制转运蛋白IolT1/IolT2对葡萄糖的转运过程，同时会抑制激酶对葡萄糖的磷酸化过程，不利于底物吸收利用。敲除转录调控因子基因能够解除IolR对葡萄糖转运及磷酸化的抑制。转入透明颤菌血红蛋白基因能够促进谷氨酸棒杆菌主动摄取培养基中的氧气，促进产物合成。本专利的核心一方面在于在重组菌株中通过高表达心磷脂合酶来强化心磷脂合成；进一步地，结合敲除IolR，促进底物吸收利用，或结合转入透明颤菌血红蛋白基因，促进重组菌摄取氧气。相比于出发菌株，以上单个或两个、三个组合策略均能提高重组谷氨酸棒杆菌异源合成透明质酸的产量。
一方面，本申请提供了一种高产透明质酸的重组菌，其特征在于，是将心磷脂合成途径中的心磷脂合酶基因、磷脂酰甘油磷酸合酶基因中的一种或一种以上转化至原始菌株构建而成。
进一步地，所述重组菌中转入心磷脂合酶基因。
进一步地，心磷脂合酶基因、磷脂酰甘油磷酸合酶基因来源于但不限于谷氨酸棒杆菌、大肠杆菌、枯草芽孢杆菌、铜绿假单胞菌等菌种。
进一步地，所述原始菌株为谷氨酸棒杆菌，优选为ATCC 13032的谷氨酸棒杆菌。
进一步地，所述原始菌株为中国专利申请号ZL201910495504.8中的C.glu-Δldh/AB、C.glu-Δldh/A-PdapB-B、C.glu-Δldh/A-PdapB-B-aF或者C.glu-Δldh-aEG/A-PdapB-B-aF-aE。
进一步地，所述心磷脂合酶蛋白序列为SEQ ID NO.1。
进一步地，所述心磷脂合酶蛋白序列也包括在SEQ ID NO.1基础上对其酶活进行突变改造的单点或多点突变所获得的突变体。
进一步地，上述突变体为将377位谷氨酰胺突变为赖氨酸、谷氨酸、天冬酰胺等；优选地，第377位谷氨酰胺突变为天冬酰胺。
进一步地，上述突变体为将459位谷氨酸突变为天冬氨酸、谷氨酰胺；优选地，第459位谷氨酸突变为天冬氨酸。
进一步地，上述突变体为将472位赖氨酸突变为苏氨酸、精氨酸、异亮氨酸；优选地，第472位赖氨酸突变为精氨酸。
本申请中所述的突变体可以通过对蛋白序列为SEQ ID NO.1的心磷脂合酶进行突变改造获得；突变改造所用的预测方法、操作方法、验证方法可以为本领域中已知或研究中的，本领域技术人员认为可用的方法；本申请中的突变体保持有心磷脂合酶的活性。
进一步地，所述重组菌中失活了转录调控因子IolR，和/或转入透明颤菌血红蛋白基因。
进一步地，所述转录调控因子IolR的核酸序列为SEQ ID NO.2，和/或所述透明颤菌血红蛋白基因蛋白序列为SEQ ID NO.3。
进一步地，所述失活为通过基因敲除的方式失活；所述转入是通过基因组整合的方式转入。
进一步地，所述重组菌中，所述CLS蛋白受强启动子控制，所述VHb蛋白受自身组成型启动子控制。
另一方面，本申请提供了上述菌株在制备透明质酸中的应用。
进一步地，所述应用中包括将重组菌接入培养基中，进行扩大培养，得到基因工程重组菌菌液；按照1-20％体积百分比将上述步骤获得的工程菌菌液接入发酵培养基中，进行发酵培养，得到含有透明质酸的发酵液。
进一步地，所述的扩大培养的方法为：在20-40℃、摇床转速为100-250rpm的条件下培养10-20h。
进一步地，所述的发酵培养的方法为：在20-40℃、摇床转速为100-250rpm的条件下培养培养1.5-4h时加入0.5-1.5mM IPTG诱导剂后继续培养40-72h。
进一步地，所述发酵培养基的组分为：可发酵糖类10-100g/L，无机氮源5-60g/L，有机氮源1-50g/L，KH2PO4 0.1-6g/L，K2HPO4 0.1-6g/L，MgSO4·7H2O 0.1-10g/L，MnSO40.001-0.1g/L，FeSO4·7H2O 0.001-0.1g/L，谷氨酰胺0.5-5g/L，3-吗啉丙磺酸(MOPS)20-50g/L，pH 6.0-8.0。
上述发酵培养基中的糖可选葡萄糖、果糖、蔗糖、麦芽糖、淀粉、淀粉水解液、纤维素、纤维素预处理液、半纤维素、木聚糖等；无机氮源可选硝酸铵、硝酸钠、硝酸钾、硫酸铵、氯化铵等；有机氮源可选玉米浆粉、玉米浆、大豆粉、酵母膏、酵母粉、牛肉膏、蛋白胨等。
另一方面，本申请提供了上述重组菌的制备方法，其特征在于，包括；敲除iolR基因或者将VHb基因(vgb)整合到原始基因组中；扩增得到心磷脂合酶基因cls序列，插入到穿梭质粒，构建表达质粒，将表达质粒导入菌株。
进一步地，所述方法具体为：
(1)构建iolR基因敲除重组质粒pk18mobsacB-ΔiolR，转入出发菌株构建iolR基因敲除重组菌株C.glu-ΔiolR；或者
(2)构建基因组整合vgb的重组质粒pk18mobsacB-vgb，转入出发菌株，构建基因组整合vgb的重组菌株C.glu-VHb；以及
(3)构建带有心磷脂合酶基因cls的重组质粒pEC-XK99E-cls，转入上述步骤(1)或步骤(2)所得到的重组菌株，得到转化有pEC-XK99E-cls重组表达质粒的基因工程重组菌。
进一步地，所述方法具体为：
(1)以IolR-up-F/Iol-up-R为上下游引物，以谷氨酸棒杆菌基因组为模板进行聚合酶链式反应，扩增iolR基因上游同源臂序列iolR-up。以IolR-down-F/IolR-down-R为上下游引物，以谷氨酸棒杆菌基因组为模板进行聚合酶链式反应，扩增iolR基因下游同源臂序列iolR-down。上游同源臂iolR-up核酸序列如SEQ ID NO.4所示。下游同源臂iolR-down核酸序列如SEQ ID NO.5所示；
(2)利用限制性内切酶EcoRI/XbaI将pk18mobsacB载体双酶切，并回收纯化，利用Gibson组装试剂盒将iolR上下游同源臂和pk18mobscB载体连接，得到连接产物；
(3)将连接产物化至大肠杆菌E.coli TOP10感受态细胞，进行抗性筛选阳性克隆，得到带有iolR上下游同源臂的重组质粒pk18mobsacB-ΔiolR；
(4)将重组质粒转入出发菌株中，涂布于含有5μg/mL氯霉素和25μg/mL卡那霉素的LBHIS固体平板，筛选阳性克隆；对阳性克隆扩增培养，涂布于无抗的含有150g/L蔗糖的LB固体平板，筛选得到iolR敲除的重组菌C.glu-ΔiolR；或者
(5)以ldh-up-F/ldh-up-R为上下游引物，以谷氨酸棒杆菌基因组为模板进行聚合酶链式反应，扩增ldh基因上游同源臂序列ldh-up。以ldh-down-F/ldh-down-R为上下游引物，以谷氨酸棒杆菌基因组为模板进行聚合酶链式反应，扩增ldh基因下游同源臂序列ldh-down。上游同源臂ldh-up核酸序列如SEQ ID NO.6所示。下游同源臂ldh-down核酸序列如SEQ ID NO.7所示；
(6)利用限制性内切酶EcoRI/XbaI将pk18mobsacB载体双酶切，并回收纯化，利用Gibson组装试剂盒将ldh上下游同源臂，vgb基因序列和pk18mobscB载体连接，得到连接产物。其中，vgb基因序列为带有自身启动子的人工合成序列，优选的，其核酸序列如SEQ IDNO.8所示；
(7)将连接产物化至大肠杆菌E.coli TOP10感受态细胞，进行抗性筛选阳性克隆，得到带有ldh上下游同源臂、vgb基因序列的重组质粒pk18mobsacB-vgb；
(8)将重组质粒转入出发菌株中，涂布于含有5μg/mL氯霉素和25μg/mL卡那霉素的LBHIS固体平板，筛选阳性克隆；对阳性克隆扩增培养，涂布于无抗的含有150g/L蔗糖的LB固体平板，筛选得到基因组整合vgb的重组菌C.glu-VHb；以及
(9)以cls-F/cls-R为上下游引物，以谷氨酸棒杆菌基因组为模板进行聚合酶链式反应，扩增cls基因，其核酸序列如SEQ ID NO.9所示；
(10)利用限制性内切酶KpnI/SalI对pEC-XK99E载体和步骤9中获得的cls基因双酶切，并回收纯化，利用连接酶将两个酶切产物进行连接，得到连接产物；
(11)将连接产物化至大肠杆菌E.coli TOP10感受态细胞，进行抗性筛选阳性克隆，得到带有cls基因序列的重组质粒pEC-XK99E-cls；
(12)将重组质粒转入出发菌株、iolR敲除的重组菌C.glu-ΔiolR、基因组整合vgb的重组菌C.glu-VHb，获得转化有pEC-XK99E-cls质粒过表达心磷脂合酶基因的重组菌。
除了谷氨酸棒杆菌ATCC 13032及以其为基础的基因工程菌株外，其他有产透明质酸能力的谷氨酸棒杆菌、兽疫链球菌、枯草芽孢杆菌或乳酸菌等菌株及以其为基础的基因工程菌株经过适当的验证也可用于本申请。
申请人的在先专利申请ZL201910495504.8中对C.glu-Δldh/AB、C.glu-Δldh/A-PdapB-B、C.glu-Δldh/A-PdapB-B-aF或者C.glu-Δldh-aEG/A-PdapB-B-aF-aE。的制备方法有详细的记载：
所述谷氨酸棒杆菌C.glu-Δldh/AB，是指在谷氨酸棒杆菌ATCC13032菌中敲除乳酸脱氢酶基因，转入透明质酸合成酶基因和UDP-葡萄糖脱氢酶基因。
所述谷氨酸棒杆菌C.glu-Δldh/A-PdapB-B，是指在C.glu-Δldh/AB中，将UDP-葡萄糖脱氢酶基因同时置于两个启动子控制下表达。
所述谷氨酸棒杆菌C.glu-Δldh/A-PdapB-B-aF，是指在C.glu-Δldh/A-PdapB-B中，下调了果糖二磷酸醛缩酶基因的表达量，来弱化糖酵解途径。
所述谷氨酸棒杆菌C.glu-Δldh-aEG/A-PdapB-B-aF-aE，是指在C.glu-Δldh/A-PdapB-B-aF中，下调了丙酮酸脱氢酶基因的表达量。
有益效果：
本发明采用基因工程技术手段构建重组菌，在生产透明质酸的重组谷氨酸棒杆菌细胞中扩增并表达了心磷脂合成途径中的心磷脂合酶CLS，通过强化心磷脂供应来增强透明质酸合成酶的活力。进一步敲除谷氨酸棒杆菌中的转录抑制调控因子IolR的基因，增强碳源吸收利用，或转入透明颤菌血红蛋白基因，增强宿主摄取氧气的能力，构建得到高产透明质酸的基因工程谷氨酸棒杆菌。本发明所采用的谷氨酸棒杆菌宿主是食品级安全菌株，对人、畜均无致病性；所构建的重组菌株透明质酸产量高，与出发菌株相比摇瓶产量提高2.15倍，具有良好的产业化应用前景，摇瓶发酵所得发酵液中透明质酸产量平均为9-13g/L。
附图说明
图1为用于基因过表达质粒pEC-XK99E的示意图。
图2为用于基因敲除以及基因组整合的自杀质粒pk18mobsacB的示意图。
图3为携带有心磷脂合酶基因的谷氨酸棒杆菌-大肠杆菌穿梭质粒pEC-XK99E-cls的示意图。
图4为携带有iolR上下游同源臂序列的自杀质粒pk18mobsacB-ΔiolR的示意图。
图5为携带有ldh上下游同源臂序列、vgb基因的自杀质粒pk18mobsacB-vgb的示意图。
图6为通过双交换同源重组构建iolR敲除的重组菌株C.glu-ΔiolR的PCR验证。其中最左边泳道为DNA分子量标准，用泳道1-2为原始菌株野生型谷氨酸棒杆菌使用引物iolR-test-F/R进行PCR扩增的结果，约2.1kb；泳道3-4为iolR敲除的重组菌株C.glu-ΔiolR使用引物iolR-test-F/R进行PCR扩增的结果，约1.4kb。
图7为通过双交换同源重组进行基因组整合vgb重组菌株C.glu-VHb的PCR验证。其中最左边泳道为DNA分子量标准，用泳道1-2为原始菌株野生型谷氨酸棒杆菌使用引物VHb-test-F/R进行PCR扩增的结果，约1.7kb；泳道3-4为基因组整合vgb重组菌株C.glu-VHb使用引物VHb-test-F/R进行PCR扩增的结果，约2.4kb。
图8为原始菌株菌C.glu-Δldh/A-PdapB-B-aF(本发明中记为C.glu-0)和基因工程菌C.glu-CLS，C.glu-dR-CLS，C.glu-VHb-CLS在500mL摇瓶中培养时的透明质酸的产量比较。
具体实施方式
下面结合附图和具体实施案例对本发明做进一步说明，如未特别指明，实施例中的方法均为常规方法，下面描述的试剂如无特殊说明均可通过商业途径获得。
实施例1：携带有心磷脂合酶CLS基因(cls)的重组质粒pEC-XK99E-cls构建
1.以cls-F/cls-R为引物，以谷氨酸棒杆菌基因组DNA为模板，PCR反应扩增心磷脂合成酶基因cls，将PCR产物进行回收纯化。利用限制性内切酶KpnI/SalI将pEC-XK99E质粒和cls基因双酶切后，将线性化重组质粒与cls基因再次进行回收纯化，使用T4连接酶将cls基因连接入pEC-XK99E质粒中，反应温度4-16℃，反应时间4-16h，得到连接产物。
通过化学转化法将连接产物转化至大肠杆菌TOP10感受态细胞(购买于全式金，具体操作步骤如下：将10微升连接体系加入到100微升感受态细胞中，置于冰浴上放置30分钟，42℃热激90秒后，继续冰浴2分钟，加入900微升无抗LB培养基，置于摇床中复苏60分钟，条件为200rpm，37℃。复苏结束后涂布于含有50μg/mL卡那霉素的LB固体培养基平板，挑选阳性克隆，并进行PCR验证，得到重组质粒pEC-XK99E-cls。
LB培养基配方：蛋白胨10g/L，酵母粉5g/L，氯化钠10g/L，pH＝7.0，固体平板培养基含有琼脂1.5％-2％。
引物由苏州金唯智生物科技有限公司合成，用无菌水溶解稀释至10μM使用。PCR扩增过程中使用的DNA聚合酶采用购自于南京vazyme公司的Phanta Max Master 2×mix。
引物的序列如下：
cls-F：5’-CGCGGTACCAAAGGAGGACACATATGAATTTGCAGATCAACC-3’(SEQ ID NO.10)
cls-R：5’-CGCGACCTCAGCACTGCAATAAGTCGAC-3’(SEQ ID NO.11)
PCR反应体系为：
PCR反应条件为：
实施例2：携带有心磷脂合酶突变体的重组质粒构建
1.以K472R-F/K472R-R引物为上下游引物，以实施例1中所获得的质粒为模板，进行聚合酶链式反应，获得带有突变位点的线性化质粒，并进行纯化回收
2.利用Gibson Assembly试剂盒(Clonesmarter，购买于中美泰和生物技术有限公司)进行连接，并通过化学转化法转化至大肠杆菌TOP10感受态细胞(购买于全式金，具体操作步骤见实施例1)。得到重组质粒pEC-XK99E-cls(K472R)
引物的序列如下：
K472R-F：5’-CGAGACGCAAGCCGAGAACTCACTCTGG-3’(SEQ ID NO.12)
K472R-R：5’-GAGTTCTCGGCTTGCGTCTCGGTAGCG-3’(SEQ ID NO.13)
利用类似的方法，利用以下引物构建带有E459D突变体的重组质粒pEC-XK99E-cls(E459D)、带有Q377N突变体的重组质粒pEC-XK99E-cls(Q377N)。
E459D-F：5’-CAACCTCATCCAAGACCTCAACACACTCAC-3’(SEQ ID NO.14)
E459D-R：5’-GTTGAGGTCTTGGATGAGGTTGCCTTTG-3’(SEQ ID NO.15)
Q377N-F：5’-TGTCTCTGAAAACGCCGACCAATTTGC-3’(SEQ ID NO.16)
Q377N-R：5’-TGGTCGGCGTTTTCAGAGACAAATAG-3’(SEQ ID NO.17)
实施例3：iolR敲除的重组菌C.glu-ΔiolR构建
1.谷氨酸棒杆菌自杀质粒pk18mobsacB-ΔiolR的构建
以谷氨酸棒杆菌ATCC13032的基因组DNA为模板，通过IolR-up-F/IolR-up-R引物扩增iolR基因上游的同源臂序列，其序列如SEQ ID NO.4所示，通过IolR-down-F/IolR-down-R引物扩增iolR基因下游同源臂，其序列如SEQ ID NO.5所示。将这两段DNA片段进行纯化，作为双交换的上、下游同源臂；
利用限制性内切酶EcoRI/XbaI对pk18mobsacB载体进行双酶切，并对酶切得到的线性化质粒进行纯化；
将上述三个片段利用Gibson Assembly试剂盒(Clonesmarter，购买于中美泰和生物技术有限公司)进行连接，并通过化学转化法转化至大肠杆菌TOP10感受态细胞(购买于全式金，具体操作步骤见实施例1)。得到重组质粒pk18mobsacB-ΔiolR。
引物由苏州金唯智生物科技有限公司合成，用无菌水溶解稀释至10μM使用。PCR扩增过程中使用的DNA聚合酶采用购自于南京Vazyme公司的Phanta Max Master 2×mix。
引物的序列如下：
IolR-up-F：5’-TGATTACGAATTCGGTCTGGCTATCTACATCC-3’(SEQ ID NO.18)
IolR-up-R：5’-AACGAAAGGAGACATCGAAATAAACCAAAGAGCCC-3’(SEQ ID NO.19)
IolR-down-F:5’-TCGATGTCTCCTTTCGTTGCCCACC-3’(SEQ ID NO.20)
IolR-down-R：5’-TGCAGGTCGACTCTAGACAGAAACGTTTGCTGCGC-3’(SEQ ID NO.21)
PCR反应体系为：
PCR反应条件为：
2.将构建得到了重组质粒pk18mobsacB-ΔiolR通过电转化法转入出发菌株C.glu-Δldh/A-PdapB-B-aF感受态细胞(出发重组菌株构建方法详见中国专利CN110305827A，在本专利中记为C.glu-0)。具由于pk18mobsacB质粒没有谷氨酸棒杆菌中的复制起点，因此只能通过同源重组的方法整合到基因组上。上述的电转化的具体操作为：将10μL上述重组质粒加入到100μL C.glu-Δldh/A-PdapB-B-aF感受态细胞中，混合均匀之后冰浴30min，移入1mm电转杯；调节电穿孔仪的电压为1.8kV，将电转杯装入电穿孔仪，按下电击键；电击结束之后马上向电击杯中加入900μL LBHIS复苏培养基，重悬细胞，并转入1.5mL离心管。30℃，200rpm复苏2h。复苏结束后，13000rpm高速离心并除去800μL上清液，将离心下来的细胞重新悬浮，并涂布于LBHIS固体培养基上(卡那霉素25μg/mL，氯霉素5μg/mL)，置于30℃培养箱倒置培养16-24h，挑取阳性克隆进行第一次筛选。将挑选得到的阳性重组子在无抗性的LB液体培养基中培养16h，然后涂布于含有150g/L蔗糖的LB固体培养基上进行第二次筛选，利用iolR-test-F和iolR-test-R两条引物进行验证。IolR的基因敲除菌株会扩增出一条大小约为1.4kb的条带(如图1所示)。得到iolR敲除的重组菌株C.glu-ΔiolR。
引物序列：
iolR-test-F：5’-ATCGGTTGGACTCTCATCGCC-3’(SEQ ID NO.22)
iolR-test-R：5’-GCAACGTACTGGTTGTCCACG-3’(SEQ ID NO.23)
实施例4：基因组整合vgb重组菌株C.glu-VHb构建
1.谷氨酸棒杆菌自杀质粒pk18mobsacB-VHb的构建
将透明颤菌血红蛋白基因(vgb)整合到谷氨酸棒杆菌基因组上的乳酸脱氢酶(ldh)位点，引入透明颤菌血红蛋白基因。以谷氨酸棒杆菌ATCC13032的基因组DNA为模板，通ldh-up-F/ldh-up-R引物扩增ldh基因上游的同源臂序列，其序列如SEQ ID NO.6所示，通过ldh-down-F/ldh-down-R引物扩增ldh基因下游同源臂序列，其序列如SEQ ID NO.7所示。将这两段DNA片段进行纯化，作为双交换的上下游同源臂；
利用限制性内切酶EcoRI/XbaI对pk18mobsacB载体进行双酶切，并对酶切得到的线性化质粒进行纯化；
将ldh基因上游同源臂、ldh基因下游同源臂、透明颤菌血红蛋白基因(vgb，SEQ IDNO.8，订购于苏州金唯智生物科技有限公司，由化学合成法获得)以及线性化的pk18mobsacB载体通过将上述三个片段利用Gibson Assembly试剂盒(Clonesmarter，购买于中美泰和生物技术有限公司)进行连接，并通过化学转化法转化至大肠杆菌TOP10感受态细胞。转化方法同实施例1。
引物的序列如下：
ldh-up-F：5’-CTATGACCATGATTACGAATTCAGGTGCCGACACTAATGC-3’(SEQ ID NO.24)
ldh-up-R：5’-ACCGACGGTTTCTTTCATTTTCGATC-3’(SEQ ID NO.25)
ldh-down-F:5’-CTTCAGGTGTGCCTTGGCATAACTTTTTGGTTTACGGGCACAATG-3’
(SEQ ID NO.26)
ldh-down-R：5’-8-3’
(SEQ ID NO.27)
PCR反应体系为：
PCR反应条件为：
2.将构建得到了重组质粒pk18mobsacB-VHb通过电转化法转入谷氨酸棒杆菌C.glu-Δldh/A-PdapB-B-aF(C.glu-0)感受态细胞。具体的电转化方法见实施例2。利用VHb-test-F和VHb-test-R两条引物进行验证。VHb的基因敲除菌株会扩增出一条大小约为3.2kb的条带(如图2所示)。得到重组菌株。
引物序列如下：
VHb-test-F：5’-CTGCGCCTGGGGCGATTTCGCC-3’(SEQ ID NO.28)
VHb-test-R：5’-TGTAATGACCGAAACCGCTGCG-3’(SEQ ID NO.29)
实施例5：过表达心磷脂合酶基因的基因工程重组菌C.glu-CLS构建
将实施例1中所构建的重组质粒pEC-XK99E-cls电转化至出发菌株C.glu-Δldh/A-PdapB-B-aF(C.glu-0)感受态细胞(出发重组菌株构建方法详见中国专利CN110305827A)。挑选阳性克隆后，得到过表达心磷脂合酶基因的基因工程重组菌C.glu-CLS。
实施例6：过表达心磷脂合酶基因的基因工程重组菌C.glu-dR-CLS构建
将实施例1中所构建的重组质粒pEC-XK99E-cls电转化至实施例3中所构建的重组菌株C.glu-ΔiolR感受态细胞。挑选阳性克隆后，得到过表达心磷脂合酶基因的基因工程重组菌C.glu-dR-CLS。
实施例7：过表达心磷脂合酶基因的基因工程重组菌C.glu-VHb-CLS构建
将实施例1中所构建的重组质粒pEC-XK99E-cls电转化至实施例4中所构建的重组菌株C.glu-VHb感受态细胞。挑选阳性克隆后，得到过表达心磷脂合酶基因的基因工程重组菌C.glu-VHb-CLS。
实施例8：过表达心磷脂合酶突变体的基因工程菌株构建
将实施例1-2中所构建的重组质粒pEC-XK99E-cls、pEC-XK99E-cls(K472R)、pEC-XK99E-cls(E459D)、pEC-XK99E-cls(Q377N)电转化至实施例3-4中所构建的重组菌株感受态细胞。挑选阳性克隆后，得到过表达心磷脂合酶突变体的基因工程重组菌株。在发酵产透明质酸的过程中，过表达心磷脂合酶突变体和野生型心磷脂合酶效果相当。
实施例9：使用过表达心磷脂合酶基因的重组菌株发酵生产透明质酸
将实施例4-6所构建的重组菌接种于LB液体培养基(含有5μg/mL氯霉素，25μg/mL卡那霉素)中，30℃、200rpm条件下培养12-16h后，以体积比5％的比例接入发酵培养基中。30℃、200rpm条件下培养3h，加入IPTG(终浓度1mM)，继续培养48h。发酵期间，每12h用6MNaOH调节pH至7.2.。在第24h时补加碳源，维持糖浓度在5-10g/L。
发酵培养基的配方为：葡萄糖40g/L，硫酸铵30g/L，玉米浆粉20g/L，KH2PO4 1g/L，K2HPO4 0.5g/L，MnSO4 0.01g/L，FeSO4·7H2O 0.01g/L，MgSO4 5g/L。
取1mL上述所得的发酵液加入3mL的无水乙醇，4℃下放置1h；13000rpm离心10min，除去上清后室温下倒置1h，待乙醇完全挥发之后用去离子水重悬，得到透明质酸溶液。稀释适当倍数后，加入到700μL的乙酸缓冲液(0.2mol/L醋酸钠、0.15mol/L氯化钠，用乙酸调节pH至6.0)和2mLCTAB溶液(0.5mol/L，使用NaOH溶液溶解)，反应5min后测定OD400。
测定结果显示，实施例5-8所构建的重组菌株的透明质酸摇瓶产量均达到10g/L，其中C.glu-dR-CLS的产量最高，达到13.3g/L，为出发菌株的2.15倍。所得到的重组菌摇瓶透明质酸平均产量为9-13g/L。
以上的各个实施例仅仅是用以说明本发明的技术方案，而非对其进行限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解；其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中的部分或者全部技术特征进行等同替换；而这些修改或者替换，并不能使得相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 清华大学
<120> 一种高产透明质酸的方法、重组菌构建及其应用
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gaagaataca tccacgtcga gatctacatc atggcctggg ataaatacac ccaacctttt 480
ttcacagcac tagaaaacgc ccacaaccga ggcgttaaag tccgtttact cttcgaccac 540
gtcggcagct ggaaataccc cggctaccga cgcctgaaaa aagaactcaa ccgcatggga 600
tttgcttggt acctcatgct ccccttacaa ccatggcgcc tacgcttccg acgccctgac 660
ctgcgcaacc accgaaaaat gctcatcatc gacggccaca ccggcttcat gggctcccaa 720
aacctcatcg cacccagcta cctgcaacgc aaaaacatca aactaggccg cgaatggaaa 780
gacctcatgg tggaactgtc cggccccatc gtctcctcca tggaaatgat cttcgccgga 840
gactggtacg tcgaatccaa cgaagcacta gacatccgcg accacgcaga agcccacggc 900
tacatcggca acactcaaaa agattccgcg accaacctcg tgcagctcat cccctccggc 960
cctggttaca ccaccgagcc caacctgcgc atgttcaact ccatcgttca ccacgccaaa 1020
gaacgactca ttttgtgcag tccctacttc atccccgacg aatccctcct cgaagccgtc 1080
acctcagcct gctaccgcgg agtaaccgtc gaactatttg tctctgaaca agccgaccaa 1140
tttgccatcg accacgccca atcctcctac taccaggcac tccttgaagc cggcgtccgc 1200
atttaccaat tccccaaacc cgacgtactc cataccaagt acatgatcgc cgacccccac 1260
gacaccgacg gcaacgaagc actcgccgta ctcggatcct ccaacctcga tatccgcagc 1320
tttggcctca actacgaaat ctcattgatg atcgccaaag gcaacctcat ccaagaactc 1380
aacacactca ccgaccgcta ccgagacgca agcaaagaac tcactctgga aaggtggaat 1440
cagcgcagct ggcgacgccg ctacgttgac aacgtcatgc gtttgacctc agcactgcaa 1500
taa 1503
<210> 10
<211> 42
<212> DNA
<213> artificial sequence
<400> 10
cgcggtacca aaggaggaca catatgaatt tgcagatcaa cc 42
<210> 11
<211> 28
<212> DNA
<213> artificial sequence
<400> 11
cgcgacctca gcactgcaat aagtcgac 28
<210> 12
<211> 28
<212> DNA
<213> artificial sequence
<400> 12
cgagacgcaa gccgagaact cactctgg 28
<210> 13
<211> 27
<212> DNA
<213> artificial sequence
<400> 13
gagttctcgg cttgcgtctc ggtagcg 27
<210> 14
<211> 30
<212> DNA
<213> artificial sequence
<400> 14
caacctcatc caagacctca acacactcac 30
<210> 15
<211> 27
<212> DNA
<213> artificial sequence
<400> 15
gttgaggtct tggatgaggt tgccttt 27
<210> 16
<211> 27
<212> DNA
<213> artificial sequence
<400> 16
tgtctctgaa aacgccgacc aatttgc 27
<210> 17
<211> 26
<212> DNA
<213> artificial sequence
<400> 17
tggtcggcgt tttcagagac aaatag 26
<210> 18
<211> 32
<212> DNA
<213> artificial sequence
<400> 18
tgattacgaa ttcggtctgg ctatctacat cc 32
<210> 19
<211> 35
<212> DNA
<213> artificial sequence
<400> 19
aacgaaagga gacatcgaaa taaaccaaag agccc 35
<210> 20
<211> 25
<212> DNA
<213> artificial sequence
<400> 20
tcgatgtctc ctttcgttgc ccacc 25
<210> 21
<211> 35
<212> DNA
<213> artificial sequence
<400> 21
tgcaggtcga ctctagacag aaacgtttgc tgcgc 35
<210> 22
<211> 21
<212> DNA
<213> artificial sequence
<400> 22
atcggttgga ctctcatcgc c 21
<210> 23
<211> 21
<212> DNA
<213> artificial sequence
<400> 23
gcaacgtact ggttgtccac g 21
<210> 24
<211> 40
<212> DNA
<213> artificial sequence
<400> 24
ctatgaccat gattacgaat tcaggtgccg acactaatgc 40
<210> 25
<211> 26
<212> DNA
<213> artificial sequence
<400> 25
accgacggtt tctttcattt tcgatc 26
<210> 26
<211> 45
<212> DNA
<213> artificial sequence
<400> 26
cttcaggtgt gccttggcat aactttttgg tttacgggca caatg 45
<210> 27
<211> 41
<212> DNA
<213> artificial sequence
<400> 27
tgcctgcagg tcgactctag agcttccaga cggtttcatc g 41
<210> 28
<211> 22
<212> DNA
<213> artificial sequence
<400> 28
ctgcgcctgg ggcgatttcg cc 22
<210> 29
<211> 22
<212> DNA
<213> artificial sequence
<400> 29
tgtaatgacc gaaaccgctg cg 22
Claims (8)
1.一种高产透明质酸的重组菌，其特征在于，所述重组菌是通过在出发菌株的基础上失活转录调控因子IolR并转入谷氨酸棒杆菌心磷脂合酶基因获得的，所述出发菌株为中国专利申请ZL201910495504.8中的C.glu-Δldh/AB、C.glu-Δldh/A-PdapB-B、C.glu-Δldh/A-PdapB-B-aF或者C.glu-Δldh-aEG/A-PdapB-B-aF-aE；
所述C.glu-Δldh/AB是在谷氨酸棒杆菌ATCC13032中敲除乳酸脱氢酶基因，转入透明质酸合成酶基因和UDP-葡萄糖脱氢酶基因获得的；
所述C.glu-Δldh/A-PdapB-B是在C.glu-Δldh/AB中将UDP-葡萄糖脱氢酶基因同时置于两个启动子控制下表达获得的；
所述C.glu-Δldh/A-PdapB-B-aF是在C.glu-Δldh/A-PdapB-B中下调果糖二磷酸醛缩酶基因的表达量，来弱化糖酵解途径获得的；
所述C.glu-Δldh-aEG/A-PdapB-B-aF-aE是在C.glu-Δldh/A-PdapB-B-aF中下调丙酮酸脱氢酶基因的表达量获得的。
2. 根据权利要求1所述的重组菌，其中所述心磷脂合酶蛋白序列为SEQ ID NO.1。
3. 根据权利要求1所述的重组菌，其中所述心磷脂合酶蛋白序列为在SEQ ID NO.1基础上进行突变改造获得的突变体K472R、E459D或者Q377N。
4. 根据权利要求1所述的重组菌，其中所述转录调控因子IolR的核苷酸序列为SEQ IDNO.2。
5.根据权利要求1-4任一项所述的重组菌在制备透明质酸中的应用。
6.根据权利要求5所述的应用，其中所述应用包括将重组菌接入培养基中，进行扩大培养，得到基因工程重组菌菌液；按照1-20％体积百分比将基因工程菌菌液接入发酵培养基中，进行发酵培养，得到含有透明质酸的发酵液。
7.根据权利要求1所述的重组菌的制备方法，其特征在于，包括；(A)敲除iolR基因；(B)扩增得到心磷脂合酶基因cls序列，插入到穿梭质粒，构建表达质粒，将表达质粒导入步骤(A)所得的菌株。
8.根据权利要求7所述的方法，所述方法包括：
(1)构建iolR基因敲除重组质粒pk18mobsacB-ΔiolR，转入出发菌株构建iolR基因敲除重组菌株C.glu-ΔiolR；
(2)构建带有心磷脂合酶基因cls的重组质粒pEC-XK99E-cls，转入上述步骤(1)所得到的重组菌株，得到转化有pEC-XK99E-cls重组表达质粒的基因工程重组菌。
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