CN108517321B - 棒状杆菌诱导型启动子及含有该启动子的表达载体与应用 - Google Patents
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Abstract
本发明涉及一种棒状杆菌诱导型启动子及含有该启动子的表达载体与应用，所述表达载体转化至谷氨酸棒状杆菌获得的5‑氨基乙酰丙酸生产菌株并利用该菌株发酵法生产5‑氨基乙酰丙酸。用本发明获得的启动子构建的表达载体，通过肌醇诱导实现基因的可控表达，克服了现用诱导剂异丙基‑β‑D‑硫代半乳糖苷(Isopropylβ‑D‑Thiogalactoside，IPTG)对菌体细胞生长的抑制作用以及因其价格高昂造成的生产成本高等不足，同时具有稳定性等特性。利用该启动子整合于宿主基因组或将表达载体转化至宿主谷氨酸棒状杆菌构建获得5‑氨基乙酰丙酸生产菌株，其5‑氨基乙酰丙酸产量达到24.2g/L。
Description
技术领域
本发明涉及生物技术生产领域，尤其是棒状杆菌诱导型启动子及含有该启动子的表达载体与应用。
背景技术
目前工业化生产中多用异丙基-β-D-硫代半乳糖苷IPTG诱导的表达质粒，需要在生产过程中添加IPTG，价格昂贵，不利于工业化生产，并且IPTG对菌体细胞生长影响严重同时发酵液中含有大量残余IPTG，为后期产物的分离提取带来困难。尽管乳糖能够代谢IPTG，但对于大多数棒状杆菌而言，乳糖不能够进入胞内，进一步限制了其在棒状杆菌中的应用。
肌醇，也称为环己六醇，属于维生素类，是合成细胞质膜中丰富的磷脂——磷脂酰肌醇所必需的，又是棒状杆菌如脂多糖和脂褐素单胞菌中更复杂的细胞糖脂的前体。此外，肌醇也是咪唑硫醇的结构单元，它是一种对棒状杆菌特异性的低分子量硫醇，对于保护活性氧的类似于真核生物和革兰氏阴性细菌中的谷胱甘肽的破坏作用是必需的。
5-氨基乙酰丙酸作为一种具有高附加值的氨基酸衍生物，在医疗和农业方面具有重要的应用价值与开发前景。近年来，作为一种安全、选择性和渗透性好的光动力学药物在医学领域逐渐得到了重视。目前，己经应用于多种癌症的诊断和治疗。在农业上，已经成为一种无公害的绿色农药，作为除草剂、植物生长调节剂等。同时，也是工业上一些有机物合成的中间体。化学合成法反应历程比较长，其反应历程中要不可避免的采取一些有毒且价格昂贵的原料，同时也不可避免的造成环境污染。此外，5-氨基乙酰丙酸的结构中含有活泼的氨基和羧基，合成过程中必须将其保护起来，这样就增加了反应的步骤、副产物和成本，导致得率不高。而目前生物法因生产效率低不能工业化生产需求。
发明内容
本发明所要解决的技术问题在于提供棒状杆菌诱导型启动子。
本发明所要解决的另一技术问题在于提供含有上述棒状杆菌诱导型启动子的棒状杆菌诱导型表达质粒。
本发明所要解决的另一技术问题在于提供利用上述棒状杆菌诱导型表达质粒得到的用于生产5-氨基乙酰丙酸的质粒。
为解决上述技术问题，本发明的技术方案是：
一种棒状杆菌诱导型启动子Piol，受肌醇诱导调控，其序列如序列表<400>1所示。
一种棒状杆菌诱导型表达质粒pIol，含有上述棒状杆菌诱导型启动子。
优选的，上述棒状杆菌诱导型表达质粒pIol，是将上述棒状杆菌诱导型启动子插入至质粒pXMJ19的Hpa Ⅰ和Hind Ⅲ酶切位点得到的，其序列如序列表<400>2所示。
一种用于生产5-氨基乙酰丙酸的质粒pIolhemAL，在上述棒状杆菌诱导型表达质粒的BamH I和Kpn I酶切位点酶切位点插入谷氨酰-tRNA还原编码基因hemA和谷氨酸-1-半醛-氨基转移酶hemL，其序列如序列表<400>3所示，hemA序列如序列表<400>4所示，hemL序列如序列表<400>5所示。
上述用于生产5-氨基乙酰丙酸的质粒pIolhemAL的构建方法如下：
(1)克隆谷氨酰-tRNA还原编码基因hemA和谷氨酸-1-半醛-氨基转移酶hemL，hemA序列如<400>4所示，hemL序列如<400>5所示；
(2)分别将hemA和hemL转接至pIol，pIolhemAL获得其序列如<400>3所示；
(3)将pIolhemAL转化至所需大肠杆菌Escherichia coli DH5α。
一株5-氨基乙酰丙酸生产菌株，将上述棒状杆菌诱导型启动子连同其过表达的关键基因整合至宿主基因组，以及含有上述用于生产5-氨基乙酰丙酸的质粒。
优选的，上述5-氨基乙酰丙酸生产菌株，关键基因为柠檬酸合酶编码基因gltA、谷氨酸脱氢酶编码基因gdh、苏氨酸输出载体rhtA以及转氢酶pntAB中的一种或几种；其中，gltA序列如序列表<400>6所示，gdh序列如序列表<400>7所示，rhtA序列如序列表<400>8所示，pntAB序列如序列表<400>9所示。
优选的，上述5-氨基乙酰丙酸生产菌株，是由下述方法构建得到的：
(1)构建含启动子Piol的关键基因整合片段，关键基因包括柠檬酸合酶编码基因gltA、谷氨酸脱氢酶编码基因gdh、苏氨酸输出载体rhtA以及转氢酶pntAB中的一种或几种。gltA序列如<400>6所示，gdh序列如<400>7所示，rhtA序列如<400>8所示，pntAB序列如<400>9所示。
(2)将上述整合片段连接至pK18mobsacB获得重组质粒；
(3)将上述重组质粒转化至谷氨酸棒杆菌，获得关键基因过表达重组菌株；
(4)将pIolhemAL转化至上述重组菌株获得-氨基乙酰丙酸生产菌株。
一种应用上述菌株发酵法生产5-氨基乙酰丙酸的方法，利用所述5-氨基乙酰丙酸生产菌株生产5-氨基乙酰丙酸，具体步骤如下：
(1)种子培养：将权利要求5的菌株活化后，接种至装有1L种子培养基的5L发酵罐，流加25％W/V的氨水调节发酵液pH至6.8-7.2，溶氧维持在10-20％，通风量1-3m3/h，搅拌转速200-600rpm，32-37℃培养6-8h；
(2)发酵罐发酵：以5％-10％接种量将步骤(1)的种子培养物接至装有6L发酵培养基的10L发酵罐进行发酵培养，发酵温度32-37℃，通风量3-5m3/h，搅拌转速100-3000rpm，溶氧维持在10-20％，流加浓度为60-80％W/V的葡萄糖溶液，维持残糖浓度为0.1-0.5％W/V，流加25％W/V的氨水调节发酵液pH至6.8-7.2，发酵周期30-48h；
(3)种子培养基成分为：蔗糖10-15g/L，玉米浆15-25mL/L，酵母粉1-6g/L，(NH4)2SO4 1-4，KH2PO4 0.5-1.5g/L，MgSO4 0.1-0.5g/L，FeSO4·7H2O 0.01-0.05g/L，MnSO4·H2O0.010.05g/L，pH 7.0，0.075MPa高压蒸汽灭菌15min；
(4)发酵培养基成分为：葡萄糖15-45g/L，玉米浆5-15mL/L，豆饼水解液5-15mL/L，(NH4)2SO4 8-20g/L，生物素0.00004g/L，pH 7.0-7.2，121℃高温灭菌20min。
本发明的有益效果是：
用本发明所述启动子构建的表达载体，通过肌醇诱导实现基因的可控表达，克服了现用诱导剂异丙基-β-D-硫代半乳糖苷(Isopropyl β-D-Thiogalactoside，IPTG)对菌体细胞生长的抑制作用以及因其价格高昂和IPTG于发酵液中残留致使分离提取困难造成的生产成本高等不足，同时具有稳定性等特性。利用该启动子整合于宿主基因组或将表达载体转化至宿主谷氨酸棒状杆菌构建获得5-氨基乙酰丙酸生产菌株，将其应用于发酵法生产5-氨基乙酰丙酸中，其5-氨基乙酰丙酸产量达到24.2g/L，解决了发酵法效率低下的问题。
附图说明
图1肌醇诱导型表达质粒pIol构建示意图。
图2重组质粒pIolhemAL酶切验证图谱，其中M为GeneRuler 1kb，1泳道为hemA-hemL基因片段，2泳道为pIolhemAL BamH I单酶切，3泳道为pIolhemAL Kpn I单酶切，4泳道为pIolhemAL BamH I和Kpn I双酶切。
具体实施方式
为了使本领域的技术人员更好的理解本发明的技术方案，下面结合具体实施方式对本发明所述技术方案作进一步的详细说明。
实施例1：肌醇诱导启动子Piol的构建
采用PCR技术以谷氨酸棒杆菌13032基因组DNA为模板，
利用引物iolR-F和iolR-R以及iolT-F和iolT-R分别扩增iolR基因和iolT上游500bp序列PiolT500，PCR条件为94℃5min 1个循环，94℃30s、55℃30s、72℃40s 30个循环，72℃10min 1个循环，反应体系为50μL。将PCR产物经1％琼脂糖凝胶电泳后纯化回收。
iolR-F：5'TCAGTTAACATGACCACCGAAGCTCCCA3'
iolR-R：5'GCGGGTTCAGGGGGTGATTACCTGGCCACCAGAGTGG3'
iolt-F：5'TCACCCCCTGAACCCGC 3'
iolt-R：5'CTAGAAGCTTCTTGTCTCCTAAGTTTGTCGTGCC 3'
以上述序列为模板，利用引物iolR-F和iolT-R进行重叠PCR，条件为94℃5min 1个循环，94℃30s、55℃30s、72℃40s 30个循环，72℃10min1个循环，反应体系为50μL。将PCR产物经1％琼脂糖凝胶电泳后切胶回收，获得肌醇诱导启动子Piol。
实施例2：肌醇诱导表达质粒pIol的构建
将实施例1利用限制内切酶Hpa Ⅰ和Hind Ⅲ双切后，将pXMJ19用限制内切酶Hpa Ⅰ和Hind Ⅲ双切后去除其原始启动子Ptac和lacIq，然后将二者采用T4连接酶连接后转化后大肠杆菌E.coli DH5α，经筛选后获得肌醇诱导表达质粒pIol。肌醇诱导表达质粒pIol构建示意图如图1所示。
实施例3：5-氨基乙酰丙酸生产质粒pIolhemAL的构建
以谷氨酸棒杆菌13032基因组DNA为模板，利用引物hemA-F、hemA-R、hemL-F和hemL-R，扩增hemA、hemL基因片段。PCR条件为94℃5min 1个循环，94℃30s、55℃30s、72℃40s 30个循环，72℃10min1个循环，反应体系为50μL。将PCR产物经1％琼脂糖凝胶电泳后纯化回收，获得hemA和hemL。
hemA-F：5'CGCGGATCCATGGTGAGTGTACTCATCGTAGGG 3'
hemA-R：5'TTACTCCCTCGTTTGTGTGGC 3'
hemL-F：
5'GCCACACAAACGAGGGAGTAAGCACCCAAAAACACTATTGACCA3'
hemL-R：5'CGGGGTACCTCATGATGCCTTCGCTTCTGC 3'
以基因片段hemA、hemL为模板，利用引物hemA-F和hemL-R进行重叠PCR，获得hemA-hemL片段。
将hemA-hemL经BamH I和Kpn I双切后，将pXMJ19用限制内切酶BamH I和Kpn I双切，然后将二者采用T4连接酶连接后转化后大肠杆菌E.coli DH5α，经筛选后获得5-氨基乙酰丙酸生产质粒pIolhemAL。重组质粒pIolhemAL酶切验证图谱如图2所示。
实施例4：5-氨基乙酰丙酸生产菌株的构建
以谷氨酸棒杆菌13032基因组DNA为模板，利用引物gltA-1和gltA-2扩增柠檬酸合酶编码基因gltA，利用引物gdh-1和gdh-2扩增谷氨酸脱氢酶编码基因gdh，利用引物cg-1和cg-2以及cg-3和cg-4扩增cg1890上游500bp序列PU500和下游500bp序列PD500。以pIol为模板，利用引物P-1和P-2扩增PiolT400。
cg-1：5'GCCAGTGCCAAGCTTGCATGCAAACACAGGGTGCTCGTGGC 3'
cg-2：5'
TGGGAGCTTCGGTGGTCATAATCTTCTTCCCTTAAAAAGTGTCG 3'
cg-3：
5'TGGCACAGGGCGTCATCTAATTAATACCTAGTTCTTAGATGAAGTT TCAAA 3'
cg-4：5'TATGACCATGATTACGAATTCCCACTCATTACCGCCCGAT 3'
P-1：5'ATGACCACCGAAGCTCCCA 3'
P-2：
5'TTTGTTCGGAAAAAAACTCTTCCCTTGTCTCCTAAGTTTGTCGTGCC3'
gltA-1：5'GGAAGAGTTTTTTTCCGAACAAA 3'
gltA-2：5'CTGTCATGATTTCCTCGTTCCCGGAAGCCAAGGTTGCCATT3'
gdh-1：5'GGGAACGAGGAAATCATGACAG 3'
gdh-2：5'TTAGATGACGCCCTGTGCCA 3'
采用重叠PCR的方法，利用引物扩增PU500-PiolT400-gltA-gdh-PD500，将上述片段采用同源重组的方法连接至pK18mobsacB，获得pK-1。
将pK-1转化至谷氨酸棒杆菌13032，经筛选获得PiolT400-gltA-gdh整合至基因组的菌株，将实施例3所述的5-氨基乙酰丙酸生产质粒pIolhemAL转化至上述菌株，经筛选获得5-氨基乙酰丙酸生产菌株。
实施例5：5-氨基乙酰丙酸生产菌株的构建
以谷氨酸棒杆菌13032基因组DNA为模板，利用引物gltA-1和gltA-2扩增柠檬酸合酶编码基因gltA，利用引物gdh-1和gdh-2扩增谷氨酸脱氢酶编码基因gdh，利用引物cg-1和cg-2以及cg-5和cg-4扩增cg1890上游500bp序列PA500和下游500bp序列PB500。
cg-1：5'GCCAGTGCCAAGCTTGCATGCAAACACAGGGTGCTCGTGGC 3'
cg-2：5'
TGGGAGCTTCGGTGGTCATAATCTTCTTCCCTTAAAAAGTGTCG 3'
cg-4：5'TATGACCATGATTACGAATTCCCACTCATTACCGCCCGAT 3'
cg-5：
5'GAGCAAAATAAAAGAATTAGACATTAATTAATTAATACCTAGTTCT TAGATGAAGTTTCAAA 3'
以大肠杆菌E.coli K-12MG1655基因组DNA为模板，利用引物pntAB-1和pntAB-2扩增转氢酶pntAB，利用引物rhtA-1和rhtA-2扩增苏氨酸输出载体rhtA；以pIol为模板，利用引物P-1和P-2扩增PiolT400。
pntAB-1：5'
TGGCACAGGGCGTCATCTAAATGGAAGGGAATATCATGCGAA 3'
pntAB-2：5'TTACAGAGCTTTCAGGATTGCATC 3'
rhtA-1：
5'GATGCAATCCTGAAAGCTCTGTAAGTGGGAGAAAGGATGCCTGG3'
rhtA-2：5'TTAATTAATGTCTAATTCTTTTATTTTGCTC3'
采用重叠PCR的方法，利用引物扩增
PA500-PiolT400-gltA-gdh-pntAB-rhtA-PB500，将上述片段采用同源重组的方法连接至pK18mobsacB，获得pK-2。
将pK-2转化至谷氨酸棒杆菌13032，经筛选获得
PiolT400-gltA-gdh-pntAB-rhtA整合至基因组的菌株，将实施例3所述的5-氨基乙酰丙酸生产质粒pIolhemAL转化至上述菌株，经筛选获得5-氨基乙酰丙酸生产菌株。
实施例6：5-氨基乙酰丙酸生产菌株pK-1的10L发酵罐发酵
(1)种子培养：将权利5-氨基乙酰丙酸生产菌株pK-1活化后，接种至装有1L种子培养基的5L发酵罐，流加25％W/V的氨水调节发酵液pH至6.8-7.2，溶氧维持在10-20％，通风量1-3m3/h，搅拌转速200-600rpm，32-37℃培养6-8h；
(2)发酵罐发酵：以5％-10％接种量将步骤(1)的种子培养物接至装有6L发酵培养基的10L发酵罐进行发酵培养，发酵温度32-37℃，通风量3-5m3/h，搅拌转速100-3000rpm，溶氧维持在10-20％，流加浓度为60-80％W/V的葡萄糖溶液，维持残糖浓度为0.1-0.5％W/V，流加25％W/V的氨水调节发酵液pH至6.8-7.2，发酵周期30-48h；
(3)种子培养基成分为：蔗糖10-15g/L，玉米浆15-25mL/L，酵母粉1-6g/L，(NH4)2SO4 1-4，KH2PO4 0.5-1.5g/L，MgSO4 0.1-0.5g/L，FeSO4·7H2O0.01-0.05g/L，MnSO4·H2O0.010.05g/L，pH 7.0，0.075MPa高压蒸汽灭菌15min；
(4)发酵培养基成分为：葡萄糖15-45g/L，玉米浆5-15mL/L，豆饼水解液5-15mL/L，(NH4)2SO4 8-20g/L，生物素0.00004g/L，pH 7.0-7.2，121℃高温灭菌20min。
(5)发酵液中5-氨基乙酰丙酸(ALA)的检测：发酵液经8000×g离心10min后取上清液并用去离子水稀释10倍后，采用UltiMate 3000(Thermo Scientific)高效液相色谱仪测定5-氨基乙酰丙酸(ALA)的含量。检测条件为：色谱柱REzex RoA-organic Acid H+，流动相5mmol/L H2SO4，流速0.5mL/min，柱温30℃，检测波长215nm，进样量为20μL。检测结果表明，5-氨基乙酰丙酸(ALA)的出峰时间约为3.1min，产量约为6.2g/L。
实施例7：5-氨基乙酰丙酸生产菌株pK-2的10L发酵罐发酵
(1)种子培养：将权利5-氨基乙酰丙酸生产菌株pK-2活化后，接种至装有1L种子培养基的5L发酵罐，流加25％W/V的氨水调节发酵液pH至6.8-7.2，溶氧维持在10-20％，通风量1-3m3/h，搅拌转速200-600rpm，32-37℃培养6-8h；
(2)发酵罐发酵：以5％-10％接种量将步骤(1)的种子培养物接至装有6L发酵培养基的10L发酵罐进行发酵培养，发酵温度32-37℃，通风量3-5m3/h，搅拌转速100-3000rpm，溶氧维持在10-20％，流加浓度为60-80％W/V的葡萄糖溶液，维持残糖浓度为0.1-0.5％W/V，流加25％W/V的氨水调节发酵液pH至6.8-7.2，发酵周期30-48h；
(3)种子培养基成分为：蔗糖10-15g/L，玉米浆15-25mL/L，酵母粉1-6g/L，(NH4)2SO4 1-4，KH2PO4 0.5-1.5g/L，MgSO4 0.1-0.5g/L，FeSO4·7H2O0.01-0.05g/L，MnSO4·H2O0.010.05g/L，pH 7.0，0.075MPa高压蒸汽灭菌15min；
(4)发酵培养基成分为：葡萄糖15-45g/L，玉米浆5-15mL/L，豆饼水解液5-15mL/L，(NH4)2SO4 8-20g/L，生物素0.00004g/L，pH 7.0-7.2，121℃高温灭菌20min。
(5)发酵液中5-氨基乙酰丙酸(ALA)的检测：发酵液经8000×g离心10min后取上清液并用去离子水稀释10倍后，采用UltiMate 3000(Thermo Scientific)高效液相色谱仪测定5-氨基乙酰丙酸(ALA)的含量。检测条件为：色谱柱REzex RoA-organic Acid H+，流动相5mmol/L H2SO4，流速0.5mL/min，柱温30℃，检测波长215nm，进样量为20μL。检测结果表明，5-氨基乙酰丙酸(ALA)的出峰时间约为3.1min，产量约为24.2g/L。
上述参照具体实施方式对该棒状杆菌诱导型启动子及含有该启动子的表达载体与应用进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。
序列表
<110> 天津科技大学
<120> 棒状杆菌诱导型启动子及含有该启动子的表达载体与应用
<150> 2017113335859
<151> 2017-12-14
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1943
<212> DNA
<213> 肌醇诱导启动子Piol(棒状杆菌)
<220>
<221> promoter
<222> (1)..(1943)
<400> 1
accctcacga tcgcatgtca tgacaataac acattgtcct gacaaagcaa tacatttccg 60
aaaattttct aaaacgattc ccgaaaacgc gcaaaacccc agggcaacgc cccggttttc 120
attcaaagat tttgcttgtc gacgaaaacc ccctcgcttt ggaaggtgcg gggttaacaa 180
agttgcacgt tatgacctgc aaactctgcc cttttcacta agtttcgcta ctcattccct 240
aatgcaagtg ataatgtcag atcaataaaa gccctggatg acacaaaagt cctgcatgaa 300
cacggattta ccaagaccac cacccgcaac tcagttacat tgttcaaatg tcctaacaca 360
tttacatgag cttgttgggt gggcaacgaa aggagacatc atgaccaccg aagctcccat 420
ttggccagcc gaactcttcg aagacctcga ccgcaacgga ccaatccccc tctacttcca 480
agtagcccaa cgcctcgaag acggcatccg cagcggagtc ctcccacccg gagcacgcct 540
agaaaacgag atctccgtgg cgaaacacct caacgtatcc cgccccaccg tccgacgcgc 600
catccaagaa gtcgtagaca aaggcctctt agttcgccgc cgcggtgttg gcacccaggt 660
cgtccaaagc cacgtcaccc gcccagtcga actgaccagt ttcttcaacg acctcaaaaa 720
cgccaacctg gaccccaaaa cccgagtcct cgagcaccgc ctccttgcag caagttccgc 780
catcgcagaa aaactcggag tttccgcagg tgacgaagtc ctcctcatcc gccgcctccg 840
ctccaccgga gacatccccg tagcgatcct ggaaaactac ctccccccag cgttcaacga 900
cgtctccctc gacgaactag aaaagggtgg actctacgat gcgctgcgca gccgaggtgt 960
tgtcttaaaa atcgccaacc agaaaatcgg tgcgcgccga gcagtcggtg aagaaagcac 1020
cctcctcgac atcgaagacg gcggaccact tctcaccgtc gaacgcgttg cattggataa 1080
ttccggccaa gtaatcgagt tgggaagcca ctgctaccgc ccagatatgt acaactttga 1140
aaccactctg gtggccaggt aagaaataaa ccaaagagcc cttctgaact ggagctcttg 1200
gtctgatatg gcattttcaa ggctggaaaa ctgctctatc agaccaaacg gatgctgaat 1260
tttcaccccc tgaacccgcc tagggtttga tgcaaaaatt cgttcgactt tatggccaga 1320
cctcacgcct gtggtgaaaa attgatcagc aaacacccag gtttcacatt cgccccacca 1380
gtcccaaaat gatgacgttc cagagcgccg cctgacggca atttgtgccc actttgacac 1440
aagtggtcga tcgacgtctc gagccgctta aacgggcgat tatcgcccca ccattcccga 1500
tgtccgctcc tcgcacgctt tttgtaatga cattaggatc tttaagcagt gaatgaggtg 1560
acaatgtcac ctaacaaagg tgtcaaacag ccccaatcac taccccctcc acccccgcac 1620
ccttatccag aaactcccat gctccaacat ttccagaggg ggcagtttct gacattaacc 1680
acataactcc tgcatcaaac cgcagctaac agccacaccc ctgctgaaaa tcccgaatgg 1740
aaaaccatac ccaagcagac acccccaccc ctaagtatta ccaattactc aaaagtattc 1800
aaaaaaagtt tgttatgtac gattgacggg acatatcgtg tctgccacga ttaaagacat 1860
tggtgatgtg aatcactgcc tactacatcg tgtttcgtga ccctgcacct ccaagtaagg 1920
gcacgacaaa cttaggagac aag 1943
<210> 2
<211> 7286
<212> DNA
<213> 肌醇诱导表达质粒pIol(棒状杆菌)
<220>
<221> gene
<222> (1)..(7286)
<400> 2
cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct 60
gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc 120
catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg 180
ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc 240
gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc 300
ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt 360
tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 420
aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 480
tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 540
aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 600
aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 660
tgatgagcac ttttgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 720
gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 780
caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 840
tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 900
gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 960
ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 1020
cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg 1080
tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 1140
tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 1200
cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 1260
agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 1320
agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 1380
gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 1440
aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 1500
ggattttggt catgagatta tcaaaaagga tcttcaccta gatccttttg gggtgggcga 1560
agaactccag catgagatcc ccgcgctgga ggatcatcca gccattcggg gtcgttcact 1620
ggttcccctt tctgatttct ggcatagaag aacccccgtg aactgtgtgg ttccgggggt 1680
tgctgatttt tgcgagactt ctcgcgcaat tccctagctt aggtgaaaac accatgaaac 1740
actagggaaa cacccatgaa acacccatta gggcagtagg gcggcttctt cgtctagggc 1800
ttgcatttgg gcggtgatct ggtctttagc gtgtgaaagt gtgtcgtagg tggcgtgctc 1860
aatgcactcg aacgtcacgt catttaccgg gtcacggtgg gcaaagagaa ctagtgggtt 1920
agacattgtt ttcctcgttg tcggtggtgg tgagcttttc tagccgctcg gtaaacgcgg 1980
cgatcatgaa ctcttggagg ttttcaccgt tctgcatgcc tgcgcgcttc atgtcctcac 2040
gtagtgccaa aggaacgcgt gcggtgacca cgacgggctt agcctttgcc tgcgcttcta 2100
gtgcttcgat ggtggcttgt gcctgcgctt gctgcgcctg tagtgcctgt tgagcttctt 2160
gtagttgctg ttctagctgt gccttggttg ccatgcttta agactctagt agctttcctg 2220
cgatatgtca tgcgcatgcg tagcaaacat tgtcctgcaa ctcattcatt atgtgcagtg 2280
ctcctgttac tagtcgtaca tactcatatt tacctagtct gcatgcagtg catgcacatg 2340
cagtcatgtc gtgctaatgt gtaaaacatg tacatgcaga ttgctggggg tgcagggggc 2400
ggagccaccc tgtccatgcg gggtgtgggg cttgccccgc cggtacagac agtgagcacc 2460
ggggcaccta gtcgcggata ccccccctag gtatcggaca cgtaaccctc ccatgtcgat 2520
gcaaatcttt aacattgagt acgggtaagc tggcacgcat agccaagcta ggcggccacc 2580
aaacaccact aaaaattaat agtccctaga caagacaaac ccccgtgcga gctaccaact 2640
catatgcacg ggggccacat aacccgaagg ggtttcaatt gacaaccata gcactagcta 2700
agacaacggg cacaacaccc gcacaaactc gcactgcgca accccgcaca acatcgggtc 2760
taggtaacac tgagtaacac tgaaatagaa gtgaacacct ctaaggaacc gcaggtcaat 2820
gagggttcta aggtcactcg cgctagggcg tggcgtaggc aaaacgtcat gtacaagatc 2880
accaatagta aggctctggc ggggtgccat aggtggcgca gggacgaagc tgttgcggtg 2940
tcctggtcgt ctaacggtgc ttcgcagttt gagggtctgc aaaactctca ctctcgctgg 3000
gggtcacctc tggctgaatt ggaagtcatg ggcgaacgcc gcattgagct ggctattgct 3060
actaagaatc acttggcggc gggtggcgcg ctcatgatgt ttgtgggcac tgttcgacac 3120
aaccgctcac agtcatttgc gcaggttgaa gcgggtatta agactgcgta ctcttcgatg 3180
gtgaaaacat ctcagtggaa gaaagaacgt gcacggtacg gggtggagca cacctatagt 3240
gactatgagg tcacagactc ttgggcgaac ggttggcact tgcaccgcaa catgctgttg 3300
ttcttggatc gtccactgtc tgacgatgaa ctcaaggcgt ttgaggattc catgttttcc 3360
cgctggtctg ctggtgtggt taaggccggt atggacgcgc cactgcgtga gcacggggtc 3420
aaacttgatc aggtgtctac ctggggtgga gacgctgcga aaatggcaac ctacctcgct 3480
aagggcatgt ctcaggaact gactggctcc gctactaaaa ccgcgtctaa ggggtcgtac 3540
acgccgtttc agatgttgga tatgttggcc gatcaaagcg acgccggcga ggatatggac 3600
gctgttttgg tggctcggtg gcgtgagtat gaggttggtt ctaaaaacct gcgttcgtcc 3660
tggtcacgtg gggctaagcg tgctttgggc attgattaca tagacgctga tgtacgtcgt 3720
gaaatggaag aagaactgta caagctcgcc ggtctggaag caccggaacg ggtcgaatca 3780
acccgcgttg ctgttgcttt ggtgaagccc gatgattgga aactgattca gtctgatttc 3840
gcggttaggc agtacgttct cgattgcgtg gataaggcta aggacgtggc cgctgcgcaa 3900
cgtgtcgcta atgaggtgct ggcaagtctg ggtgtggatt ccaccccgtg catgatcgtt 3960
atggatgatg tggacttgga cgcggttctg cctactcatg gggacgctac taagcgtgat 4020
ctgaatgcgg cggtgttcgc gggtaatgag cagactattc ttcgcaccca ctaaaagcgg 4080
cataaacccc gttcgatatt ttgtgcgatg aatttatggt caatgtcgcg ggggcaaact 4140
atgatgggtc ttgttgttgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa 4200
ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc 4260
gaagggcacc aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtac 4320
tgttgtaatt cattaagcat tctgccgaca tggaagccat cacagacggc atgatgaacc 4380
tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa 4440
acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc 4500
cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg 4560
ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg 4620
tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa 4680
gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg gaactccgga 4740
tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt 4800
ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat 4860
tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg 4920
gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgtc 4980
gaagctcggc ggatttgtcc tactcaagct gatccgacaa aatccacaca ttatcccagg 5040
tgtccggatc ggtcaaatac gctgccagct catagaccgt atccaaagca tccggggctg 5100
atccccggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc 5160
ttcaccgcct gccctgagag agttgcagca agcggtccac gtggtttgcc ccagcaggcg 5220
aaaatcctgt ttgatggtgg ttaacaccct cacgatcgca tgtcatgaca ataacacatt 5280
gtcctgacaa agcaatacat ttccgaaaat tttctaaaac gattcccgaa aacgcgcaaa 5340
accccagggc aacgccccgg ttttcattca aagattttgc ttgtcgacga aaaccccctc 5400
gctttggaag gtgcggggtt aacaaagttg cacgttatga cctgcaaact ctgccctttt 5460
cactaagttt cgctactcat tccctaatgc aagtgataat gtcagatcaa taaaagccct 5520
ggatgacaca aaagtcctgc atgaacacgg atttaccaag accaccaccc gcaactcagt 5580
tacattgttc aaatgtccta acacatttac atgagcttgt tgggtgggca acgaaaggag 5640
acatcatgac caccgaagct cccatttggc cagccgaact cttcgaagac ctcgaccgca 5700
acggaccaat ccccctctac ttccaagtag cccaacgcct cgaagacggc atccgcagcg 5760
gagtcctccc acccggagca cgcctagaaa acgagatctc cgtggcgaaa cacctcaacg 5820
tatcccgccc caccgtccga cgcgccatcc aagaagtcgt agacaaaggc ctcttagttc 5880
gccgccgcgg tgttggcacc caggtcgtcc aaagccacgt cacccgccca gtcgaactga 5940
ccagtttctt caacgacctc aaaaacgcca acctggaccc caaaacccga gtcctcgagc 6000
accgcctcct tgcagcaagt tccgccatcg cagaaaaact cggagtttcc gcaggtgacg 6060
aagtcctcct catccgccgc ctccgctcca ccggagacat ccccgtagcg atcctggaaa 6120
actacctccc cccagcgttc aacgacgtct ccctcgacga actagaaaag ggtggactct 6180
acgatgcgct gcgcagccga ggtgttgtct taaaaatcgc caaccagaaa atcggtgcgc 6240
gccgagcagt cggtgaagaa agcaccctcc tcgacatcga agacggcgga ccacttctca 6300
ccgtcgaacg cgttgcattg gataattccg gccaagtaat cgagttggga agccactgct 6360
accgcccaga tatgtacaac tttgaaacca ctctggtggc caggtaagaa ataaaccaaa 6420
gagcccttct gaactggagc tcttggtctg atatggcatt ttcaaggctg gaaaactgct 6480
ctatcagacc aaacggatgc tgaattttca ccccctgaac ccgcctaggg tttgatgcaa 6540
aaattcgttc gactttatgg ccagacctca cgcctgtggt gaaaaattga tcagcaaaca 6600
cccaggtttc acattcgccc caccagtccc aaaatgatga cgttccagag cgccgcctga 6660
cggcaatttg tgcccacttt gacacaagtg gtcgatcgac gtctcgagcc gcttaaacgg 6720
gcgattatcg ccccaccatt cccgatgtcc gctcctcgca cgctttttgt aatgacatta 6780
ggatctttaa gcagtgaatg aggtgacaat gtcacctaac aaaggtgtca aacagcccca 6840
atcactaccc cctccacccc cgcaccctta tccagaaact cccatgctcc aacatttcca 6900
gagggggcag tttctgacat taaccacata actcctgcat caaaccgcag ctaacagcca 6960
cacccctgct gaaaatcccg aatggaaaac catacccaag cagacacccc cacccctaag 7020
tattaccaat tactcaaaag tattcaaaaa aagtttgtta tgtacgattg acgggacata 7080
tcgtgtctgc cacgattaaa gacattggtg atgtgaatca ctgcctacta catcgtgttt 7140
cgtgaccctg cacctccaag taagggcacg acaaacttag gagacaaggt cgactctaga 7200
ggatccccgg gtaccgagct cgaattcagc ttggctgttt tggcggatga gagaagattt 7260
tcagcctgat acagattaaa tcagaa 7286
<210> 3
<211> 10094
<212> DNA
<213> 5-氨基乙酰丙酸的质粒pIolhemAL(5-氨基乙酰丙酸)
<220>
<221> gene
<222> (1)..(10094)
<400> 3
cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct 60
gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc 120
catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg 180
ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc 240
gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc 300
ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt 360
tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 420
aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 480
tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 540
aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 600
aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 660
tgatgagcac ttttgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 720
gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 780
caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 840
tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 900
gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 960
ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 1020
cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg 1080
tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 1140
tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 1200
cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 1260
agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 1320
agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 1380
gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 1440
aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 1500
ggattttggt catgagatta tcaaaaagga tcttcaccta gatccttttg gggtgggcga 1560
agaactccag catgagatcc ccgcgctgga ggatcatcca gccattcggg gtcgttcact 1620
ggttcccctt tctgatttct ggcatagaag aacccccgtg aactgtgtgg ttccgggggt 1680
tgctgatttt tgcgagactt ctcgcgcaat tccctagctt aggtgaaaac accatgaaac 1740
actagggaaa cacccatgaa acacccatta gggcagtagg gcggcttctt cgtctagggc 1800
ttgcatttgg gcggtgatct ggtctttagc gtgtgaaagt gtgtcgtagg tggcgtgctc 1860
aatgcactcg aacgtcacgt catttaccgg gtcacggtgg gcaaagagaa ctagtgggtt 1920
agacattgtt ttcctcgttg tcggtggtgg tgagcttttc tagccgctcg gtaaacgcgg 1980
cgatcatgaa ctcttggagg ttttcaccgt tctgcatgcc tgcgcgcttc atgtcctcac 2040
gtagtgccaa aggaacgcgt gcggtgacca cgacgggctt agcctttgcc tgcgcttcta 2100
gtgcttcgat ggtggcttgt gcctgcgctt gctgcgcctg tagtgcctgt tgagcttctt 2160
gtagttgctg ttctagctgt gccttggttg ccatgcttta agactctagt agctttcctg 2220
cgatatgtca tgcgcatgcg tagcaaacat tgtcctgcaa ctcattcatt atgtgcagtg 2280
ctcctgttac tagtcgtaca tactcatatt tacctagtct gcatgcagtg catgcacatg 2340
cagtcatgtc gtgctaatgt gtaaaacatg tacatgcaga ttgctggggg tgcagggggc 2400
ggagccaccc tgtccatgcg gggtgtgggg cttgccccgc cggtacagac agtgagcacc 2460
ggggcaccta gtcgcggata ccccccctag gtatcggaca cgtaaccctc ccatgtcgat 2520
gcaaatcttt aacattgagt acgggtaagc tggcacgcat agccaagcta ggcggccacc 2580
aaacaccact aaaaattaat agtccctaga caagacaaac ccccgtgcga gctaccaact 2640
catatgcacg ggggccacat aacccgaagg ggtttcaatt gacaaccata gcactagcta 2700
agacaacggg cacaacaccc gcacaaactc gcactgcgca accccgcaca acatcgggtc 2760
taggtaacac tgagtaacac tgaaatagaa gtgaacacct ctaaggaacc gcaggtcaat 2820
gagggttcta aggtcactcg cgctagggcg tggcgtaggc aaaacgtcat gtacaagatc 2880
accaatagta aggctctggc ggggtgccat aggtggcgca gggacgaagc tgttgcggtg 2940
tcctggtcgt ctaacggtgc ttcgcagttt gagggtctgc aaaactctca ctctcgctgg 3000
gggtcacctc tggctgaatt ggaagtcatg ggcgaacgcc gcattgagct ggctattgct 3060
actaagaatc acttggcggc gggtggcgcg ctcatgatgt ttgtgggcac tgttcgacac 3120
aaccgctcac agtcatttgc gcaggttgaa gcgggtatta agactgcgta ctcttcgatg 3180
gtgaaaacat ctcagtggaa gaaagaacgt gcacggtacg gggtggagca cacctatagt 3240
gactatgagg tcacagactc ttgggcgaac ggttggcact tgcaccgcaa catgctgttg 3300
ttcttggatc gtccactgtc tgacgatgaa ctcaaggcgt ttgaggattc catgttttcc 3360
cgctggtctg ctggtgtggt taaggccggt atggacgcgc cactgcgtga gcacggggtc 3420
aaacttgatc aggtgtctac ctggggtgga gacgctgcga aaatggcaac ctacctcgct 3480
aagggcatgt ctcaggaact gactggctcc gctactaaaa ccgcgtctaa ggggtcgtac 3540
acgccgtttc agatgttgga tatgttggcc gatcaaagcg acgccggcga ggatatggac 3600
gctgttttgg tggctcggtg gcgtgagtat gaggttggtt ctaaaaacct gcgttcgtcc 3660
tggtcacgtg gggctaagcg tgctttgggc attgattaca tagacgctga tgtacgtcgt 3720
gaaatggaag aagaactgta caagctcgcc ggtctggaag caccggaacg ggtcgaatca 3780
acccgcgttg ctgttgcttt ggtgaagccc gatgattgga aactgattca gtctgatttc 3840
gcggttaggc agtacgttct cgattgcgtg gataaggcta aggacgtggc cgctgcgcaa 3900
cgtgtcgcta atgaggtgct ggcaagtctg ggtgtggatt ccaccccgtg catgatcgtt 3960
atggatgatg tggacttgga cgcggttctg cctactcatg gggacgctac taagcgtgat 4020
ctgaatgcgg cggtgttcgc gggtaatgag cagactattc ttcgcaccca ctaaaagcgg 4080
cataaacccc gttcgatatt ttgtgcgatg aatttatggt caatgtcgcg ggggcaaact 4140
atgatgggtc ttgttgttgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa 4200
ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc 4260
gaagggcacc aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtac 4320
tgttgtaatt cattaagcat tctgccgaca tggaagccat cacagacggc atgatgaacc 4380
tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa 4440
acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc 4500
cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg 4560
ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg 4620
tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa 4680
gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg gaactccgga 4740
tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt 4800
ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat 4860
tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg 4920
gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgtc 4980
gaagctcggc ggatttgtcc tactcaagct gatccgacaa aatccacaca ttatcccagg 5040
tgtccggatc ggtcaaatac gctgccagct catagaccgt atccaaagca tccggggctg 5100
atccccggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc 5160
ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgtggtttgc cccagcaggc 5220
gaaaatcctg tttgatggtg gttaacaccc tcacgatcgc atgtcatgac aataacacat 5280
tgtcctgaca aagcaataca tttccgaaaa ttttctaaaa cgattcccga aaacgcgcaa 5340
aaccccaggg caacgccccg gttttcattc aaagattttg cttgtcgacg aaaaccccct 5400
cgctttggaa ggtgcggggt taacaaagtt gcacgttatg acctgcaaac tctgcccttt 5460
tcactaagtt tcgctactca ttccctaatg caagtgataa tgtcagatca ataaaagccc 5520
tggatgacac aaaagtcctg catgaacacg gatttaccaa gaccaccacc cgcaactcag 5580
ttacattgtt caaatgtcct aacacattta catgagcttg ttgggtgggc aacgaaagga 5640
gacatcatga ccaccgaagc tcccatttgg ccagccgaac tcttcgaaga cctcgaccgc 5700
aacggaccaa tccccctcta cttccaagta gcccaacgcc tcgaagacgg catccgcagc 5760
ggagtcctcc cacccggagc acgcctagaa aacgagatct ccgtggcgaa acacctcaac 5820
gtatcccgcc ccaccgtccg acgcgccatc caagaagtcg tagacaaagg cctcttagtt 5880
cgccgccgcg gtgttggcac ccaggtcgtc caaagccacg tcacccgccc agtcgaactg 5940
accagtttct tcaacgacct caaaaacgcc aacctggacc ccaaaacccg agtcctcgag 6000
caccgcctcc ttgcagcaag ttccgccatc gcagaaaaac tcggagtttc cgcaggtgac 6060
gaagtcctcc tcatccgccg cctccgctcc accggagaca tccccgtagc gatcctggaa 6120
aactacctcc ccccagcgtt caacgacgtc tccctcgacg aactagaaaa gggtggactc 6180
tacgatgcgc tgcgcagccg aggtgttgtc ttaaaaatcg ccaaccagaa aatcggtgcg 6240
cgccgagcag tcggtgaaga aagcaccctc ctcgacatcg aagacggcgg accacttctc 6300
accgtcgaac gcgttgcatt ggataattcc ggccaagtaa tcgagttggg aagccactgc 6360
taccgcccag atatgtacaa ctttgaaacc actctggtgg ccaggtaaga aataaaccaa 6420
agagcccttc tgaactggag ctcttggtct gatatggcat tttcaaggct ggaaaactgc 6480
tctatcagac caaacggatg ctgaattttc accccctgaa cccgcctagg gtttgatgca 6540
aaaattcgtt cgactttatg gccagacctc acgcctgtgg tgaaaaattg atcagcaaac 6600
acccaggttt cacattcgcc ccaccagtcc caaaatgatg acgttccaga gcgccgcctg 6660
acggcaattt gtgcccactt tgacacaagt ggtcgatcga cgtctcgagc cgcttaaacg 6720
ggcgattatc gccccaccat tcccgatgtc cgctcctcgc acgctttttg taatgacatt 6780
aggatcttta agcagtgaat gaggtgacaa tgtcacctaa caaaggtgtc aaacagcccc 6840
aatcactacc ccctccaccc ccgcaccctt atccagaaac tcccatgctc caacatttcc 6900
agagggggca gtttctgaca ttaaccacat aactcctgca tcaaaccgca gctaacagcc 6960
acacccctgc tgaaaatccc gaatggaaaa ccatacccaa gcagacaccc ccacccctaa 7020
gtattaccaa ttactcaaaa gtattcaaaa aaagtttgtt atgtacgatt gacgggacat 7080
atcgtgtctg ccacgattaa agacattggt gatgtgaatc actgcctact acatcgtgtt 7140
tcgtgaccct gcacctccaa gtaagggcac gacaaactta ggagacaaga tggtgagtgt 7200
actcatcgta gggatgtcgc acaggtctgc gcctgtgtcg cttcttgaac gtctgagcat 7260
ggatgattca gtacgtggtg aaacaactca agcactcctg ggtagggcgt ctttaagcga 7320
ggccctcatt gtctctacgt gtaaccgcct ggaggtctac accgtcacta gcagtttcca 7380
tactggtgtt aatgatgtgg tggaggttct ccatgaggca agtggcgtag atattgaaac 7440
tttgcgcgga tatctttatg tccgttacgc cgatgctgct gctgaacaca tgttggtggt 7500
gacttccggg ttggattcca tggtgttggg tgagcagcag atcattggtc aggtgcgcac 7560
tgcgtaccaa gcagctaatg aatatggttc tgtcggtcct gctttgcatt cacttaccca 7620
gaccgcgctg cataccggca agcgcgtgca ttcggagact gctattgatg atgctggtgc 7680
atcgatggtg tctttcgctg tggatcgcgc gttggtgcag atgggtcttg attcggaggc 7740
agaagcccca ctatctggca agacagcctt ggtgttgggc gctggcgcga tgagttctct 7800
tgcagccact caccttggtc gcgctggaat ttccaacttg atcatggcca accgcactct 7860
ggaacgtgcc gaaaggcttg cggagcattc cctagaagcc ggagttcctg cagaggttgt 7920
ggaatacgat cagcgagctt ccgcctacaa tcgcgttgac ctggtagttt ccgccacggg 7980
agcggatgat ttcaccgtga agcctgagga tattccagaa ggtgcttcgt tgatgttggt 8040
ggatttgtcc atgccacgag acatcgatga tgcttgtgcg gatctgccgg gcgttgattt 8100
ggtgaacatc gaacgcctgc acaaggcctc ccgtgagggt ggatcgggca tggcgccaag 8160
cgaggaagaa gctttggcga ttgttcggga agagttggat tctttcacct ctgagcagcg 8220
cattcgcgat atcgttccag ctgtgtccgc gttgcgcagg caggccgcgt cggtgggaag 8280
cgatgaattg gatcgactcc gccaacgtgc ccccgggatt tccgaggtgg aatgggggga 8340
agtggagaaa acagtgagac gggtcgtcga taagcttctt catgaaccca ctgtgcgcgt 8400
caaggaactg gcggcccggt ccggcagcat ctcttatgat tcagctctgc aagagctgtt 8460
cggtttggag tcgctggcga gcaccgcagc accggcaacc acgtccgtca acgcgtcaga 8520
actgccggat gcgggtatcg tcgcattcgt gaacgcacct tctgccacac aaacgaggga 8580
gtaaatgacg cacatgacat cgtccaatac ggctcgatcc gcagagtggt ttgaaaaggc 8640
tcagaagctc acccctggtg gtgtgaattc tcctgttcgc gctttcggtt cagttggcgg 8700
acaagcccgt ttcatcgaaa aagctcacgg ttcaacgctg atcgatgtgg acggaaatga 8760
atacgttgac ctggtctgtt cttggggccc catgctgatg ggtcacgctc acccagcagt 8820
ggtcgaggct gtgcagaagg ccgtcgtgga tggtctttct ttcggcgctc ccaccatcgg 8880
tgaggttgag ttggcccaag atatcgtcaa gcgcacttct gtggaggaag tccgcctggt 8940
caactccggc actgaggcca ccatgtcggc ggttcgtctg gcgcgcggtt acactcagcg 9000
ttccaagatt ttgaagtttg agggctgcta ccacggccac gtcgatgcgc tgctcgcatc 9060
tgctggttct ggtgtcgcaa ctttcgctct gcctgattcc ccaggcatca ccggcgctca 9120
gacttctgac actattgttg ttccttacaa cgacattgaa gccgtgcgca acgcttttgc 9180
ggagtaccca ggcgagatcg cctgcatcat cgcagaggca gccggtggca acatgggcac 9240
cgtcgctcca aaggacaact ttaacgacaa gcttctcgcg atcgctcacg ctgacggcgc 9300
gctgctgatc ctcgatgaag tcatgaccgg cttccgcacc tcttaccgtg gctggttcgg 9360
cgtagacaag gttgccgctg acctggtcac cttcggcaag gtcgtctccg gcggcctacc 9420
tgccgcagcg tttggcggca aggctgaaat catgaacatg ctggccccac agggccccgt 9480
ctaccaagca ggcacactgt ccggcaaccc ggttgcggtc gcagctggtc gggcatcgct 9540
taagcttgcc gacgaatccc tctacacaac catcaacgcc aacgcagatc gtctccacgg 9600
tttgatctct gatgccttaa cccacgaagg cgtagcccac cacattcagc gtgcctcaaa 9660
catgctgtct atccgttttg cagaaggtga gggccacaac ttctctgata tgaaggcagc 9720
cgacatcttc cgcttcgcac cgttcttcca cactttgctg gacaacggcg tctacgcacc 9780
accaagcgtt ttcgaaacct ggtttgtgtc ttccgctctc acggacgatg atttctccaa 9840
gatcgagcag gcactcaagc ccgccgcacg tgcagcagca gaagcgaagg catcatgagt 9900
cgactctaga ggatccccgg gtaccgagct cgaattcagc ttggctgttt tggcggatga 9960
gagaagattt tcagcctgat acagattaaa tcagaagtcg actctagagg atccccgggt 10020
accgagctcg aattcagctt ggctgttttg gcggatgaga gaagattttc agcctgatac 10080
agattaaatc agaa 10094
<210> 4
<211> 1395
<212> DNA
<213> 谷氨酰-tRNA还原编码基因hemA(谷氨酰-tRNA还原编码基因)
<220>
<221> gene
<222> (1)..(1395)
<400> 4
atggtgagtg tactcatcgt agggatgtcg cacaggtctg cgcctgtgtc gcttcttgaa 60
cgtctgagca tggatgattc agtacgtggt gaaacaactc aagcactcct gggtagggcg 120
tctttaagcg aggccctcat tgtctctacg tgtaaccgcc tggaggtcta caccgtcact 180
agcagtttcc atactggtgt taatgatgtg gtggaggttc tccatgaggc aagtggcgta 240
gatattgaaa ctttgcgcgg atatctttat gtccgttacg ccgatgctgc tgctgaacac 300
atgttggtgg tgacttccgg gttggattcc atggtgttgg gtgagcagca gatcattggt 360
caggtgcgca ctgcgtacca agcagctaat gaatatggtt ctgtcggtcc tgctttgcat 420
tcacttaccc agaccgcgct gcataccggc aagcgcgtgc attcggagac tgctattgat 480
gatgctggtg catcgatggt gtctttcgct gtggatcgcg cgttggtgca gatgggtctt 540
gattcggagg cagaagcccc actatctggc aagacagcct tggtgttggg cgctggcgcg 600
atgagttctc ttgcagccac tcaccttggt cgcgctggaa tttccaactt gatcatggcc 660
aaccgcactc tggaacgtgc cgaaaggctt gcggagcatt ccctagaagc cggagttcct 720
gcagaggttg tggaatacga tcagcgagct tccgcctaca atcgcgttga cctggtagtt 780
tccgccacgg gagcggatga tttcaccgtg aagcctgagg atattccaga aggtgcttcg 840
ttgatgttgg tggatttgtc catgccacga gacatcgatg atgcttgtgc ggatctgccg 900
ggcgttgatt tggtgaacat cgaacgcctg cacaaggcct cccgtgaggg tggatcgggc 960
atggcgccaa gcgaggaaga agctttggcg attgttcggg aagagttgga ttctttcacc 1020
tctgagcagc gcattcgcga tatcgttcca gctgtgtccg cgttgcgcag gcaggccgcg 1080
tcggtgggaa gcgatgaatt ggatcgactc cgccaacgtg cccccgggat ttccgaggtg 1140
gaatgggggg aagtggagaa aacagtgaga cgggtcgtcg ataagcttct tcatgaaccc 1200
actgtgcgcg tcaaggaact ggcggcccgg tccggcagca tctcttatga ttcagctctg 1260
caagagctgt tcggtttgga gtcgctggcg agcaccgcag caccggcaac cacgtccgtc 1320
aacgcgtcag aactgccgga tgcgggtatc gtcgcattcg tgaacgcacc ttctgccaca 1380
caaacgaggg agtaa 1395
<210> 5
<211> 1314
<212> DNA
<213> 谷氨酸-1-半醛-氨基转移酶hemL(谷氨酸-1-半醛-氨基转移酶)
<220>
<221> gene
<222> (1)..(1314)
<400> 5
atgacgcaca tgacatcgtc caatacggct cgatccgcag agtggtttga aaaggctcag 60
aagctcaccc ctggtggtgt gaattctcct gttcgcgctt tcggttcagt tggcggacaa 120
gcccgtttca tcgaaaaagc tcacggttca acgctgatcg atgtggacgg aaatgaatac 180
gttgacctgg tctgttcttg gggccccatg ctgatgggtc acgctcaccc agcagtggtc 240
gaggctgtgc agaaggccgt cgtggatggt ctttctttcg gcgctcccac catcggtgag 300
gttgagttgg cccaagatat cgtcaagcgc acttctgtgg aggaagtccg cctggtcaac 360
tccggcactg aggccaccat gtcggcggtt cgtctggcgc gcggttacac tcagcgttcc 420
aagattttga agtttgaggg ctgctaccac ggccacgtcg atgcgctgct cgcatctgct 480
ggttctggtg tcgcaacttt cgctctgcct gattccccag gcatcaccgg cgctcagact 540
tctgacacta ttgttgttcc ttacaacgac attgaagccg tgcgcaacgc ttttgcggag 600
tacccaggcg agatcgcctg catcatcgca gaggcagccg gtggcaacat gggcaccgtc 660
gctccaaagg acaactttaa cgacaagctt ctcgcgatcg ctcacgctga cggcgcgctg 720
ctgatcctcg atgaagtcat gaccggcttc cgcacctctt accgtggctg gttcggcgta 780
gacaaggttg ccgctgacct ggtcaccttc ggcaaggtcg tctccggcgg cctacctgcc 840
gcagcgtttg gcggcaaggc tgaaatcatg aacatgctgg ccccacaggg ccccgtctac 900
caagcaggca cactgtccgg caacccggtt gcggtcgcag ctggtcgggc atcgcttaag 960
cttgccgacg aatccctcta cacaaccatc aacgccaacg cagatcgtct ccacggtttg 1020
atctctgatg ccttaaccca cgaaggcgta gcccaccaca ttcagcgtgc ctcaaacatg 1080
ctgtctatcc gttttgcaga aggtgagggc cacaacttct ctgatatgaa ggcagccgac 1140
atcttccgct tcgcaccgtt cttccacact ttgctggaca acggcgtcta cgcaccacca 1200
agcgttttcg aaacctggtt tgtgtcttcc gctctcacgg acgatgattt ctccaagatc 1260
gagcaggcac tcaagcccgc cgcacgtgca gcagcagaag cgaaggcatc atga 1314
<210> 6
<211> 1338
<212> DNA
<213> 柠檬酸合酶编码基因gltA(柠檬酸合酶)
<220>
<221> gene
<222> (1)..(1338)
<400> 6
ggaagagttt ttttccgaac aaatatgttt gaaagggata tcgtggctac tgataacaac 60
aaggctgtcc tgcactaccc cggtggcgag ttcgaaatgg acatcatcga ggcttctgag 120
ggtaacaacg gtgttgtcct gggcaagatg ctgtctgaga ctggactgat cacttttgac 180
ccaggttatg tgagcactgg ctccaccgag tcgaagatca cctacatcga tggcgatgcg 240
ggaatcctgc gttaccgcgg ctatgacatc gctgatctgg ctgagaatgc caccttcaac 300
gaggtttctt acctacttat caacggtgag ctaccaaccc cagatgagct tcacaagttt 360
aacgacgaga ttcgccacca cacccttctg gacgaggact tcaagtccca gttcaacgtg 420
ttcccacgcg acgctcaccc aatggcaacc ttggcttcct cggttaacat tttgtctacc 480
tactaccagg accagctgaa cccactcgat gaggcacagc ttgataaggc aaccgttcgc 540
ctcatggcaa aggttccaat gctggctgcg tacgcacacc gcgcacgcaa gggtgctcct 600
tacatgtacc cagacaactc cctcaatgcg cgtgagaact tcctgcgcat gatgttcggt 660
tacccaaccg agccatacga gatcgaccca atcatggtca aggctctgga caagctgctc 720
atcctgcacg ctgaccacga gcagaactgc tccacctcca ccgttcgtat gatcggttcc 780
gcacaggcca acatgtttgt ctccatcgct ggtggcatca acgctctgtc cggcccactg 840
cacggtggcg caaaccaggc tgttctggag atgctcgaag acatcaagag caaccacggt 900
ggcgacgcaa ccgagttcat gaacaaggtc aagaacaagg aagacggcgt ccgcctcatg 960
ggcttcggac accgcgttta caagaactac gatccacgtg cagcaatcgt caaggagacc 1020
gcacacgaga tcctcgagca cctcggtggc gacgatcttc tggatctggc aatcaagctg 1080
gaagaaattg cactggctga tgattacttc atctcccgca agctctaccc gaacgtagac 1140
ttctacaccg gcctgatcta ccgcgcaatg ggcttcccaa ctgacttctt caccgtattg 1200
ttcgcaatcg gtcgtctgcc aggatggatc gctcactacc gcgagcagct cggtgcagca 1260
ggcaacaaga tcaaccgccc acgccaggtc tacaccggca acgaatcccg caagttggtt 1320
cctcgcgagg agcgctaa 1338
<210> 7
<211> 1359
<212> DNA
<213> 谷氨酸脱氢酶编码基因gdh(谷氨酸脱氢酶)
<220>
<221> gene
<222> (1)..(1359)
<400> 7
gggaacgagg aaatcatgac agttgatgag caggtctcta actattacga catgcttctg 60
aagcgcaatg ctggcgagcc tgaatttcac caggcagtgg cagaggtttt ggaatctttg 120
aagatcgtcc tggaaaagga ccctcattac gctgattacg gtctcatcca gcgcctgtgc 180
gagcctgagc gtcagctcat cttccgtgtg ccttgggttg atgaccaggg ccaggtccac 240
gtcaaccgtg gtttccgcgt gcagttcaac tctgcacttg gaccatacaa gggcggcctg 300
cgcttccacc catctgtaaa cctgggcatt gtgaagttcc tgggctttga gcagatcttt 360
aaaaactccc taaccggcct gccaatcggt ggtggcaagg gtggatccga cttcgaccct 420
aagggcaagt ccgatctgga aatcatgcgt ttctgccagt ccttcatgac cgagctacac 480
cgccacatcg gtgagtaccg cgacgttcct gcaggtgaca tcggagttgg tggccgcgag 540
atcggttacc tgtttggcca ctaccgtcgc atggctaacc agcacgagtc cggcgttttg 600
accggtaagg gcctgacctg gggtggatcc ctggtccgca ccgaggcaac tggctacggc 660
tgcgtttact tcgtgagtga aatgatcaag gctaagggcg agagcatcag cggccagaag 720
atcatcgttt ccggttccgg caacgtagca acctacgcga ttgaaaaggc tcaggaactc 780
ggcgcaaccg ttattggttt ctccgattcc agcggttggg ttcatacccc taacggcgtt 840
gacgtggcta agctccgcga aatcaaggaa gttcgtcgcg cacgcgtatc cgtgtacgcc 900
gacgaagttg aaggcgcaac ctaccacacc gacggttcca tctgggatct caagtgcgat 960
atcgctcttc cttgtgcaac tcagaacgag ctcaacggcg agaacgctaa gactcttgca 1020
gacaacggct gccgtttcgt tgctgaaggc gcgaacatgc cttccacccc tgaggctgtt 1080
gaggtcttcc gtgagcgcga catccgcttc ggaccaggca aggcagctaa cgctggtggc 1140
gttgcaacct ccgctctgga gatgcagcag aacgcttcgc gcgattcctg gagcttcgag 1200
tacaccgacg agcgcctcca ggtgatcatg aagaacatct tcaagacctg tgcagagacc 1260
gcagcagagt atggacacga gaacgattac gttgtcggcg ctaacattgc tggcttcaag 1320
aaggtagctg acgcgatgct ggcacagggc gtcatctaa 1359
<210> 8
<211> 900
<212> DNA
<213> 苏氨酸输出载体rhtA(苏氨酸)
<220>
<221> gene
<222> (1)..(900)
<400> 8
gtgggagaaa ggatgcctgg ttcattacgt aaaatgccgg tctggttacc aatagtcata 60
ttgctcgttg ccatggcgtc tattcagggt ggagcctcgt tagctaagtc actttttcct 120
ctggtgggcg caccgggtgt cactgcgctg cgtctggcat taggaacgct gatcctcatc 180
gcgttcttta agccatggcg actgcgcttt gccaaagagc aacggttacc gctgttgttt 240
tacggcgttt cgctgggtgg gatgaattat cttttttatc tttctattca gacagtaccg 300
ctgggtattg cggtggcgct ggagttcacc ggaccactgg cggtggcgct gttctcttct 360
cgtcgcccgg tagatttcgt ctgggttgtg ctggcggttc ttggtctgtg gttcctgcta 420
ccgctggggc aagacgtttc ccatgtcgat ttaaccggct gtgcgctggc actgggggcc 480
ggggcttgtt gggctattta cattttaagt gggcaacgcg caggagcgga acatggccct 540
gcgacggtgg caattggttc gttgattgca gcgttaattt tcgtgccaat tggagcgctt 600
caggctggtg aagcactctg gcactggtcg gttattccat tgggtctggc tgtcgctatt 660
ctctcgaccg ctctgcctta ttcgctggaa atgattgccc tcacccgttt gccaacacgg 720
acatttggta cgctgatgag catggaaccg gcgctggctg ccgtttccgg gatgattttc 780
ctcggagaaa cactgacacc catacagcta ctggcgctcg gcgctatcat cgccgcttca 840
atggggtcta cgctgacagt acgcaaagag agcaaaataa aagaattaga cattaattaa 900
<210> 9
<211> 2947
<212> DNA
<213> 转氢酶pntAB(转氢酶)
<220>
<221> gene
<222> (1)..(2947)
<400> 9
atggaaggga atatcatgcg aattggcata ccaagagaac ggttaaccaa tgaaacccgt 60
gttgcagcaa cgccaaaaac agtggaacag ctgctgaaac tgggttttac cgtcgcggta 120
gagagcggcg cgggtcaact ggcaagtttt gacgataaag cgtttgtgca agcgggcgct 180
gaaattgtag aagggaatag cgtctggcag tcagagatca ttctgaaggt caatgcgccg 240
ttagatgatg aaattgcgtt actgaatcct gggacaacgc tggtgagttt tatctggcct 300
gcgcagaatc cggaattaat gcaaaaactt gcggaacgta acgtgaccgt gatggcgatg 360
gactctgtgc cgcgtatctc acgcgcacaa tcgctggacg cactaagctc gatggcgaac 420
atcgccggtt atcgcgccat tgttgaagcg gcacatgaat ttgggcgctt ctttaccggg 480
caaattactg cggccgggaa agtgccaccg gcaaaagtga tggtgattgg tgcgggtgtt 540
gcaggtctgg ccgccattgg cgcagcaaac agtctcggcg cgattgtgcg tgcattcgac 600
acccgcccgg aagtgaaaga acaagttcaa agtatgggcg cggaattcct cgagctggat 660
tttaaagagg aagctggcag cggcgatggc tatgccaaag tgatgtcgga cgcgttcatc 720
aaagcggaaa tggaactctt tgccgcccag gcaaaagagg tcgatatcat tgtcaccacc 780
gcgcttattc caggcaaacc agcgccgaag ctaattaccc gtgaaatggt tgactccatg 840
aaggcgggca gtgtgattgt cgacctggca gcccaaaacg gcggcaactg tgaatacacc 900
gtgccgggtg aaatcttcac tacggaaaat ggtgtcaaag tgattggtta taccgatctt 960
ccgggccgtc tgccgacgca atcctcacag ctttacggca caaacctcgt taatctgctg 1020
aaactgttgt gcaaagagaa agacggcaat atcactgttg attttgatga tgtggtgatt 1080
cgcggcgtga ccgtgatccg tgcgggcgaa attacctggc cggcaccgcc gattcaggta 1140
tcagctcagc cgcaggcggc acaaaaagcg gcaccggaag tgaaaactga ggaaaaatgt 1200
acctgctcac cgtggcgtaa atacgcgttg atggcgctgg caatcattct ttttggctgg 1260
atggcaagcg ttgcgccgaa agaattcctt gggcacttca ccgttttcgc gctggcctgc 1320
gttgtcggtt attacgtggt gtggaatgta tcgcacgcgc tgcatacacc gttgatgtcg 1380
gtcaccaacg cgatttcagg gattattgtt gtcggagcac tgttgcagat tggccagggc 1440
ggctgggtta gcttccttag ttttatcgcg gtgcttatag ccagcattaa tattttcggt 1500
ggcttcaccg tgactcagcg catgctgaaa atgttccgca aaaattaagg ggtaacatat 1560
gtctggagga ttagttacag ctgcatacat tgttgccgcg atcctgttta tcttcagtct 1620
ggccggtctt tcgaaacatg aaacgtctcg ccagggtaac aacttcggta tcgccgggat 1680
ggcgattgcg ttaatcgcaa ccatttttgg accggatacg ggtaatgttg gctggatctt 1740
gctggcgatg gtcattggtg gggcaattgg tatccgtctg gcgaagaaag ttgaaatgac 1800
cgaaatgcca gaactggtgg cgatcctgca tagcttcgtg ggtctggcgg cagtgctggt 1860
tggctttaac agctatctgc atcatgacgc gggaatggca ccgattctgg tcaatattca 1920
cctgacggaa gtgttcctcg gtatcttcat cggggcggta acgttcacgg gttcggtggt 1980
ggcgttcggc aaactgtgtg gcaagatttc gtctaaacca ttgatgctgc caaaccgtca 2040
caaaatgaac ctggcggctc tggtcgtttc cttcctgctg ctgattgtat ttgttcgcac 2100
ggacagcgtc ggcctgcaag tgctggcatt gctgataatg accgcaattg cgctggtatt 2160
cggctggcat ttagtcgcct ccatcggtgg tgcagatatg ccagtggtgg tgtcgatgct 2220
gaactcgtac tccggctggg cggctgcggc tgcgggcttt atgctcagca acgacctgct 2280
gattgtgacc ggtgcgctgg tcggttcttc gggggctatc ctttcttaca ttatgtgtaa 2340
ggcgatgaac cgttccttta tcagcgttat tgcgggtggt ttcggcaccg acggctcttc 2400
tactggcgat gatcaggaag tgggtgagca ccgcgaaatc accgcagaag agacagcgga 2460
actgctgaaa aactcccatt cagtgatcat tactccgggg tacggcatgg cagtcgcgca 2520
ggcgcaatat cctgtcgctg aaattactga gaaattgcgc gctcgtggta ttaatgtgcg 2580
tttcggtatc cacccggtcg cggggcgttt gcctggacat atgaacgtat tgctggctga 2640
agcaaaagta ccgtatgaca tcgtgctgga aatggacgag atcaatgatg actttgctga 2700
taccgatacc gtactggtga ttggtgctaa cgatacggtt aacccggcgg cgcaggatga 2760
tccgaagagt ccgattgctg gtatgcctgt gctggaagtg tggaaagcgc agaacgtgat 2820
tgtctttaaa cgttcgatga acactggcta tgctggtgtg caaaacccgc tgttcttcaa 2880
ggaaaacacc cacatgctgt ttggtgacgc caaagccagc gtggatgcaa tcctgaaagc 2940
tctgtaa 2947
Claims (7)
1.一种棒状杆菌诱导型启动子Piol，其特征在于：受肌醇诱导调控，其序列如SEQ IDNO:1所示。
2.一种棒状杆菌诱导型表达质粒pIol，其特征在于：含有权利要求1所述的启动子。
3.根据权利要求2所述的棒状杆菌诱导型表达质粒pIol，其特征在于：是将权利要求1所述的启动子插入至质粒pXMJ19的HpaⅠ和Hind Ⅲ酶切位点得到的，所述质粒pIol序列如SEQ ID NO:2所示。
4.一种用于生产5-氨基乙酰丙酸的质粒pIolhemAL，其特征在于：在权利要求2所述的表达质粒的BamHI和Kpn I酶切位点酶切位点插入谷氨酰-tRNA还原编码基因hemA和谷氨酸-1-半醛-氨基转移酶hemL，所述质粒pIolhemAL序列如SEQ ID NO:3所示，hemA序列如SEQID NO:4所示，hemL序列如SEQ ID NO:5所示。
5.一株5-氨基乙酰丙酸生产菌株，其特征在于：是由下述方法制备得到的：将权利要求1所述启动子连同过表达的关键基因整合至宿主基因组，所述关键基因包括柠檬酸合酶编码基因gltA、谷氨酸脱氢酶编码基因gdh、转氢酶基因pntAB以及苏氨酸输出载体rhtA，所述宿主为棒状杆菌，并且，所述菌株含有权利要求4所述的用于生产5-氨基乙酰丙酸的质粒pIolhemAL。
6.权利要求5所述的5-氨基乙酰丙酸生产菌株，其特征在于：关键基因为柠檬酸合酶编码基因gltA、谷氨酸脱氢酶编码基因gdh、苏氨酸输出载体rhtA以及转氢酶pntAB；其中，gltA序列如SEQ ID NO:6所示，gdh序列如SEQ ID NO:7所示，rhtA序列如SEQ ID NO:8所示，pntAB序列如SEQ ID NO:9所示。
7.一种应用权利要求5所述菌株发酵法生产5-氨基乙酰丙酸的方法，其特征在于：利用所述5-氨基乙酰丙酸生产菌株生产5-氨基乙酰丙酸，具体步骤如下：
(1) 种子培养：将权利要求5所述的菌株活化后，接种至装有1L 种子培养基的5L发酵罐，流加25% W/V的氨水调节发酵液pH至6.8-7.2，溶氧维持在10-20%，通风量1-3m3/h，搅拌转速200-600rpm，32-37℃培养6-8h；
(2) 发酵罐发酵：以5%-10%接种量将步骤(1)的种子培养物接至装有6L发酵培养基的10L发酵罐进行发酵培养，发酵温度32-37℃，通风量3-5m3/h，搅拌转速100-3000rpm，溶氧维持在10-20%，流加浓度为60-80% W/V的葡萄糖溶液，维持残糖浓度为0.1-0.5% W/V ，流加25% W/V的氨水调节发酵液pH至6.8-7.2，发酵周期30-48 h；
(3) 种子培养基成分为 ：蔗糖 10-15g/L，玉米浆 15-25mL/L，酵母粉 1-6g/L，(NH4)2SO4 1-4 g/L，KH2PO4 0.5-1.5g/L，MgSO4 0.1-0.5g/L， FeSO4·7H2O 0.01-0.05g/L，MnSO4·H2O 0.01-0.05g/L，pH 7.0，0.075MPa 高压蒸汽灭菌15min；
(4) 发酵培养基成分为 ：葡萄糖 15-45g/L，玉米浆 5-15mL/L，豆饼水解液 5-15mL/L，(NH4)2SO4 8-20 g/L，生物素0.00004 g/L， pH 7.0-7.2，121℃高温灭菌20min。
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