CN107227286A - 一株高产琥珀酸的基因工程菌及其构建方法与应用 - Google Patents
一株高产琥珀酸的基因工程菌及其构建方法与应用 Download PDFInfo
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- CN107227286A CN107227286A CN201710443617.4A CN201710443617A CN107227286A CN 107227286 A CN107227286 A CN 107227286A CN 201710443617 A CN201710443617 A CN 201710443617A CN 107227286 A CN107227286 A CN 107227286A
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Abstract
本发明公开了一株高产琥珀酸的基因工程菌，它是在宿主菌中导入甲醇脱氢酶基因mdh2、3‑己糖‑6‑磷酸合酶基因hps和3‑己糖‑6‑磷酸异构酶基因phi。利用本发明的方法构建的重组大肠杆菌在以葡萄糖和甲醇为共同碳源的条件下，可提高胞内的还原力水平，琥珀酸的产量和收率明显提高，具有工业应用前景。本发明的方法为琥珀酸的高效合成以及工业化生产奠定了基础，为其他还原性产物的高效合成提供了一种新途径。
Description
技术领域
本发明属于生物工程领域，特别涉及一株高产琥珀酸的基因工程菌及其构建方法与应用。
背景技术
琥珀酸，又名丁二酸，是三羧酸循环(TCA)的中间产物，同时也是许多厌氧微生物能量代谢的主要末端产物。作为一种优秀的C4平台化合物，琥珀酸具有重要的应用价值，广泛应用于食品、医药、染料、香料、油漆、塑料等行业，同时也可合成一些重要的化工产品如丁二醇、四氢呋喃、γ-丁内酯、2-吡咯烷酮等；另外，琥珀酸还可用来合成可降解的生物聚合物，如聚丁烯琥珀酸酯(PBS)和聚酰胺。
微生物法生产丁二酸具有高效率和环保性等特点，因而被广泛研究。在琥珀酸生产菌株中，大肠杆菌由于其遗传背景清楚，易操作易调控，培养基要求简单等优点，近年来被广泛用于研究以获得高产琥珀酸生产菌株。目前，利用重组E.coli发酵产琥珀酸发酵模式主要包括有氧发酵、有氧-厌氧两阶段发酵以及一步厌氧发酵三种模式。与其他两种相比，采用一步厌氧发酵时，琥珀酸的比生产强度及理论最大收率均高于其它两种模式，并且可全过程固定CO2。然而目前生产菌株的琥珀酸收率仅为80％左右，远低于理论收率113％。这是由于在厌氧合成琥珀酸的代谢过程中，1mol葡萄糖经糖酵解至C3产生2mol的NADH，而从C3至形成2mol琥珀酸需要消耗4mol的NADH，还原力不足是厌氧条件下琥珀酸收率较低的主要原因。此外，生物法制备琥珀酸主要是以糖类作为碳源，但这些糖类物质的成本较高，限制了微生物法制备丁二酸的工业化。因此，若能以廉价的还原性底物为原料，不仅能有效提高还原力的供给，还能在一定程度上降低成本。
甲醇是煤化工产业中的重要产品，近年来，随着甲醇生产工艺的发展，使得甲醇的价格持续走低，因而利用甲醇作为发酵原料成为生物转化过程降低成本的重要突破口。此外，在甲醇利用的磷酸核酮糖循环途径中每同化一分子甲醇即可产生一分子NADH，因而可以为琥珀酸的合成提供足够的还原力，进而提高代谢速率，增加产物的浓度。因此，若能通过合成生物学手段，将甲醇代谢模块引入大肠杆菌中，以葡萄糖和甲醇作为共同碳源，可在细胞水平上增强还原力的供给，驱动琥珀酸的高效合成。
技术方案
本发明要解决的技术问题是提供一种利用合成生物学方法构建可以利用甲醇作为碳源进行代谢的菌株，并利用该菌株厌氧发酵生产琥珀酸，解决了传统发酵存在的琥珀酸收率低的技术问题。
为解决上述技术问题，本发明采用如下技术方案：
一株高产琥珀酸的基因工程菌，它是在宿主菌中导入甲醇脱氢酶基因mdh2、3-己糖-6-磷酸合酶基因hps和3-己糖-6-磷酸异构酶基因phi；其中甲醇脱氢酶基因mdh2将甲醇氧化成甲醛，并产生NADH，甲醛和核酮糖-5磷酸在3-己糖-6-磷酸合酶基因hps的催化下转化成己糖-6-磷酸，随后己糖-6-磷酸在3-己糖-6-磷酸异构酶基因phi的催化下异构成果糖-6-磷酸，进而进入糖酵解途径参与物质循环和能力代谢。
其中，所述的宿主菌为大肠杆菌E.coli BL21或大肠杆菌CCTCC NO:M 2012351，优选大肠杆菌CCTCC NO:M 2012351。本发明中使用的大肠埃希氏菌(Escherichia coli)BER208的保藏日期为2012年09月14日，保藏单位全称为中国典型培养物保藏中心，简称CCTCC，地址为：中国.武汉.武汉大学，其保藏编号为CCTCC NO:M 2012351，其具体信息已经在申请号为201210392035.5的中国专利中详细公开。
其中，所述甲醇脱氢酶基因mdh2来源于甲醇芽孢杆菌Bacillus methanolicus，其基因序列如SEQ ID NO:3所示。
其中，所述3-己糖-6-磷酸合酶基因hps来源于枯草芽孢杆菌Bacillus subtilis168，其GenBank登记号为CAB12140.1。
其中，所述3-己糖-6-磷酸异构酶基因phi来源于枯草芽孢杆菌Bacillussubtilis 168，其GenBank登记号为CAB12139.1。
上述高产琥珀酸的基因工程菌的构建方法，包括如下步骤：
(1)将SEQ ID NO:3和SEQ ID NO:8所示的序列克隆至表达载体中，得到重组质粒；
(2)将步骤(1)得到的重组质粒转化至宿主细胞中，得到高产琥珀酸的基因工程菌。
优选，所述表达载体为opt pTrc99A。
优选，所述宿主菌为大肠杆菌CCTCC NO:M212315。
具体的构建方法如下：
(1S)以表达载体pTrc99A为模板，设计具有一系列同尾酶酶切位点的一对引物，将PCR产物自连，获得符合Biobrick标准的表达载体opt pTrc99A；
(2S)利用基因合成的方法合成甲醇芽孢杆菌Bacillus methanolicus来源的甲醇脱氢酶基因mdh2(SEQ ID NO:3)，并对其进行密码子优化；设计引物，将其连接到表达载体opt pTrc99A中，构建质粒opt pTrc99A-mdh2；
(3S)以枯草芽孢杆菌Bacillus subtilis 168基因组为模板，设计引物扩增3-己糖-6-磷酸合酶基因hps和3-己糖-6-磷酸异构酶基因phi，将其连接到质粒opt pTrc99A，构建质粒opt pTrc99A-si；
(4S)对质粒opt pTrc99A-mdh2进行NheI、SalI双酶切，质粒opt pTrc99A-si进行AvrII、SalI双酶切，将酶切后的片段做T4连接，转化至E.coli DH5α中，经质粒酶切和菌落PCR验证，获得opt pTrc99A-msi。
(5S)将质粒opt pTrc99A-msi和opt pTrc99A分别导入到实验室自主优化的大肠杆菌BER208中，获得重组大肠杆菌BER308和重组大肠杆菌BER408，以实现基因的同时表达，进而实现甲醇的代谢，以大肠杆菌BER308作为对照菌株进行发酵性能考察。
上述高产琥珀酸的基因工程菌在发酵产琥珀酸中的应用。
优选地，发酵产琥珀酸包括以下步骤：
(1a)种子培养：将高产琥珀酸的基因工程菌接种到种子培养基中，培养10～12h，获得种子培养液；
(2a)发酵产琥珀酸：将种子培养液接种到发酵培养基中，培养至OD550＝0.8～1.2，添加终浓度为0.1mM的诱导剂IPTG和终浓度为200mM的甲醇，再厌氧发酵培养时间为48h。
优选地，所述种子培养基为NBS培养基，其配方如下：甜菜碱0.12g/L，三水磷酸氢二钾6.54g/L，磷酸二氢钾3.5g/L，磷酸氢二铵3.5g/L，七水硫酸镁0.25g/L，二水氯化钙15.0mg/L，维生素B1 0.5mg/L，六水氯化铁1.6mg/L，六水氯化钴0.2mg/L，二水氯化铜0.1mg/L，四水氯化锌0.2mg/L，二水钼酸钠0.2mg/L，硼酸0.05mg/L，葡萄糖30g/L，溶剂为水。
优选地，所述发酵培养基的配方如下：磷酸二氢铵0.87g/L，磷酸氢二铵2.6g/L，氯化钾0.15g/L，七水硫酸镁0.37g/L，六水氯化铁2.4mg/L，六水氯化钴0.3mg/L，二水氯化铜0.15mg/L，四水氯化锌0.5mg/L，二水钼酸钠0.5mg/L，硼酸0.075mg/L，四水氯化锰0.5mg/L，葡萄糖30g/L，溶剂为水。
具体的种子液培养过程如下：将重组大肠杆菌BER208/opt pTrc99A-msi按1～2％(v/v)接种量从冻存管接种到种子培养基中，100mL三角瓶装液量20mL，35～37℃有氧培养10～12h，获得种子培养液。
摇瓶发酵产琥珀酸具体过程如下：将种子培养液按10％(v/v)接种到发酵培养基中，37℃，200rpm培养至OD550＝1.0，添加终浓度为0.1mM的诱导剂IPTG和200mM的甲醇，充二氧化碳3～5min，发酵温度37℃，发酵培养时间为48h。
发酵罐中发酵生产琥珀酸的具体过程如下：将种子培养液接种到装有1.5L发酵培养基的3L发酵罐中，接种量10％(v/v)，初始葡萄糖100g/L，连续通入二氧化碳，发酵温度37℃，至OD 550＝1.0时添加终浓度为0.1mM的IPTG和200mM的甲醇，连续通入二氧化碳，发酵温度37℃，发酵培养时间为106h。
有益效果：
通过本发明的方法可实现大肠杆菌以非食品级原料甲醇作为辅助碳源生产琥珀酸，并为琥珀酸的生物合成提供充足的还原力，不仅可提高琥珀酸的产量和收率，还在一定程度上降低了生产成本，具有重大的社会意义和经济价值。
附图说明：
图1重组质粒opt pTrc99A-msi构建图。
图2原始菌和重组菌在3L发酵罐中原料消耗趋势图。
图3原始菌和重组菌在3L发酵罐中产物合成趋势图。
图4不同甲醇浓度下，重组菌株的生长和产酸情况。
具体实施方式
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径获得。
pTrc99A载体：本实验室自主保存。
大肠杆菌BER208保藏日期为2012年09月14日，保藏单位全称为中国典型培养物保藏中心，简称CCTCC，地址为：中国.武汉.武汉大学，其保藏编号为CCTCC NO:M 2012351，其具体信息已经在申请号为201210392035.5的中国专利中详细公开。
实施例1：表达载体opt pTrc99A的获得
为了能够快速有效的实现多基因的表达，本实施方式根据Biobrick标准设计一对引物P1(SEQ ID No.1)、P2(SEQ ID No.2)，以载体opt pTrc99APCR为模板，将扩增获得的片段做T4连接，导入E.coli DH5α后筛选获得优化后的表达载体opt pTrc99A。实施例2：利用合成生物学方法构建重组大肠杆菌BER208-opt pTrc99A-msi。
1、甲醇脱氢酶mdh2来源于甲醇芽孢杆菌Bacillus methanolicus，并根据大肠杆菌密码子偏好性对基因进行密码子优化，其优化后的核苷酸序列如SEQ ID NO.3所示，送至苏州金唯智生物科技有限公司进行基因合成,。
2、根据人工合成获得密码子优化的甲醇脱氢酶基因，设计上游引物P3(SEQ IDNo.4)、下游引物P4(SEQ ID No.5)，经PCR扩增后利用One Step Cloning Kit(Vazyme,C112-02)连接到表达载体opt pTrc99A上，转化至E.coli DH5α中，经质粒酶切和菌落PCR验证，获得质粒opt pTrc99A-mdh2。
3、3-己糖-6-磷酸合酶(Hps)和3-己糖-6-磷酸异构酶(Phi)总是相辅相成的，两者的表达与作用会相互影响，而且两者的基因也紧邻，因此本实施方式将二者作为一个整体来设计引物。根据NCBI上公布的基因序列，设计上游引物P5(SEQ ID No.6)、下游引物P6(SEQ ID No.7)，以枯草芽孢杆菌Bacillus subtilis 168基因组为模板，PCR扩增得到目的基因，其核苷酸序列如SEQ ID NO.8所示。利用One Step Cloning Kit(Vazyme,C112-02)将PCR产物连接到表达载体opt pTrc99A上，转化至E.coli DH5α中，经质粒酶切和菌落PCR验证，获得质粒opt pTrc99A-si。
4、对质粒opt pTrc99A-mdh2进行NheI、SalI双酶切，质粒opt pTrc99A-si进行AvrII、SalI双酶切，将酶切后的片段做T4连接，转化至E.coli DH5α中，经质粒酶切和菌落PCR验证，获得opt pTrc99A-msi。
5、将opt pTrc99A-msi导入大肠杆菌BER208中，经质粒酶切和菌落PCR验证，获得重组大肠杆菌BER208/opt pTrc99A-msi(大肠杆菌BER 308)。
实施例3：重组菌株的发酵实验。
1.种子培养：将重组大肠杆菌BER 308按1～2％(v/v)接种量从冻存管接种到种子培养基中，100mL三角瓶装液量20mL，35～37℃有氧培养10～12h，获得种子培养液。
2.发酵产琥珀酸：将种子培养液按10％(v/v)接种到发酵培养基中，37℃，200rpm培养至OD550＝1.0，添加终浓度为0.1mM的诱导剂IPTG和200mM的甲醇，充二氧化碳3～5min，发酵温度37℃，发酵培养时间为48h。
3.重组大肠杆菌BER 308在3L发酵罐中生产琥珀酸：将种子培养液接种到装有1.5L发酵培养基的3L发酵罐中，接种量10％(v/v)，初始葡萄糖100g/L，连续通入二氧化碳，发酵温度37℃，至OD 550＝1.0时添加终浓度为0.1mM的IPTG和200mM的甲醇，连续通入二氧化碳，发酵温度37℃，发酵培养106h，发酵结果如图2和图3所示。结果表明，重组大肠杆菌BER308的甲醇消耗量、琥珀酸产量和收率为25mM、68.7g/L和0.976g/g，分别是对照菌株大肠杆菌BER208的1.92倍、1.12和1.08倍。
实施例4：重组菌株的甲醇耐受实验。
1.种子培养：将大肠杆菌BER308、大肠杆菌BER208按1～2％(v/v)接种量从冻存管分别接种到种子培养基中，100mL三角瓶装液量20mL，35～37℃有氧培养10～12h，获得种子培养液。
2.发酵产琥珀酸：将种子培养液按10％(v/v)接种到发酵培养基中，37℃，200rpm培养至OD550＝1.0，添加终浓度为0.1mM的诱导剂IPTG和不同浓度的甲醇，充二氧化碳3～5min，发酵温度37℃，发酵培养时间为48h。结果如图4所示，在不同甲醇浓度条件下，表达甲醇代谢途径的重组大肠杆菌BER308的最大细胞密度和琥珀酸产量都明显高于对照菌株，并具有较好的甲醇耐受性，即使甲醇浓度达到600mM，菌体仍具有良好的生长性能。
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atcaccgttt taggggctac agacgatgca acgattaaag gcgcagtaga agaagccaaa 300
aaacaaaaga agaaaatctt agtggacatg attaacgtga aagatatcga atcccgtgcg 360
aaagaaattg acgcactcgg tgttgactac atctgcgtcc acactggcta tgatcttcaa 420
gcagagggca aaaactcttt cgaagaatta acgacaatca aaaataccgt aaaaaacgca 480
aaaaccgcaa tcgcgggcgg catcaaactt gatacactgc cagaagtgat ccagcaaaag 540
cctgaccttg tcattgtcgg gggcggaatt acaagcgcag ctgataaggc ggaaacagct 600
tcaaaaatga agcagctgat tgtccaagga taactccgat gaaaacgact gaatacgtag 660
cggaaattct caatgagtta cacaattcag cagcttatat ttctaatgaa gaagctgacc 720
agcttgccga tcacattctt tcatcccacc aaattttcac cgcgggtgcg gggcggtctg 780
gcctgatggc aaaatccttc gcgatgagac tgatgcacat gggcttcaac gcccatatag 840
taggtgagat tctcactccg ccgctcgccg aaggagatct agttattatc ggctcaggat 900
caggcgagac aaagagcttg attcataccg cagcaaaagc aaaaagctta cacggaattg 960
ttgccgcttt aaccatcaat ccggaatcaa gcatcggaaa acaagcggac ctcatcatca 1020
gaatgcctgg ttcccctaaa gaccagtcta acggaagcta taaaaccatt cagccaatgg 1080
gatcattatt tgaacaaact ttgctgctct tctatgatgc agtgatttta aaactcatgg 1140
agaaaaaagg tctcgattct gaaactatgt tcactcacca cgcaaacctt gaatag 1196
Claims (10)
1.一株高产琥珀酸的基因工程菌，其特征在于，它是在宿主菌中导入甲醇脱氢酶基因mdh2、3-己糖-6-磷酸合酶基因hps和3-己糖-6-磷酸异构酶基因phi。
2.根据权利要求1所述的高产琥珀酸的基因工程菌，其特征在于，所述的宿主菌为大肠杆菌E.coli BL21或大肠杆菌CCTCC NO:M2012351。
3.根据权利要求1所述的高产琥珀酸的基因工程菌，其特征在于，所述甲醇脱氢酶基因mdh2来源于甲醇芽孢杆菌Bacillus methanolicus，其基因序列如SEQ ID NO:3所示。
4.根据权利要求1所述的高产琥珀酸的基因工程菌，其特征在于，所述3-己糖-6-磷酸合酶基因hps和3-己糖-6-磷酸异构酶基因phi来源于枯草芽孢杆菌Bacillus subtilis168，其GenBank登记号分别为CAB12140.1和CAB12139.1。
5.权利要求1～4任一所述高产琥珀酸的基因工程菌的构建方法，其特征在于，包括如下步骤：
(1)将SEQ ID NO:3和SEQ ID NO:8所示的序列克隆至表达载体中，得到重组质粒；
(2)将步骤(1)得到的重组质粒转化至宿主细胞中，得到高产琥珀酸的基因工程菌。
6.根据权利要求5所述高产琥珀酸的基因工程菌的构建方法，其特征在于，所述表达载体为opt pTrc99A，所述宿主菌为大肠杆菌CCTCC NO:M212315。
7.权利要求1～4任一所述高产琥珀酸的基因工程菌在发酵产琥珀酸中的应用。
8.根据权利要求7所述的应用，包括以下步骤：
(1a)种子培养：将高产琥珀酸的基因工程菌接种到种子培养基中，培养10～12h，获得种子培养液；
(2a)发酵产琥珀酸：将种子培养液接种到发酵培养基中，培养至OD550＝0.8～1.2，添加终浓度为0.1mM的诱导剂IPTG和终浓度为200mM的甲醇，再厌氧发酵培养时间为48h。
9.根据权利要求7所述的应用，其特征在于，所述种子培养基为NBS培养基，其配方如下：甜菜碱0.12g/L，三水磷酸氢二钾6.54g/L，磷酸二氢钾3.5g/L，磷酸氢二铵3.5g/L，七水硫酸镁0.25g/L，二水氯化钙15.0mg/L，维生素B1 0.5mg/L，六水氯化铁1.6mg/L，六水氯化钴0.2mg/L，二水氯化铜0.1mg/L，四水氯化锌0.2mg/L，二水钼酸钠0.2mg/L，硼酸0.05mg/L，葡萄糖30g/L，溶剂为水。
10.根据权利要求7所述的应用，其特征在于，所述发酵培养基的配方如下：磷酸二氢铵0.87g/L，磷酸氢二铵2.6g/L，氯化钾0.15g/L，七水硫酸镁0.37g/L，六水氯化铁2.4mg/L，六水氯化钴0.3mg/L，二水氯化铜0.15mg/L，四水氯化锌0.5mg/L，二水钼酸钠0.5mg/L，硼酸0.075mg/L，四水氯化锰0.5mg/L，葡萄糖30g/L，溶剂为水。
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| CN115058376A (zh) * | 2022-06-17 | 2022-09-16 | 福建师范大学 | 重组菌株及其在利用甲酸或其衍生物中的应用和方法 |
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| CN112239738B (zh) * | 2020-10-29 | 2022-11-25 | 江南大学 | 一株产琥珀酸的大肠杆菌及其应用 |
| CN115058376A (zh) * | 2022-06-17 | 2022-09-16 | 福建师范大学 | 重组菌株及其在利用甲酸或其衍生物中的应用和方法 |
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