CN107267472B - 提高大肠杆菌甲醇代谢途径中限速酶活性的方法 - Google Patents

提高大肠杆菌甲醇代谢途径中限速酶活性的方法 Download PDF

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CN107267472B
CN107267472B CN201710476350.9A CN201710476350A CN107267472B CN 107267472 B CN107267472 B CN 107267472B CN 201710476350 A CN201710476350 A CN 201710476350A CN 107267472 B CN107267472 B CN 107267472B
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陈可泉
陆晓璐
王昕�
张博文
毛静文
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Abstract

本发明公开了一种提高大肠杆菌甲醇代谢途径中限速酶活性的方法,将大肠杆菌过表达基因Mdh和Hps‑Phi后,通过添加激活蛋白nudF,增强甲醇脱氢酶的酶活,从而提高甲醇的代谢量;其中,所述基因Mdh和Hps‑Phi为来源于甲醇芽孢杆菌MGA3的Mdh2和Hps‑Phi;所述激活蛋白nudF来源于E.coli MG1655。本发明在大肠杆菌中进行甲醇代谢路径的组装,实现甲醇代谢,并通过分子手段改造提高甲醇脱氢酶Mdh的酶活,实现了甲醇代谢的提高。

Description

提高大肠杆菌甲醇代谢途径中限速酶活性的方法
技术领域
本发明涉及大肠杆菌甲醇代谢途径的组装与调控,具体涉及一种增强甲醇代谢通路中限速酶甲醇脱氢酶活性的方法,从而提高甲醇在大肠杆菌中的代谢量。
背景技术
随着代谢工程的迅猛发展及生物合成学的崛起,人类改造微生物作为细胞工厂进行生物制造的能力显著提高。当前生物制造的原料仍以粮食为主,随着粮食危机日益家具以及其价格不断攀升,以粮食为原料的生物制造正面临严峻考验。因此,为了解决原料来源问题,实现生物制造可持续发展,目前逐渐将“甲醇经济”发展为合理的可替代能源——以甲醇为原料,缓解化石能源的紧缺。
与其他原料相比,甲醇具备以下优势:1)价格低廉,当前甲醇的价格是葡萄糖价格的1/3;2)来源广泛,从天然气、煤化工、生物质等均可合成;3)含能高,为目标产物的合成提供充足还原力。因此,以甲醇替代粮食作为原料,可以大幅度降低生物制造的而成本,实现生物制造的可持续发展。
自然界中存在能够代谢甲醇的微生物,如甲基杆菌(MethylobacteriumExtorquens)、甲醇芽孢杆菌(Bacillus methanolicus)、甲醇营养型酵母(Methylotrophicyeasts)等。尽管这些微生物可以利用甲醇作为原料,但其利用效率较低,究其原因,一方面是大部分的甲醇营养菌是好氧型,另一方面,菌株遗传操作低效落后。故,在模式菌株中构建甲醇代谢途径是实现甲醇高效生物转化的有效途径。
在甲醇营养菌株中,甲醇经甲醇脱氢酶(Mdh)氧化为甲醛,后经过磷酸核酮糖途径(Rump途径)完成中心糖代谢。其中,甲醇代谢的Rump途径由两个模块组成,第1模块由异源基因组成,第2模块由内源磷酸戊糖途径的相关基因组成。甲醇经第1模块中的甲醇脱氢酶作用氧化为甲醛,与第2模块中的5-磷酸核酮糖一起经第1模块生成6-磷酸果糖,进而部分通过模块1流向糖酵解路径生成产品,另一部分通过模块2再生5-磷酸核酮糖促进甲醛同化。
发明内容
本发明的目的在于克服现有技术的缺陷,提供一种提高大肠杆菌甲醇代谢途径中限速酶活性的方法,来提高菌株对甲醇利用率。
为实现上述技术目的,本发明采用如下技术方案:
一种提高大肠杆菌甲醇代谢途径中限速酶活性的方法,将大肠杆菌过表达基因Mdh和Hps-Phi后,通过添加激活蛋白nudF,增强甲醇脱氢酶的酶活,从而提高甲醇的代谢量;
其中,所述基因Mdh和Hps-Phi为来源于甲醇芽孢杆菌MGA3的Mdh2和Hps-Phi;
所述激活蛋白nudF来源于E.coli MG1655。
本发明所述的方法,其具体包括如下步骤:
(1)将来源于甲醇芽孢杆菌MGA3的Mdh2和Hps-Phi,合成基因后通过基因克隆至质粒pETDuet,构建得到质粒pETDuet-Mdh2-RuMP;
(2)将质粒pETDuet-Mdh2-RuMP导入大肠杆菌BL21(DE3),获得菌株BL21(DE3)/pETDuet-Mdh2-RuMP;
(3)添加来源于E.coliMG1655的同源激活蛋白nudF,构建菌株
BL21(DE3)/pETDuet-nudF-Mdh2-RuMP。
本发明的另一目的在于提供上述方法制备的菌株。
本发明的又一目的在于提供上述方法制备的菌株在甲醇代谢中的应用。
上述应用中,采用菌株发酵代谢甲醇,培养基成分为:17.1g/LNa2HPO4·12H2O、3g/L KH2PO4、10g/LNH4Cl、0.5g/LNaCl,微量元素;其中添加50mM甲醇和10g/L葡萄糖为混合碳源。
优选的,采用菌株发酵代谢甲醇,在培养基中添加有机氮源,所述有机氮源选自为麦芽浸出粉、酵母粉、蛋白胨、玉米浆中的一种,其终浓度均为1g/L。
本发明在大肠杆菌中进行甲醇代谢路径的组装,实现甲醇代谢,并通过分子手段改造提高甲醇脱氢酶Mdh的酶活,以实现甲醇代谢的提高。本发明中高通量筛选甲醇脱氢酶所用培养基为M9培养基,并在其中添加一定量的甲醇,即以葡萄糖和甲醇为混合碳源,通过甲醇转化为甲醛的量表达甲醇脱氢酶的活力。另本发明中,定量测定甲醇消耗所用的培养基为M9培养基,在摇瓶发酵过程中添加一定量的葡萄糖和甲醇作为碳源,通过检测甲醇的消耗量表达大肠杆菌内甲醇代谢能力。本发明还对菌株发酵的培养基进行了优化,采用麦芽浸出粉氮源进一步提高了菌株的甲醇代谢效果。
附图说明
图1是甲醛浓度随时间变化曲线图;
图2是菌株生长状况随时间变化曲线图;
图3是菌株甲醇消耗量柱形图;
图4是不同氮源下甲醇消耗量柱形图。
具体实施方式
下面结合附图说明对本发明的具体实施方式做进一步阐述。
实施例中选用的载体质粒为pETDuet,购自宝日医生物技术公司;所用表达基因的宿主菌为大肠杆菌BL21(DE3),购自北京全式金生物技术有限公司。
实施例1:大肠杆菌甲醇代谢路径组装
本发明在大肠杆菌中组装依赖NAD甲醇脱氢酶和RuMP途径的甲醇代谢途径。通过对比不同来源的甲醇脱氢酶Mdh和3-己糖-6-磷酸合酶Hps以及3-己糖-6-磷酸异构酶Phi,选定来源于甲醇芽孢杆菌(Bacillus methanolicus)MGA3的Mdh2和Hps-Phi,合成基因后通过基因克隆至质粒pETDuet,构建得到质粒pETDuet-Mdh2和质粒pETDuet-Mdh2-RuMP。将该质粒通过常规方法分别导入大肠杆菌BL21(DE3)中,获得的菌株分别命名为M和MR,并以甘油管形式保存,其中菌株M只过表达基因Mdh2,且连接至载体的酶切位点为NcoI和BamHI;菌株MR过表达基因Mdh2、Hps-Phi,且Mdh2的酶切位点与上述相同,Hps-Phi的酶切位点为NdeI和XhoI,均以T7为启动子。
实施例2:增强甲醇脱氢酶酶活
为了增强甲醇脱氢酶Mdh的活力,本实施例中采取了三种方案:
方案一为通过定向进化增强甲醇脱氢酶的酶活,但酶活提高不明显;
方案二为通过添加激活蛋白增强甲醇脱氢酶的酶活,其中激活蛋白采用两种来源:一是添加来源于E.coliMG1655的同源激活蛋白nudF,构建成菌株BL21(DE3)/pETDuet-nudF-Mdh2和BL21(DE3)/pETDuet-nudF-Mdh2-RuMP,菌株分别命名为nudF-M和nudF-MR;
二是添加来源于Bacillus.methanolicusPB1异源激活蛋白ACT,构建成菌株BL21(DE3)/pETDuet-ACT-Mdh2,菌株命名为ACT-M;
方案三为替换甲醇脱氢酶Mdh2,替换的甲醇脱氢酶来源于CupriavidusnecatorN-1,且在第26、31、169位点进行点突变,构建成菌株BL21(DE3)/PETDuet-MDH2,菌株命名为Mdh2-M。其中添加激活蛋白的目的是促进甲醇脱氢酶Mdh的辅因子NADH的氧化,从而提高甲醇脱氢酶Mdh的转化速率。
在进行甲醇脱氢酶酶活定量检测时,以空菌BL21(DE3)/pETDuet为对照,该空菌命名为B。
实施例3:甲醇脱氢酶酶活的检测
本发明中检测甲醇脱氢酶Mdh的酶活采用Nash试剂(即乙酰丙酮试剂)与甲醛的显色反应,通过生成的甲醛的量检测甲醇脱氢酶的活力(即甲醇氧化为甲醛的能力)。
M9培养基:17.1g/LNa2HPO4·12H2O、3g/LKH2PO4、10g/LNH4Cl、0.5g/LNaCl,微量元素,及10g/LGlucose。
Nash试剂:150g/L醋酸铵、3ml/L乙酸、2ml/L乙酰丙酮。
甘油管中菌株接至摇管使菌株复壮,37℃,200rpm,过夜培养,收集菌,5000g,7min离心,M9重悬,转接至9ml的M9培养基中,添加0.5M甲醇开始反应,不同时间取样,与Nash试剂1:1反应混合,在58℃反应10min,于412nm检测吸光度,对应标曲计算甲醛浓度。
结果如图1所示,在相同时间90min,添加同源激活蛋白nudF的菌株nudF-M生成0.056mM甲醛,添加异源激活蛋白ACT的菌株ACT-M生成0.019mM,而未经改造的菌株M生成甲醛0.014mM,即添加同源激活蛋白nudF的菌株生成甲醛是未经改造菌株生成甲醛的4倍。更直观地比较甲醇脱氢酶Mdh的变化,将定义1U定义为每分钟生成1μmol甲醛的量,即酶活,酶活结果如表3。
表3
菌株 Activity(mU)
B 0
M 166.42
ACT-M 219.29
nudF-M 593.25
实施例4:甲醇代谢菌株摇瓶发酵
M9培养基:17.1g/LNa2HPO4·12H2O、3g/LKH2PO4、10g/LNH4Cl、0.5g/LNaCl,微量元素。
本实施例中,定量检测甲醇消耗采用摇瓶发酵,以M9培养基添加50mM甲醇和10g/L葡萄糖为混合碳源,其步骤具体如下:
将上述实施例1、2构建的菌株B、MR、nudF-MR在LB种子培养液中过夜培养,检测其OD600,转接至50MLM9培养基中,且该培养基中葡萄糖浓度为10g/L,使其初始OD600为0.3,取其相应的种子液,5000rpm,离心7min,于无菌条件下弃上清液,用发酵培养基重悬转接至摇瓶,于37℃,200rpm恒温培养摇床培养4小时左右,加入0.5mMIPTG和50mM甲醇,于一定时间取样测其OD600以及检测甲醇的量。
菌株生长状况结果如图2所示,在添加50mM甲醇后,一定时间采用分光光度计检测菌株在OD为600nm处的生长状况,在0-11h时,为三菌株的延滞期;11-24h,为菌株的指数生长期;24-36h,为菌株的稳定期。三株菌的生长趋势相似,在11h左右,添加激活蛋白的菌株与对照菌株及未添加激活蛋白的菌株相比,生长状况较佳,故添加激活蛋白的菌株在含甲醇的培养基中生长并未产生不佳影响。
甲醇消耗结果如图3所示,在甲醇添加量为3g/L时,与对照菌株B相比,未添加激活代表的菌株MR甲醇消耗为0.36g/L,添加激活蛋白后的菌株nudF-MR甲醇消耗为0.57g/L,即大肠杆菌内甲醇代谢路径组装后甲醇有消耗,且在添加同源激活蛋白提高限速酶的酶活后,甲醇代谢量得以提高约1.5倍。
实施例5:摇瓶发酵培养基的优化
通过实施例4中的结果显示,菌株nudF-MR的甲醇代谢量最佳,以该菌株为基础,将实施例4中的发酵培养基M9培养基添加不同的有机氮源,分别为麦芽浸出粉、酵母粉、蛋白胨、玉米浆,其终浓度均为1g/L。按照实施例4中的发酵方法,在一定时间检测甲醇的代谢量。
结果如图4显示,通过添加不同的无机氮源,定量检测甲醇消耗,以确定最佳的发酵条件。结果显示,添加1g/L麦芽浸出粉时,甲醇的消耗量为最高,约为0.6g/L,而最新报道在添加酵母粉的条件下,摇瓶发酵甲醇消耗约为0.3g/L。

Claims (2)

1.一种提高大肠杆菌甲醇代谢途径中限速酶活性的方法,其特征在于,将大肠杆菌过表达基因Mdh和Hps-Phi后,通过添加激活蛋白nudF,增强甲醇脱氢酶的酶活,从而提高甲醇的代谢量;具体包括如下步骤:
(1)将来源于甲醇芽孢杆菌MGA3的Mdh2和Hps-Phi,合成基因后通过基因克隆至质粒pETDuet,构建得到质粒pETDuet-Mdh2-RuMP;其中Mdh2连接至载体的NcoI和BamHI酶切位点;Hps-Phi连接至NdeI和XhoI酶切位点,均以T7为启动子;
(2)将质粒pETDuet-Mdh2-RuMP导入大肠杆菌BL21(DE3),获得菌株BL21(DE3)/pETDuet-Mdh2-RuMP;
(3)添加来源于E.coli MG1655的同源激活蛋白nudF,构建菌株BL21(DE3)/pETDuet-nudF-Mdh2-RuMP;
(4)采用步骤(3)构建的菌株发酵代谢甲醇,在培养基中添加1g/L麦芽浸出粉。
2.根据权利要求1所述的方法,其特征在于,采用菌株发酵代谢甲醇,培养基成分为:17.1g/L Na2HPO4·12H2O、3g/L KH2PO4、10g/L NH4Cl、0.5g/L NaCl,微量元素;其中添加50mM甲醇和10g/L葡萄糖为混合碳源。
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