CN114150009B - 用于制备麻黄碱的工程菌的构建及麻黄碱的制备方法 - Google Patents
用于制备麻黄碱的工程菌的构建及麻黄碱的制备方法 Download PDFInfo
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Abstract
本发明涉及一种用于制备麻黄碱的工程菌的构建及麻黄碱的制备方法，其构建方法包括：向大肠杆菌或酿酒酵母中转化含有羟基扁桃酸合成酶基因HmaS和乙酰羟酸合成酶基因AHAS的重组质粒以及含有扁桃酸脱氢酶基因SMDH和苯甲酰甲酸脱羧酶基因BFD的重组质粒。采用上述方法构建得到的基于质粒的表达工程菌被证实可实现生物合成与制备麻黄碱，具有可规模化的生产、产量高、环境友好等工业应用前景，未来可通过基因组整合相应的基因模块而获得更为稳定的表达菌株，以及可以通过投苯丙氨酸、苯甲醛等补料方式进一步提高产量。
Description
技术领域
本发明涉及生物工程技术领域，具体涉及一种用于制备麻黄碱的工程菌的构建及麻黄碱的制备方法。
背景技术
麻黄碱，存在于***茶、麻黄属等植物中，是具有独特生理活性及药理特性的次级代谢产物。针对不同的分子靶点，麻黄碱具有多种多样的生物活性，被广泛使用于治疗哮喘等神经性疾病。
对于麻黄碱的生产，主要有植物提取法和化学合成法。植物提取法是直接从***中提取麻黄碱，但是***资源限制，提取的方法价格昂贵且产率低。化学合成法是先生产L-麻黄碱和D-伪麻黄碱的外消旋体，然后将生产得到的外消旋体进行拆分，但是其产生过多三废，不符合我国环保国情。
与植物提取法和化学合成法相比，生物转化法天然具有原料来源广以及生产成本低的优势。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的目的在于提供一种产量高、环境友好的麻黄碱生物合成方法。
为此，在本发明的一方面，本发明提出了一种用于制备麻黄碱的工程菌构建方法，其包括：
向大肠杆菌或酿酒酵母中转化含有羟基扁桃酸合成酶基因HmaS和乙酰羟酸合成酶基因AHAS的重组质粒以及含有扁桃酸脱氢酶基因SMDH和苯甲酰甲酸脱羧酶基因BFD的重组质粒。
根据本发明实施例的一种用于制备麻黄碱的工程菌构建方法，该方法构建得到的大肠杆菌工程菌或酿酒酵母工程菌中同时包含了麻黄碱生物合成通路中相关酶对应的四个基因：羟基扁桃酸合成酶基因HmaS、扁桃酸脱氢酶基因SMDH、苯甲酰甲酸脱羧酶基因BFD、乙酰羟酸合成酶基因AHAS；四个基因在大肠杆菌工程菌或者酿酒酵母工程菌中构建形成了麻黄碱的生物合成通路，通过简单的发酵工艺，实现以葡萄糖、甘油等碳源作为原料直接生产麻黄碱。未来可通过基因组整合相应的基因模块而获得更为稳定的表达菌株，以及可以通过投苯丙氨酸、苯甲醛等补料方式进一步提高产量。
另外，根据本发明上述实施例提出的用于制备麻黄碱的工程菌构建方法，还可以具有如下附加的技术特征：
可选地，所述乙酰羟酸合成酶基因AHAS，以大肠杆菌基因组为模板，其核苷酸序列如SEQ ID NO:1所示，经PCR扩增得到；或以酿酒酵母基因组为模板，其核苷酸序列如SEQ IDNO:2所示，经PCR扩增得到。
可选地，所述羟基扁桃酸合成酶基因HmaS，以东方拟无枝酸菌来源的HmaS基因为模板，其核苷酸序列如SEQ ID NO:3所示，经PCR扩增得到。
可选地，所述扁桃酸脱氢酶基因SMDH，以恶臭假单胞菌KT2440基因组为模板，其核苷酸序列如SEQ ID NO:4所示，经PCR扩增得到。
可选地，所述苯甲酰甲酸脱羧酶基因BFD，以恶臭假单胞菌KT2440基因组为模板，其核苷酸序列如SEQ ID NO:5所示，经PCR扩增得到。
可选地，所述大肠杆菌或酿酒酵母具备积累苯甲醛的能力。
进一步地，以大肠杆菌MG1655(DE3)ΔendAΔrecA衍生菌株ΔdkgBΔyeaEΔ(yqhC-dkgA)ΔyahKΔyjgB作为生产宿主。
进一步地，以CEN.PK2-1C苯甲醛积累酿酒酵母作为生产宿主。具体地，以CEN.PK2-1C衍生菌株Δadh6Δadh7Δsfa1Δgre2Δhfd1Δgre3Δgcy1Δydl124wΔypr1作为生产宿主。
在本发明的第二个方面，本发明提出由上述工程菌构建方法构建得到的用于制备麻黄碱的工程菌。
在本发明的第三个方面，本发明提出麻黄碱的制备方法，其通过将上述的工程菌接种于培养基中发酵培养，即得。
根据本发明实施例的麻黄碱的制备方法，通过简单的发酵工艺，实现以葡萄糖、甘油等碳源作为原料直接生产麻黄碱，有利于麻黄碱的工业化、规模化生产，麻黄碱的产量高、纯度高、具有广阔的工业化应用前景。同时，避免了麻黄碱半合成的制备方法中大量消耗植物材料和化学试剂，具有环境友好的特点。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
图1为根据本发明实施例的麻黄碱的合成流程图；
图2为根据本发明实施例的质粒示意图；
图3为根据本发明实施例的大肠杆菌发酵液LC-QTOF/MS检测结果；
图4为根据本发明实施例的酿酒酵母发酵液LC-QTOF/MS检测结果。
具体实施方式
以下通过特定的具体实例说明本发明的技术方案。应理解，本发明提到的一个或多个方法步骤并不排斥在所述组合步骤前后还存在其他方法步骤或在这些明确提到的步骤之间还可以***其他方法步骤；还应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。而且，除非另有说明，各方法步骤的编号仅为鉴别各方法步骤的便利工具，而非为限制各方法步骤的排列次序或限定本发明可实施的范围，其相对关系的改变或调整，在无实质变更技术内容的情况下，当亦视为本发明可实施的范畴。
为了更好的理解上述技术方案，下面更详细地描述本发明的示例性实施例。虽然显示了本发明的示例性实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
本发明采用的试材皆为普通市售品，皆可于市场购得；涉及的实验如无特别说明，均为常规实验方法。
所用材料来源：酿酒酵母CEN.PK2-1C、大肠杆菌菌株MG1655 RARE均为市售。酿酒酵母表达载体pESC-URA，pESC-LEU，来源于Agilent Technologies。大肠杆菌表达载体pRSFDuet-1，pCDFDuet-1来源于Novagen。Phusion高保真DNA聚合酶、限制性内切酶购买自厦门鹭隆生物科技发展有限公司。质粒提取试剂盒、DNA纯化试剂盒、胶回收试剂盒和基因组DNA提取试剂盒购买自上海生物工程有限公司。
本发明实施例的麻黄碱生物合成路径见图1：
以葡萄糖、甘油等碳源为底物通过莽草酸途径生产苯丙酮酸，经羟基扁桃酸合酶HmaS的催化作用下合成扁桃酸，再经扁桃酸脱氢酶SMDH(或对羟基扁桃酸氧化酶HMO)、苯甲酰甲酸脱羧酶BFD的催化下合成苯甲醛，在乙酰羟酸合成酶AHAS或(丙酮酸脱羧酶PDC)作用下与丙酮酸反应合成L-苯乙酰基甲醇，再经由大肠杆菌或酿酒酵母内源氨基转移酶(AT)及N-甲基转移酶(NMT)催化作用下合成麻黄碱。
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任何方式限制本发明。
实施例1
1、基因克隆：
大肠杆菌的乙酰羟酸合成酶基因AHAS：以大肠杆菌基因组为模板，其核苷酸序列如SEQ ID NO:1所示，以表1的AHAS fwd和AHAS rev为引物，经高保真酶PCR扩增得到ilvB基因AHAS目的条带，将AHAS目的条带经过DNA纯化试剂盒回收，得到胶回收产物。PCR扩增条件：98℃2min，98℃10s，56℃30s，72℃1min，循环30次；72℃2min。
东方拟无枝酸菌的羟基扁桃酸合成酶基因HmaS：以合成HmaS基因为模板，其核苷酸序列如SEQ ID NO:3所示，以表1的HmaS fwd和HmaS rev为引物，经PCR扩增得到HmaS基因片段。PCR扩增条件：98℃2min，98℃10s，56℃30s，72℃1min，循环30次；72℃2min。
恶臭假单胞菌的扁桃酸脱氢酶基因SMDH：以恶臭假单胞菌KT2440基因组为模板，其核苷酸序列如SEQ ID NO:4所示，以表1的SMDH fwd和SMDH rev为引物，经PCR扩增得到SMDH片段。PCR扩增条件：98℃2min，98℃10s，56℃30s，72℃1min，循环30次；72℃2min。
恶臭假单胞菌的苯甲酰甲酸脱羧酶基因BFD：以恶臭假单胞菌KT2440基因组为模板，其核苷酸序列如SEQ ID NO:5所示，以表1的BFD fwd和BFD rev为引物，经PCR扩增得到BFD片段。PCR扩增条件：98℃2min，98℃10s，56℃30s，72℃1min，循环30次；72℃2min。
酿酒酵母的乙酰羟酸合成酶基因AHAS：以酿酒酵母基因组为模板，其核苷酸序列如SEQ ID NO:2所示，以表2的AHAS fwd和AHAS rev为引物，经PCR扩增得到ilv2基因AHAS目的片段，其中ilv2基因N端54个氨基酸线粒体定位信号被移除，以便其在酿酒酵母细胞胞质表达，将AHAS目的条带经过DNA纯化试剂盒回收，得到胶回收产物。PCR扩增条件：98℃ 2分钟；98℃ 10秒，56℃ 30秒，72℃ 1分钟，循环30次；72℃ 2分钟。
构建酿酒酵母工程菌的其余基因(HmaS，SMDH，BFD)均采用表2的引物，采用与上述相同的PCR扩增条件获得对应的基因片段。
表1：大肠杆菌生产麻黄碱菌株所需的PCR扩增引物列表
表2：酿酒酵母生产麻黄碱菌株所需的PCR扩增引物列表
| 引物名称： | 引物5'-3 |
| AHAS_fwd | TTGGTCTCATGAAAACAATGCCAGAGCCTGCTCCAAG |
| AHAS_rev | TTGGTCTCAATCTAGTGCTTACCGCCTGTACG |
| HmaS_fwd | TTGGTCTCAAACCAATGCAGAATTTCGAAATCGAC |
| HmaS_rev | TTGGTCTCATCTTAACGCCTCGCGGCTCCAAAC |
| SMDH_fwd | TTGGTCTCATGAAAACAATGAGCCGGAATCTCTTTAAC |
| SMDH_rev | TTGGTCTCAATCTATGCGTGTGTTCCTTTAC |
| BFD_fwd | TTGGTCTCAAACCAATGGCTTCGGTACACGGC |
| BFD_rev | TTGGTCTCATCTTACTTCACCGGGCTTACGG |
2、表达载体构建
将羟基扁桃酸合成酶基因HmaS和乙酰羟酸合成酶基因AHAS在37℃，BsaI酶切2小时后；PCR纯化回收酶切片段；将纯化回收的片段与经过BamHI和XhoI双酶切的表达载体pRSFDuet-1连接，16℃连接1小时转入大肠杆菌感受态细胞中，提取对应的大肠杆菌pRSF-HmaS-AHAS表达质粒。
同上所述，相似的方法也被应用于构建大肠杆菌表达载体pCDFDuet-1衍生pCDF-SMDH-BFD表达质粒，以及酿酒酵母表达载体pESC-URA衍生pESC-URA-HmaS-AHAS与表达载体pESC-LEU衍生pESC-LEU-SMDH-BFD表达质粒。
3、大肠杆菌重组菌株
将上述2获取的pRSF-HmaS-AHAS质粒和pCDF-SMDH-BFD质粒电击法导入对应的大肠杆菌表达菌株中，以含有卡纳霉素和壮观霉素抗生素的固体LB培养基中筛选，从而得到重组大肠杆菌。
4、酿酒酵母重组菌株
将上述2获取的pESC-URA-HmaS-AHAS和pESC-LEU-SMDH-BFD质粒电击法导入对应的酿酒酵母表达菌株中，以尿嘧啶和亮氨酸缺失的固体酵母培养基中筛选，从而得到重组酿酒酵母菌。
实施例2
将实施例1制备的重组大肠杆菌接种在LB培养液中，过夜培养后按1:100比例接入M9培养液中扩大培养2-3h后(OD600达0.4-06)，加入0.1mM IPTG持续诱导目标蛋白表达，后续通过液相色谱或质谱检测麻黄碱。
产品定性与定量分析：采用岛津高效液相色谱仪检测分析，采用光电二极管阵列检测器(工作波长280nm)；C18色谱柱(4.6mm×250mm，5μm)，流速1ml/min、柱温40℃、进样量10μL；流动相：68％双蒸水，0.5％乙酸，32％乙腈。LC-QTOF-MS检测样品则采用Triple TOF5600+QTOF-MS***(SCIEX)与LC-30AD UPLC***(岛津)。
结果如图3所示，大肠杆菌发酵液利用LC-QTOF/MS在正离子模式下检测麻黄碱对应信号。
实施例3
将实施例1制备的重组酿酒酵母菌接种在酵母氮源基础培养基中，过夜培养后按1:100比例接入酵母氮源基础培养基并使用20μM铜离子诱导蛋白表达，后续通过液相色谱或质谱检测麻黄碱。
产品定性与定量分析：采用岛津高效液相色谱仪检测分析，采用光电二极管阵列检测器(工作波长280nm)；C18色谱柱(4.6mm×250mm，5μm)，流速1ml/min、柱温40℃、进样量10μL；流动相：68％双蒸水，0.5％乙酸，32％乙腈。LC-QTOF-MS检测样品则采用Triple TOF5600+QTOF-MS***(SCIEX)与LC-30AD UPLC***(岛津)。
结果如图4所示，酿酒酵母发酵液利用LC-QTOF/MS在负离子模式下检测麻黄碱对应信号。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不应理解为必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。此外，本领域的技术人员可以将本说明书中描述的不同实施例或示例进行接合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
序列表
<110> 厦门大学
<120> 用于制备麻黄碱的工程菌的构建及麻黄碱的制备方法
<130> 无
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1689
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggcaagtt cgggcacaac atcgacgcgt aagcgcttta ccggcgcaga atttatcgtt 60
catttcctgg aacagcaggg cattaagatt gtgacaggca ttccgggcgg ttctatcctg 120
cctgtttacg atgccttaag ccaaagcacg caaatccgcc atattctggc ccgtcatgaa 180
cagggcgcgg gctttatcgc tcagggaatg gcgcgcaccg acggtaaacc ggcggtctgt 240
atggcctgta gcggaccggg tgcgactaac ctggtgaccg ccattgccga tgcgcggctg 300
gactccatcc cgctgatttg catcactggt caggttcccg cctcgatgat cggcaccgac 360
gccttccagg aagtggacac ctacggcatc tctatcccca tcaccaaaca caactatctg 420
gtcagacata tcgaagaact cccgcaggtc atgagcgatg ccttccgcat tgcgcaatca 480
ggccgcccag gcccggtgtg gatagacatt cctaaggatg tgcaaacggc agtttttgag 540
attgaaacac agcccgctat ggcagaaaaa gccgccgccc ccgcctttag cgaagaaagc 600
attcgtgacg cagcggcgat gattaacgct gccaaacgcc cggtgcttta tctgggcggc 660
ggtgtgatca atgcgcccgc acgggtgcgt gaactggcgg agaaagcgca actgcctacc 720
accatgactt taatggcgct gggcatgttg ccaaaagcgc atccgttgtc gctgggtatg 780
ctggggatgc acggcgtgcg cagcaccaac tatattttgc aggaggcgga tttgttgata 840
gtgctcggtg cgcgttttga tgaccgggcg attggcaaaa ccgagcagtt ctgtccgaat 900
gccaaaatca ttcatgtcga tatcgaccgt gcagagctgg gtaaaatcaa gcagccgcac 960
gtggcgattc aggcggatgt tgatgacgtg ctggcgcagt tgatcccgct ggtggaagcg 1020
caaccgcgtg cagagtggca ccagttggta gcggatttgc agcgtgagtt tccgtgtcca 1080
atcccgaaag cgtgcgatcc gttaagccat tacggcctga tcaacgccgt tgccgcctgt 1140
gtcgatgaca atgcaattat caccaccgac gttggtcagc atcagatgtg gaccgcgcaa 1200
gcttatccgc tcaatcgccc acgccagtgg ctgacctccg gtgggctggg cacgatgggt 1260
tttggcctgc ctgcggcgat tggcgctgcg ctggcgaacc cggatcgcaa agtgttgtgt 1320
ttctccggcg acggcagcct gatgatgaat attcaggaga tggcgaccgc cagtgaaaat 1380
cagctggatg tcaaaatcat tctgatgaac aacgaagcgc tggggctggt gcatcagcaa 1440
cagagtctgt tctacgagca aggcgttttt gccgccacct atccgggcaa aatcaacttt 1500
atgcagattg ccgccggatt cggcctcgaa acctgtgatt tgaataacga agccgatccg 1560
caggcttcat tgcaggaaat catcaatcgc cctggcccgg cgctgatcca tgtgcgcatt 1620
gatgccgaag aaaaagttta cccgatggtg ccgccaggtg cggcgaatac tgaaatggtg 1680
ggggaataa 1689
<210> 2
<211> 1689
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggcaagtt cgggcacaac atcgacgcgt aagcgcttta ccggcgcaga atttatcgtt 60
catttcctgg aacagcaggg cattaagatt gtgacaggca ttccgggcgg ttctatcctg 120
cctgtttacg atgccttaag ccaaagcacg caaatccgcc atattctggc ccgtcatgaa 180
cagggcgcgg gctttatcgc tcagggaatg gcgcgcaccg acggtaaacc ggcggtctgt 240
atggcctgta gcggaccggg tgcgactaac ctggtgaccg ccattgccga tgcgcggctg 300
gactccatcc cgctgatttg catcactggt caggttcccg cctcgatgat cggcaccgac 360
gccttccagg aagtggacac ctacggcatc tctatcccca tcaccaaaca caactatctg 420
gtcagacata tcgaagaact cccgcaggtc atgagcgatg ccttccgcat tgcgcaatca 480
ggccgcccag gcccggtgtg gatagacatt cctaaggatg tgcaaacggc agtttttgag 540
attgaaacac agcccgctat ggcagaaaaa gccgccgccc ccgcctttag cgaagaaagc 600
attcgtgacg cagcggcgat gattaacgct gccaaacgcc cggtgcttta tctgggcggc 660
ggtgtgatca atgcgcccgc acgggtgcgt gaactggcgg agaaagcgca actgcctacc 720
accatgactt taatggcgct gggcatgttg ccaaaagcgc atccgttgtc gctgggtatg 780
ctggggatgc acggcgtgcg cagcaccaac tatattttgc aggaggcgga tttgttgata 840
gtgctcggtg cgcgttttga tgaccgggcg attggcaaaa ccgagcagtt ctgtccgaat 900
gccaaaatca ttcatgtcga tatcgaccgt gcagagctgg gtaaaatcaa gcagccgcac 960
gtggcgattc aggcggatgt tgatgacgtg ctggcgcagt tgatcccgct ggtggaagcg 1020
caaccgcgtg cagagtggca ccagttggta gcggatttgc agcgtgagtt tccgtgtcca 1080
atcccgaaag cgtgcgatcc gttaagccat tacggcctga tcaacgccgt tgccgcctgt 1140
gtcgatgaca atgcaattat caccaccgac gttggtcagc atcagatgtg gaccgcgcaa 1200
gcttatccgc tcaatcgccc acgccagtgg ctgacctccg gtgggctggg cacgatgggt 1260
tttggcctgc ctgcggcgat tggcgctgcg ctggcgaacc cggatcgcaa agtgttgtgt 1320
ttctccggcg acggcagcct gatgatgaat attcaggaga tggcgaccgc cagtgaaaat 1380
cagctggatg tcaaaatcat tctgatgaac aacgaagcgc tggggctggt gcatcagcaa 1440
cagagtctgt tctacgagca aggcgttttt gccgccacct atccgggcaa aatcaacttt 1500
atgcagattg ccgccggatt cggcctcgaa acctgtgatt tgaataacga agccgatccg 1560
caggcttcat tgcaggaaat catcaatcgc cctggcccgg cgctgatcca tgtgcgcatt 1620
gatgccgaag aaaaagttta cccgatggtg ccgccaggtg cggcgaatac tgaaatggtg 1680
ggggaataa 1689
<210> 3
<211> 1074
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgcagaatt tcgaaatcga ctatgttgag atgtatgtgg agaatctgga agtggcggct 60
ttctcatggg tcgacaagta cgcattcgcc gtggctggta cgagccgttc agcggaccac 120
aggagcatcg cgttgagaca agggcaggtg acacttgttc ttactgaacc gacgtcagat 180
agacatccgg ccgccgcgta tttgcagact catggcgatg gtgtagcgga tattgctatg 240
gccacctccg acgttgctgc ggcctacgag gcggcagtac gtgccggtgc tgaagctgtt 300
agagcaccag ggcaacacag tgaagctgct gtgacgaccg cgaccatagg tgggtttgga 360
gatgtcgtcc atactctgat ccaaagggac ggcactagcg ctgaattacc ccccggattt 420
accggctcca tggacgttac gaaccatggt aaaggagacg tagatcttct ggggatagat 480
cactttgcga tatgtcttaa cgccggagat ctgggaccta ctgtggaata ctacgagaga 540
gctttaggtt ttaggcaaat atttgacgag catatagttg ttggcgctca ggcgatgaac 600
tcaactgtcg tgcaaagtgc gagtggggcg gtaacactaa ccttgataga acccgatcgt 660
aatgccgacc ccgggcaaat tgatgagttc ctaaaagatc accagggtgc gggtgtgcag 720
cacatcgcct ttaattctaa tgacgctgtg cgtgcagtga aagcactgtc agagagaggg 780
gtcgagtttt tgaaaacccc gggggcgtat tacgatcttc taggagagag aataaccctg 840
caaacgcata gtctggatga tttgagagcg accaatgttt tggcagatga ggatcacgga 900
ggtcaacttt ttcaaatctt cacagcgagt acgcacccaa gacacaccat tttttttgaa 960
gtcatcgaaa gacaaggagc gggcactttt ggttccagta atatcaaggc tttatatgag 1020
gcagtggaac tagaacgtac aggtcaaagt gagtttggag ccgcgaggcg ttag 1074
<210> 4
<211> 1182
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgagccgga atctctttaa cgttgaggac tatcgcaagc tcgcgcaaaa gcgcttgcca 60
aagatggtgt acgactatct ggaaggtgga gctgaagacg aatacggggt gaaacacaac 120
cgcgacgtct tccagcaatg gcgattcaaa ccgaagcggc tggtagatgt cagccgccgc 180
agccttcaag cggaagtact tggaaagagg cagtcaatgc ctctcttgat tggcccaact 240
gggctgaatg gtgcgctctg gcctaaaggg gatctcgccc ttgctcaagc ggcaaccaag 300
gccggcatcc cgttcgtgct gtcgaccgcc tccaacatgt ccattgaaga cctcgctcgt 360
cagtgtgatg gcgatctatg gttccagctc tatgtgatcc accgagagat cgcgcagggg 420
atggtgctca aagccctgca ctccggttac accacgctgg tactcacaac agacgtcgcg 480
gttaatggct atcgcgagcg ggacctgcac aaccgattca agatgccgat gagctacacc 540
ccaaaggtga tgctggacgg atgcctgcat ccacgctggt cgctcgatct ggtgcgccac 600
ggcatgccgc aactggccaa cttcgtcagc agtcaaacgt ccagcttaga gatgcaggca 660
gcattgatga gccgccaaat ggatgccagt ttcaactggg aggcattgag atggctgcgt 720
gacctctggc cgcacaaact cctcgtaaag gggttgctca gtgctgagga cgccgatcgg 780
tgcatcgctg aaggtgccga cggcgtgatc ctgtcaaacc acggcggtcg ccaactcgat 840
tgcgcggtat cgccaatgga agttttggct caatcggtag cgaaaactgg aaaaccagtg 900
cttatcgata gcggcttccg acggggttcg gacatcgtta aagcgcttgc gctaggtgct 960
gaggctgtac tcctgggacg tgcaactttg tatggccttg cagcacgagg tgaaacgggt 1020
gttggcgagg tgctaaccct cctcaaagcg gatatcgacc gcactctggc ccagattgga 1080
tgccctgaca tcacctccct ttctcctgat tacctccaaa gcgagggagt gactaacacc 1140
gctccagtcg atcacctcat tggtaaagga acacacgcat ga 1182
<210> 5
<211> 1587
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggcttcgg tacacggcac cacatatgaa ctcctgcgac gtcagggcat cgacacggtc 60
ttcggcaatc ctggttcgaa cgagctcccg tttttgaagg actttccaga agactttcga 120
tacatcctgg ctttgcagga agcgtgtgtg gtgggcattg cagacggcta cgcgcaagcc 180
agccggaagc cagctttcat taacctgcat tctgctgctg gtaccggcaa tgccatgggt 240
gcactcagca acgcctggaa ctcacattcc cctctgatcg tcactgccgg ccagcagacc 300
agggcgatga ttggcgtcga agctttgctc accaacgtcg atgcagcgaa cctgccacga 360
ccactcgtca aatggagcta cgagcccgca agcgcggcag aagtacctca tgcaatgagc 420
cgggctatcc atatggcgag catggcgcca caaggccctg tctatctctc ggtaccatat 480
gacgattggg ataaagatgc tgatcctcag tcccaccacc tttttgatcg ccatgtcagt 540
tcggcagtac gcctgaacga ccaggatctt gagattctgg tgaaggcact gaacagcgcg 600
tccaacccgg cgatcgtact gggcccggac gtcgacgcag caaatgcgaa cgcagactgc 660
gtccagttgg ccgaacgcct caaagctccg gtttgggttg caccatccgc tccacgctgc 720
ccattcccta cccgtcatcc ttgcttccgt ggattgatgc cagctggcat cgcagcgatt 780
tctcagttgc tggaaggtca cgatttggtt ttggtgatcg gcgctccagt tttccgctac 840
caccaatacg acccaggaca atatctcaaa cctggcacgc gattgatttc ggtgacctgt 900
gacccgctcg aagcagcacg cgcgccaatg ggcgatgcga tcgtggcaga cattggcgcg 960
atggctagcg ctcttgccaa ctcggttgaa gagtgcagcc gcccgctccc aactgccgcc 1020
ccggaacctg cgaaggttga ccaagacgct ggccgactcc acccggagac tgtgttcgac 1080
acattgaatg acatggcccc ggagaatgcg atttacctga acgagtcgac ttcaacgacc 1140
gcccaaatgt ggcagcgcct gaacatgcgc aaccctggta gctattactt ctgtgccgct 1200
ggtggactgg gcttcgccct gcctgcagca attggggtgc aactcgcaga acccgaacga 1260
caagtcatcg cagtcattgg cgacggttcg gcgaactaca gcatcagtgc gttgtggact 1320
gcagcgcagt acaacatccc caccatcttc gtgatcatga acaacggcac ctacggtgcg 1380
ttgcgatggt ttgccggcgt tctcgaagca gaaaacgttc ccgggctgga tgtaccgggc 1440
atcgacttct gcgcactcgc caagggctat ggtgtccaag cgctgaaagc caacaacctt 1500
gagcagctca agggttcgtt acaagaagcg ctttctgcca aaggcccggt acttatcgaa 1560
gtaagcaccg taagcccggt gaagtga 1587
Claims (4)
1.一种用于制备麻黄碱的工程菌构建方法，其特征在于，包括：
向大肠杆菌MG1655 RARE或酿酒酵母CEN.PK2-1C中转化含有羟基扁桃酸合成酶基因HmaS和乙酰羟酸合成酶基因AHAS的重组质粒以及含有扁桃酸脱氢酶基因SMDH和苯甲酰甲酸脱羧酶基因BFD的重组质粒；所述乙酰羟酸合成酶基因AHAS是以核苷酸序列如SEQ IDNO:1所示的大肠杆菌基因组为模板，或以核苷酸序列如SEQ ID NO:2所示的酿酒酵母基因组为模板，经PCR扩增得到；所述羟基扁桃酸合成酶基因HmaS是以核苷酸序列如SEQ ID NO:3所示的东方拟无枝酸菌来源的HmaS基因为模板，经PCR扩增得到；所述扁桃酸脱氢酶基因SMDH，以核苷酸序列如SEQ ID NO:4所示的恶臭假单胞菌KT2440基因组为模板，经PCR扩增得到；所述苯甲酰甲酸脱羧酶基因BFD是以核苷酸序列如SEQ ID NO:5所示的恶臭假单胞菌KT2440基因组为模板，经PCR扩增得到。
2.如权利要求1所述的工程菌的构建方法，其特征在于，所述大肠杆菌或酿酒酵母具备积累苯甲醛的能力。
3.一种用于制备麻黄碱的工程菌，其特征在于，由如权利要求1所述的工程菌的构建方法构建得到。
4.一种麻黄碱的制备方法，其特征在于，包括以下步骤：
将权利要求3所述的用于制备麻黄碱的工程菌接种于培养基中发酵培养，即得。
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