CN114134092A - 一种可高效利用甲醇的重组微生物及其应用 - Google Patents
一种可高效利用甲醇的重组微生物及其应用 Download PDFInfo
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Abstract
本发明涉及基因工程和生物发酵技术领域，具体公开了一种可高效利用甲醇的重组微生物及其应用。本发明提供了一种重组微生物，所述重组微生物与出发菌株相比，具有升高的甲醇脱氢酶和二羟基丙酮合成酶的表达和/或酶活性和降低的甲醛异化基因frmAB表达量和6‑磷酸果糖激酶的表达和/或酶活性；所述出发菌株为大肠杆菌。具体同时敲除甲醛异化基因frmAB、6‑磷酸果糖激酶基因pfkAB和α‑酮戊二酸脱氢酶基因sucA，抑制3‑磷酸甘油醛脱氢酶基因gapA后可进一步提高甲醇利用率，降低中间体甲醛的积累。
Description
技术领域
本发明涉及基因工程和生物发酵技术领域，具体地说，涉及一种可高效利用甲醇的重组微生物及其应用。
背景技术
发展绿色生物制造技术是解决能源环境问题、实现“碳中和”目标的有效途径之一，其核心是构建微生物细胞工厂，高效合成大宗及高值化学品，现已在医药、化工、食品等领域有重要应用。但大多数微生物法生产化学品都依赖糖基原料，存在与人争粮、价格竞争等问题。液态甲醇作为一种基础化工原料，可从天然气、页岩气等制备，价格低廉、储存及运输条件简单，作为生物制造原料有巨大潜力。且甲醇平均碳原子还原度高，为醇、酸等有机化合物的合成提供更强还原力。开发基于甲醇的生物制造工业对于环境保护及经济产能都具有重大意义。
目前基于甲醇的生物利用主要集中在改造天然甲基营养菌、在模式生物中引入外源甲醇利用途径两大类。如何使得改造菌能更高效地利用甲醇仍有必要进行进一步研究。
发明内容
本发明的目的是提供一种可高效利用甲醇的重组微生物。
本发明的技术方案如下：
一种重组微生物，所述重组微生物与出发菌株相比，具有升高的甲醇脱氢酶和二羟基丙酮合成酶的表达和/或酶活性和降低的甲醛异化基因frmAB表达量和6-磷酸果糖激酶的表达和/或酶活性；所述出发菌株为大肠杆菌。
优选，所述升高的甲醇脱氢酶和二羟基丙酮合成酶的表达和/或酶活性，通过过表达mdh基因和DAS基因实现；所述mdh基因的核苷酸序列如SEQ ID No：1所示，所述DAS基因的核苷酸序列如SEQ ID No：2所示。
优选，所述降低的甲醛异化基因frmAB表达量和6-磷酸果糖激酶的表达和/或酶活性，通过敲除所述甲醛异化基因frmAB和6-磷酸果糖激酶基因pfkAB实现；所述甲醛异化基因frmAB的核苷酸序列如SEQ ID No：3-4所示，所述6-磷酸果糖激酶基因pfkAB的核苷酸序列如SEQ ID No：5-6所示。
本发明在大肠杆菌中通过组合引入甲醇脱氢酶和二羟基丙酮合成酶基因，实现了甲醇的初步利用。并通过减少甲醛异化途径(敲除甲醛异化基因frmAB)，敲除大肠杆菌底盘原有竞争途径(糖酵解途径中的6-磷酸果糖激酶基因pfkAB)，进一步提高甲醇利用。
本发明中，所述重组微生物进一步具有降低的α-酮戊二酸脱氢酶的表达和/或酶活性。
优选，所述降低的α-酮戊二酸脱氢酶的表达和/或酶活性，通过敲除α-酮戊二酸脱氢酶基因sucA实现，所述α-酮戊二酸脱氢酶基因sucA的核苷酸序列如SEQ ID No：7所示。
本发明中，所述重组微生物还具有降低的3-磷酸甘油醛脱氢酶的表达和/或酶活性。
优选，所述降低的3-磷酸甘油醛脱氢酶的表达和/或酶活性，通过抑制3-磷酸甘油醛脱氢酶基因gapA实现，所述3-磷酸甘油醛脱氢酶基因gapA的核苷酸序列如SEQ ID No：8所示。
本发明还通过进一步敲除或弱化大肠杆菌底盘原有竞争途径(敲除三羧酸循环中的α-酮戊二酸脱氢酶基因，抑制3-磷酸甘油醛脱氢酶基因)，进一步提高甲醇利用率。
本发明还提供一种上述重组微生物的如下任一种应用：
(1)在生物法转化甲醇中的应用；
(2)在用于转化甲醇的微生物遗传育种中的应用；
(3)在提高甲醇合成生物量的效率中的应用。
本发明另提供一种生物法转化甲醇的方法，其包括培养上述的重组微生物的步骤。
本发明的有益效果至少在于：
本发明通过对重组大肠杆菌进行系统的改造和创新，通过特定设计引入甲醇代谢反应并进行底盘适配改造，使得改造的大肠杆菌利用甲醇的生物效率显著提高，降低中间体甲醛积累，实现从甲醇的高效生物转化，且本发明重组微生物可显著提高生物量，具有十分广阔的应用前景。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1在大肠杆菌中引入甲醇转化路径
本实施例在大肠杆菌W3110(购自中国工业微生物菌种保藏中心)中通过引入质粒pTrc99a-mdh-das获得能够利用甲醇的底盘菌株X1/mdh-das。
质粒pTrc99a-mdh-das的构建方法：人工合成mdh和das基因，其基因序列分别如SEQ ID No：1、SEQ ID No：2所示。利用Gibson组装的方法将mdh和das基因插入到质粒pTrc99a(购自addgene)的EcoRI/SmaI酶切位点处，获得质粒pTrc99a-mdh-das。
大肠杆菌X1/mdh-das的构建方法：将质粒pTrc99a-mdh-das电转入大肠杆菌W3110中，在包含100mg/L氨苄青霉素的LB平板上获得抗性菌株，命名为X1/mdh-das。
将大肠杆菌X1/mdh-das在500mL的摇瓶进行培养，培养基为包含甲醇的M9培养基(无水磷酸氢二钠6.8g/L，磷酸二氢钾3g/L，氯化钠0.5g/L，氯化铵1g/L，酵母粉1g/L，甲醇400mmol/L，七水合硫酸镁2.0mmol/L，氯化钙0.1mmol/L，六水合氯化铁0.00675g/L，四水合氯化锌0.0005g/L，六水合氯化钴0.0005g/L，二水合锰酸钠0.0005g/L，六水合氯化铜0.000325g/L，硼酸0.000125g/L，氨苄青霉素100mg/L，IPTG 1mmol/L)和不包含甲醇的M9培养基(无水磷酸氢二钠6.8g/L，磷酸二氢钾3g/L，氯化钠0.5g/L，氯化铵1g/L，酵母粉1g/L，七水合硫酸镁2.0mmol/L，氯化钙0.1mmol/L,六水合氯化铁0.00675g/L，四水合氯化锌0.0005g/L，六水合氯化钴0.0005g/L，二水合锰酸钠0.0005g/L，六水合氯化铜0.000325g/L，硼酸0.000125g/L，氨苄青霉素100mg/L，IPTG 1mmol/L)，培养温度为37度，转速200rpm，发酵每隔12小时取样，利用可见光分光光度计在600nm下检测不同条件下生长情况OD值。在包含甲醇的M9培养基中，菌体24小时、48小时OD600nm分别可达1.211、1.301；在不含甲醇的M9培养基中，菌体24小时、48小时生长OD600nm分别可达0.937、1.008。因此证实通过引入mdh和DAS基因可以有效提高大肠杆菌生物量，证明了非糖基原料在大肠杆菌中利用的可行性。
实施例2通过改造大肠杆菌自身的代谢进一步提高甲醇利用
甲醇利用途径受胞内原有甲醛异化途径限制，大部分转化为二氧化碳而非进入中央碳代谢。本实施例利用Red重组技术首先敲除甲醛异化基因frmAB(frmA基因核酸序列如SEQ ID No：3所示，frmB基因核酸序列如SEQ ID No：4所示)，可提高甲醇整合进入中央代谢循环比例。此外通过敲除6-磷酸果糖激酶基因pfkAB(pfkA基因核酸序列如SEQ ID No：5所示，pfkB基因核酸序列如SEQ ID No：6所示)和α-酮戊二酸脱氢酶基因sucA(sucA基因核酸序列如SEQ ID No：7所示)，弱化3-磷酸甘油醛脱氢酶基因gapA(gapA基因核酸序列如SEQID No：8所示)，可进一步提高甲醇利用。
甲醛异化基因(frmAB)的敲除方法：以frmAB-F(acgccagcaccag cgcgcccagcccgcccatagaatgaccagagatagacgtgtaggctggagctgcttc，SEQ ID No：9)和frmAB-R(cgtgaccaccggtattggcgcggtacacaacacagctaaagtccagc cagctgatcagtgataagctgtcaaacatgag，SEQ IDNo：10)为引物，以质粒pKD13(购自Addgene)为模板扩增获得1.4Kb的PCR片段，将该片段通过电转转入到包含质粒pSIJ8(购自Addgene)的大肠杆菌W3110中，在包含50mg/L的卡那霉素的LB平板上筛选获得抗性的菌株。挑取单克隆菌株在包含1％鼠李唐的LB培养基中过夜培养(42℃)，获得抗性消失的菌株，命名为X2。
6-磷酸果糖激酶基因1(pfkA)的敲除方法：以pfkA-F(caatggccgcagccaacgtcagatctccacaataacggcccatcacttccgtgtaggctggagctgcttc，SEQ ID No：11)和pfkA-R(agacttccggcaacagatttcattttgcattccaaagttcagaggtagtcctgatcagtgataagctgtcaaacatgag,SEQID No：12)为引物，以质粒pKD13(购自Addgene)为模板扩增获得1.4Kb的PCR片段，将该片段通过电转转入到包含质粒pSIJ8(购自Addgene)的大肠杆菌X2中，在包含50mg/L的卡那霉素的LB平板上筛选获得抗性的菌株。挑取单克隆菌株在包含1％鼠李唐的LB培养基中过夜培养(42℃)，获得抗性消失的菌株，命名为X3。
6-磷酸果糖激酶基因2(pfkB)的敲除方法：以pfkB-F(actttccgctgattcggtgccagactgaaatcagcctataggaggaaatgctgatcagtgataagctgtcaaacatgag，SEQ ID No：13)和pfkB-R(ccaactcgatgttaccaattgccagtgctgcacttaacgcttcgccagaagtgtaggctggagctgcttc，SEQID No：14)为引物，以质粒pKD13(购自Addgene)为模板扩增获得1.4Kb的PCR片段，将该片段通过电转转入到包含质粒pSIJ8(购自Addgene)的大肠杆菌X3中，在包含50mg/L的卡那霉素的LB平板上筛选获得抗性的菌株。挑取单克隆菌株在包含1％鼠李唐的LB培养基中过夜培养(42℃)，获得抗性消失的菌株，命名为X4。
α-酮戊二酸脱氢酶基因(sucA)的敲除方法：以sucA-F(aagatgcttaagggatcacgctgatcagtgataagctgtcaaacatgag，SEQ ID No：15)和sucA-R(gtgcatatactcggcaccaagtgtaggctggagctgcttc，SEQ ID No：16)为引物，以质粒pKD13(购自Addgene)为模板扩增获得1.4Kb的PCR片段，将该片段通过电转转入到包含质粒pSIJ8(购自Addgene)的大肠杆菌X4中，在包含50mg/L的卡那霉素的LB平板上筛选获得抗性的菌株。挑取单克隆菌株在包含1％鼠李唐的LB培养基中过夜培养(42℃)，获得抗性消失的菌株，命名为X5。
弱化3-磷酸甘油醛脱氢酶基因(gapA)质粒的构建方法：以vector-F(gagaatccacccggggatcctctagagtcg，SEQ ID No：17)和vector-R(cattatacgagccggatgattaattgtcaattaaagtttattcactttgtcgtggtttggc，SEQ ID No：18)为引物，以质粒pTrc99a-mdh-das为模板扩增获得7.5Kb的PCR片段。以fragment-F(ttgacaattaatcatccggctcgtataatgaaaaccgttgatacctactttgatagtcatcgtcccgcaaggatgcg，SEQ ID No：19)和fragment-R(gactctagaggatccccgggtggattctcaccaataaaaaacgcccg，SEQ ID No：20)为引物，以质粒pKMV-antisenRNA2(购自Addgene)为模板扩增获得0.2Kb的PCR片段。利用Gibson组装的方法将两片段组装，获得质粒pTrc99a-mdh-das-antigapA。该反义RNA基因序列与大肠杆菌W3110的gapA基因转录的RNA可特异性结合，与野生型大肠杆菌相比，导入该质粒的3-磷酸甘油醛脱氢酶活性可降低三分之一。
重组甲醇利用型大肠杆菌的构建方法：将实施例1制备的质粒pTrc99a-mdh-das分别电转入大肠杆菌X2、X4、X5中，在包含100mg/L氨苄青霉素的LB平板上获得抗性菌株，分别命名为X2/mdh-das、X4/mdh-das、X5/mdh-das。将质粒pTrc99a-mdh-das-antigapA电转入大肠杆菌X5，获得的重组菌株命名为X5/mdh-das-antigapA。
将大肠杆菌X1/mdh-das、X2/mdh-das、X4/mdh-das、X5/mdh-das、X5/mdh-das-antigapA在500mL的摇瓶进行培养，培养基为包含甲醇的M9培养基(参见实施例1)，培养温度为37度，转速200rpm，发酵每隔12小时取样。利用可见光分光光度计在600nm下检测不同条件下生长情况OD值。样品离心后取上清液与Nash试剂(5mol/L乙酸铵，50mmol/L乙酰丙酮)在37℃条件下反应一小时，利用酶标仪测得412nm处吸光值，检测甲醛的浓度。在包含甲醇的M9培养基中X1/mdh-das、X2/mdh-das、X4/mdh-das、X5/mdh-das、X5/mdh-das-antigapA菌株48小时生长OD600nm分别可达1.239、1.225、1.01、1.357、1.778，甲醛积累量分别为30.4μmol/L、59.8μmol/L、22.0μmol/L、18.4μmol/L、16.1μmol/L。这说明通过本发明的系统改造，可以将甲醛积累从59.8μmol/L显著降低至16.1μmol/L，约降低73％，可进一步促进利用甲醇进行生物量的合成。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 清华大学
<120> 一种可高效利用甲醇的重组微生物及其应用
<130> KHP211123867.1
<160> 20
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggcgttca agaacctggc ggatcagact aatggtttct acatcccgtg cgtcagcttg 60
ttcggtcctg ggtgtgcaaa agaagtgggg gccaaagcac aaaaccttgg ggctaaaaaa 120
gcgttgatcg tcaccgatgc agggcttttt aaattcggag tggcagacat catcgtaggg 180
taccttaaag atgcgggagt cgacagtcat gtgtttccgg gggcagaacc caatcccacg 240
gacattaatg tattgaatgg ggttcaggcc tataatgata atggatgcga ttttattgtt 300
tctctgggtg gaggcagctc acacgactgc gctaagggca ttgggctggt gacggctggt 360
gggggtaaca ttcgcgatta tgagggtatt gacaaatcgt cagtccccat gacaccctta 420
attgcgatca atacgaccgc tggaacagcc tcggaaatga cccgtttttg tattatcaca 480
aatacagata cccacgttaa gatggcaatc gtggattggc gctgtactcc tttggtcgct 540
attgacgacc cgaaattgat gattgctaaa ccggctgccc tgactgcagc tactggcatg 600
gatgcgctta cacatgcggt agaagcgtac gtgtcgactg cagccaaccc cattacagat 660
gcatgcgctg aaaaggcgat cagcatgatt tctgagtggt tgagttcagc agtagcaaac 720
ggggaaaaca tcgaagcgcg cgacgctatg gcttacgctc agtatcttgc gggtatggcg 780
tttaataatg cgtcgttagg atatgtgcac gccatggccc accagctggg tgggttttac 840
aatttacctc acggtgtatg taacgccatc ctgctgccac acgtatgcga gttcaatctt 900
attgcgtgcc ccgatcgttt tgcgaagatt gcacagctga tgggcgtaga tacaacaggt 960
atgacggtaa ctgaggcggg ttacgaggct attgcagcga ttcgcgagct ttcagccagt 1020
attgggattc cgagcgggct gaccgaactg ggggtaaaag ccgcagacca tgctgtaatg 1080
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atgtccatgc gcattcccaa agcggcgagc gtaaacgacg agcagcatca acgcattatc 60
aaatatggcc gcgccttggt cctggacatt gttgaacagt acgggggagg gcatcccgga 120
tcggccatgg gcgcgatggc aatcggcatt gcattatgga agtatacact taagtacgct 180
cctaatgatc caaattactt caatcgtgat cgcttcgttt tgagtaatgg tcacgtttgt 240
ctttttcagt acatcttcca gcacttgtat gggttaaagt ccatgacgat ggcgcagtta 300
aagtcttacc actcgaatga ctttcattca ttatgtcctg gccacccgga gatcgaacac 360
gatgcggttg aggtcactac cggaccgctg gggcagggaa tctctaactc ggttggtttg 420
gctatcgcca ccaagaacct tgccgcgacc tacaacaaac caggcttcga tatcattact 480
aataaagttt actgtatggt tggagatgca tgtttacaag aagggcctgc tctggaaagt 540
atcagtttgg ctggacacat ggggttggat aatttaattg tcctttacga caacaatcaa 600
gtatgctgcg atggctcggt cgacatcgca aataccgagg atatctcggc taaatttaag 660
gcgtgcaatt ggaatgtcat cgaggttgaa aatgccagtg aggacgttgc aaccatcgtc 720
aaggcgctgg aatatgcaca ggctgagaag caccgcccga ctttaattaa ttgtcgtact 780
gttatcggta gtggggcagc atttgagaac cactgtgcag ctcatgggaa cgctttaggc 840
gaagacgggg tacgcgagtt aaaaattaaa tacggaatga acccggcgca aaaattttat 900
attccgcagg atgtatatga ctttttcaaa gagaagccag ccgaaggtga caagctggtt 960
gccgagtgga aaagtcttgt ggccaagtac gtgaaggcat acccggaaga gggtcaagaa 1020
ttccttgccc gtatgcgcgg cgaattgccc aaaaactgga agtcgtttct tccccaacaa 1080
gagtttacag gagatgctcc cacacgcgcc gctgcccgtg agttggttcg cgcccttggt 1140
caaaattgta agtctgtaat cgctggatgt gccgacttgt ccgtttcggt gaatttacag 1200
tggcctggcg ttaagtactt catggatccc tcattatcca cgcagtgcgg gctttccgga 1260
gattattcgg gtcgttatat cgagtacggt attcgcgaac atgcgatgtg tgcgatcgct 1320
aacggattgg cagcctataa taaagggacg tttctgccca tcacgtcgac cttcttcatg 1380
ttctatttgt acgctgcgcc tgccattcgt atggcaggcc ttcaagagtt aaaggctatc 1440
catatcggga cgcacgactc aattaacgag ggtgaaaatg ggcccacaca tcaacccgtt 1500
gagtcaccag cgctgttccg cgctatgcca aatatttact acatgcgccc ggttgactcc 1560
gcagaagtgt tcggtctttt tcagaaggca gtcgaacttc cattctcctc catcctgtcg 1620
ctttcacgta atgaggtatt gcaatatcca gggaagtcct ccgcagagaa agcgcaacgc 1680
ggtgggtaca tcctggaaga cgcggagaat gccgaagtgc aaatcattgg agtcggggcc 1740
gaaatggaat ttgcttacaa ggctgcgaaa atccttggtc gcaagtttcg cactcgcgtt 1800
ttatcaatcc cttgcactcg tttatttgat gagcaatcga ttggataccg tcgtagcgtg 1860
cttcgtaagg atggccgcca ggtacccact gtagtagtgg atggccacgt cgcattcggc 1920
tgggaacgtt acgcaaccgc aagctattgt atgaatactt atggaaagtc attacctccc 1980
gaggtaattt atgaatactt tgggtataat ccggcgacta tcgcgaagaa ggtggaggct 2040
tacgtacgcg cttgccagcg cgacccctta ttattacatg atttcttaga cttaaaagag 2100
aagccaaacc acgacaaagt gaataaactt taa 2133
<210> 3
<211> 1110
<212> DNA
<213> 人工序列(Artificial Sequence)
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atgaaatcac gtgctgccgt tgcatttgct cccggtaaac cgctggaaat cgttgaaatt 60
gacgttgcac caccgaaaaa aggtgaagtg ctaattaaag tcacccatac cggcgtttgc 120
cataccgacg catttaccct ctccggcgat gacccggaag gtgtattccc ggtggttctc 180
ggtcacgaag gggccggcgt tgtggttgaa gtcggtgaag gcgtaaccag cgtcaaacct 240
ggcgaccatg tgatcccgct ttacaccgcg gagtgcggcg agtgtgagtt ctgtcgttct 300
ggcaaaacta acctctgtgt tgcggttcgc gaaacccagg gtaaaggcct gatgccagac 360
ggcaccaccc gtttttctta caacgggcag ccgctttatc actacatggg gtgctctaca 420
ttcagtgaat acaccgtagt cgcggaagtg tctctggcaa aaattaatcc agaagcaaac 480
catgaacacg tctgcctgct gggctgtggc gtgaccaccg gtattggcgc ggtacacaac 540
acagctaaag tccagccagg tgattctgtt gccgtgtttg gtcttggcgc gattggtctg 600
gcagtggttc agggcgcgcg tcaggcgaaa gcgggtcgga ttatcgctat cgataccaac 660
ccgaagaaat tcgatctggc gcgtcgcttc ggtgctaccg actgcattaa cccgaatgac 720
tacgacaaac cgataaaaga tgtcctgttg gatatcaaca aatggggtat cgaccatacc 780
tttgaatgca tcggtaacgt caacgtgatg cgtgcggcgc tggaaagtgc gcaccgcggc 840
tggggtcagt cggtgatcat cggggtcgcg gttgccggtc aggaaatctc cacccgtcca 900
ttccagttgg tcactggtcg cgtatggaaa ggttccgcgt ttggcggcgt gaaaggtcgt 960
tcccagttac cgggcatggt tgaagatgcg atgaaaggtg atatcgatct ggaaccgttt 1020
gtcacgcata ccatgagcct ggatgaaatt aatgacgcct tcgacctgat gcatgaaggc 1080
aaatccattc gaaccgtaat tcgttactga 1110
<210> 4
<211> 834
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggaactca ttgaaaaaca tgtcagcttt ggcggctggc aaaatatgta tcggcattat 60
tcccaatcac tgaaatgtga aatgaatgtc ggcgtctatc tcccaccaaa agccgcgaat 120
gaaaaattgc cggtgctgta ctggctttca ggcctgacct gcaacgagca gaatttcatt 180
actaaatcgg ggatgcagcg ttacgcggct gagcacaaca ttattgttgt tgcgccggac 240
accagtccgc gaggcagtca tgtcgcagat gctgaccgtt acgatctcgg gcaaggtgcc 300
gggttttacc tgaacgcgac gcaagcgccg tggaatgaac attacaaaat gtatgactat 360
atccgcaacg agctgccgga tttagtgatg catcattttc cggcaacggc caaaaagtct 420
atctctggtc attctatggg cgggctgggc gcgctggtgc tggcgttacg taacccagat 480
gaatatgtca gcgtctcggc gttttcgccc attgtctccc catcgcaagt gccgtgggga 540
cagcaagcct ttgctgcata tcttgctgaa aataaagatg cctggttgga ttacgacccg 600
gtgagtctta tttcacaagg tcaacgcgtt gcggaaatca tggttgatca ggggttgagt 660
gatgattttt acgcagaaca gctgcggact ccaaatcttg aaaagatctg ccaggagatg 720
aatatcaaga cgttaatccg ttatcacgag ggttatgatc acagctatta ttttgtctcc 780
agttttattg gcgagcatat tgcctaccac gccaataaac tgaatatgcg ttga 834
<210> 5
<211> 963
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgattaaga aaatcggtgt gttgacaagc ggcggtgatg cgccaggcat gaacgccgca 60
attcgcgggg ttgttcgttc tgcgctgaca gaaggtctgg aagtaatggg tatttatgac 120
ggctatctgg gtctgtatga agaccgtatg gtacagctag accgttacag cgtgtctgac 180
atgatcaacc gtggcggtac gttcctcggt tctgcgcgtt tcccggaatt ccgcgacgag 240
aacatccgcg ccgtggctat cgaaaacctg aaaaaacgtg gtatcgacgc gctggtggtt 300
atcggcggtg acggttccta catgggtgca atgcgtctga ccgaaatggg cttcccgtgc 360
atcggtctgc cgggcactat cgacaacgac atcaaaggca ctgactacac tatcggtttc 420
ttcactgcgc tgagcaccgt tgtagaagcg atcgaccgtc tgcgtgacac ctcttcttct 480
caccagcgta tttccgtggt ggaagtgatg ggccgttatt gtggagatct gacgttggct 540
gcggccattg ccggtggctg tgaattcgtt gtggttccgg aagttgaatt cagccgtgaa 600
gacctggtaa acgaaatcaa agcgggtatc gcgaaaggta aaaaacacgc gatcgtggcg 660
attaccgaac atatgtgtga tgttgacgaa ctggcgcatt tcatcgagaa agaaaccggt 720
cgtgaaaccc gcgcaactgt gctgggccac atccagcgcg gtggttctcc ggtgccttac 780
gaccgtattc tggcttcccg tatgggcgct tacgctatcg atctgctgct ggcaggttac 840
ggcggtcgtt gtgtaggtat ccagaacgaa cagctggttc accacgacat catcgacgct 900
atcgaaaaca tgaagcgtcc gttcaaaggt gactggctgg actgcgcgaa aaaactgtat 960
taa 963
<210> 6
<211> 930
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atggtacgta tctatacgtt gacacttgcg ccctctctcg atagcgcaac aattaccccg 60
caaatttatc ccgaaggaaa actgcgctgt accgcaccgg tgttcgaacc cgggggcggc 120
ggcatcaacg tcgcccgcgc cattgcccat cttggaggca gtgccacagc gatcttcccg 180
gcgggtggcg cgaccggcga acacctggtt tcactgttgg cggatgaaaa tgtccccgtc 240
gctactgtag aagccaaaga ctggacccgg cagaatttac acgtacatgt ggaagcaagc 300
ggtgagcagt atcgttttgt tatgccaggc gcggcattaa atgaagatga gtttcgccag 360
cttgaagagc aagttctgga aattgaatcc ggggccatcc tggtcataag cggaagcctg 420
ccgccaggtg tgaagctgga aaaattaacc caactgattt ccgctgcgca aaaacaaggg 480
atccgctgca tcgtcgacag ttctggcgaa gcgttaagtg cagcactggc aattggtaac 540
atcgagttgg ttaagcctaa ccaaaaagaa ctcagtgcgc tggtgaatcg cgaactcacc 600
cagccggacg atgtccgcaa agccgcgcag gaaatcgtta atagcggcaa ggccaaacgg 660
gttgtcgttt ccctgggtcc acaaggagcg ctgggtgttg atagtgaaaa ctgtattcag 720
gtggtgccac caccggtgaa aagccagagt accgttggcg ctggtgacag catggtcggc 780
gcgatgacac tgaaactggc agaaaatgcc tctcttgaag agatggttcg ttttggcgta 840
gctgcgggga gtgcagccac actcaatcag ggaacacgtc tgtgctccca tgacgatacg 900
caaaaaattt acgcttacct ttcccgctaa 930
<210> 7
<211> 2802
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgcagaaca gcgctttgaa agcctggttg gactcttctt acctctctgg cgcaaaccag 60
agctggatag aacagctcta tgaagacttc ttaaccgatc ctgactcggt tgacgctaac 120
tggcgttcga cgttccagca gttacctggt acgggagtca aaccggatca attccactct 180
caaacgcgtg aatatttccg ccgcctggcg aaagacgctt cacgttactc ttcaacgatc 240
tccgaccctg acaccaatgt gaagcaggtt aaagtcctgc agctcattaa cgcataccgc 300
ttccgtggtc accagcatgc gaatctcgat ccgctgggac tgtggcagca agataaagtg 360
gccgatctgg atccgtcttt ccacgatctg accgaagcag acttccagga gaccttcaac 420
gtcggttcat ttgccagcgg caaagaaacc atgaaactcg gcgagctgct ggaagccctc 480
aagcaaacct actgcggccc gattggtgcc gagtatatgc acattaccag caccgaagaa 540
aaacgctgga tccaacagcg tatcgagtct ggtcgcgcga ctttcaatag cgaagagaaa 600
aaacgcttct taagcgaact gaccgccgct gaaggtcttg aacgttacct cggcgcaaaa 660
ttccctggcg caaaacgctt ctcgctggaa ggcggtgacg cgttaatccc gatgcttaaa 720
gagatgatcc gccacgctgg caacagcggc acccgcgaag tggttctcgg gatggcgcac 780
cgtggtcgtc tgaacgtgct ggtgaacgtg ctgggtaaaa aaccgcaaga cttgttcgac 840
gagttcgccg gtaaacataa agaacacctc ggcacgggtg acgtgaaata ccacatgggc 900
ttctcgtctg acttccagac cgatggcggc ctggtgcacc tggcgctggc gtttaacccg 960
tctcaccttg agattgtaag cccggtagtt atcggttctg ttcgtgcccg tctggacaga 1020
cttgatgagc cgagcagcaa caaagtgctg ccaatcacca tccacggtga cgccgcagtg 1080
accgggcagg gcgtggttca ggaaaccctg aacatgtcga aagcgcgtgg ttatgaagtt 1140
ggcggtacgg tacgtatcgt tatcaacaac caggttggtt tcaccacctc taatccgctg 1200
gatgcccgtt ctacgccgta ctgtactgat atcggtaaga tggttcaggc cccgattttc 1260
cacgttaacg cggacgatcc ggaagccgtt gcctttgtga cccgtctggc gctcgatttc 1320
cgtaacacct ttaaacgtga tgtcttcatc gacctggtgt gctaccgccg tcacggccac 1380
aacgaagccg acgagccgag cgcaacccag ccgctgatgt atcagaaaat caaaaaacat 1440
ccgacaccgc gcaaaatcta cgctgacaag ctggagcagg aaaaagtggc gacgctggaa 1500
gatgccaccg agatggttaa cctgtaccgc gatgcgctgg atgctggcga ttgcgtagtg 1560
gcagagtggc gtccgatgaa catgcactct ttcacctggt cgccgtacct caaccacgaa 1620
tgggacgaag agtacccgaa caaagttgag atgaagcgcc tgcaggagct ggcgaaacgc 1680
atcagcacgg tgccggaagc agttgaaatg cagtctcgcg ttgccaagat ttatggcgat 1740
cgccaggcga tggctgccgg tgagaaactg ttcgactggg gcggtgcgga aaacctcgct 1800
tacgccacgc tggttgatga aggcattccg gttcgcctgt cgggtgaaga ctccggtcgc 1860
ggtaccttct tccaccgcca cgcggtgatc cacaaccagt ctaacggttc cacttacacg 1920
ccgctgcaac atatccataa cgggcagggc gcgttccgtg tctgggactc cgtactgtct 1980
gaagaagcag tgctggcgtt tgaatatggt tatgccaccg cagaaccacg cactctgacc 2040
atctgggaag cgcagttcgg tgacttcgcc aacggtgcgc aggtggttat cgaccagttc 2100
atctcctctg gcgaacagaa atggggccgg atgtgtggtc tggtgatgtt gctgccgcac 2160
ggttacgaag ggcaggggcc ggagcactcc tccgcgcgtc tggaacgtta tctgcaactt 2220
tgtgctgagc aaaacatgca ggtttgcgta ccgtctaccc cggcacaggt ttaccacatg 2280
ctgcgtcgtc aggcgctgcg cgggatgcgt cgtccgctgg tcgtgatgtc gccgaaatcc 2340
ctgctgcgtc atccgctggc ggtttccagc ctcgaagaac tggcgaacgg caccttcctg 2400
ccagccatcg gtgaaatcga cgagcttgat ccgaagggcg tgaagcgcgt agtgatgtgt 2460
tctggtaagg tttattacga cctgctggaa cagcgtcgta agaacaatca acacgatgtc 2520
gccattgtgc gtatcgagca actctacccg ttcccgcata aagcgatgca ggaagtgttg 2580
cagcagtttg ctcacgtcaa ggattttgtc tggtgccagg aagagccgct caaccagggc 2640
gcatggtact gcagccagca tcatttccgt gaagtgattc cgtttggggc ttctctgcgt 2700
tatgcaggcc gcccggcctc cgcctctccg gcggtagggt atatgtccgt tcaccagaaa 2760
cagcaacaag atctggttaa tgacgcgctg aacgtcgaat aa 2802
<210> 8
<211> 996
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgactatca aagtaggtat caacggtttt ggccgtatcg gtcgcattgt tttccgtgct 60
gctcagaaac gttctgacat cgagatcgtt gcaatcaacg acctgttaga cgctgattac 120
atggcataca tgctgaaata tgactccact cacggccgtt tcgacggtac cgttgaagtg 180
aaagacggtc atctgatcgt taacggtaaa aaaatccgtg ttaccgctga acgtgatccg 240
gctaacctga aatgggacga agttggtgtt gacgttgtcg ctgaagcaac tggtctgttc 300
ctgactgacg aaactgctcg taaacacatc accgctggtg cgaagaaagt ggttatgact 360
ggtccgtcta aagacaacac tccgatgttc gttaaaggcg ctaacttcga caaatatgct 420
ggccaggaca tcgtttccaa cgcttcctgc accaccaact gcctggctcc gctggctaaa 480
gttatcaacg ataacttcgg catcatcgaa ggtctgatga ccaccgttca cgctactacc 540
gctactcaga aaaccgttga tggcccgtct cacaaagact ggcgcggcgg ccgcggcgct 600
tcccagaaca tcatcccgtc ctctaccggt gctgctaaag ctgtaggtaa agtactgcca 660
gaactgaatg gcaaactgac tggtatggcg ttccgcgttc cgaccccgaa cgtatctgta 720
gttgacctga ccgttcgtct ggaaaaagct gcaacttacg agcagatcaa agctgccgtt 780
aaagctgctg ctgaaggcga aatgaaaggc gttctgggct acaccgaaga tgacgtagta 840
tctaccgatt tcaacggcga agtttgcact tccgtgttcg atgctaaagc tggtatcgct 900
ctgaacgaca acttcgtgaa actggtatcc tggtacgaca acgaaaccgg ttactccaac 960
aaagttctgg acctgatcgc tcacatctcc aaataa 996
<210> 9
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
acgccagcac cagcgcgccc agcccgccca tagaatgacc agagatagac gtgtaggctg 60
gagctgcttc 70
<210> 10
<211> 79
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cgtgaccacc ggtattggcg cggtacacaa cacagctaaa gtccagccag ctgatcagtg 60
ataagctgtc aaacatgag 79
<210> 11
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
caatggccgc agccaacgtc agatctccac aataacggcc catcacttcc gtgtaggctg 60
gagctgcttc 70
<210> 12
<211> 79
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
agacttccgg caacagattt cattttgcat tccaaagttc agaggtagtc ctgatcagtg 60
ataagctgtc aaacatgag 79
<210> 13
<211> 79
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
actttccgct gattcggtgc cagactgaaa tcagcctata ggaggaaatg ctgatcagtg 60
ataagctgtc aaacatgag 79
<210> 14
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ccaactcgat gttaccaatt gccagtgctg cacttaacgc ttcgccagaa gtgtaggctg 60
gagctgcttc 70
<210> 15
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
aagatgctta agggatcacg ctgatcagtg ataagctgtc aaacatgag 49
<210> 16
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gtgcatatac tcggcaccaa gtgtaggctg gagctgcttc 40
<210> 17
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gagaatccac ccggggatcc tctagagtcg 30
<210> 18
<211> 61
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cattatacga gccggatgat taattgtcaa ttaaagttta ttcactttgt cgtggtttgg 60
c 61
<210> 19
<211> 77
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ttgacaatta atcatccggc tcgtataatg aaaaccgttg atacctactt tgatagtcat 60
cgtcccgcaa ggatgcg 77
<210> 20
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gactctagag gatccccggg tggattctca ccaataaaaa acgcccg 47
Claims (9)
1.一种重组微生物，其特征在于，所述重组微生物与出发菌株相比，具有升高的甲醇脱氢酶和二羟基丙酮合成酶的表达和/或酶活性和降低的甲醛异化基因frmAB表达量和6-磷酸果糖激酶的表达和/或酶活性；所述出发菌株为大肠杆菌。
2.根据权利要求1所述的重组微生物，其特征在于，所述升高的甲醇脱氢酶和二羟基丙酮合成酶的表达和/或酶活性，通过过表达mdh基因和DAS基因实现；所述mdh基因的核苷酸序列如SEQ ID No：1所示，所述DAS基因的核苷酸序列如SEQ ID No：2所示。
3.根据权利要求1或2所述的重组微生物，其特征在于，所述降低的甲醛异化基因frmAB表达量和6-磷酸果糖激酶的表达和/或酶活性，通过敲除所述甲醛异化基因frmAB和6-磷酸果糖激酶基因pfkAB实现；所述甲醛异化基因frmAB的核苷酸序列如SEQ ID No：3-4所示，所述6-磷酸果糖激酶基因pfkAB的核苷酸序列如SEQ ID No：5-6所示。
4.根据权利要求1-3任一项所述的重组微生物，其特征在于，所述重组微生物具有降低的α-酮戊二酸脱氢酶的表达和/或酶活性。
5.根据权利要求4所述的重组微生物，其特征在于，所述降低的α-酮戊二酸脱氢酶的表达和/或酶活性，通过敲除α-酮戊二酸脱氢酶基因sucA实现，所述α-酮戊二酸脱氢酶基因sucA的核苷酸序列如SEQ ID No：7所示。
6.根据权利要求4或5所述的重组微生物，其特征在于，所述重组微生物具有降低的3-磷酸甘油醛脱氢酶的表达和/或酶活性。
7.根据权利要求6所述的重组微生物，其特征在于，所述降低的3-磷酸甘油醛脱氢酶的表达和/或酶活性，通过抑制3-磷酸甘油醛脱氢酶基因gapA实现，所述3-磷酸甘油醛脱氢酶基因gapA的核苷酸序列如SEQ ID No：8所示。
8.权利要求1-7任一项所述的重组微生物的如下任一种应用：
(1)在生物法转化甲醇中的应用；
(2)在用于转化甲醇的微生物遗传育种中的应用；
(3)在提高甲醇合成生物量的效率中的应用。
9.一种生物法转化甲醇的方法，其特征在于，包括培养权利要求1-7任一项所述的重组微生物的步骤。
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| CN115927453B (zh) * | 2023-01-31 | 2023-11-24 | 昆明理工大学 | 苹果酸脱氢酶基因在提高植物甲醛吸收代谢能力中的应用 |
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