CN114107147B - 一种可利用甲醇生产光学纯1,3-丁二醇的重组微生物及其应用 - Google Patents
一种可利用甲醇生产光学纯1,3-丁二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，具体公开了一种可利用甲醇生产光学纯1,3‑丁二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，过表达mdh基因和DAS基因，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；所述出发菌株为可生产1，3‑丁二醇的大肠杆菌。利用本发明的重组微生物可以同时发酵利用葡萄糖和甲醇，显著提高1,3‑丁二醇的产量，具有重要的工业应用潜力。
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，涉及一种可利用甲醇生产光学纯1,3-丁二醇的重组微生物及其应用。
背景技术
1,3-丁二醇是一种具有重要工业应用价值的二元醇，其可以被用作化妆品的溶剂，同时可以用作单体来合成聚酯、聚氨酯及生物增塑剂。同时光学纯的1,3-丁二醇可以用作医药中间体来合成许多药物。但是化学法合成的1,3-丁二醇都是外消旋体，无法直接用作医药中间体。因此，利用生物法生产光学纯1，3-丁二醇具有重要应用价值。
生物法合成1，3-丁二醇过程需要消耗大量的还原力NAD(P)H，因此提高NAD(P)H的供给对于提高1，3-丁二醇的产量至关重要。进而有必要对生物法生产1，3-丁二醇进行进一步研究。
发明内容
本发明的目的是提供一种可提高光学纯1，3-丁二醇产量的重组微生物。
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，过表达mdh基因(甲醇脱氢酶基因)和DAS基因(二羟基丙酮合成酶基因)；所述出发菌株为可生产1，3-丁二醇的大肠杆菌。
甲醇是一种还原度高且价格低廉的底物，本发明通过在可以合成1，3-丁二醇的大肠杆菌中引入了特定的甲醇代谢途径，使其能利用甲醇和葡萄糖更多地提供还原力NAD(P)H，从而显著提高了1，3-丁二醇的产量和得率。
本发明中，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。
本发明中，所述出发菌株可生产1，3-丁二醇的能力来源于过表达了phaA基因、phaB基因、bld基因和yqhD基因。
其中，所述phaA基因为乙酰CoA酰基转移酶基因，来源于Cupriavidus necator，其为编码基因如SEQ ID No：3所示；
所述phaB基因为3-氧酰基-(酰基载体蛋白)还原酶基因，来源于Cupriavidusnecator，其为编码基因如SEQ ID No：4所示；
所述bld基因来源于Clostridium saccharoperbutylacetonicum，其编码基因如SEQ ID No：5所示；
所述yqhD基因为醇脱氢酶基因，来源于大肠杆菌(Escherichia coli)，其为编码基因如SEQ ID NO:6所示。
本发明中，所述出发菌株还过表达pntAB基因。用于强化NADPH的供给。
优选，所述pntAB基因的核苷酸序列如SEQ ID No：8所示。
甲醇为底物进行代谢时，主要产生的还原力是NADH，而本发明研究发现通过具体的改进将其产生的NADH转化为NADPH(通过转氢酶pntAB)，能使其被更好地用于丁二醇的合成。
本发明还提供一种上述重组微生物的如下任一种应用：
(1)在发酵生产光学纯1,3-丁二醇中的应用；
(2)在用于生产光学纯1,3-丁二醇的微生物遗传育种中的应用；
(3)在提高生物法合成光学纯1，3-丁二醇的产量中的应用。
本发明另提供一种发酵生产光学纯1，3-丁二醇的方法，其包括培养上述的重组微生物的步骤。
培养所述重组微生物时的碳源可为甲醇和葡萄糖。
可选地，发酵时的发酵培养基为M9Y：葡萄糖20g/L，甲醇5g/L，Na2HPO4 6g/L，KH2PO4 3g/L，NaCl 0.5g/L，NH4Cl 1g/L，MgSO4 0.5g/L，CaCl2 15mg/L,酵母粉2g/L，氨苄青霉素100mg/L，以水配制。
培养条件为：37℃，100rpm。
本发明又提供一种构建生产光学纯1，3-丁二醇的重组微生物的方法，其包括使出发菌株过表达mdh基因和DAS基因的步骤，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，所述出发菌株为可生产1，3-丁二醇的大肠杆菌。
本发明的有益效果至少在于：
利用本发明的大肠杆菌工程菌在微氧条件下可同时发酵廉价葡萄糖和甲醇，1，3-丁二醇得率较单独利用葡萄糖可以提高20％以上，且R型1，3-丁二醇纯度达到99％以上，具有重要的工业应用潜力。
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。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW，MolecularCloning:a Laboratory Manual，2001)，或按照制造厂商说明书建议的条件进行。
本发明具体实施方式包括：(1)构建1，3-丁二醇合成质粒；(2)强化NADPH的供给；(3)引入甲醇的代谢途径。
实施例1在大肠杆菌中构建1,3-丁二醇的生物合成途径
大肠杆菌本身不能直接合成1,3-丁二醇，为了引入1,3-丁二醇的合成途径，本实施例对1,3-丁二醇合成过程的关键基因进行了设计和优化。具体方法如下：
首先构建包含来源于Cupriavidus necator的phaA、phaB基因，来源于Clostridium saccharoperbutylacetonicum的醛脱氢酶基因bld基因，来源于大肠杆菌的yqhD基因的质粒ptrc99a–bld-yqhD-phaAB。具体将ptrc99a(购自Addgene公司)用EcoRI/XbaI进行双酶切，将人工合成的phaA(SEQ ID NO:3)和phaB基因(SEQ ID NO:4)，bld(SEQID NO:5)和yqhD基因(SEQ ID NO:6)利用Gibson组装试剂盒插入到ptrc99a质粒骨架上EcoRI/XbaI双酶切位点之间，获得的质粒命名为ptrc99a-bld-yqhD-phaAB。
将质粒ptrc99a-bld-yqhD-phaAB转化到大肠杆菌MG1655中，获得的重组菌命名为E.coli/ptrc99a-bld-yqhD-phaAB。将该菌株在500mL的不带挡板摇瓶中进行培养，装液量为200ml，培养基为不包含甲醇的M9Y(葡萄糖20g/L,Na2HPO4 6g/L，KH2PO4 3g/L，NaCl0.5g/L，NH4Cl 1g/L，MgSO4 0.5g/L,CaCl2 15mg/L，酵母粉2g/L，氨苄青霉素100mg/L)和包含甲醇的M9Y(葡萄糖20g/L，甲醇5g/L，Na2HPO4 6g/L，KH2PO4 3g/L，NaCl 0.5g/L，NH4Cl 1g/L，MgSO4 0.5g/L，CaCl2 15mg/L，酵母粉2g/L，氨苄青霉素100mg/L)，培养温度为37℃，转速100rpm，待菌液OD600＝0.6时，加入0.1mM IPTG进行诱导，发酵48h时取样，利用高效液相色谱检测菌株生产1，3-丁二醇的情况。结果显示，大肠杆菌E.coli/ptrc99a-bld-yqhD-phaAB在不包含甲醇的M9Y培养基中可以生产2.3g/L的1，3-丁二醇，其R构型的光学纯度达到99％以上，在包含甲醇的M9Y培养基中可以生产2.2g/L的1，3-丁二醇，其R构型的光学纯度达到99％以上。
实施例2通过强化NADPH的合成提高1，3-丁二醇的产量
1,3-丁二醇合成途径需要消耗大量的NADPH，本实施例通过过表达pntAB基因可以显著提高细胞的1，3-丁二醇产量。
将实施例1中获得的质粒ptrc99a-bld-yqhD-phaAB用SbfI进行双酶切，利用Gibson组装试剂盒将大肠杆菌的pntAB基因(SEQ ID No：8)插入到ptrc99a-bld-yqhD-phaAB质粒骨架上，获得的质粒命名为ptrc99a-bld-yqhD-phaAB-pntAB。将该质粒转化到不包含甲醇代谢途径的大肠杆菌MG1655中，获得的重组菌命名为E.coli/ptrc99a-bld-yqhD-phaAB-pntAB。将该菌株在不包含甲醇和包含甲醇的M9Y培养基中进行培养(具体培养基配方同实施例1中所述)，发酵条件同上所述，48h时取样，利用高效液相色谱检测1,3-丁二醇的产量。结果显示，E.coli/ptrc99a-bld-yqhD-phaAB-pntAB在不包含甲醇M9Y培养基中的1，3-丁二醇的产量达到3.4g/L，其R构型的光学纯度达到99％以上，在包含甲醇的M9Y培养基中可以生产3.2g/L的1，3-丁二醇，其R构型的光学纯度达到99％以上。
实施例3构建甲醇利用途径
为了进一步提高1，3-丁二醇的产量，需要进一步提高胞内还原力NAD(P)H的供给。为此，本实施例在上述1，3-丁二醇生产菌株中引入甲醇的代谢途径。人工合成包含mdh基因(SEQ ID NO:1)和DAS基因(SEQ ID NO:2)的操纵子(SEQ ID NO:7，包含trc启动子)，将pET28a基因(购自Addgene公司)用EcoRI进行单酶切，将上述基因片段插入到pET28a中，获得的重组质粒命名为pET-mdh-DAS。将该质粒转化到实施例2中构建的重组菌E.coli/ptrc99a-bld-yqhD-phaAB-pntAB中，之后将该菌株在不包含甲醇和包含甲醇的M9Y培养基中进行培养(具体培养基配方同实施例1中所述)，发酵条件同上所述，48h时取样，利用高效液相色谱检测1，3-丁二醇的产量。结果显示，引入甲醇代谢途径的E.coli/ptrc99a-bld-yqhD-phaAB-pntAB在不包含甲醇M9Y培养基中的1，3-丁二醇的产量达到3.4g/L，其R构型的光学纯度达到99％以上，在包含甲醇的M9Y培养基中可以生产4.4g/L的1,3-丁二醇，其R构型的光学纯度达到99％以上。因此，通过引入甲醇的利用途径，可以显著提高光学纯1，3-丁二醇的产量。
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> 一种可利用甲醇生产光学纯1,3-丁二醇的重组微生物及其应用
<130> KHP211123841.3
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1173
<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
acgtctaacg cgcaaaaaga tgcttgcatg ctgacaaatc ctcgcaaagc gactgacgcg 1140
caggtgattg ctattttcga agcagctatg taa 1173
<210> 2
<211> 2133
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
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> 1182
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgactgacg ttgtcatcgt atccgccgcc cgcaccgcgg tcggcaagtt tggcggctcg 60
ctggccaaga tcccggcacc ggaactgggt gccgtggtca tcaaggccgc gctggagcgc 120
gccggcgtca agccggagca ggtgagcgaa gtcatcatgg gccaggtgct gaccgccggt 180
tcgggccaga accccgcacg ccaggccgcg atcaaggccg gcctgccggc gatggtgccg 240
gccatgacca tcaacaaggt gtgcggctcg ggcctgaagg ccgtgatgct ggccgccaac 300
gcgatcatgg cgggcgacgc cgagatcgtg gtggccggcg gccaggaaaa catgagcgcc 360
gccccgcacg tgctgccggg ctcgcgcgat ggtttccgca tgggcgatgc caagctggtc 420
gacaccatga tcgtcgacgg cctgtgggac gtgtacaacc agtaccacat gggcatcacc 480
gccgagaacg tggccaagga atacggcatc acacgcgagg cgcaggatga gttcgccgtc 540
ggctcgcaga acaaggccga agccgcgcag aaggccggca agtttgacga agagatcgtc 600
ccggtgctga tcccgcagcg caagggcgac ccggtggcct tcaagaccga cgagttcgtg 660
cgccagggcg ccacgctgga cagcatgtcc ggcctcaagc ccgccttcga caaggccggc 720
acggtgaccg cggccaacgc ctcgggcctg aacgacggcg ccgccgcggt ggtggtgatg 780
tcggcggcca aggccaagga actgggcctg accccgctgg ccacgatcaa gagctatgcc 840
aacgccggtg tcgatcccaa ggtgatgggc atgggcccgg tgccggcctc caagcgcgcc 900
ctgtcgcgcg ccgagtggac cccgcaagac ctggacctga tggagatcaa cgaggccttt 960
gccgcgcagg cgctggcggt gcaccagcag atgggctggg acacctccaa ggtcaatgtg 1020
aacggcggcg ccatcgccat cggccacccg atcggcgcgt cgggctgccg tatcctggtg 1080
acgctgctgc acgagatgaa gcgccgtgac gcgaagaagg gcctggcctc gctgtgcatc 1140
ggcggcggca tgggcgtggc gctggcagtc gagcgcaaat aa 1182
<210> 4
<211> 741
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgactcagc gcattgcgta tgtgaccggc ggcatgggtg gtatcggaac cgccatttgc 60
cagcggctgg ccaaggatgg ctttcgtgtg gtggccggtt gcggccccaa ctcgccgcgc 120
cgcgaaaagt ggctggagca gcagaaggcc ctgggcttcg atttcattgc ctcggaaggc 180
aatgtggctg actgggactc gaccaagacc gcattcgaca aggtcaagtc cgaggtcggc 240
gaggttgatg tgctgatcaa caacgccggt atcacccgcg acgtggtgtt ccgcaagatg 300
acccgcgccg actgggatgc ggtgatcgac accaacctga cctcgctgtt caacgtcacc 360
aagcaggtga tcgacggcat ggccgaccgt ggctggggcc gcatcgtcaa catctcgtcg 420
gtgaacgggc agaagggcca gttcggccag accaactact ccaccgccaa ggccggcctg 480
catggcttca ccatggcact ggcgcaggaa gtggcgacca agggcgtgac cgtcaacacg 540
gtctctccgg gctatatcgc caccgacatg gtcaaggcga tccgccagga cgtgctcgac 600
aagatcgtcg cgacgatccc ggtcaagcgc ctgggcctgc cggaagagat cgcctcgatc 660
tgcgcctggt tgtcgtcgga ggagtccggt ttctcgaccg gcgccgactt ctcgctcaac 720
ggcggcctgc atatgggctg a 741
<210> 5
<211> 1407
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgatcaagg acaccctggt ttcgattaca aaagacttaa agttgaaaac aaacgtagaa 60
aacgccaacc ttaaaaacta taaagatgac tcgtcttgct tcggggtgtt tgagaacgtg 120
gaaaatgcga tttcgaacgc agtccacgcc cagaagattt tatcgcttca ctacactaag 180
gagcagcgcg aaaagattat cacggagatt cgtaaagctg cacttgagaa caaagagatt 240
ttggcaacca tgatcttgga ggagactcat atggggcgtt atgaagataa aattttaaag 300
catgaacttg tcgctaaata cactccaggg actgaagact taacgaccac ggcatggagc 360
ggagataatg ggcttactgt tgttgaaatg agcccctacg gggtgattgg ggcaatcact 420
cccagcacaa atcctaccga gactgtgatt tgtaattcta ttggcatgat tgcggctggc 480
aatacggtgg tcttcaatgg acatccgggg gccaagaagt gtgttgcatt tgctgttgag 540
atgatcaaca aagccattat ttcatgtggt gggcccgaga atttggttac aaccattaag 600
aatccaacta tggactctct ggacgctatt attaaacacc cgtcgattaa acttttatgc 660
ggaaccggag gaccgggaat ggtgaaaacc cttctgaatt ccgggaaaaa ggcgatcggt 720
gcgggtgccg gcaacccacc tgtcattgtt gatgacacag cagacattga aaaggcgggc 780
aagagcatca tcgagggctg tagttttgat aacaacacgc cctgcattgc tgaaaaggaa 840
gttttcgtct tcgagaatgt tgcggacgac cttatttcca atatgcttaa gaacaacgcg 900
gtaatcatta acgaagacca agtatcaaag ttaatcgatt tggtcctgca gaagaacaat 960
gagactcaag agtatagcat taataaaaaa tgggttggga aggacgcaaa attgtttctt 1020
gatgagatcg atgtggaatc cccttcctct gtcaaatgca tcatttgcga agtatctgcg 1080
cgtcacccat tcgtcatgac agagctgatg atgcccattc tgcctattgt acgcgttaaa 1140
gatatcgacg aagctattga atatgcgaaa atcgccgagc agaaccgtaa gcactcagcg 1200
tatatttatt ccaaaaatat cgataactta aatcgttttg agcgcgaaat cgatacaact 1260
atcttcgtca agaacgccaa aagctttgct ggcgtgggct atgaagctga agggtttacc 1320
acgttcacca tcgcagggag caccggcgaa gggattacaa gtgcgcgcaa tttcactcgt 1380
caacgtcgct gcgtgttagc cggttaa 1407
<210> 6
<211> 1164
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgaacaact ttaatctgca caccccaacc cgcattctgt ttggtaaagg cgcaatcgct 60
ggtttacgcg aacaaattcc tcacgatgct cgcgtattga ttacctacgg cggcggcagc 120
gtgaaaaaaa ccggcgttct cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180
gaatttggcg gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg 240
gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt actggacggc 300
accaaattta tcgccgcagc ggctaactat ccggaaaata tcgatccgtg gcacattctg 360
caaacgggcg gtaaagagat taaaagcgcc atcccgatgg gctgtgtgct gacgctgcca 420
gcaaccggtt cagaatccaa cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480
caggcgttcc attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc 540
tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt acacaccgtg 600
gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg accgtttcgc agaaggcatt 660
ttgctgacgc taatcgaaga tggtccgaaa gccctgaaag agccagaaaa ctacgatgtg 720
cgcgccaacg tcatgtgggc ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780
ccgcaggact gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat 840
cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg cgataccaag 900
cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca tcactgaagg ttccgatgat 960
gagcgtattg acgccgcgat tgccgcaacc cgcaatttct ttgagcaatt aggcgtgccg 1020
acccacctct ccgactacgg tctggacggc agctccatcc cggctttgct gaaaaaactg 1080
gaagagcacg gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc 1140
cgtatatacg aagccgcccg ctaa 1164
<210> 7
<211> 3414
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcac 60
acaggaaaca gaccatggaa gaaggagata tacatggcgt tcaagaacct ggcggatcag 120
actaatggtt tctacatccc gtgcgtcagc ttgttcggtc ctgggtgtgc aaaagaagtg 180
ggggccaaag cacaaaacct tggggctaaa aaagcgttga tcgtcaccga tgcagggctt 240
tttaaattcg gagtggcaga catcatcgta gggtacctta aagatgcggg agtcgacagt 300
catgtgtttc cgggggcaga acccaatccc acggacatta atgtattgaa tggggttcag 360
gcctataatg ataatggatg cgattttatt gtttctctgg gtggaggcag ctcacacgac 420
tgcgctaagg gcattgggct ggtgacggct ggtgggggta acattcgcga ttatgagggt 480
attgacaaat cgtcagtccc catgacaccc ttaattgcga tcaatacgac cgctggaaca 540
gcctcggaaa tgacccgttt ttgtattatc acaaatacag atacccacgt taagatggca 600
atcgtggatt ggcgctgtac tcctttggtc gctattgacg acccgaaatt gatgattgct 660
aaaccggctg ccctgactgc agctactggc atggatgcgc ttacacatgc ggtagaagcg 720
tacgtgtcga ctgcagccaa ccccattaca gatgcatgcg ctgaaaaggc gatcagcatg 780
atttctgagt ggttgagttc agcagtagca aacggggaaa acatcgaagc gcgcgacgct 840
atggcttacg ctcagtatct tgcgggtatg gcgtttaata atgcgtcgtt aggatatgtg 900
cacgccatgg cccaccagct gggtgggttt tacaatttac ctcacggtgt atgtaacgcc 960
atcctgctgc cacacgtatg cgagttcaat cttattgcgt gccccgatcg ttttgcgaag 1020
attgcacagc tgatgggcgt agatacaaca ggtatgacgg taactgaggc gggttacgag 1080
gctattgcag cgattcgcga gctttcagcc agtattggga ttccgagcgg gctgaccgaa 1140
ctgggggtaa aagccgcaga ccatgctgta atgacgtcta acgcgcaaaa agatgcttgc 1200
atgctgacaa atcctcgcaa agcgactgac gcgcaggtga ttgctatttt cgaagcagct 1260
atgtaaaaga aggagatata catgtccatg cgcattccca aagcggcgag cgtaaacgac 1320
gagcagcatc aacgcattat caaatatggc cgcgccttgg tcctggacat tgttgaacag 1380
tacgggggag ggcatcccgg atcggccatg ggcgcgatgg caatcggcat tgcattatgg 1440
aagtatacac ttaagtacgc tcctaatgat ccaaattact tcaatcgtga tcgcttcgtt 1500
ttgagtaatg gtcacgtttg tctttttcag tacatcttcc agcacttgta tgggttaaag 1560
tccatgacga tggcgcagtt aaagtcttac cactcgaatg actttcattc attatgtcct 1620
ggccacccgg agatcgaaca cgatgcggtt gaggtcacta ccggaccgct ggggcaggga 1680
atctctaact cggttggttt ggctatcgcc accaagaacc ttgccgcgac ctacaacaaa 1740
ccaggcttcg atatcattac taataaagtt tactgtatgg ttggagatgc atgtttacaa 1800
gaagggcctg ctctggaaag tatcagtttg gctggacaca tggggttgga taatttaatt 1860
gtcctttacg acaacaatca agtatgctgc gatggctcgg tcgacatcgc aaataccgag 1920
gatatctcgg ctaaatttaa ggcgtgcaat tggaatgtca tcgaggttga aaatgccagt 1980
gaggacgttg caaccatcgt caaggcgctg gaatatgcac aggctgagaa gcaccgcccg 2040
actttaatta attgtcgtac tgttatcggt agtggggcag catttgagaa ccactgtgca 2100
gctcatggga acgctttagg cgaagacggg gtacgcgagt taaaaattaa atacggaatg 2160
aacccggcgc aaaaatttta tattccgcag gatgtatatg actttttcaa agagaagcca 2220
gccgaaggtg acaagctggt tgccgagtgg aaaagtcttg tggccaagta cgtgaaggca 2280
tacccggaag agggtcaaga attccttgcc cgtatgcgcg gcgaattgcc caaaaactgg 2340
aagtcgtttc ttccccaaca agagtttaca ggagatgctc ccacacgcgc cgctgcccgt 2400
gagttggttc gcgcccttgg tcaaaattgt aagtctgtaa tcgctggatg tgccgacttg 2460
tccgtttcgg tgaatttaca gtggcctggc gttaagtact tcatggatcc ctcattatcc 2520
acgcagtgcg ggctttccgg agattattcg ggtcgttata tcgagtacgg tattcgcgaa 2580
catgcgatgt gtgcgatcgc taacggattg gcagcctata ataaagggac gtttctgccc 2640
atcacgtcga ccttcttcat gttctatttg tacgctgcgc ctgccattcg tatggcaggc 2700
cttcaagagt taaaggctat ccatatcggg acgcacgact caattaacga gggtgaaaat 2760
gggcccacac atcaacccgt tgagtcacca gcgctgttcc gcgctatgcc aaatatttac 2820
tacatgcgcc cggttgactc cgcagaagtg ttcggtcttt ttcagaaggc agtcgaactt 2880
ccattctcct ccatcctgtc gctttcacgt aatgaggtat tgcaatatcc agggaagtcc 2940
tccgcagaga aagcgcaacg cggtgggtac atcctggaag acgcggagaa tgccgaagtg 3000
caaatcattg gagtcggggc cgaaatggaa tttgcttaca aggctgcgaa aatccttggt 3060
cgcaagtttc gcactcgcgt tttatcaatc ccttgcactc gtttatttga tgagcaatcg 3120
attggatacc gtcgtagcgt gcttcgtaag gatggccgcc aggtacccac tgtagtagtg 3180
gatggccacg tcgcattcgg ctgggaacgt tacgcaaccg caagctattg tatgaatact 3240
tatggaaagt cattacctcc cgaggtaatt tatgaatact ttgggtataa tccggcgact 3300
atcgcgaaga aggtggaggc ttacgtacgc gcttgccagc gcgacccctt attattacat 3360
gatttcttag acttaaaaga gaagccaaac cacgacaaag tgaataaact ttaa 3414
<210> 8
<211> 2932
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgcgaattg gcataccaag agaacggtta accaatgaaa cccgtgttgc agcaacgcca 60
aaaacagtgg aacagctgct gaaactgggt tttaccgtcg cggtagagag cggcgcgggt 120
caactggcaa gttttgacga taaagcgttt gtgcaagcgg gcgctgaaat tgtagaaggg 180
aatagcgtct ggcagtcaga gatcattctg aaggtcaatg cgccgttaga tgatgaaatt 240
gcgttactga atcctgggac aacgctggtg agttttatct ggcctgcgca gaatccggaa 300
ttaatgcaaa aacttgcgga acgtaacgtg accgtgatgg cgatggactc tgtgccgcgt 360
atctcacgcg cacaatcgct ggacgcacta agctcgatgg cgaacatcgc cggttatcgc 420
gccattgttg aagcggcaca tgaatttggg cgcttcttta ccgggcaaat tactgcggcc 480
gggaaagtgc caccggcaaa agtgatggtg attggtgcgg gtgttgcagg tctggccgcc 540
attggcgcag caaacagtct cggcgcgatt gtgcgtgcat tcgacacccg cccggaagtg 600
aaagaacaag ttcaaagtat gggcgcggaa ttcctcgagc tggattttaa agaggaagct 660
ggcagcggcg atggctatgc caaagtgatg tcggacgcgt tcatcaaagc ggaaatggaa 720
ctctttgccg cccaggcaaa agaggtcgat atcattgtca ccaccgcgct tattccaggc 780
aaaccagcgc cgaagctaat tacccgtgaa atggttgact ccatgaaggc gggcagtgtg 840
attgtcgacc tggcagccca aaacggcggc aactgtgaat acaccgtgcc gggtgaaatc 900
ttcactacgg aaaatggtgt caaagtgatt ggttataccg atcttccggg ccgtctgccg 960
acgcaatcct cacagcttta cggcacaaac ctcgttaatc tgctgaaact gttgtgcaaa 1020
gagaaagacg gcaatatcac tgttgatttt gatgatgtgg tgattcgcgg cgtgaccgtg 1080
atccgtgcgg gcgaaattac ctggccggca ccgccgattc aggtatcagc tcagccgcag 1140
gcggcacaaa aagcggcacc ggaagtgaaa actgaggaaa aatgtacctg ctcaccgtgg 1200
cgtaaatacg cgttgatggc gctggcaatc attctttttg gctggatggc aagcgttgcg 1260
ccgaaagaat tccttgggca cttcaccgtt ttcgcgctgg cctgcgttgt cggttattac 1320
gtggtgtgga atgtatcgca cgcgctgcat acaccgttga tgtcggtcac caacgcgatt 1380
tcagggatta ttgttgtcgg agcactgttg cagattggcc agggcggctg ggttagcttc 1440
cttagtttta tcgcggtgct tatagccagc attaatattt tcggtggctt caccgtgact 1500
cagcgcatgc tgaaaatgtt ccgcaaaaat taaggggtaa catatgtctg gaggattagt 1560
tacagctgca tacattgttg ccgcgatcct gtttatcttc agtctggccg gtctttcgaa 1620
acatgaaacg tctcgccagg gtaacaactt cggtatcgcc gggatggcga ttgcgttaat 1680
cgcaaccatt tttggaccgg atacgggtaa tgttggctgg atcttgctgg cgatggtcat 1740
tggtggggca attggtatcc gtctggcgaa gaaagttgaa atgaccgaaa tgccagaact 1800
ggtggcgatc ctgcatagct tcgtgggtct ggcggcagtg ctggttggct ttaacagcta 1860
tctgcatcat gacgcgggaa tggcaccgat tctggtcaat attcacctga cggaagtgtt 1920
cctcggtatc ttcatcgggg cggtaacgtt cacgggttcg gtggtggcgt tcggcaaact 1980
gtgtggcaag atttcgtcta aaccattgat gctgccaaac cgtcacaaaa tgaacctggc 2040
ggctctggtc gtttccttcc tgctgctgat tgtatttgtt cgcacggaca gcgtcggcct 2100
gcaagtgctg gcattgctga taatgaccgc aattgcgctg gtattcggct ggcatttagt 2160
cgcctccatc ggtggtgcag atatgccagt ggtggtgtcg atgctgaact cgtactccgg 2220
ctgggcggct gcggctgcgg gctttatgct cagcaacgac ctgctgattg tgaccggtgc 2280
gctggtcggt tcttcggggg ctatcctttc ttacattatg tgtaaggcga tgaaccgttc 2340
ctttatcagc gttattgcgg gtggtttcgg caccgacggc tcttctactg gcgatgatca 2400
ggaagtgggt gagcaccgcg aaatcaccgc agaagagaca gcggaactgc tgaaaaactc 2460
ccattcagtg atcattactc cggggtacgg catggcagtc gcgcaggcgc aatatcctgt 2520
cgctgaaatt actgagaaat tgcgcgctcg tggtattaat gtgcgtttcg gtatccaccc 2580
ggtcgcgggg cgtttgcctg gacatatgaa cgtattgctg gctgaagcaa aagtaccgta 2640
tgacatcgtg ctggaaatgg acgagatcaa tgatgacttt gctgataccg ataccgtact 2700
ggtgattggt gctaacgata cggttaaccc ggcggcgcag gatgatccga agagtccgat 2760
tgctggtatg cctgtgctgg aagtgtggaa agcgcagaac gtgattgtct ttaaacgttc 2820
gatgaacact ggctatgctg gtgtgcaaaa cccgctgttc ttcaaggaaa acacccacat 2880
gctgtttggt gacgccaaag ccagcgtgga tgcaatcctg aaagctctgt aa 2932
Claims (5)
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，过表达mdh基因和DAS基因；所述出发菌株为可生产1，3-丁二醇的大肠杆菌；所述mdh基因的核苷酸序列如SEQID No：1所示，所述DAS基因的核苷酸序列如SEQ ID No：2所示；
所述出发菌株中过表达了phaA基因、phaB基因、bld基因、yqhD基因和pntAB基因；所述phaA基因的核苷酸序列如SEQ ID No：3所示；所述phaB基因的核苷酸序列如SEQ ID No：4所示；所述bld基因的核苷酸序列如SEQ ID No：5所示；所述yqhD基因的核苷酸序列如SEQIDNo：6所示；所述pntAB基因的核苷酸序列如SEQ ID No：8所示。
2.权利要求1所述的重组微生物的如下任一种应用：
(1)在发酵生产光学纯1,3-丁二醇中的应用；
(2)在用于生产光学纯1,3-丁二醇的微生物遗传育种中的应用；
(3)在提高生物法合成光学纯1,3-丁二醇的产量中的应用。
3.一种发酵生产光学纯1,3-丁二醇的方法，其特征在于，包括培养权利要求1所述的重组微生物的步骤。
4.根据权利要求3所述的方法，其特征在于，培养所述重组微生物时的碳源为甲醇和葡萄糖。
5.一种构建生产光学纯1,3-丁二醇的重组微生物的方法，其特征在于，包括使出发菌株过表达mdh基因和DAS基因的步骤，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，所述出发菌株为可生产1，3-丁二醇的大肠杆菌；
所述方法还包括使出发菌株过表达phaA基因、phaB基因、bld基因、yqhD基因和pntAB基因的步骤；所述phaA基因的核苷酸序列如SEQ ID No：3所示；所述phaB基因的核苷酸序列如SEQ ID No：4所示；所述bld基因的核苷酸序列如SEQ ID No：5所示；所述yqhD基因的核苷酸序列如SEQ ID No：6所示；所述pntAB基因的核苷酸序列如SEQ ID No：8所示。
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