CN113106047B - 一种重组食甲基丁酸杆菌及其构建方法与应用 - Google Patents
一种重组食甲基丁酸杆菌及其构建方法与应用 Download PDFInfo
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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Abstract
本发明公开了一种重组食甲基丁酸杆菌及其构建方法与应用。以食甲基丁酸杆菌为来源，构建过表达甲醇利用和丁酸合成基因工程菌，即BM/pXY1‑mtaA/mtaB/mtaC2和BM/pXY1‑atoB/paaH/crt/bcd。重组菌株对甲醇利用和丁酸合成的能力显著增强，在PB培养基中，甲醇的消耗能力分别提升了69％，14％。丁酸的产量分别提升了38％，28.6％。为进一步提高甲醇的消耗能力奠定了基础。
Description
技术领域
本发明属于微生物基因工程领域，具体涉及一种重组食甲基丁酸杆菌及其构建方法与应用。
背景技术
甲醇，是一种有刺激性气味的无色透明液体。甲醇作为生物制造原料具有以下较明显的优势：甲醇并且不需要压缩和减压安全性更高，更低的运输成本；甲醇还原力高于葡萄糖，使用甲醇作为唯一的或辅助的碳源来生产包括有机酸在内的还原性化学品，则有望获得更高的产品产量。当前生产甲醇的主要方法是化学合成法，即加压催化从化石燃料而来的合成气、直接氧化甲烷、还原大气中二氧化碳和氢气来合成甲醇，由于生产甲醇的原材料丰富，建设项目大批涌现，甲醇产能持续快速增长。因此，近年来甲醇产能压力过剩等问题日益加剧。寻找一种能够将甲醇作为底物生产各种化学品的方法，对于缓解甲醇产量过剩与需求不足之间的矛盾是至关重要的。
食甲基丁酸杆菌(Butyribacterium methylotrophicum)，细菌学分类为梭菌属，是一种专性厌氧革兰氏阳性芽孢杆菌。B.methylotrophicum可同时利用甲醇、CO、CO2等碳一化合物进行发酵。当提供还原型基质时，如CH3OH-CO2、CH3OH-HCOOH等，即合成丁酸。因此，食甲基丁酸杆菌作为天然利用甲醇菌株，与好氧甲基营养菌有所不同，甲醇代谢途径还原力消耗较少，主要用于代谢产物的积累，同时菌株具有对甲醇的耐受性高，代谢甲醇速率较快等优势，其发酵主要产物是羧酸、丁酸和相应的醇类等，在生物制造业上有一定研究价值。
然而，目前的研究主要集中在食甲基丁酸杆菌的发酵条件和培养基筛选等方面，并未涉及基因改造。食甲基丁酸杆菌中甲醇代谢主要分为三个模块，第一个模块为甲基转移酶系统，该系统能将甲醇的甲基转移到甲基四氢叶酸中，该模块的三个主要基因为mtaA,mtaB和mtaC2. 随后通过WLP途径的甲基分支，甲基四氢叶酸和二氧化碳被转化乙酰-CoA。随后，涉及丁酸合成途径的几个基因:atoB,paaH,crt和bcd等基因能将乙酰-CoA进一步转化为丁酸。但食甲基丁酸杆菌对甲醇消耗速率以及丁酸生产效率方面仍有进一步提高的空间。
发明内容
针对现有技术的不足，本发明提供了一种重组食甲基丁酸杆菌及其构建方法与应用，通过在食甲基丁酸杆菌中过表达甲醇利用途径和丁酸合成途径，提高了该菌株对于甲醇消耗能力以及丁酸合成能力。
为解决现有技术问题，本发明采取的技术方案为：
一种重组食甲基丁酸杆菌，所述重组食甲基丁酸杆菌通过转入含有甲醇利用途径相关基因或丁酸合成途径相关基因的质粒，获得过表达甲醇利用途径或丁酸合成途径重组菌株；所述甲醇利用途径相关基因和丁酸合成途径相关基因的核苷酸序列分别如SEQ NO.1和SEQ NO.2 所示。
基于上述重组食甲基丁酸杆菌的构建方法包括如下步骤：(1)分别构建重组质粒pXY1-mtaA/mtaB/mtaC2和重组质粒pXY1-atoB/paaH/crt/bcd；(2)对重组质粒 pXY1-mtaA/mtaB/mtaC2和重组质粒pXY1-atoB/paaH/crt/bcd分别进行甲基化修饰的重组质粒；(3)再将分别甲基化修饰的重组质粒电转化进入食甲基丁酸杆菌，构建重组食甲基丁酸杆菌。
作为改进的是，所述基因matA,mtaB,mtaC2,atoB,paaH,crt,bcd的Gene ID分别为BUME_RS16910,BUME_RS16905,BUME_RS16900,BUME_RS03485,BUME_RS03475, BUME_RS03480,BUME_RS0347。基因matA,mtaB,mtaC2,atoB,paaH,crt,bcd为食甲基丁酸杆菌内源基因，所述食甲基丁酸杆菌购自ATCC菌种保藏中心。
作为改进的是，所述重组质粒pXY1-mtaA/mtaB/mtaC2的构建方法为：使用引物MT3-BamHⅠ-F/MT3-NdeⅠ-R进行PCR扩增来源于食甲基丁酸杆菌的甲基转移酶相关基因mtaA/mtaB/mtaC2，分别使用BamHⅠ和NdeⅠ双酶切扩增片段和载体pXY1，酶连后获得重组质粒pXY1-mtaA/mtaB/mtaC2。
作为改进的是，所述重组质粒pXY1-atoB/paaH/crt/bcd的构建方法为：使用引物Tchb-HR-F/Tchb-HR-R进行PCR扩增来源于食甲基丁酸杆菌的丁酸合成相关基因atoB/paaH/crt/bcd，将载体线性化后，通过同源重组的方法连接到载体pXY1上，获得重组质粒pXY1-atoB/paaH/crt/bcd。
作为改进的是，将步骤1构建的重组质粒pXY1-mtaA/mtaB/mtaC2和重组质粒pXY1-atoB/paaH/crt/bcd，分别以热激法转化进含有pMCljs质粒的大肠杆菌Top10感受态中，随后培养含有pMCljs和重组质粒的大肠杆菌，并提取总质粒，完成重组质粒pXY1-mtaA/mtaB/mtaC2和pXY1-atoB/paaH/crt/bcd的甲基化修饰。
作为改进的是，将甲基化修饰的重组质粒通过电转的方式转化进食甲基丁酸杆菌中，电转程序为2200v，400Ω；在厌氧箱中以37℃培养3-4天，菌落PCR验证后获得重组菌株。
上述过表达甲醇利用和丁酸合成途径的食甲基丁酸杆菌重组菌株在甲醇发酵产丁酸上的应用。
上述应用，具体步骤如下：在平板上挑取重组食甲基丁酸杆菌的单菌落，接种至含有红霉素的1ml YTF培养基中，培养12-16h后，将离心管中的菌液全部转接至安剖瓶中，生长至OD600为1-1.2，将菌液倒入50ml离心管中，4000rpm离心10min，弃去上清，用PB培养基重悬后以OD600＝0.1的接种量接种至50ml PB培养基中，再加入100mM甲醇,每隔一定时间，吸取2ml菌液，离心后将上清液转移至新的离心管保存，用于高效液相色谱检测甲醇、丁酸，用2ml超纯水重悬后检测其OD600。
有益效果：
与现有技术，本发明一种重组食甲基丁酸杆菌及其构建方法与应用，具有如下优势：
本发明通过构建重组食甲基丁酸杆菌，B.methylotrophicum/pXY1-mtaA/mtaB/mtaC2，该菌株能够过表达甲醇利用途径，与含有空质粒的重组菌株相比，当以甲醇和CO2作为碳源时，其生物量，甲醇消耗量以及丁酸的产量分别增加了25％，69％，38％，这些结果表明甲醇利用途径在调节食甲基丁酸杆菌的甲醇生物转化效率中起着重要作用。甲醇产量的提高能进一步增加丁酸的产量，进而验证了甲醇到丁酸的合成途径。
本发明通过构建食甲基丁酸杆菌重组菌株， B.methylotrophicum/pXY1-atoB/paaH/crt/bcd，该菌株能够过表达丁酸合成途径，与含有空质粒的重组菌株相比，当以甲醇和CO2作为碳源时，其甲醇消耗量以及丁酸的产量分别增加了 14％，28.6％。表明过表达丁酸合成途径能够提高丁酸产量，从而进一步提高甲醇的消耗。表明通过代谢改造提高甲醇的生物转化是可行的。
附图说明
图1为重组菌株BM/pXY1-mtaA/mtaB/mtaC2与BM/pXY1生长情况对比结果；
图2为重组菌株BM/pXY1-mtaA/mtaB/mtaC2与BM/pXY1甲醇消耗对比结果；
图3为重组菌株BM/pXY1-mtaA/mtaB/mtaC2与BM/pXY1丁酸生成对比结果；
图4为重组菌株BM/pXY1-atoB/paaH/crt/bcd与BM/pXY1甲醇消耗对比结果；
图5为重组菌株BM/pXY1-atoB/paaH/crt/bcd与BM/pXY1丁酸生成对比结果。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
以下实施例中如无特别说明，所用酶及质粒均为购买所得。
实施例1：构建重组质粒pXY1-mtaA/mtaB/mtaC2
实施例中所用到的基因matA,mtaB,mtaC2,atoB,paaH,crt,bcd的Gene ID分别为来自BUME_RS16910,BUME_RS16905,BUME_RS16900,BUME_RS03485,BUME_RS03475, BUME_RS03480,BUME_RS0347。基因matA,mtaB,mtaC2,atoB,paaH,crt,bcd为食甲基丁酸杆菌内源基因，所述食甲基丁酸杆菌购自ATCC菌种保藏中心。
甲醇过表达基因mtaB/mtaC2/mtaA碱基序列，即(甲醇利用途径相关基因SEQ NO.1所示)：
atggcaaagaaatttgataaactggcaattaataatctggacgattttatttatggctcttgtccgaaccctgtcaccac caggagcggcatggtcatcggcggcggcaccatctatccggaaatcaacttcacactgccgggcatggatgtcaatgatcagaccattgacaaggccttgggcatttattccaatatcatcgacggtgtgctcaagagagcggcagagctctacgcgccc ggcgtgctggtagaatttgaaaccgtgccggactttaccgagcatccaaaatatgggattgacgccaaccgcattttattaaatggcatcaaggaagccgcagacaagtacggcctcaaggccgccctgcggaccacccccaacgacctgcgcgaaatga gccgtcctccggttatgcgcggcggcaagtactgggataccatgctggagctgtacgaacagtgcgccaaggatggttcagactttttatccatcgaatcgaccgggggcaaggaaatcaatgacgaagccctcgtaaaggccgatatccgcaaagccat cttcgccatgggcgtgctgggctgccgcgacatggaatacctctggggcaatctggttaaattatccgatgctaatggctgcttcgccgctggcgactctgcctgtggctttgccaacaccgccatggttctggccgaaaaaggctttatcccccatgtg ttcgcagcggttatgcgtgttgtggcagtgccgagagccctggtggcctttgaacagggcgcggttggcccgagcaaggactgcgcctatgaaggcccatacctcaaggccattaccggcagtcccatcgccatggaaggtaagagcgcggctggcgccc atttaagcccagttggcaacatcgcggcagccgtggccgatacctggagtaatgaatccatccagcaggtcaagctcttatccgagatggcccctgtggtgggcatggaacagctggtatatgactgccgtctcatgaacgtggccaaggaaaaaggcca gggccttatgatgcgcgacctgctcgttgaatctgacgcgccgctggatgtccaggcatgggttttaagacccgatgttgtgcttaagatcgccggcgagctggtgaaagagcaggataacttcctgagaaccaaactggccgccaaattaaccattaacgagctgcgcgacgccatcaaggccgaaaaggttaaggctgaccgccgcgacatgaaatggctcgacaagatggaaaaagcagtggacaagattccagacgatccggaacagttctacgcagaaatcaagcccgagctggacatggacaagtggcatccta aaggctatggcttaaaggcctgatcttaatattcttaaaacagaaattcactgttaaaaattataaaaatttggacaattccctccggtgcggctcagtacacactgcgcgccggaagagcaggtaaacagctttaatcaggaataaaaaaatcggagga tataaaaatggcaattttagaagatattcaaaactgtgtgttggacggcgaactggatgagatcaaggacttggtgcagaaggcagtggatgaggggatcgaccccgccgccatcatcaacgacggcctcatcggcggcatgaacattgtggccccgctg tttaagagcggtgaaatgtttgtcccggaagttatggaatccgcggataccatgaacgaaggcatgcaggtggttaagcccctgatcaccgacgcggatatgcccaccaagggcaaggtcatcatcggcactgtcaacggtgacctgcacgatatcggta aaaacctggtggtcttaatgatggaaagccgcggctacacagttattgacatgggcgtagatgtgaaggaagaacagtttgtggaagccatcaaagaacacaagccagacattgtgggcatgtcctcactgctcaccaccaccatgatgaagattgacga taccatcaagatgattaacgagtccggcctgcgtgaccaggttaagatcatcatcggcggcgcacccatctctcaggaattcgccgatgatatcggggcggacggctattcagaggacgcctccaccgcggttgagctctgcgaccgcatgatggccatg tagcagacagcgaataaaattttgacgaacaggaggcatttcagatgctgacaattgttggtgaactcattaacaccagccgccctccggtcaaggaggcggtacagaataaagacgaagcgttcatccgggagcttgccaaaaaacaggcagacgccgg cgcgacctatatcgacgtaaactgcggcaatatggtcaagaatgaactggaaataatggaatggcttgtgaacattgttcaggacgaggtcgacacccccctgtgcattgacagccccaacgccaaagcactggacgtgggtctggccctgtgcaggaac ggccgtcccatgatcaattccatctccgacgaggacgggcgttatgaatccgttcttcccttaattaaaaaatataacgccaaaatcgttgtattgtgcatggactccactggtatgcccgaaacctcagccgaccgcatgaaggtggtcaataaccttt atgccaagctgaaggccgagggcatcgccgatgacgacatgtactttgacccgctggtcaagcccatcagcagcgtgaccagtgccggagcagaggtgctcgacaccatccgccagatcaagcaggattaccccgatgtacacttcatgtgcggtttgag caacatttcctatggcctgccgaaccgcagcatcctcaatcggctgtttgttgtccagacaatgaccctggggatggacggctacgtccttgaccccaccaatggcaaaatgatggcggacatcatcaccgccaccgcgctcctgggcagagacagctat tgcagtaaatacattaaagcccatagaaagggcaaacttgacgcatcgtctgatgcgtaa
重组质粒pXY1-mtaA/mtaB/mtaC2的构建：以食甲基丁酸杆菌的全基因组为模板，通过常规 PCR扩增甲基转移酶操纵子mtaA/mtaB/mtaC2，
所用的上游引物MT3-BamHⅠ-F带有BamHI酶切位点，序列如下：
CGGGATCCATGGCAAAGAAATTTGATAAACTGG；
所用的下游引物MT3-NdeⅠ-R带有NdeⅠ酶切位点，序列如下:
GGAATTCCATATGTTACGCATCAGACGATGCG,
反应条件为：95℃3min，95℃15s，57℃15s，72℃3min，共30个循环；72℃5min。得到的基因片段经1％琼脂糖凝胶电泳后回收相应片段。将该序列与载体pXY1用Takara公司的BamHI和NdeⅠ酶切，酶切反应体系为：10×buffer 5μL，BamHI 5μL，NdeⅠ5μL，基因片段或pXY1载体30μL，ddH2O 5μL。酶切体系于30℃条件下反应1小时后再于37℃条件下反应1小时。将酶切产物经1％琼脂糖凝胶电泳后回收相应片段。将纯化后的酶切产物进行连接，反应体系为：10×Ligase buffer 1μL，T4 DNA Ligase(Takara)1μL，基因片段7μL，载体1μL。连接于25℃下反应1-2小时。将连接产物转化大肠杆菌Trans1-T1。 PCR筛选阳性菌株pXY1-mtaA/mtaB/mtaC2并进行DNA测序，验证重组质粒构建正确。
实施例2：构建重组菌株BM/pXY1-mtaA/mtaB/mtaC2
重组菌株BM/pXY1-mtaA/mtaB/mtaC2的构建：将质粒pMCljs热激转化进入大肠杆菌 Trans-T1中,得到重组菌株Trans-T1/pMCljs,将该重组菌株制备成感受态细胞，具体的步骤如下所示：
从LB平板上挑取新活化的Trans-T1/pMCljs单菌落，接种于5ml含有盐酸壮双霉素的 LB培养基中，37℃下培养至对数生长期，将菌株接种于50ml液体培养基中，37℃培养至OD600＝0.4-0.5；将菌液倒入离心管中，冰上放置10min，于4℃下4000rpm离心10min；弃去上清，用预冷的0.1mol/L的CaCl2溶液10ml轻轻悬浮细胞，放置冰上15min，4℃下4000rpm 离心10min，重复该步骤2次；弃去上清，加入5ml预冷含15％甘油的0.1mol/L CaCl2溶液，轻轻悬浮细胞，冰上放置5min，即得到含有pMCljs质粒的Trans-T1感受态细胞。
将实施例1得到的重组质粒转化进该感受态，将得到的重组菌株Trans-T1/pMCljs/ pXY1-mtaA/mtaB/mtaC2接种于5ml含有氨苄抗性和盐酸壮双霉素的LB培养基中，37℃培养 12小时后提取质粒，即得到甲基化质粒pXY1-mtaA/mtaB/mtaC2。随后制作食甲基丁酸杆菌感受态细胞，在厌氧箱里，从YTF平板挑点接入1ml YTF培养基；12-16h后，将离心管中的菌液全部转接至安剖瓶中，生长至OD600为0.3-0.4，加入细胞壁弱化剂(20mM Thr溶液和270mM蔗糖溶液)；生长至OD600为0.8左右，将菌液倒入50ml离心管中，取出冰浴 30min-1h；4000rpm离心10min，弃去上清，用冰浴后的SMP缓冲液重悬细胞(每次10ml)； 4000rpm离心10min，弃去上清，重复3次；加入1ml SMP缓冲液，再加入细胞膜增溶剂-1％丁醇，轻轻重悬细胞混匀，即得到感受态细胞。
食甲基丁酸杆菌电转步骤如下所示：吸取100μl感受态细胞至电转杯中，加入甲基化后的混合质粒，拿出电转；电转程序为2200v，400Ω；电转后，在电击杯中加入1ml YTF，混合后吸入2ml离心管，厌氧箱中复苏6h；5000rpm离心4min，吸出900μl，剩余菌液混匀涂板；正放至厌氧箱，1小时后翻板，生长3-4天，挑取转化子，即得到重组菌株 BM/pXY1-mtaA/mtaB/mtaC2。
实施例3：重组菌株BM/pXY1-mtaA/mtaB/mtaC2发酵实验
在平板上挑取重组菌株BM/pXY1-mtaA/mtaB/mtaC2单菌落，接种至含有红霉素的1ml YTF 培养基(蛋白胨16g/L，酵母粉12g/L，氯化钠4g/L，葡萄糖5g/L)中，培养12-16h后，将离心管中的菌液全部转接至安剖瓶中，生长至OD600为1左右，将菌液倒入50ml离心管中，4000rpm离心10min，弃去上清，用PB培养基重悬后以OD600＝0.1的接种量接种至50ml PB培养基中，再加入100mM甲醇,每隔24h，吸取2ml菌液，离心后将上清液转移至新的离心管保存，用于高效液相色谱检测甲醇、丁酸。用2ml超纯水重悬后检测其OD600，相较于重组菌株BM/pXY1，该重组菌株的最终OD600提高了25％。
其中，PB培养基的配方为：磷酸二氢钾4g/L，磷酸氢二钾6g/L，氯化铵1g/L，六水合氯化镁0.1g/L，二水合氯化钙0.1g/L，酵母粉3g/L，微量元素溶液2％(V/V)，维生素溶液2％(V/V)；
微量元素溶液：次氮基三乙酸1.5g，七水合硫酸镁3g，氯化钠1g，一水合硫酸锰0.5g，七水合硫酸亚铁0.1g，七水合硫酸钴0.18g，二水合氯化钙0.1g，七水合硫酸锌0.18 g，五水合硫酸铜0.01g，十二水和硫酸铝钾0.02g，硼酸0.01g，二水合钼酸钠0.01g，六水合氯化镍0.025g，五水合亚硒酸钠0.3mg，超纯水定容至1L。
维生素溶液：生物素2mg，叶酸2mg，吡哆醇盐酸10mg，二水合硫铵盐酸盐5mg，核黄素5mg，烟酸5mg，泛酸钙5mg，钴胺素0.1mg，对氨基苯甲酸5mg，硫辛酸5mg，超纯水定容至1L。
高效液相色谱法检测方法为：安捷伦高效液相色谱，色谱柱为Biorad HPX-87H，流动相为0.8g/L的H2SO4溶液，柱温为60℃，进样体积为20μL，流速为0.5mL/min，甲醇检测器为示差检测器。丁酸检测器为紫外检测器。
实施例4：构建重组质粒pXY1-atoB/paaH/crt/bcd
丁酸合成途径基因(atoB/paaH/crt/bcd)碱基序列(即丁酸合成途径相关基因的核苷酸序，如SEQ NO.2 所示)：
gtggcaaaagaagtagtattagctggtgctgtacgtacagcgattggtagttttggcggttctttagcaaacgttccggt agtcgatcttggaacaatcgttattaaagaagctttaaaccgtgctggcgttaaaccggaagacgttgatgaagtgttaatggggtgtgtattacaggcggctcagggacagagtgttgcccgtcagtctgctgtaaatgctggtattcctgttgaggtt cctgctttaacccttaacaatttatgtggttctggtctcaaatgtatcaatcttgcagctgctatgatccaggctggagaagcagatattattgttgctggtggtatggaaagcatgtccggcgctgcttacgctgttcctaagggacgctatggctaca gaatgggcgatggccagttcatcgacaccatgatcaaagacggtttaaccgatgccttcaatcactatcacatgggtatcaccgctgaaaatgtagcagaacagtatgatgtaacccgcgaagatcaggatgatttcgccgctaagagccagcagaaatg tgaagccgctcaggcagctggccgttttgatgatgaaatcgtaccggttccggttaaagttaaaaaagaaattgttgaattcaaagttgatgaattcccaagaaaaggcgtaaccgctgaaggcatcagcaaaatgcgtccggctttcaaaaaagacggt accgtaaccgctgcaaatgcttctggtatcaatgacggcgctgctgccatcgttgtgatgtctgctgaaaaagcaaaagaattaggtgttaagccaatggctaaatttgttgtcggcgcttccgctggtgttgatccatccatcatgggtgttggaccaa tcttctcaagccgtaaagctttagaaaaagctggtttaaccattgacgatatggatttagttgaagcaaacgaagctttcgcagcacagtcctgtgctgtaggcaaaactttaaatattcctgaagataaattaaatgtaaacggcggcgcgatcgctct gggtcacccggttggtgcttctggctgccgtatcatggtaaccttactgcacgaaatgcagaaacgcggtgctaaaaaaggtcttgcaaccttatgcgtaggcggcggtatgggtgtatctaccatcgtagaaatggactaatttttctaaggagagaaggtaattacaatgggttttgttaaatatgaaccacagggtgcagtggctgttatcaccatcgaccgtgaaaaggctttaaatgctttaaatagtgaagtgcttgaagatctggacaaggttattgacggtgttgatttagagaccattcgctgcctgatta ttacaggtgccggtcagaaatcttttgttgccggtgcggacatcggtgaaatgagcagcctgactcaggctgaaggcgaagcttttggtaaaaaaggaaacgctgttttcagaaagatcgagactttaccaattccggttatcgctgcggttaacggctt tgcacttggcggcggctgcgaattatccatgtcctgcgacattcgtctggcttctgaaaatgcaacctttggccagcctgaagttggccttggcatcactgctggtttcggtggcactcagcgtcttgcacgtctgatcccgaccggtaaagcaaaggaa atgctctatgcctgcaccaatatcaaagcggccgacgctttgagctggggtctggttaatgctgtctacccggcagacgaattaatgcctgctgcattgaaattggctggtaaaattgctaacaacgctccaattgctgtccgtaacaccaaaaaagcca tcaatgacggccttgaaatgggtatggacgacgcaattgcctttgaagcaaaacagtttggcggatgttttgaatccgctgaccaaaaagaaggcatggctgccttcctggaaaaacgtaaacacgaacctttccaaaacaaataaatatgattttcaac tcaagggtatttttgtataaaaatacccttgagtttgggcttttcgcctgtatatacaaatctgtatatacaagaatacaaaaaagtttttaatttatctaggaggacttaaaatgaaagttggcgttattggtgccggtacaatgggatcaggtattgc tcaggttttcgcttctaccgatggttatgaagttgtactttgcgatatcaaacaggaatttgccgatggtgggaaagctaaaatcgaaaaagcattagcaaaacaggttgctaagggccgtatcgatcaggctaaaatggatgcaaccttagcaaaaatc acaacaggtttaagagatgctgttgcggattgcgatttggttgttgaagctgttttagaacagatggaaatgaaacatgaattattccaggcattacagggaatctgtaaaccagaatgtattttcgcttccaatacttcttctttatctttaaccgaaa tgtctcagggcgttgaccgtccagtgattggtatgcatttctttaatccggttccggctatgaaactggttgaagttattgctggttaccatacttctcaggaaaccgttgataccatcaaaaagattgcaacagatatcggcaaaactccggtacaggt taacgaagctgcaggttttgttgtaaacagaatcttagttccaatgatcaacgaaggtatcgaagtttatgctgctggtactgcttccgctgcggatatcgacactgctatgaaattaggcgcaaaccacccaatgggaccactggctttaggcgactta attggtctggacgttgttctggcaattatggaagtattacaggcagaaactggctctgacaaatacgctccgtctccactgcttcgcaaaatggtacgcgcaggcgttttaggtatgaaaacaggaaaaggattctttgattacacaaaataagcttgtg tgacaaatatatttaggaggaacagtatggacttcaatctgagtaaggaacatcaaatgttgcgcacactctacagagagtttgcagaaaatgaagcaaaaccaatcgcgcaggaagttgacgaagaagaacgcttcccgcaggaaaccgttgataaaat ggtcaaaaatggctttatgggcattccatttgccaaagaagtcggtggacagggctgtgatacattagcttatattttagctgttgaagagttatcccgcgtttgcggtactaccggcgttatcctttccgcacacacttcactgggaacagacccaatc cgtaaattcggtacaccggaacaaaaagaaaaatacttaccgcgtttagcaagcggtgaattattaggcgctttcggcttaactgaaccaggcgctggtactgatgcttccggacagcagacaaaggctgttttagaaggcgaccactatgtattaaacg gtacgaaaatcttcattaccaacggtggtaaagcagatgtttatatcatcttcgcaatgacagataagagcaaaggcaccaaggggatctctgcattcatcgtagaaaaagattatcctggcttctcaatcggtacaaaagaaaagaaaatgggtatccg tggttcttccacaaccgaattaatttttgaagactgcatcgttccaaaagaaaatcttcttggtaaagaaggtaaaggctttggaatcgcgatgcagactctggacggcggccgtatcggtatcgctgctcaggctttaggtctggctcagggcgctttc gacgaaaccgttgcttacgttaaagaaagaaaacagtttggtcgctcaattgctaaattccagaacacacagtttaaattagccgatatgtacgcacgtatcgaagctgcccgtaacctggtttacaaagcagctattgctaaggatactcagaaagtattctctgtagaagcagcaactgctaaacttttcgcagctgaaactgctatggctgttaccacagaatgtgtacagttacttggtggttatggctacaccagagactatccagttgaacgtatgatgcgtgatgctaagattaccgaaatttatgaaggaac aagcgaggtacaacgtatggttatatctggcaacgttctgaaatag
重组质粒pXY1-atoB/paaH/crt/bcd的构建：以食甲基丁酸杆菌的全基因组为模板，通过常规 PCR扩增丁酸合成途径操纵子atoB/paaH/crt/bcd，
所用的上游引物Tchb-HR-F扩增产物物上游含有同源臂，序列如下： GTTAGTTAGAGGATCGTGGCAAAAGAAGTAGTATTAGCT；
所用的下游引物Tchb-HR-R使扩增产物下游含有同源臂，序列如下: TGAGAGTGCACCATACTATTTCAGAACGTTGCCAGA,
反应条件为：95℃3min，95℃15s，57℃15s，72℃4min，共30个循环；72℃5min。得到的基因片段经1％琼脂糖凝胶电泳后回收相应片段。将载体pXY1用Takara公司的BamHI和NdeⅠ酶切，酶切反应体系为：10×buffer 5μL，BamHI 5μL，NdeⅠ5μL，pXY1载体30 μL，ddH2O5μL。酶切体系于30℃条件下反应1小时后再于37℃条件下反应1小时。将酶切产物经1％琼脂糖凝胶电泳后回收得到线性化的片段。将线性化的质粒和纯化的目的片段通过同源重组连接，同源重组所需试剂和酶来自Vazyme公司，体系为线性化载体pXY1 0.5 μL，目的基因1μL，5×CE II Buffer 4μL，Exnase II 2μL,ddH2O 12.5μL.同源重组反应在37℃的情况下反应1h。将连接产物转化大肠杆菌Trans1-T1。PCR筛选阳性菌株 pXY1-atoB/paaH/crt/bcd并进行DNA测序，验证重组质粒构建正确。重组菌株BM/pXY1-atoB/paaH/crt/bcd的构建和发酵参考案例2和3。相较于重组菌株BM/pXY1，其甲醇消耗量以及丁酸的产量分别增加了14％和28.6％。
综上所述，这表明针对特定的代谢途径改造，例如甲醇利用途径和丁酸合成途径，能够提高甲醇的消耗和丁酸的合成。这表明在食甲基丁酸杆菌种进行代谢工程是可行的。为将来利用甲醇和CO2生产高附加值产物提供了一个潜在的底盘细胞和基因改造工具。
以上所述，仅为本发明较佳的具体实施方式，本发明的保护范围不限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
序列表
<110> 南京工业大学
<120> 一种重组食甲基丁酸杆菌及其构建方法与应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3020
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggcaaaga aatttgataa actggcaatt aataatctgg acgattttat ttatggctct 60
tgtccgaacc ctgtcaccac caggagcggc atggtcatcg gcggcggcac catctatccg 120
gaaatcaact tcacactgcc gggcatggat gtcaatgatc agaccattga caaggccttg 180
ggcatttatt ccaatatcat cgacggtgtg ctcaagagag cggcagagct ctacgcgccc 240
ggcgtgctgg tagaatttga aaccgtgccg gactttaccg agcatccaaa atatgggatt 300
gacgccaacc gcattttatt aaatggcatc aaggaagccg cagacaagta cggcctcaag 360
gccgccctgc ggaccacccc caacgacctg cgcgaaatga gccgtcctcc ggttatgcgc 420
ggcggcaagt actgggatac catgctggag ctgtacgaac agtgcgccaa ggatggttca 480
gactttttat ccatcgaatc gaccgggggc aaggaaatca atgacgaagc cctcgtaaag 540
gccgatatcc gcaaagccat cttcgccatg ggcgtgctgg gctgccgcga catggaatac 600
ctctggggca atctggttaa attatccgat gctaatggct gcttcgccgc tggcgactct 660
gcctgtggct ttgccaacac cgccatggtt ctggccgaaa aaggctttat cccccatgtg 720
ttcgcagcgg ttatgcgtgt tgtggcagtg ccgagagccc tggtggcctt tgaacagggc 780
gcggttggcc cgagcaagga ctgcgcctat gaaggcccat acctcaaggc cattaccggc 840
agtcccatcg ccatggaagg taagagcgcg gctggcgccc atttaagccc agttggcaac 900
atcgcggcag ccgtggccga tacctggagt aatgaatcca tccagcaggt caagctctta 960
tccgagatgg cccctgtggt gggcatggaa cagctggtat atgactgccg tctcatgaac 1020
gtggccaagg aaaaaggcca gggccttatg atgcgcgacc tgctcgttga atctgacgcg 1080
ccgctggatg tccaggcatg ggttttaaga cccgatgttg tgcttaagat cgccggcgag 1140
ctggtgaaag agcaggataa cttcctgaga accaaactgg ccgccaaatt aaccattaac 1200
gagctgcgcg acgccatcaa ggccgaaaag gttaaggctg accgccgcga catgaaatgg 1260
ctcgacaaga tggaaaaagc agtggacaag attccagacg atccggaaca gttctacgca 1320
gaaatcaagc ccgagctgga catggacaag tggcatccta aaggctatgg cttaaaggcc 1380
tgatcttaat attcttaaaa cagaaattca ctgttaaaaa ttataaaaat ttggacaatt 1440
ccctccggtg cggctcagta cacactgcgc gccggaagag caggtaaaca gctttaatca 1500
ggaataaaaa aatcggagga tataaaaatg gcaattttag aagatattca aaactgtgtg 1560
ttggacggcg aactggatga gatcaaggac ttggtgcaga aggcagtgga tgaggggatc 1620
gaccccgccg ccatcatcaa cgacggcctc atcggcggca tgaacattgt ggccccgctg 1680
tttaagagcg gtgaaatgtt tgtcccggaa gttatggaat ccgcggatac catgaacgaa 1740
ggcatgcagg tggttaagcc cctgatcacc gacgcggata tgcccaccaa gggcaaggtc 1800
atcatcggca ctgtcaacgg tgacctgcac gatatcggta aaaacctggt ggtcttaatg 1860
atggaaagcc gcggctacac agttattgac atgggcgtag atgtgaagga agaacagttt 1920
gtggaagcca tcaaagaaca caagccagac attgtgggca tgtcctcact gctcaccacc 1980
accatgatga agattgacga taccatcaag atgattaacg agtccggcct gcgtgaccag 2040
gttaagatca tcatcggcgg cgcacccatc tctcaggaat tcgccgatga tatcggggcg 2100
gacggctatt cagaggacgc ctccaccgcg gttgagctct gcgaccgcat gatggccatg 2160
tagcagacag cgaataaaat tttgacgaac aggaggcatt tcagatgctg acaattgttg 2220
gtgaactcat taacaccagc cgccctccgg tcaaggaggc ggtacagaat aaagacgaag 2280
cgttcatccg ggagcttgcc aaaaaacagg cagacgccgg cgcgacctat atcgacgtaa 2340
actgcggcaa tatggtcaag aatgaactgg aaataatgga atggcttgtg aacattgttc 2400
aggacgaggt cgacaccccc ctgtgcattg acagccccaa cgccaaagca ctggacgtgg 2460
gtctggccct gtgcaggaac ggccgtccca tgatcaattc catctccgac gaggacgggc 2520
gttatgaatc cgttcttccc ttaattaaaa aatataacgc caaaatcgtt gtattgtgca 2580
tggactccac tggtatgccc gaaacctcag ccgaccgcat gaaggtggtc aataaccttt 2640
atgccaagct gaaggccgag ggcatcgccg atgacgacat gtactttgac ccgctggtca 2700
agcccatcag cagcgtgacc agtgccggag cagaggtgct cgacaccatc cgccagatca 2760
agcaggatta ccccgatgta cacttcatgt gcggtttgag caacatttcc tatggcctgc 2820
cgaaccgcag catcctcaat cggctgtttg ttgtccagac aatgaccctg gggatggacg 2880
gctacgtcct tgaccccacc aatggcaaaa tgatggcgga catcatcacc gccaccgcgc 2940
tcctgggcag agacagctat tgcagtaaat acattaaagc ccatagaaag ggcaaacttg 3000
acgcatcgtc tgatgcgtaa 3020
<210> 2
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cgggatccat ggcaaagaaa tttgataaac tgg 33
<210> 3
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggaattccat atgttacgca tcagacgatg cg 32
<210> 4
<211> 4126
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gtggcaaaag aagtagtatt agctggtgct gtacgtacag cgattggtag ttttggcggt 60
tctttagcaa acgttccggt agtcgatctt ggaacaatcg ttattaaaga agctttaaac 120
cgtgctggcg ttaaaccgga agacgttgat gaagtgttaa tggggtgtgt attacaggcg 180
gctcagggac agagtgttgc ccgtcagtct gctgtaaatg ctggtattcc tgttgaggtt 240
cctgctttaa cccttaacaa tttatgtggt tctggtctca aatgtatcaa tcttgcagct 300
gctatgatcc aggctggaga agcagatatt attgttgctg gtggtatgga aagcatgtcc 360
ggcgctgctt acgctgttcc taagggacgc tatggctaca gaatgggcga tggccagttc 420
atcgacacca tgatcaaaga cggtttaacc gatgccttca atcactatca catgggtatc 480
accgctgaaa atgtagcaga acagtatgat gtaacccgcg aagatcagga tgatttcgcc 540
gctaagagcc agcagaaatg tgaagccgct caggcagctg gccgttttga tgatgaaatc 600
gtaccggttc cggttaaagt taaaaaagaa attgttgaat tcaaagttga tgaattccca 660
agaaaaggcg taaccgctga aggcatcagc aaaatgcgtc cggctttcaa aaaagacggt 720
accgtaaccg ctgcaaatgc ttctggtatc aatgacggcg ctgctgccat cgttgtgatg 780
tctgctgaaa aagcaaaaga attaggtgtt aagccaatgg ctaaatttgt tgtcggcgct 840
tccgctggtg ttgatccatc catcatgggt gttggaccaa tcttctcaag ccgtaaagct 900
ttagaaaaag ctggtttaac cattgacgat atggatttag ttgaagcaaa cgaagctttc 960
gcagcacagt cctgtgctgt aggcaaaact ttaaatattc ctgaagataa attaaatgta 1020
aacggcggcg cgatcgctct gggtcacccg gttggtgctt ctggctgccg tatcatggta 1080
accttactgc acgaaatgca gaaacgcggt gctaaaaaag gtcttgcaac cttatgcgta 1140
ggcggcggta tgggtgtatc taccatcgta gaaatggact aatttttcta aggagagaag 1200
gtaattacaa tgggttttgt taaatatgaa ccacagggtg cagtggctgt tatcaccatc 1260
gaccgtgaaa aggctttaaa tgctttaaat agtgaagtgc ttgaagatct ggacaaggtt 1320
attgacggtg ttgatttaga gaccattcgc tgcctgatta ttacaggtgc cggtcagaaa 1380
tcttttgttg ccggtgcgga catcggtgaa atgagcagcc tgactcaggc tgaaggcgaa 1440
gcttttggta aaaaaggaaa cgctgttttc agaaagatcg agactttacc aattccggtt 1500
atcgctgcgg ttaacggctt tgcacttggc ggcggctgcg aattatccat gtcctgcgac 1560
attcgtctgg cttctgaaaa tgcaaccttt ggccagcctg aagttggcct tggcatcact 1620
gctggtttcg gtggcactca gcgtcttgca cgtctgatcc cgaccggtaa agcaaaggaa 1680
atgctctatg cctgcaccaa tatcaaagcg gccgacgctt tgagctgggg tctggttaat 1740
gctgtctacc cggcagacga attaatgcct gctgcattga aattggctgg taaaattgct 1800
aacaacgctc caattgctgt ccgtaacacc aaaaaagcca tcaatgacgg ccttgaaatg 1860
ggtatggacg acgcaattgc ctttgaagca aaacagtttg gcggatgttt tgaatccgct 1920
gaccaaaaag aaggcatggc tgccttcctg gaaaaacgta aacacgaacc tttccaaaac 1980
aaataaatat gattttcaac tcaagggtat ttttgtataa aaataccctt gagtttgggc 2040
ttttcgcctg tatatacaaa tctgtatata caagaataca aaaaagtttt taatttatct 2100
aggaggactt aaaatgaaag ttggcgttat tggtgccggt acaatgggat caggtattgc 2160
tcaggttttc gcttctaccg atggttatga agttgtactt tgcgatatca aacaggaatt 2220
tgccgatggt gggaaagcta aaatcgaaaa agcattagca aaacaggttg ctaagggccg 2280
tatcgatcag gctaaaatgg atgcaacctt agcaaaaatc acaacaggtt taagagatgc 2340
tgttgcggat tgcgatttgg ttgttgaagc tgttttagaa cagatggaaa tgaaacatga 2400
attattccag gcattacagg gaatctgtaa accagaatgt attttcgctt ccaatacttc 2460
ttctttatct ttaaccgaaa tgtctcaggg cgttgaccgt ccagtgattg gtatgcattt 2520
ctttaatccg gttccggcta tgaaactggt tgaagttatt gctggttacc atacttctca 2580
ggaaaccgtt gataccatca aaaagattgc aacagatatc ggcaaaactc cggtacaggt 2640
taacgaagct gcaggttttg ttgtaaacag aatcttagtt ccaatgatca acgaaggtat 2700
cgaagtttat gctgctggta ctgcttccgc tgcggatatc gacactgcta tgaaattagg 2760
cgcaaaccac ccaatgggac cactggcttt aggcgactta attggtctgg acgttgttct 2820
ggcaattatg gaagtattac aggcagaaac tggctctgac aaatacgctc cgtctccact 2880
gcttcgcaaa atggtacgcg caggcgtttt aggtatgaaa acaggaaaag gattctttga 2940
ttacacaaaa taagcttgtg tgacaaatat atttaggagg aacagtatgg acttcaatct 3000
gagtaaggaa catcaaatgt tgcgcacact ctacagagag tttgcagaaa atgaagcaaa 3060
accaatcgcg caggaagttg acgaagaaga acgcttcccg caggaaaccg ttgataaaat 3120
ggtcaaaaat ggctttatgg gcattccatt tgccaaagaa gtcggtggac agggctgtga 3180
tacattagct tatattttag ctgttgaaga gttatcccgc gtttgcggta ctaccggcgt 3240
tatcctttcc gcacacactt cactgggaac agacccaatc cgtaaattcg gtacaccgga 3300
acaaaaagaa aaatacttac cgcgtttagc aagcggtgaa ttattaggcg ctttcggctt 3360
aactgaacca ggcgctggta ctgatgcttc cggacagcag acaaaggctg ttttagaagg 3420
cgaccactat gtattaaacg gtacgaaaat cttcattacc aacggtggta aagcagatgt 3480
ttatatcatc ttcgcaatga cagataagag caaaggcacc aaggggatct ctgcattcat 3540
cgtagaaaaa gattatcctg gcttctcaat cggtacaaaa gaaaagaaaa tgggtatccg 3600
tggttcttcc acaaccgaat taatttttga agactgcatc gttccaaaag aaaatcttct 3660
tggtaaagaa ggtaaaggct ttggaatcgc gatgcagact ctggacggcg gccgtatcgg 3720
tatcgctgct caggctttag gtctggctca gggcgctttc gacgaaaccg ttgcttacgt 3780
taaagaaaga aaacagtttg gtcgctcaat tgctaaattc cagaacacac agtttaaatt 3840
agccgatatg tacgcacgta tcgaagctgc ccgtaacctg gtttacaaag cagctattgc 3900
taaggatact cagaaagtat tctctgtaga agcagcaact gctaaacttt tcgcagctga 3960
aactgctatg gctgttacca cagaatgtgt acagttactt ggtggttatg gctacaccag 4020
agactatcca gttgaacgta tgatgcgtga tgctaagatt accgaaattt atgaaggaac 4080
aagcgaggta caacgtatgg ttatatctgg caacgttctg aaatag 4126
<210> 5
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gttagttaga ggatcgtggc aaaagaagta gtattagct 39
<210> 6
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tgagagtgca ccatactatt tcagaacgtt gccaga 36
Claims (4)
1.一种重组食甲基丁酸杆菌在甲醇发酵产丁酸上的应用，其特征在于，所述重组食甲基丁酸杆菌的构建方法，其特征在于，由如下步骤组成：(1)构建含有丁酸合成途径相关基因的质粒pXY1-atoB/paaH/crt/bcd；(2)对重组质粒pXY1-atoB/paaH/crt/bcd进行甲基化修饰；(3)再将甲基化修饰的重组质粒电转化进入食甲基丁酸杆菌(Butyribacterium methylotrophicum)，构建重组食甲基丁酸杆菌；
所述基因atoB,paaH,crt,bcd的Gene ID分别为BUME_RS03485, BUME_RS03475, BUME_RS03480, BUME_RS0347；
所述丁酸合成途径相关基因的核苷酸序列如SEQ NO.2所示；
所述步骤（2）为：将步骤1构建的重组质粒pXY1-atoB/paaH/crt/bcd以热激法转化进含有pMCljs质粒的大肠杆菌Top10感受态中，随后培养含有pMCljs和重组质粒的大肠杆菌，并提取总质粒，完成重组质粒pXY1-atoB/paaH/crt/bcd的甲基化修饰。
2.根据权利要求1所述的应用，其特征在于，所述步骤（1）重组质粒pXY1-atoB/paaH/crt/bcd的构建方法为：使用引物Tchb-HR-F/Tchb-HR-R进行PCR扩增来源于食甲基丁酸杆菌的甲基转移酶相关基因atoB/paaH/crt/bcd，将载体线性化后，通过同源重组的方法连接到载体pXY1上，获得重组质粒pXY1-atoB/paaH/crt/bcd；其中，所述引物Tchb-HR-F扩增产物物上游含有同源臂，序列如下：GTTAGTTAGAGGATCGTGGCAAAAGAAGTAGTATTAGCT；所述引物Tchb-HR-R使扩增产物下游含有同源臂，序列如下: TGAGAGTGCACCATACTATTTCAGAACGTTGCCAGA。
3.根据权利要求1所述的应用，其特征在于，将甲基化修饰的重组质粒通过电转的方式转化进食甲基丁酸杆菌中，电转程序为2200v，400Ω；在厌氧箱中以37℃培养3-4天，菌落PCR验证后获得重组菌株。
4.根据权利要求1所述的应用，其特征在于，在平板上挑取重组食甲基丁酸杆菌的单菌落，接种至含有红霉素的1ml YTF培养基中，培养12-16h后，将离心管中的菌液全部转接至安剖瓶中，生长至OD600为1-1.2，将菌液倒入50mL离心管中，4000rpm离心10min，弃去上清，用PB培养基重悬后以OD600＝0.1的接种量接种至50mL PB培养基中，再加入100mM甲醇,每隔一定时间，吸取2mL菌液，离心后将上清液转移至新的离心管保存，用于高效液相色谱检测甲醇、丁酸，用2mL超纯水重悬后检测其OD600。
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