CN114990036A - 一种全细胞催化生产2-吡喃酮-4,6-二羧酸的方法 - Google Patents
一种全细胞催化生产2-吡喃酮-4,6-二羧酸的方法 Download PDFInfo
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Abstract
本发明提供一种用于全细胞催化生产2‑吡喃酮‑4,6‑二羧酸的基因工程重组菌及其生产方法,属于生物工程技术领域。本发明以共表达原儿茶酸‑4,5‑二氧化酶复合体和4‑羧基‑2‑羟基粘康酸‑6‑半醛脱氢酶基因的基因工程重组菌作为全细胞催化剂,以原儿茶酸作为底物,实现了原儿茶酸到2‑吡喃酮‑4,6‑二羧酸的高效全细胞催化转化,整个催化过程无需添加辅因子NADP+。本发明提供的全细胞催化生产2‑吡喃酮‑4,6‑二羧酸的方法具有合成效率高、无需添加辅因子、操作简单的特点,具有重要的工业应用价值。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种全细胞催化生产2-吡喃酮-4,6-二羧酸的基因工程重组菌及其生产方法。
背景技术
2-吡喃酮-4,6-二羧酸(2-pyrone-4,6-dicarboxylic acid或alpha-pyrone-4,6-dicarboxylic acid,英文缩写PDC,CAS号72698-24-9)是一种含有吡喃环的拟芳香二元羧酸化合物。2-吡喃酮-4,6-二羧酸具有和对苯二甲酸(TPA)相类似的二羧酸分子结构,可以与各种二元醇或羟基酸发生脱水缩聚反应生成聚酯类高分子材料(Tsuyoshi Michinobu,MasakiyoHishida, Masae Sato, Yoshihiro Katayama, Eiji Masai, Masaya Nakamura,Yuichiro Otsuka, Seiji Ohara and KiyotakaShigehara. Polyesters of 2-Pyrone-4,6-Dicarboxylic Acid (PDC) Obtained from a Metabolic Intermediate of Lignin.Polymer Journal. 2008, 40(1): 68-75.;MasakiyoHishida, Kazuhiro Shikinaka,Yoshihiro Katayama, Shinya Kajita, Eiji Masai, Masaya Nakamura, YuichiroOtsuka, Seiji Ohara and KiyotakaShigehara. Polyesters of 2-Pyrone-4,6-dicarboxylic Acid (PDC) as Bio-based Plastics Exhibiting Strong AdheringProperties. Polymer Journal. 2009, 41(4): 297–302.)。与聚对苯二甲酸乙二醇酯(PET)相比,2-吡喃酮-4,6-二羧酸衍生聚合材料具有良好的热稳定性,并且在环境中更容易被微生物降解,因此可以作为一种优良的生物可降解聚合材料的单体(TsuyoshiMichinobu, Masami Bito, Yoshiko Yamada, Miki Tanimura, Yoshihiro Katayama,Eiji Masai, Masaya Nakamura, Yuichiro Otsuka, Seiji Ohara andKiyotakaShigehara. Fusible, Elastic, and Biodegradable Polyesters of 2-Pyrone-4,6-Dicarboxylic Acid (PDC). Polymer Journal. 2009, 41(12): 1111-1116.)。鉴于这些特性,2-吡喃酮-4,6-二羧酸被认为是芳香族聚酯原材料中对苯二甲酸的潜在替代化学品。此外,2-吡喃酮-4,6-二羧酸还可以作为锂离子电池电解液的添加剂和含有放射性元素铯废水处理的络合剂。
目前,2-吡喃酮-4,6-二羧酸很难通过传统的石油化工手段进行合成。已报道的合成方法主要有微生物分解木质素合成、以原儿茶酸为底物催化合成和以葡萄糖为原料从头合成。在自然界中,某些微生物在降解木质素来源的芳香族化合物过程中可以生成2-吡喃酮-4,6-二羧酸,但是产量较低,例如红球菌RhodococcusjostiiRHA1(Zoe Mycroft, MariaGomis, Paul Mines, Paul Law and Timothy D.H. Bugg. Biocatalytic Conversion ofLignin to Aromatic Dicarboxylic Acids in RhodococcusjostiiRHA1 by Re-RoutingAromatic Degradation Pathways. Green Chemistry. 2015,17: 4974-4979.)、恶臭假单胞菌Pseudomonas putida KT2440(Sandra Notonier, Allison Z Werner, EugeneKuatsjah, Linda Dumalo, Paul E Abraham, E Anne Hatmaker, Caroline B Hoyt,Antonella Amore, Kelsey J Ramirez, Sean P Woodworth, Dawn M Klingeman,Richard J Giannone, Adam M Guss, Robert L Hettich, Lindsay D Eltis,Christopher W Johnson, Gregg T Beckham. Metabolism of syringyl lignin-derivedcompounds in Pseudomonas putida enables convergent production of 2-pyrone-4,6-dicarboxylic acid. Metabolic Engineering. 2021, 65:111–122.)等等。Otsuka等利用共表达鞘氨醇单胞菌Sphingomonaspaucimobilis SYK-6来源的原儿茶酸-4,5-二氧化酶复合体LigAB和4-羧基-2-羟基粘康酸-6-半醛脱氢酶LigC的基因工程恶臭假单胞菌作为全细胞催化剂,可以将原儿茶酸催化为2-吡喃酮-4,6-二羧酸,但是产量只有10 g/L(Yuichiro Otsuka, Masaya Nakamura, KiyotakaShigehara, Kosuke Sugimura, EijiMasai, Seiji Ohara, Yoshihiro Katayama. Efficient production of 2-pyrone 4,6-dicarboxylic acid as a novel polymer-based material from protocatechuate bymicrobial function. Applied Microbiology and Biotechnology. 2006, 71(5):608-614.)。国外学者通过代谢工程手段,利用大肠杆菌(Escherichia coli)内源莽草酸途径合成3-脱氢莽草酸,再整合外源合成途径基因,包括3-脱氢莽草酸脱水酶基因quiC或asbF、原儿茶酸-4,5-二氧化酶基因ligAB或pmdAB、4-羧基-2-羟基粘康酸-6-半醛脱氢酶基因ligC或pmdC,以葡萄糖为碳源,实现了从头合成2-吡喃酮-4,6-二羧酸,但是最高产量只有16.7g/L(Masahiro Nakajima, Yukari Nishino, Masatsugu Tamura, Kohei Mase, EijiMasai, Yuichiro Otsuka, Masaya Nakamura, Kanna Sato, Masao Fukuda,KiyotakaShigehara, Seiji Ohara, Yoshihiro Katayama, Shinya Kajita. Microbialconversion of glucose to a novel chemical building block, 2-pyrone-4,6-dicarboxylic acid. Metabolic Engineering. 2009,11: 213–220.; Zi Wei Luo, WonJun Kim, Sang Yup Lee. Metabolic Engineering of Escherichia coli forEfficient Production of 2-Pyrone-4,6-dicarboxylic Acid from Glucose. ACSSynthetic Biology. 2018, 7: 2296−2307.)。由此可见,利用微生物合成2-吡喃酮-4,6-二羧酸的效率仍然较低,难以满足大规模工业化发酵生产的要求。本发明公开的生产方法可以有效解决2-吡喃酮-4,6-二羧酸合成效率低的问题。
发明内容
有鉴于此,本发明提供了一种高效全细胞催化生产2-吡喃酮-4,6-二羧酸的方法及所用到的基因工程重组菌。
所述基因工程重组菌中共表达原儿茶酸-4,5-二氧化酶复合体(EC1.13.11.8)和4-羧基-2-羟基粘康酸-6-半醛脱氢酶(EC1.1.1.312)基因的基因工程重组菌,例如大肠杆菌、枯草芽孢杆菌(Bacillus subtilis)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、需钠弧菌(Vibrio natriegens)等原核生物作为全细胞催化剂,以原儿茶酸作为底物,实现了原儿茶酸到2-吡喃酮-4,6-二羧酸的全细胞催化合成(图1)。
在一种实施方式中,所述基因工程重组菌的构建选取大肠杆菌作为宿主细胞,通过载体共表达原儿茶酸-4,5-二氧化酶和4-羧基-2-羟基粘康酸-6-半醛脱氢酶,获得全细胞催化剂。
在一种实施方式中,所述原儿茶酸-4,5-二氧化酶复合体的αβ亚基(命名为AB)和4-羧基-2-羟基粘康酸-6-半醛脱氢酶(命名为C)大部分来源于变形菌门(Proteobacteria)细菌,少部分来源于放线菌门(Actinobacteria)细菌;
示例性地,通过蛋白同源性比对,在NCBI数据库中选取16组不同物种来源ABC催化酶,并将这些酶的编码基因序列按照大肠杆菌密码子偏爱性进行密码子优化。具体地,原儿茶酸-4,5-二氧化酶复合体α亚基来源于NCBI登录号为TIX48797.1、MBO9517659.1、MBB5734100.1、QWT16175.1、MBE1527979.1、RIV77917.1、TCU95342.1、KAB0542660.1、ACB35890.1、MBQ0919761.1、AXF85167.1、KQP37453.1、SKA71457.1、NHO66815.1;原儿茶酸-4,5-二氧化酶复合体β亚基来源于NCBI登录号为TIX48798.1、MBO9517658.1、MBB5734099.1、QWT16174.1、MBE1527978.1、RIV77918.1、TCU95343.1、KAB0542661.1、ACB35891.1、MBQ0919762.1、AXF85168.1、KQP37452.1、SKA71463.1、NHO66816.1;单亚基原儿茶酸-4,5-二氧化酶的NCBI登录号分别为EZP27614.1和AYG79827.1;4-羧基-2-羟基粘康酸-6-半醛脱氢酶的NCBI登录号为TIX48799.1、MBO9517657.1、MBB5734098.1、QWT16173.1、MBE1527977.1、RIV77919.1、TCU95344.1、KAB0542662.1、ACB35892.1、MBQ0919763.1、AXF85169.1、KQP37451.1、SKA71468.1、NHO66817.1、EZP27613.1、AYG79828.1。
更优选地,原儿茶酸-4,5-二氧化酶复合体αβ亚基编码基因AB和4-羧基-2-羟基粘康酸-6-半醛脱氢酶编码基因C在基因组上以基因簇的形式排列,分别来源于NCBI中登录号为SSHH01000004.1、JAGIBN010000003.1、JACIJL010000010.1、CP076557.1、JADBDT010000001.1、QXFK01000016.1、SMBU01000015.1、VZPC01000007.1、CP001013.1、JAGPWB010000023.1、CP031124.1、LMNA01000027.1、FUYB01000003.1、JAAONZ010000012.1、JFYO01000005.1、CP032698.1基因组上对应基因簇内的核苷酸序列。
优选地,将所述基因序列按照大肠杆菌密码子偏爱性进行密码子优化,更优选的优化后的AB基因的核苷酸序列分别如SEQ ID NO:4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34所示;优化后的C基因的核苷酸序列分别如SEQ ID NO:5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35所示。
在一种实施方式中,所述基因工程重组大肠杆菌采用pETDuet-1或pRSFDuet-1或pACYCDue-1或pCOLADuet-1或pCDFDuet-1中任意一种质粒表达原儿茶酸-4,5-二氧化酶复合体αβ亚基(1AB-16AB)和4-羧基-2-羟基粘康酸-6-半醛脱氢酶(1C-16C)基因,连接顺序可以是AB基因在前,C基因在后,或者C基因在前,AB基因在后;
示例性地,所述原儿茶酸-4,5-二氧化酶复合体αβ亚基(AB)基因连接至pRSFDuet-1载体上的NcoI和HindⅢ酶切位点之间;AB连接成功之后,将4-羧基-2-羟基粘康酸-6-半醛脱氢酶(C)基因连接至pRSFDuet-AB载体上的NdeI和XhoI酶切位点之间,形成ABC三个基因的共表达载体pRSFDuet-ABC(图2)。
在一种实施方式中,所述基因工程重组大肠杆菌表达的原儿茶酸-4,5-二氧化酶复合体αβ亚基,其中1AB-14AB中的AB基因串联在一起进行表达,AB基因编码框中间包含一段RBS序列,如SEQ ID NO:1所示核苷酸序列GGATCCGAAGGAGATATACC。
在一种实施方式中,将所述表达载体pRSFDuet-ABC转化至大肠杆菌BL21(DE3)或BL21(DE3)pLysS或Rosetta(DE3)或Rosetta (DE3)pLysS中的任意一种菌株中,获得基因工程重组大肠杆菌;
示例性地,选取大肠杆菌BL21(DE3)作为表达ABC基因的宿主细胞。
本发明还提供催化生产2-吡喃酮-4,6-二羧酸的全细胞催化剂,其是培养所述基因工程重组菌并表达所述AB基因和所述C基因,得到全细胞催化剂。在一种实施方式中,所述重组大肠杆菌在异丙基-β-D-硫代半乳糖苷(IPTG)或乳糖诱导下共表达原儿茶酸-4,5-二氧化酶和4-羧基-2-羟基粘康酸-6-半醛脱氢酶,获得全细胞催化剂。
在一种实施方式中,所述全细胞催化体系的原儿茶酸浓度为1-100 g/L,采用分批补加的方式,每次补加浓度小于20 g/L,并且催化底物可以为原儿茶酸纯品,也可以为微生物发酵获得的原儿茶酸发酵液。
在一种实施方式中,所述催化体系的全细胞催化剂用量为1-100 OD600,反应pH为5-8,反应温度为30-43℃,溶氧1%-100%,反应时间0.5-24 h。
本发明的优点在于提供了一种将原儿茶酸高效全细胞催化合成2-吡喃酮-4,6-二羧酸的生产方法,具体为利用一种共表达原儿茶酸-4,5-二氧化酶和4-羧基-2-羟基粘康酸-6-半醛脱氢酶的基因工程重组菌作为全细胞催化剂,将原儿茶酸高效催化转化为2-吡喃酮-4,6-二羧酸,整个催化过程中无需添加辅因子NADP+(图1)。本发明提供的全细胞催化生产2-吡喃酮-4,6-二羧酸的方法具有合成效率高、无需添加辅因子、操作简单的特点,具有重要的工业应用价值。
附图说明
图1为全细胞催化原儿茶酸合成2-吡喃酮-4,6-二羧酸示意图(PCA,原儿茶酸;CHMS,4-羧基-2-羟基粘康酸-6-半醛;PDC,2-吡喃酮-4,6-二羧酸;O2,氧气;NADP+,氧化型烟酰胺腺嘌呤二核苷酸磷酸/氧化型辅酶II;NADPH,还原型烟酰胺腺嘌呤二核苷酸磷酸/还原型辅酶II;AB,原儿茶酸-4,5-二氧化酶复合体;C,4-羧基-2-羟基粘康酸-6-半醛脱氢酶)。
图2为pRSFDuet-ABC质粒图谱。
具体实施方式
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚明白,以下结合实施例,对本发明进行详细、完整地描述。应当说明的是,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域技术人员在没有做出创造性劳动前提下所获得的所有其他实施例都属于本发明保护的范围。
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、仪器设备等,如无特殊说明,均可从商业途径得到。
实施例1:不同物种来源原儿茶酸-4,5-二氧化酶和4-羧基-2-羟基粘康酸-6-半醛脱氢酶基因表达载体的构建
基因的筛选
以已报道的原儿茶酸-4,5-二氧化酶α亚基LigA和PmdA作为模板序列(YuichiroOtsuka, Masaya Nakamura, KiyotakaShigehara, Kosuke Sugimura, Eiji Masai,Seiji Ohara, Yoshihiro Katayama. Efficient production of 2-pyrone 4,6-dicarboxylic acid as a novel polymer-based material from protocatechuate bymicrobial function. Applied Microbiology and Biotechnology. 2006, 71(5):608-614.Zi Wei Luo, Won Jun Kim, Sang Yup Lee. Metabolic Engineering ofEscherichia coli for Efficient Production of 2-Pyrone-4,6-dicarboxylic Acidfrom Glucose. ACS Synthetic Biology. 2018, 7: 2296−2307.),在NCBI中进行蛋白序列同源比对,发现比对结果中的大部分序列来源于变形菌门细菌,少部分来源于放线菌门细菌,并且原儿茶酸-4,5-二氧化酶复合体的αβ亚基和4-羧基-2-羟基粘康酸-6-半醛脱氢酶的编码基因在每个物种基因组上均是以基因簇的形式串联在一起,因此可以同时获得ABC三个基因的编码框。
随机选取16组不同物种来源的催化酶,其中原儿茶酸-4,5-二氧化酶复合体α亚基(1A-14A)的NCBI登录号依次为TIX48797.1、MBO9517659.1、MBB5734100.1、QWT16175.1、MBE1527979.1、RIV77917.1、TCU95342.1、KAB0542660.1、ACB35890.1、MBQ0919761.1、AXF85167.1、KQP37453.1、SKA71457.1、NHO66815.1;原儿茶酸-4,5-二氧化酶复合体β亚基(1B-14B)的NCBI登录号依次为TIX48798.1、MBO9517658.1、MBB5734099.1、QWT16174.1、MBE1527978.1、RIV77918.1、TCU95343.1、KAB0542661.1、ACB35891.1、MBQ0919762.1、AXF85168.1、KQP37452.1、SKA71463.1、NHO66816.1;15AB和16AB均为单亚基的原儿茶酸-4,5-二氧化酶,NCBI登录号分别为EZP27614.1和AYG79827.1;4-羧基-2-羟基粘康酸-6-半醛脱氢酶(1C-16C)的NCBI登录号依次为TIX48799.1、MBO9517657.1、MBB5734098.1、QWT16173.1、MBE1527977.1、RIV77919.1、TCU95344.1、KAB0542662.1、ACB35892.1、MBQ0919763.1、AXF85169.1、KQP37451.1、SKA71468.1、NHO66817.1、EZP27613.1、AYG79828.1。
所述16组催化酶的编码基因依次来源于NCBI中登录号为SSHH01000004.1、JAGIBN010000003.1、JACIJL010000010.1、CP076557.1、JADBDT010000001.1、QXFK01000016.1、SMBU01000015.1、VZPC01000007.1、CP001013.1、JAGPWB010000023.1、CP031124.1、LMNA01000027.1、FUYB01000003.1、JAAONZ010000012.1、JFYO01000005.1、CP032698.1基因组上的核苷酸序列,并将这些基因序列按照大肠杆菌密码子偏爱性进行密码子优化,优化后的序列如SEQ ID NO:4-SEQ ID NO:35所示。
基因表达载体的构建
(1)基因合成:将密码子优化后的AB和C基因在北京擎科生物科技有限公司进行全基因合成,其中1AB-14AB中的AB基因串联(1A和1B串联,2A和2B串联,……以此类推)在一起进行表达,两个基因编码框中间包含一段RBS序列,如SEQ ID NO:1所示核苷酸序列GGATCCGAAGGAGATATACC。基因合成结束后,委托北京擎科生物科技有限公司将1AB-16AB基因分别连接至pRSFDuet-1载体上的NcoI和HindⅢ酶切位点之间,将1C-16C分别基因连接至pRSFDuet-1载体上的NdeI和XhoI酶切位点之间,分别形成AB基因和C基因的单独表达载体。
(2)酶切:将1AB-16AB和1C-16C的表达载体分别用NewEnglandBiolabs公司的限制性核酸内切酶NdeI和XhoI进行双酶切,获得1AB-16AB表达载体片段和1C-16C基因片段。酶切反应体系为:10×CutSmart Buffer3 μL,表达载体1000 ng,内切酶NdeI (20 U/μL) 0.5μL,内切酶XhoI (20 U/μL) 0.5 μL,补足灭菌水至30μl。酶切反应条件为37℃ 1-2 h。酶切结束后用1%的琼脂糖凝胶进行电泳检测,用干净的手术刀片在紫外灯下将目的片段切下,利用北京博迈德基因技术有限公司的琼脂糖凝胶纯化试剂盒回收载体和片段。
(3)酶连:利用Thermo Scientific公司的T4 DNA ligase将载体与目的片段进行连接。酶连反应体系为:10× T4 DNA Ligase Buffer 2 μL,载体片段 20-100 ng,目的片段20-100 ng,T4 DNA Ligase (5 U/μL)0.5 μL,补足灭菌水至20μl。酶连反应条件为22℃0.5-1 h。
(4)感受态细胞制备:挑取大肠杆菌DH5α单菌落接种于3 mL液体LB培养基中,37℃250 rpm培养8-12 h作为种子液;取100 μL种子液接种于50 mL液体LB培养基中,37℃ 250rpm培养至OD600约0.3-0.4;冰浴15 min,然后将菌液转移至预冷的50 mL离心管中;2000 g离心5 min,弃上清,用15 mL预冷的100 mM CaCl2溶液重悬菌体,重复此步骤一次;2000 g离心5 min,弃上清,用2 mL预冷的10% (v/v)甘油-100 mM CaCl2溶液重悬菌体即为感受态细胞。
(5)转化:将5-10 μL酶连产物加入至50 μL大肠杆菌DH5α感受态细胞中,轻轻混匀,冰浴30 min;42℃热激45 s,冰浴2 min,加入500 μL LB液体培养基,37℃ 250 rpm复苏培养40-60 min;8000 rpm离心2 min,去掉大部分上清,将剩余菌液涂布于含有50 μg/mL卡那霉素的固体LB培养基上,37℃培养过夜即可获得单菌落。
其中,所提及LB培养基配方为:胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L。
(6)转化子验证:挑取若干单菌落利用康为世纪生物科技有限公司的2×Es TaqMasterMix (Dye)进行菌落PCR验证。PCR扩增体系为:2×Es Taq MasterMix(Dye)12.5 μl,DuetUP2引物(10 μM) 1 μl,T7 terminator引物(10 μM) 1 μl,菌液0.5 μl,灭菌水10μl,总体积25μl。PCR扩增程序为:①94℃ 3 min;②94℃ 30 sec,55℃ 30 sec,72℃ 1 min,重复步骤②30次;③72℃ 5 min。其中正向引物DuetUP2序列为TTGTACACGGCCGCATAATC(SEQID NO:2),反向引物T7 terminator序列为TGCTAGTTATTGCTCAGCGG(SEQ ID NO:3)。PCR产物送北京擎科生物科技有限公司进行测序,测序正确的即为1ABC-16ABC基因(1AB与1C组合,2AB与2C组合,……以此类推)的共表达载体pRSFDuet-ABC,其质粒图谱如(图2)所示。
实施例2:不同物种来源原儿茶酸-4,5-二氧化酶和4-羧基-2-羟基粘康酸-6-半醛脱氢酶的全细胞催化效果比较
本实施例的目的一方面是为了筛选到催化反应速率快,并且产量高的组合用于后续研究,另一方面是建立高效的全细胞催化反应体系。
(1)感受态细胞制备:挑取大肠杆菌BL21(DE3)单菌落接种于3 mL液体LB培养基中,37℃ 250 rpm培养8-12 h作为种子液;取100 μL种子液接种于50 mL液体LB培养基中,37℃ 250 rpm培养至OD600约0.3-0.4;冰浴15 min,然后将菌液转移至预冷的50 mL离心管中;2000 g离心5 min,弃上清,用15 mL预冷的100 mM CaCl2溶液重悬菌体,重复此步骤一次;2000 g离心5 min,弃上清,用2 mL预冷的10% (v/v)甘油-100 mM CaCl2溶液重悬菌体即为感受态细胞。
(2)转化:分别将1μL16种ABC组合的共表达载体加入至50 μL大肠杆菌BL21(DE3)感受态细胞中,轻轻混匀,冰浴30 min;42℃热激45 s,冰浴2 min,加入500 μL LB液体培养基,37℃ 250 rpm复苏培养40-60 min;8000 rpm离心2 min,去掉大部分上清,将剩余菌液涂布于含有50 μg/mL卡那霉素的固体LB培养基上,37℃培养过夜即可获得重组大肠杆菌。
(3)蛋白表达:根据经典的重组大肠杆菌培养及诱导表达方案,挑取重组大肠杆菌单菌落接种于3 mL含有50 μg/mL卡那霉素的LB培养基中,37℃ 250 rpm培养10-12 h作为种子液;将种子液按0.5%接种量转接到50 mL含有50 μg/mL卡那霉素的LB培养基中,37℃250 rpm培养至OD600达到0.5-0.7,加入终浓度为0.2 mM的异丙基-β-D-硫代半乳糖苷(IPTG),在28℃ 220 rpm条件下诱导表达10-12 h;诱导表达结束后,5000 rpm离心5 min,收集细胞即为全细胞催化剂。
其中,所提及LB培养基配方为:胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L。
(4)全细胞催化:在100 mL三角瓶中依次加入10 mL含有5 g/L原儿茶酸的M9培养基(pH 7.0),5 OD600全细胞催化剂,在37℃250 rpm摇床培养条件下反应0.5-12 h,定时取样检测发酵液中原儿茶酸和2-吡喃酮-4,6-二羧酸的含量。
其中,所提及M9培养基配方为:葡萄糖4g/L,Na2HPO4•12H2O17.1 g/L,KH2PO43 g/L,NaCl0.5 g/L,NH4Cl1 g/L,MgSO4•7H2O0.5 g/L,CaCl210 mg/L,pH 7.0。
其中,所提及反应时间为0.5-12 h,具体可为0.5 h或1.5 h或3.0 h或4.5 h或6.0h或9.0 h或12.0 h。
(5)高效液相色谱分析:将发酵液样品12000 rpm离心10 min,上清液用蒸馏水稀释一定倍数,用0.22 μm水系微孔滤膜过滤;采用Agilent 1200高效液相色谱仪,配备VWD紫外检测器,色谱柱为Phenomenex公司的Rezex RFQ-Fast Acid H+ (8%) (LC Column 100× 7.8 mm);流动相为5 mM H2SO4水溶液,上样量5 μL,流速0.6 mL/min,柱温55℃,检测波长313 nm,测定发酵液中原儿茶酸和2-吡喃酮-4,6-二羧酸的含量。
(6)结果:如表1所示,在16种候选ABC组合(1ABC是1A、1B与1C组合,2ABC是2A、2B与2C组合,以此类推)中,当反应3 h时,2ABC、14ABC、15ABC的反应速率较快,其中14ABC的反应速率最快,底物原儿茶酸已经反应完全,2-吡喃酮-4,6-二羧酸产量达到5.02 g/L。随着反应的进行,当反应9 h时,大部分组合的原儿茶酸均已反应完全,其中2ABC、3ABC、4ABC、9ABC和12ABC的2-吡喃酮-4,6-二羧酸产量较高,达到6 g/L左右,而14ABC和15ABC的2-吡喃酮-4,6-二羧酸产量分别达到5.21 g/L和5.11 g/L。通过筛选,我们既获得了催化反应速率较快的3种组合2ABC、14ABC和15ABC,又获得了2-吡喃酮-4,6-二羧酸产量较高的5种组合2ABC、3ABC、4ABC、9ABC和12ABC。
表1不同催化酶组合发酵液中2-吡喃酮-4,6-二羧酸(PDC)和原儿茶酸(PCA)的含量比较
不同催化酶组合 | 3 h的PDC产量g/L | 3 h的PCA残余量g/L | 9 h的PDC产量g/L | 9 h的PCA残余量g/L |
1ABC | 2.19 | 2.89 | 4.85 | 0.63 |
2ABC | 4.51 | 1.03 | 6.00 | 0.00 |
3ABC | 3.00 | 2.51 | 5.93 | 0.00 |
4ABC | 2.32 | 2.92 | 6.23 | 0.00 |
5ABC | 3.13 | 1.83 | 5.60 | 0.00 |
6ABC | 1.74 | 1.88 | 2.11 | 1.55 |
7ABC | 1.75 | 3.07 | 5.29 | 0.28 |
8ABC | 2.63 | 0.89 | 3.89 | 0.00 |
9ABC | 2.17 | 2.99 | 5.91 | 0.00 |
10ABC | 2.62 | 1.29 | 4.37 | 0.00 |
11ABC | 2.41 | 1.72 | 4.60 | 0.11 |
12ABC | 2.14 | 3.02 | 6.10 | 0.00 |
13ABC | 2.11 | 2.17 | 4.40 | 0.50 |
14ABC | 5.02 | 0.00 | 5.21 | 0.00 |
15ABC | 4.75 | 0.25 | 5.11 | 0.00 |
16ABC | 1.45 | 3.10 | 2.34 | 2.23 |
实施例3:不同反应温度对全细胞催化效果的影响
(1)蛋白表达:根据经典的重组大肠杆菌培养及诱导表达方案,挑取14ABC重组大肠杆菌单菌落接种于3 mL含有50 μg/mL卡那霉素的LB培养基中,37℃ 250 rpm培养10-12h作为种子液;将种子液按0.5%接种量转接到50 mL含有50 μg/mL卡那霉素的LB培养基中,37℃ 250 rpm培养至OD600达到0.5-0.7,加入终浓度为0.2 mM的IPTG,在28℃ 220 rpm条件下诱导表达10-12 h;诱导表达结束后,5000 rpm离心5 min,收集细胞即为全细胞催化剂。
其中,所提及LB培养基配方为:胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L。
(2)全细胞催化:在100 mL三角瓶中依次加入10 mL含有5 g/L原儿茶酸的简化M9培养基(pH 7.0),5 OD600全细胞催化剂,在30-40℃250 rpm摇床培养条件下反应0.5-9 h,定时取样检测发酵液中原儿茶酸和2-吡喃酮-4,6-二羧酸的含量。
其中,所提及简化M9培养基配方为:葡萄糖4g/L,Na2HPO4•12H2O 17.1 g/L,KH2PO4 3 g/L,NH4Cl 1 g/L,pH 7.0。
其中,所提及反应温度为30-40℃,具体可为30℃或34℃或37℃或37℃或40℃。
其中,所提及反应时间为0.5-9 h,具体可为0.5 h或1.5 h或3.0 h或4.5 h或6.0h或9.0 h。
(3)高效液相色谱分析:将发酵液样品12000 rpm离心10 min,上清液用蒸馏水稀释一定倍数,用0.22 μm水系微孔滤膜过滤;采用Agilent 1200高效液相色谱仪,配备VWD紫外检测器,色谱柱为Phenomenex公司的Rezex RFQ-Fast Acid H+ (8%) (LC Column 100× 7.8 mm);流动相为5 mM H2SO4水溶液,上样量5 μL,流速0.6 mL/min,柱温55℃,检测波长313 nm,测定发酵液中原儿茶酸和2-吡喃酮-4,6-二羧酸的含量。
(4)结果:如表2所示,当反应3 h时,随着反应温度的升高,2-吡喃酮-4,6-二羧酸产量由3.36 g/L逐渐升高至5.78 g/L;底物原儿茶酸残余量逐渐减少,其中37℃和40℃条件下的原儿茶酸已经反应完全。
表2不同反应温度下全细胞催化反应速率比较
反应温度℃ | PDC产量g/L | PCA残余量g/L |
30 | 3.36 | 1.47 |
34 | 4.49 | 0.72 |
37 | 5.47 | 0.00 |
40 | 5.78 | 0.00 |
实施例4:不同反应pH对全细胞催化效果的影响
(1)蛋白表达:根据经典的重组大肠杆菌培养及诱导表达方案,挑取14ABC重组大肠杆菌单菌落接种于3 mL含有50 μg/mL卡那霉素的LB培养基中,37℃ 250 rpm培养10-12h作为种子液;将种子液按0.5%接种量转接到50 mL含有50 μg/mL卡那霉素的LB培养基中,37℃ 250 rpm培养至OD600达到0.5-0.7,加入终浓度为0.2 mM的IPTG,在28℃ 220 rpm条件下诱导表达10-12 h;诱导表达结束后,8000 rpm离心3 min,收集细胞即为全细胞催化剂。
其中,所提及LB培养基配方为:胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L。
(2)全细胞催化:在100 mL三角瓶中依次加入10 mL含有1 g/L原儿茶酸的不同pH磷酸缓冲液(100 mM,pH 5.0-8.0),1 OD600全细胞催化剂,在37℃250 rpm摇床培养条件下反应0.5-9 h,定时取样检测发酵液中原儿茶酸和2-吡喃酮-4,6-二羧酸的含量。
其中,所提及磷酸缓冲液pH范围为5.0-8.0,具体可为5.0或5.5或6.0或6.5或7.0或7.5或8.0。
其中,所提及反应时间为0.5-9 h,具体可为0.5 h或1.5 h或3.0 h或4.5 h或6.0h或9.0 h。
(3)高效液相色谱分析:将发酵液样品12000 rpm离心10 min,上清液用蒸馏水稀释一定倍数,用0.22 μm水系微孔滤膜过滤;采用Agilent 1200高效液相色谱仪,配备VWD紫外检测器,色谱柱为Phenomenex公司的Rezex RFQ-Fast Acid H+ (8%) (LC Column 100× 7.8 mm);流动相为5 mM H2SO4水溶液,上样量5 μL,流速0.6 mL/min,柱温55℃,检测波长313 nm,测定发酵液中原儿茶酸和2-吡喃酮-4,6-二羧酸的含量。
(4)结果:如表3所示,当反应6 h时,随着磷酸缓冲液pH的升高,2-吡喃酮-4,6-二羧酸产量呈现出先升高后降低的趋势,其中pH 5.5时的2-吡喃酮-4,6-二羧酸产量最高,达到1.28 g/L;而原儿茶酸残余量先降低后升高,只有pH 5.5时的原儿茶酸能够反应完全。
表3不同反应pH条件下全细胞催化效率比较
反应pH | PDC产量g/L | PCA残余量g/L |
5.0 | 0.22 | 0.66 |
5.5 | 1.28 | 0.00 |
6.0 | 1.07 | 0.18 |
6.5 | 0.51 | 0.58 |
7.0 | 0.23 | 0.71 |
7.5 | 0.12 | 0.55 |
8.0 | 0.05 | 0.68 |
实施例5:利用发酵罐全细胞催化生产2-吡喃酮-4,6-二羧酸
(1)全细胞催化剂制备
一级种子培养:分别挑取2ABC、14ABC、15ABC重组大肠杆菌单菌落接种于3 mL含有50 μg/mL卡那霉素的LB培养基中,37℃ 250 rpm培养10-12 h作为一级种子液。
二级种子培养:将一级种子液按0.5%接种量转接到200 mL含有50 μg/mL卡那霉素的LB培养基中,37℃ 250 rpm培养10-12 h作为二级种子液。
其中,所提及LB培养基配方为:胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L。
分批补料发酵培养:将二级种子液按8%接种量转接到装有2.3 L发酵罐培养基的发酵罐(上海保兴生物设备工程有限公司,BIOTECH-5BG发酵罐)中,添加50 μg/mL卡那霉素,初始发酵温度为37℃,通气量1 vvm,pH 7.0(用25%浓氨水调节pH),发酵过程中逐步提高搅拌浆转速使发酵液中的溶氧(DO)维持在30%以上;当溶氧回升时开始补料,控制补料速度使发酵罐中的甘油浓度始终低于3 g/L;当OD600达到30-40时,调整发酵温度至30℃,添加0.5 mM IPTG,诱导表达8 h,定时取样检测菌体OD600。诱导表达结束后,6000 rpm离心15min,收集细胞即为全细胞催化剂。
其中,所提及发酵罐培养基配方为:葡萄糖 2g/L、甘油10 g/L、酵母提取物10 g/L、胰蛋白胨16 g/L、K2HPO4•3H2O 4 g/L、NaH2PO4•2H2O 2 g/L、NaCl 3 g/L、(NH4)2SO4 2.5g/L、一水柠檬酸2 g/L、MgSO4•7H2O 0.5 g/L、FeSO4•7H2O 0.3 g/L。
其中,所提及补料培养基配方为:甘油600 g/L、酵母提取物62.5 g/L、胰蛋白胨87.5 g/L、MgSO4•7H2O 10 g/L。
(2)全细胞催化
向5 L发酵罐中依次加入1 L简化M9培养基(pH 7.0),30 OD600全细胞催化剂,初始原儿茶酸10 g/L,反应温度37℃,催化过程中用10 M NaOH控制pH为6.5,通气量2 vvm;当溶氧回升后,每次补加10 g/L原儿茶酸,以此类推,直至原儿茶酸不再消耗为止;反应时间0.5-9 h,定时取样检测发酵液中原儿茶酸和2-吡喃酮-4,6-二羧酸的含量。
其中,所提及简化M9培养基配方为:葡萄糖4g/L,Na2HPO4•12H2O17.1 g/L,KH2PO43g/L,NH4Cl1 g/L。
其中,所提及反应时间为0.5-9 h,具体可为0.5 h或1.5 h或3.0 h或4.5 h或6.0h或9.0 h。
(3)高效液相色谱分析
将发酵液样品12000 rpm离心10 min,上清液用蒸馏水稀释一定倍数,用0.22 μm水系微孔滤膜过滤;采用Agilent 1200高效液相色谱仪,配备VWD紫外检测器,色谱柱为Phenomenex公司的Rezex RFQ-Fast Acid H+ (8%) (LC Column 100 × 7.8 mm);流动相为5 mM H2SO4水溶液,上样量5 μL,流速0.6 mL/min,柱温55℃,检测波长313 nm,测定发酵液中原儿茶酸和2-吡喃酮-4,6-二羧酸的含量。
(4)结果
如表4所示,当反应6 h时,表达不同物种来源ABC全细胞催化剂的催化活力不同,其中2ABC的2-吡喃酮-4,6-二羧酸产量最高,达到52.40 g/L,合成速率达到8.73 g/L/h;而14ABC和15ABC的催化活力较差,2-吡喃酮-4,6-二羧酸产量分别为27.89 g/L和25.91 g/L。本发明公开的全细胞催化生产2-吡喃酮-4,6-二羧酸的方法,催化6 h的产量大约是文献报道最高产量(16.7 g/L)的3倍(Zi Wei Luo, Won Jun Kim, Sang Yup Lee. MetabolicEngineering of Escherichia coli for Efficient Production of 2-Pyrone-4,6-dicarboxylic Acid from Glucose. ACS Synthetic Biology. 2018, 7: 2296−2307.),PDC产量和合成效率提升显著。
表4不同全细胞催化剂在发酵罐中的催化效果比较
不同催化酶组合 | PDC产量g/L | PDC合成速率g/L/h |
2ABC | 52.40 | 8.73 |
14ABC | 27.89 | 4.65 |
15ABC | 25.91 | 4.32 |
以上所述的利用共表达原儿茶酸-4,5-二氧化酶和4-羧基-2-羟基粘康酸-6-半醛脱氢酶的基因工程重组大肠杆菌作为全细胞催化剂,以原儿茶酸作为底物,实现原儿茶酸到2-吡喃酮-4,6-二羧酸的高效全细胞催化生产,其中基因工程重组菌株的构建、全细胞催化剂的制备方法、全细胞催化方法等仅为本发明的较佳实施例而已,并不用于限制本发明。凡是在本发明的技术原则基础上所作的任何修改、等同替换和改进优化等,均包含在本发明的保护范围之内。
<110>中国科学院天津工业生物技术研究所
<120>一种全细胞催化生产2-吡喃酮-4,6-二羧酸的方法
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<212> DNA
<213>人工序列1AB
<400>4
ATGACCGAAAAAAAGAAGGAACGCATTGACATTCACGAATACCTGGCCCAGTTTGACGACATTCCGGGGACCCGTGTGTTTACCGCCAAACGCGCACGTAAAGGGTACTGGCTGAACCAGTTTGCAATGAGCCTGATGAAAGAGGATAACAGAGAACGCTTCAAAGCAGATGAAAGCGCATATCTGGACGAATGGAATCTGACCCCGGCAGCAAAACAAGCAGTGCTGGATCGCGATTATAATGCAATGATTGATGAAGGTGGTAACGTGTATTTTCTGAGCAAACTGTTTAGCACCGACGGTAAAAGCTTTCAATTTGCAGCAGGTAGTATGACCGGTATGTCTCAGGAAGATTACGCCAAAATGATGATTGATGGGGGTCGCAGTCCGGAGGGTGTTCGTAGTATTAAAGGCGGATATTAAGGATCCGAAGGAGATATACCATGGCACGGGTTACGACGGGGATTACCAGCAGCCACATTCCGGCGTTAGGTGCGGCGATGCAGACCGGGAAAGACAGAGACGACTACTGGGGGCCGGTGTTCGCGGGGTATGATGCAATTCGTGAATGGATTAAAAAGCCGGGCAATATGCCGGACGTTATTGTTTTAGTATACAATGACCACGCAAGCGCATTTGATATGAATATTATTCCTACTTTCGCCATTGGCTGTGGAGAATCTTTTAAGCCGGCAGATGAAGGGTGGGGTCCTCGTCCTGTTCCGGATGTTCAGGGCCATCCGGATTTAGCATGGCATATTGCACAGAGCCTGATTCTGGATGACTTTGATATGTGTATTTTCAACCAGATGGATGTTGATCATGGTTGTACCGTTCCGTTAAGCATGATGTTTGGTGATGTTAAGGAATGGCCTTGTAAAGTGATTCCGCTGCCTGTTAATGTTGTTACCTATCCGCCTCCTTCAGGTGCACGTTGTTTTGCGCTTGGGGATTCAATTAAAGCAGCAGTTGAGAGCTTTCCGGAGGATCTGAATGTGCATGTTTGGGGTACCGGTGGCATGAGTCATCAGTTACAGGGTCCGCGTGCAGGTTTAATTAATAAAGAATTTGACCTGGACTTCATCGAGCGGCTGATTAACGATCCGGAAGATTTAGCAAAGATGCCTCACATTGATTATCTGCGTGAAGCAGGTAGTGAGGGTATAGAGCTGGTGATGTGGCTGATTATGCGTGGAGCCCTGGGTGATAAAGTTAAAGAATTATATAACTTCTACCACATCCCGGCAAGCAATACAGCACTTGGAGCACTGATTTTACAGCCGGAAGAAACCGCCGGTGAAGCCCTGAATCCGCAGCGTGTTCTGAGCGGTCACAGTCTGGAGGCAGCCGAATAA 1355
<210>5
<211>963
<212> DNA
<213>人工序列1C
<400>5
ATGAAGATCGCACTGGCAGGTGGTGGTGCATTTGGGGAAAAACATCTGGACGGTCTGAAAAACATTGACGGTGTTGAAGTTACCGCGTTAGTTGGTAGAACCATTGACAAAACCCAGGAAATGGCAGATAAATATGGAATTCCGTATGCAACAACAGACTATGATGAAATGCTTGGCATGGAGAATGTGGATGCAGTTATCTTATGTACCCCGACCCAGATGCATGCAGAACAGGCAATAAAAGCAATGGATGCAGGCAAACATGTAGAAGTAGAAATTCCGCTGGCAGACAGCTGGGCAGACGCGGAAGCAGTTATGAAAAAGCAGGAAGAAACAGGATTAGTATGTATGGCAGGACATACCCGGCGGTTTAATCCGAGCCATCAATATGTGAAACAGCAAATAGAAGCAGGTAAATTTAACGTGCTGAGCATGGACGTGGAAACATATTTTTTTCGCCGTGAAAACAAAAACGCAAAAGGTGAACCGCGCAGCTGGACCGATCATCTGCTGTGGCATCACAGTGCACATACCATTGATATTTTTCAATACATGACCGGAAGCAAAGTTGTTAAAGCAAATATTCTGCAAGGTCCGAAACATCCTGATCTGGGGATTGCAATGGATCAGAGCATCCAAATGAAAACCGACAAAGGGCAGATTTTAACCCTGAGCCTGAGCTTCAATAATGAAGGTCCGTTTGGTACATTTTTTAGATATATTGGGGACACAGCCACCTACCTGGCACGGTACGATGACCTGTACACCGGTGCTGATGAGCCGATTGACCTGAGCGGAGTTGATGTTCCGAGTAATAATGGGATTGAGCTGCAGGATCGTGAATTTATAGCAGCCATCCGTGAAGGTCGCGAACCGAATAGCAGCGTTCATGGGGTTATTGAATGTTATCGGACAATTGGTCAGCTGGCAGCAAGCTTAGAGGAGCAAGATGGTTGGAGCTAA 963
<210>6
<211>1334
<212> DNA
<213>人工序列2AB
<400>6
ATGAGCGACAAAAAAGAACGGATTGACATTCACGAATACCTGGCGGAATTTGATGACATTCCGGGTACCCGCGTGTTCACGGCGAAAAGAGCACGCCAGGGCTACCACCTGAACCAGTTTGCAATGAGCCTGATGAAAGCCGAAAACCGTGAACGTTTCTTAGCAGATGAACGGGCATATCTGGACGAATGGCAAATCAGCGAAGCAGCAAAACAAGCAGTCTTAAATCGCGATTATAATGCAATGATTGACGAAGGTGGTAACATATATTTTCTGAGTAAGCTGTTTAGCACCGACAAAAAAAGCTTTCAGTTTGCAGCAGGTAGCATGACCGGTATGACCCAGGAAGAATATGCCGAAATGATGCTGAAGGGTGGGCGGTCCCCGCAGGGTCAGAGATCTATTAAAGGCGGATTCTAAGGATCCGAAGGAGATATACCATGGCACGGATTACGGCGGGGATTACCAGCAGCCACATTCCGGCGTTAGGCGCGGCGATTCAGACCGGGACGAGCGGTAATGATTACTGGGGGCCGGTTTTTAAGGGGTATGAACCGATTAAAGAGTGGATTCAACAGCCGGGCAATATGCCGGACGTTGTTATTTTAGTTTATAACGATCACGCAAGCGCATTTGATATGAATATTATTCCTACCTTCGCAATTGGTTGTGCAGAGACCTTTAAGAGCGCCGATGAAGGCTGGGGGCCACGGCCTGTTCCGGATGTTGTTGGTGATCCTGATTTAGCATGGCATATTGCACAGTCCCTGATTCTTGATGAGTTTGATATGACCATAATGAATCAGATGGATGTTGATCATGGTTGTACCGTTCCGTTAAGTATGATTTTTGGGGAGCCGGATAAATGGCCTTGTAAAGTTATTCCGTTTCCTGTGAATGTTGTTACCTATCCGCCGCCGAGCGGGAATCGTTGTTATAGTCTGGGCGAGTCAATCCGCGCCGCAGTTGAGAGTTATCCGGAAGATCTGAATGTGCAGGTTTGGGGTACCGGTGGTATGAGTCATCAGCTTCAGGGTCCAAGAGCCGGTCTGATTAATAAGGATTTTGATCTGGCATTTATCCGTGATTTAATTGAAGATCCGGAACGTCTGCGTAAGATGCCGCATATTGAATACCTGCGCGAAGCAGGCAGTGAAGGAATTGAGTTAGTTATGTGGCTGATTATGCGTGGTGCCCTGGGTGATAAAGGTGTTGAAGAACTGTATCGTTTTTATCATATCCCTGCAAGCAATACCGCACTGGGTGCATTAATTTTACAGCCGGAGGGTATTGGTGCCGCCCCGCCTAGTGTTCGTGAACAGGTGGATGCGTAA 1334
<210>7
<211>957
<212> DNA
<213>人工序列2C
<400>7
ATGCGCATTGCCCTGGCCGGTGCCGGTGCCTTTGGGGAAAAACACCTGGACGGGCTGAAAAACATTGACGGTGTGGAAATTACCAGCATTATTAGCCGCCGTGCCGAACAGGCCGCCGAAGTTGCAGCAAAATATGGGGCACGGCATAGCGGTACCGAATTAAGCGAAGCACTTGAAAGAGATGATGTGGATGCAGTTATTCTGTGTACCCCGACCCAAATGCATGCAGAACAGGCAATTGCATGTATGAATGCAGGTAAACACGTTCAAGTTGAAATTCCGCTGTCGGATAGCTGGGCAGATGCAGAAGCAGTTTTAAAAAAGCAACAAGAAACCGGCCTTGTATGTATGGTTGGGCATACCCGGCGCTTTAATCCGAGCCATCAGTATGTGCACAATAAAATTGTAGCAGGTGAACTGAGTATTCAGCAGATGGACGTGCAGACATATTTTTTTCGTCGTAAAAACATGAACGCAAAAGGTGAAGCCCGCAGCTGGACCGATCATCTGTTATGGCATCACGCAGCACATACCATAGATCTGTTTGCATACCAGGCAGGAAAAATTGTTACAGCAAATGCAATCCAAGGCCCGAAACATCCAGAATTAGGAATAGCAATGGACATGAGCATTCAATTAAAAAGCGAAAGCGGTGCAATTTGTACCCTGAGCCTGAGCTTCAACAATGACGGTCCGCTGGGCACCTTTTTTCGTTACATTGGTGACACCGCAACCTATATTGCACGTTATGATGATCTGGTTACCGGAAAGGAAGAACCGATTGATCTGACCGGAGTTACCGTGAGTAATAACGGCATTGAACTGCAGGATCGCGAATTTATAGCAGCAATTCGTGAGGGTCGTGAACCTAATAGCAGTGTGGCACAGGTGCTGGATTGTTATCGTGTTATTGGTGAACTGGCAGCATCATTAGAAGCACAAGATGGTTGGAGCTAA 957
<210>8
<211>1301
<212> DNA
<213>人工序列3AB
<400>8
ATGACCAAAACCATGCCGCGCGACGTTCACGCATATCTGGCCGAATTTGACGACATTCCGGGTACCCGTGTGTTTACCGCAAACCGGGCACGTAAAGGGTACCACCTGAACCAGTTCGCCATGAGCCTGATGAAACCTGAAAATCGCGAACGTTTTTTAGCAGATGAACGGGCATATCTGGACGAGTGGCCGATGACCGAAGAAGCAAAAAAAGCAGTTCTGGCCCGCGATTATAATGCAATGATTGACGAAGGTGGTAACATATATTTCCTTGCCAAACTGTTTAGCACCGACCGTCAGAGCTTTCAGGACGCAGCAGGAAGCATGACCGGTATGAGCAAAGATGAGTATGCCGCAATGATGATAGCAGGTGGGAGAAGCCCGAATGGTGTTCGCAGTAAGAAAGAAGGAAATTAAGGATCCGAAGGAGATATACCATGGCACGGATTACGCACGGGTTAACCACCAGCCACGTTCCGGCGATTGGTGCGATGATTGACCAGGGCCGCACGGACGACGACTACTGGAAACCGATTTTTGCGGGGTACGAATGGACGAAAGATTACGTGGCAAAAAATATGCCGGATGTTGTTATTCTGGTGTATAATGACCATGCAAGCGCATTTGATATGAAGGTTATTCCGACCTTTGCAATTGGTTGTGCGGATAAATTTGAACCGGCAGATGAAGGGTGGGGGCCGCGGCCTGTTCCGCCTGTGGAAGGTCATGCAGATTTAGCATGGCATATTGCACAGAGCTGTATTCTGGATGAGTTTGATATGACGATAATTAATGAGATGGACGTGGATCATGGTCTGACTGTTCCGTTAGATGTGATATTTGATAGCAAGGATAACAAAACCCCGAAAAAGTGGCCGACGAAAGTTATACCGATTGCCGTTAACGTTGTGACATACCCACCGCCGTCCGGTAATCGCTGTTGGATGCTGGGTGAAGCAATTGCAAGAGCAGTAGCAAGCTATCCGGAAGATCTGAACGTTCATATTTGGGGGACAGGTGGAATGAGTCATCAGCTGCAGGGTCCGCGGGCCGGTCTGATCAACCCTGATTGGGATAAAAGATTTTTAGATGATCTGACGAGAGATCCGCAAAGACTGCGTACCATTGAACATATTGAATACCTGCGTGAAACCGGTAGCGAAGGAATTGAAATGGTGATGTGGCTGATTATGCGCGGTGCACTGGGTCCGAATGTTAGCGAACTGCATAGACATTATCATGTTCCGTGTTCCAATACCGCCCTTGGACACATTGTTTTAAAAAATGAAGACGCGGCATAA 1301
<210>9
<211>960
<212> DNA
<213>人工序列3C
<400>9
ATGCGCATTGCCCTGGCCGGTGCCGGTGCCTTTGGGGAAAAACACCTGGACGGGCTGAAAAACATTGACGGTGTGGAAATTACCAGCATTATTAGCCGCCGTGCCGAACAGGCCGCCGAAGTTGCAGCAAAATATGGGGCAAAACATAGCGGAACCGAACTGAGCGAAGCACTTGAACGGGACGATGTGGATGCAGTGATTTTATGCACCCCCACCCAAATGCATGCAGAACAGGCAATTCAATGTATGAATGCAGGTAAACATGTTCAGGTTGAAATTCCGCTGTCCGACAGCTGGGCAGATGCACAGGCAGTACTGGACAAACAAAAAGAAACCGGGCTGGTATGTATGGTGGGTCACACGCGCCGTTTCAATCCAAGCCATCAATTTGTGAAAAATAGAATTGACGCAGGCGAATTTAATATTCAAGCAATGGACATCGAAACCTTTTTTTTTCGTCGTAAAAACATGAACGCAAAAGGTCAACCTCGCAGCTGGACCGATCATCTGCTGTGGCACCACTCAGCACATAGCATTGACATTTTCCAATATATGACCGGAAGCAAGGTTGTTACCGCAAACGCAATTCAAGGACCGCGTCACCCCGAATTAGGCATTGCAATGGATATGAGTATCCAACTTAAAACCGAAGCAGGACAGATTTTAACCCTGGCACTGAGCTTTAATAATGACGGACCACTGGGCACATTTTTTCGTTATATTGGTGACACCGCCACCTACATTGCACGCTACGACGATCTGGTTACCGGTAGAGAGGAGCCGATCGACCTGAGCGGGGTTGCAGTGAGCAGCAATGGTATTGAACTTCAGGATCGCGAATTTATTGCAGCAATCCGCGAAGGAAGAGAACCGAATAGCAGTGTTGCGAGCGTGTTAGATTGTTATCGTGTTATTGGTGAGCTGGCAGCAAGCCTGGAAGAACAGGATGGTTGGAGCTAA 960
<210>10
<211>1286
<212> DNA
<213>人工序列4AB
<400>10
ATGACCATTGAAAACCCGAAACGTCAGGATATTCACGAATACTTAGCAGAACTGGAAGACATTCCCGGCACCCGTGTTTTTACCACCGCCCGCGCCCGCAAAGGCTACTGGCTGAATCAGTTCTGCATGACCTTAATGAAACCAGAAAACCGCGAACGCTTTAAAACCGATGAACGGGCCTATCTGGACGAATGGCCGATGACCGAAGCCCAGAAACAGGCAGTTCTTAATCGCGATTACAATGCCGCACTGGACGAAGGCGGAAACATTTATTTTCTGGCAAAGGTTTTTTTCACCGACAATATAAGTTTCCTGCAGGCAGTGGGTACCATGACCGGTATGCCGGCAGAAGAATATCAGGCAATGATGATAGCGGGAGGTCGGAGCCCGGTTGGTTTACGGAGTAAGAAAGAACCGTACTAAGGATCCGAAGGAGATATACCATGGCACGGATTACGGCGGGGGTTGCGAGCAGCCACATTCCGGCGTTAGGGGCGACCATTGACCACGGCAAAACCCAGGAACCGTACTGGAAAGAGTGTTTCGCAGGGTTCGCATGGACCAGAGCATGGGAAGCAGCACAGAGACCGGATGTTGTTATATTAGTTTATAACGACCACGCAACCGCATTCGACATGGATTTTATTCCGACCTTTGCCATTGGTTGTGCAGAACGTTACAAGAGCGCGGATGAAGGTTGGGGTCCTAGACCGGTTCCGGATGTTATTGGCGATGCAGATCTGGCATGGCATATTGCACAGAGCTGTATTTTAGATGAATTTGATCTGACCATCGTTAACAGAATGGATGTTGATCATGGTTTAACCGTTCCGTTAAGTCTGATGTTTGGGCAGCCGGAAGCATGGCCGTGTAAAGTTGTTCCGCTGGCGGTTAATGTTGTTACCTATCCGCCGCCTAGCGGTAATCGTTGTTATGCACTGGGCGAAGCAATTGCACGTGCAGTTCAGTCATATCCGCAAGATCTGAATGTGCAGATTTGGGGTACCGGTGGCATGAGTCATCAGCTTCAAGGTCCGCGTGCCGGTCTGATTAATGCCGCATGGGATAATCGTTTTTTAGATGATCTGGTTAAGGATCCGCAACGGTTAAGACAGATTCCGCACATTGAGTACCTGCGTGAAACCGGTAGTGAAGGTATTGAAATGGTTATGTGGTTAATTATGCGCGGTGGTCTGGGTCCGGATGTGAGAGCATTACACAGACACTATCACGTTCCTGCAAGCAATACCGCCGTTGGACATATTGTTCTGGAACCGGGAGGCTAA1286
<210>11
<211>942
<212> DNA
<213>人工序列4C
<400>11
ATGCGCATTGCCCTGGCCGGTGCCGGTGCCTTTGGGGAAAAACACCTGGACGGGCTGCAGCAGATTGACGGGGTTGAAATTGTGAGCATTATTAGCCACACCGGGGAACAGGCCGCGGCCGTAGCAGCACGTTATGGGGCAGCACATAGCAGTGAACACCTGGAAGATGCACTGGCAAGAGACGATGTTGATGCAGTGATTCTGTGTACCCCAACCCAAATCCATGCCAGCCAGGCAATTGCATGTATGGATGCAGGTAAACATGTTCAGGTAGAAATTCCGCTGGCAGATAGCTGGGCAGATGCAGAAGCAGTGGCAGCCAAACAAAAAGAAAGCGGGTTAGTGTGTATGGTTGGGCATACGCGGCGGTTTAATCCGAGCCATCAATATGTTCACAATAAAATAGTGGCAGGGGAAATGAAAATACAGCAGATGGATGTGCAGACATATTTTTTTCGGCGTAAAAACATTAACGCAAAAGGTGAACCGCGCAGCTGGACCGATCATCTGCTTTGGCACCACGCGGCACATACCGTTGACCTGTTTGCATACCAGGCAGGCCCGATTGTGAAGGCAAATGCAATCGAGGGCCCGATTCATCCGGAATTAGGAATTGCAATGGATATGTCTATTCAGTTAAAGAGTGAAAGCGGTGCAATTTGTACCCTGAGCTTAAGTTTTAATAACGATGGTCCCTTAGGCACCTTTTTTCGTTATATTGGTGATACGGGGACCTACATTGCACGTTACGATGATCTGGTTAATGGTAAAGAAGAGCCGATTGATGTTTCCGGTGTTGACGTTAGCATGAATGGTATTGAGCTGCAGGATCGTGAGTTTATTGCAGCAATTAATGAGGGTCGTGAACCGAATAGCAGTGTGGGGAAAGTACTGGATTGTTATCGGGTTCTGGGTGATTTAGAAAAACAGCTGGTGCCGTAA 942
<210>12
<211>1277
<212> DNA
<213>人工序列5AB
<400>12
ATGACCGGGGCCCCTCACGACATTCAGGCCTATCTGGCAGAACTGGAAGATATTCCAGGAACCCTTGTTTTTACCGCAGCACGTGCCCGCGCCGGTTACCACCTGAACCAGTTTGCGATGAGCCTGATGGATCCAGATAACAGAAGTCGCTGGAAAGCAGATGAAGAAGCATATCTGGCAGATTTTCCGATGAGCGATGATCAGAGAGCAGCAGTCCGTGCAAGAGATTATAATAGATTACTGGAGCTGGGTGGGAATATTTATTTTCTTAGCAAAATCTTCGCAACCGACGGTCTGAGCTTTGTTGAAGCAGTTGCGACCATGACCGGAGCAAGCGTGGATGAATATCGCGCAATGATGAGCGCAGGGGGGCGGAGCCCGGATGGTTTACGTAGCAAAAGAGAAGGACGTTAAGGATCCGAAGGAGATATACCATGGCACACATTAGCGCCGGGGTTGCGAGCTCCCACGTTCCGCTGCTGGGGATGGCGCACGACCGCCATCAGGAACATGACCCGTGTTTTGCGCCGATTTTTGACGGGTTTGAATGGACCCGTGGCTGGGAACGTGCAAGTCCGCCAGATGTTGTTATACTTGTGTATAATGATCACGCAAGCGCATTTGATATGAAAATTATTCCGACCTTTGCAATTGGTTGTGGTGAACGTTTTCGTCCGGCCGATGAAGGTTGGGGTCGTAGACCGGTTCCGGAAGTTATTGGTCATCCTGAACTGGCATGGCATATTGCACAGAGCTTAATTCTGAGTGATTTTGATATGACCATCATTAATGAGATGGATGTTGATCATGGTTTAACGGTTCCTTTAAGCCTTATGTTTGGCGAAACACCGGCATGGCCAAGCAGAGTTATTCCTTTAGCAGTTAATGTTGTGACGTATCCGCCGCCGAGCGGTAATCGTTGTTGGAGTCTGGGCGAGGCAATTGCACGTGCCGTTGAGAGCTTTCCCGAAGATCTGGATGTGCAGATTTGGGGTACCGGTGGAATGAGCCATCAGTTACAGGGTAGCCGTGCGGGTCTGATTAATCGTGGTTGGGATGCCATGTTTCTGGATGGTTTAATTGGTGATAGCGATCATTTACGTCACATTCCGCATATTGAATATTTACGTGAAACGGGTAGTGAGGGTATTGAAATGGTTATGTGGCTGATTATGCGTGGTGCTTTAGGGCGGGAGACGCGTTGTTTACATCGTCATTATCATGTTCCTTGTAGTAATACCGCCGTTGGACATATTGTTCTGGAACCGAAAAGCTAA 1277
<210>13
<211>939
<212> DNA
<213>人工序列5C
<400>13
ATGCGCATTGCCGTTGCCGGTATTGGGGCCTTTGCGGAGAAACACATTGAAGCCTTACGGGCGATTGACGGTGTTGAAATTGTTAGCGTTGTTGGGCGGCGGGCGGAGCCGACACGTGATTTTGCGGGTCGTCATGCAATTGGTCATGCCGCAACAGAATTAGGGGAAACACTGGCATTACCGGGATTAGATGCAGTTATTTTATGTACCCCGACTCAGCTGCATGCAGCACAAGCAATTGCCTGTTTAGACGCAGGTAAACATGTACAGGTTGAAATTCCGCTGTGCGATGTTCTGGCAGATGGTGAAGCAGTTGCCCAGCGTCAGGCAGAAACCGGTCTTGTTGCAATGGTAGGGCATACCCGGCGGTTTAATCCGAGCCATCAATATCTGCATGCCAAAATTGCAGCAGGTGAAATCCATGTGCAGCAGATGGATATACAGACATATTTTTTTCGTCGTGAAAACATTAACGCAAAAGGTGAACCGCGCAGCTGGACCGATCATCTGTTATGGCACCACGCAGCACATAGCGTTGATCTGTTTGCCTGGCAGGCCGGGCCGATTGTTGCAGCATCAGCAATGGCAGGCCCTCCGCATCCGGAATTAGGAATTGCAATGGATATGTCTATTCAGTTAAAGGCAGAATCAGGTGCACTGTGTACCCTGAGCCTGAGTTTTAACAATGATGGTCCCTTAGGTACGTTTTTTCGTTATATTTGTGACGAGGGCACATGGATTGCACGTTACGATGATCTGGTTACCGGTCGTGAAGAAGCCGTTGATTTAAGCGCAGTGGCCGTTAGCCGTAATGGTATTGAGCTGCAGGATCGGGAGTTTGTTGCAGCAATTCGTGAGGGTCGTCCGCCGCGTGCTAGTGTTGCAGATGTTATGCCGTGTTATCGTCTGTTAGATAGTCTGGAAAAACAGATGCGTTAA 939
<210>14
<211>1328
<212> DNA
<213>人工序列6AB
<400>14
ATGAGCGACATTCACCAGTATCTGGCAGAATTTGACGACATTCCGGGCACCCGCGTTTACACCGCGGCCAGAGCGCGTCAGGGCTACCACTTAAACCAGTTTGCGATGAGCTTAATGAAGGAACAGAATCGCAAACGTTTTCATGCAGACGAAAGCGCCTATTTAGATGAATGGCCGATTACCCCAGAGCAAAAACAGGCAGTTCTGGAGCGCGATTATAACCGCCTGCTGGACCTGGGAGGTAACATTTATTTCCTTGCCAAAGTTTTTAGCAGCGATGGCCTGAGCTTTGTACAGGCAGTTAGCACCATGACCGGTATGTCTGTTGATGATTATCAGGCAATGATGAATGCAGGGGGGCGCAGCCCGGAGGGTGTTAGAAGTATTCGGGATGGCAATTAAGGATCCGAAGGAGATATACCATGGCACGGATTACGCACGGGATTGCGTGTTCGCACATTCCGGTTTTAGGGTATGCGTTTGACCACGGCAAAGAAGGGGAAGACTACTTCAGACCGGCGTTTGAAGGGTTCGAGTGGACGAGAAAATTTATGAAAGAGGAAAAACCGGATGTTATTGTTCTGGTTTATAATGATCACGCAAGTGCATTCGATATGAAAATTATTCCTACATTCGCAATCGGTTGTGGTGAAAGCTACCCGCCAGCGGATGAAGGTTTTGGTGCACGGCCGGTTCCGCCTGTTGAGGGACATGCAGATTTAGCATGGCATATTGCACAGAGCCTTATTTTAGATGAATTTGATATGACGATCATCAACGAAATGAAGGTGGATCATGGTTTAACCGTTCCGTTAAGTATGATGTATGGTCATGTGGATACATGGCCCGTTAAAGTAATTCCGCTGGCCGTTAATGTTGTGACCTACCCGCCGCCGAGCGGTAATCGTTGTTGGGCACTGGGCGAGGCAATTGCCCGGGCAGTTAGTAGCTTTGGGGAAGATTTAAAAGTTCAGGTTTGGGGTACCGGTGGTATGAGCCACCAGCTTCAGGGTCCGCGTGCTGGTCTTATTAATACCGAATGGGATAATATGTTCCTGGATCGCCTGATTGGTGATAGCCAGGATTTAAGACAGATTCCGCATATTGAATATCTGCGTGAAACCGGTAGTGAAGGTATTGAAATGGTTATGTGGCTGATTATGCGTGGTGCACTTGGTAGAAGCACGCGTGCATTACATAGACATTATCATGTTCCTGTTAGCAATACCGCCCTTGGACACCTGGTTTTAGAACCCGTTGATGGTAGCGTTCCTCCGAGCCCTACATTAGAACAGAGCAATGCAGCAGCACAGACCCTGGTCGCCTAA 1328
<210>15
<211>960
<212> DNA
<213>人工序列6C
<400>15
ATGAAGATCGCACTGGTTGGTGCAGGTGCATTTGGGGAAAAACATCTGGCCGGTCTGAAAAATATTGAAGGAGTGGAAATTGCCAGCGTTGTGAGCAGAAAAGCAGAACAGGCAGCAGAAGTGGCCGAAAAATATGGGGCAAGCCATAGCGGGACGGATTTAGCAGAAACATTAGCACTGCCGGATGTGGATGCAGTTATTCTGTGTACCCCGACCCAGATGCATGCACAACAAGCAATAGCATGTATGGATGCAGGAAAACATGTACAGGTTGAGATTCCGCTGGCAGATTCATGGGCAGATGCGCAGGCAGTTGCAGAAAAACAGAAAGAAACCGGGCTGGTTTGTATGGTTGGGCATACCCGCCGTTTTAATCCGAGCCATCAATGGGTGAAACAGCGTATAGATGCCGGTGAATTTAATATTCAGGCAATGGATGTGGAAACCTTTTTTTTTCGTCGGAAAAATATGAACGCCAAAGGTGAACCGCGTAGCTGGACAGATCATCTGCTGTGGCATCACAGTGCACATACAATTGATATTTTTCAGTACATGACCGATGCAAAAGTTATTGCAGCAAATGCACTGCAAGGTCCGAAACATGCAGAATTAGGTATTGCGATGGATATGAGCATTCAGATGAAAACCGACCAGGGTCAGATTCTGACCCTGGCACTGAGTTTCAATAACGATGGTCCGTTAGGTACATTTTTTCGTTATATTGGTGACACAGGGACCTACATTGCACGTTACGATGATCTGGTTACCGGTCGTGAAGAGCCCATTGATGTTTCTGGCGTTGATGTGAGCATTAATGGTATTGAGCTGCAGGATCGTGAATTTGTTGGAGCAATCCGCGAGGGTCGTGAACCGAATAGCAGTGTTGCAGGTGTGCTGGATTGTTATCGTGTTATTGGTGAACTGGCAGCGAGCCTGGAGGCACAAGATGGTTGGAGTTAA 960
<210>16
<211>1328
<212> DNA
<213>人工序列7AB
<400>16
ATGCCGCTGGACAAACCGTACAAAGACATTCCGGGGACCACCATTTTTGACGCCGAACAGAGCCGTAAGGGGTACTGGCTGAACCAGTTTTGTATGAGCCTGATGAAAGCAGACAACCGTGCACGTTTTAAAGCAGATGAAGGAACCTATCTGGACGAATGGCCGATGACCGAAGCACAGAAAGCAGCAGTTCGTGCACGCGATCTTAATGAATGTATTCGCCTGGGTGGAAATATTTATTTTCTGGCAAAAATCGGTGCAACCGACGGTAGAAGCTTTCAACAAATGGCAGGTAGCATGACAGGAATGACAGAAGCAGAATATCGCGATATGATGGTTGGTGGAGGGCGGAGCATTGAAGGAAATCGGCGCATTGGAGAAGATGGGGATGCACAGCCGCAGCACCAGCCGCAGGGAAAAAAAAAAGGCAGCTAAGGATCCGAAGGAGATATACCATGGCACGGATTACGGCGAGCGTTTACACCAGCCACGTTCCGGCGATTGGTGCGGCACTGGATCAGGGAAAAGCAGGGGACGACTACTGGCAGCCGCTGTTTCGGGGGTATGACTACTCGAAACATTGGCTGGCAGAACAGAAACCGGATGTAGTTTTTCTGGTGTATAATGATCATGCAACCGCATTCAGCCTGGATCTGATTCCGACCTTTGCACTTGGTACCGCCGCACGCTATGCACCTGCCGATGAAGGATATGGTGCACGTCCGGTTCCGGAAGTTCTGGGTCATCCTGAACTGGCAGCCCATATAGCACAAAGCGTTATCCAGGATGATTTTGATTTAACCCTGGTTAATAAGATGGATGTAGATCATGGTTTAACCGTTCCGTTAAGCCTGATGTGTGGCCAACAGGATCCGAAACAGGGTGCATGGCCGTTTAAAGTTATTCCGTTTGCAGTGAATGTTGTTCAATATCCGGCACCGAGCGGTCGTCGTTGTTTTCAGCTGGGTCAAGCCATTCGTCGTGCAGTTGAAAGCTTTGATGAAGATTTAAATGTGCAGATTTGGGGTACAGGTGGAATGAGTCATCAGCTGCAGGGTCCGCGTGCAGGTTTAATTAACCGTGAATGGGATAACGCATGGTTAGATCAGTTAATTGCAGATCCGGTTGCATGTAGTCAGGTTCCGCATATTGATTACGTTCGTGAAGCAGGTAGTGAAGGGATAGAGCTGGTTATGTGGCTGATAGCGCGGGGTGCGATGGCAGATGTTGCCGGTGGTCCTAAACCGGTTGTTAAACATCGTTTTTATCATGTTCCGGCAAGTAATACAGCAGTGGGTCACCTGATTCTGGAAAATTTACGGGATTAA 1328
<210>17
<211>960
<212> DNA
<213>人工序列7C
<400>17
ATGAGCAGCAAAATTAAGGTGGCGCTGGCGGGGGCAGGGGCGTTTGGTATTAAACATCTGGACGGGATTCGGAACATTGCGGACGTGGAAGTGGTGAGCCTGATTAGCCGTGACCTGGACAAAACACAGGAAGTGGCAAGCAAATATGGAATAGGTCATGTCACCACCGAATTAGCAGATAGCCTGGCACGCCCGGAAGTTGATGCAGTTATATTATGTACCCCAACCCAGATGCATGCAAGCCAAACACTGGCATGTCTGAAAGCAGGTAAACACGTTCAGGTTGAAATTCCGCTGTGTGATGTTTTACAGGATGGTGAAGAAGTTGTTGAGCTGGCAGCCCGCAGCGGCAAAGTAGCAATGTGCGGTCATACCCGTCGGTTTAATCCATCCCATCAGTGGGTACACCGTAAAATTGCAGCAGGTGAATTAAATGTTCAGCAGATGGATGTTCAGACCTATTTTTTTCGTCGGAGTAACATGAATGCACTGGGTCAGCCGCGCTCATGGACCGATCATCTGCTGTGGCATCACGCAGCACATACCGTGGATCTGTTTGCCTGGCAGGCACAGAGCCCTATTGTTCAAGCACACGCCCTGCAGGGTCCTATTCATCCTCAACTTGGGATTGCAATGGATATGAGCATTCAGTTAAAAGCAGCAAATGGTGCAATTTGTACCTTAAGCTTAAGCTTTAATAACGACGGACCACTGGGTACATTTTTCCGTTATATTGGTGATAGCGGTACCTACATTGCGCGCTACGATGATCTGTTTAATGGCAAAGAGGAAAAAATCGATGTGAATCAGGTTGACGTGAGCATGAACGGTATTGAACTGCAGGATCGTGAATTTTTTGCAGCAATTCGCGAGGGTCGTGAACCGAATGCAAGCGTGGCACAGGTGTTACCGTGTTATCAGGTGTTACATCAGCTGGAACAGCAGCTGAAAAATGGTTAA 960
<210>18
<211>1265
<212> DNA
<213>人工序列8AB
<400>18
ATGAGCCTGGAAAAACCGTACAGCAACGTTCCGGGGACGACCATTTTTGATGCGGACCAGAGCCGCAAAGGGTACTGGCTGAACCAGTTTTGTATGAGCCTGATGAAAGCAGAGAACCGTGATCGTTTTAAAGCAGATGAAAGAGCATATTTAGACGAATGGGATATGAGTGAAGAACAAAAACAGGCAGTTCTGGATCGCGATCTGAATCGTTGTATTGCCCTGGGTGGAAATATTTATTTCCTGGCAAAAATTGGAGCGACAGATGGAAAGAGCTTTCAGCAGATGGCAGGAAGCATGACAGGTATGAGCGAAGCAGAATATCGTGATATGATGCTGAAAGGTGGACGGAGCGTGGAGGGTAATCGCTACACCGGTGACCAGAAATAAGGATCCGAAGGAGATATACCATGGCAAAAATCACCGCGAGCGTTTACACCAGCCACGTTCCGGCGATTGGGGCGGCCCTGGACTTAAACAAACAGAACGAAGACTATTGGAAACCGGTTTTTGCGGGCTACGACTTCGGCAAAGAATGGATTAAACAAAATAAGCCAGACGTTGTGTTTCTTGTTTATAATGATCATGCAACCGCATTTAGCCTGGATTTAATTCCGACCTTTGCAATTGGTACAGGCCCGTTTTATCCGCCGGCAGATGAAGGTTGGGGTCCACGGCCGGTTCCTCCTGTTATTGGACATCCTGAACTGGCAGCCCATATTGCACAGAGCGTTATTCAGGAAGACTTTGATTTAACCATTGTTAACAAGATGGACGTAGATCATGGGTTAACCGTTCCGCTGTCACTGATGTTTGGCCAACCGGAAGCATGGCCGTGCACCGTAATTCCGTTTGCAGTTAATGTTGTTCAGTATCCGGTTCCGAGCGGTCGTAGATGTTTTGAGCTGGGCAAGGCAATCCGCCGTGCAGTTGAAAGCTTTGATAAAGATCTGAATGTTCAGATTTGGGGTACCGGTGGTATGAGTCATCAGTTACAGGGTCCGCGTGCAGGTCTTATTAATCGTGAATGGGATAATAAATTCCTGGATCGTTTAATTGCAAACCCGGACAGCCTGAGTCAGGTTCCGCACATTGAGTACGTTCGTGAAGCTGGGAGTGAAGGTATTGAACTTGTGATGTGGCTGATTGCACGGGGAGCCATGGCAGATGCGGTGGGTGGCGAAGCACCGACCGTTGCACAGCGTTTTTACCATGTTCCTGCAAGCAATACGGCGGTGGGTCACCTGATTCTGGAAGATAATTAA 1265
<210>19
<211>960
<212> DNA
<213>人工序列8C
<400>19
ATGAGCAAAACGATTAAGGTGGCGCTGGCCGGGGCAGGGGCGTTTGGTGTTAAACACCTGGACGGGATTAAAAATATTGACGGGGTGGAAGTTATTAGCCTGGTGGGGCGGGACCTGGAAAAAACAAAAGAAGTGGCGGGTAAATATGGTGTGGGTCATGTGACAACCGAACTGAGCGAAAGCCTGGCACTGCCGGAATTAGATGCAGTGATCCTGTGTACCCCGACCCAGATGCACGCAGCACAGGCAGTGCAAGCACTGAAAGCGGGGAAGCACGTTCAAGTCGAAATTCCGCTGGCAGATAGTCTGGAAGGAGCACGGGAAGTTGCACGTCTGCAGAAAGAAACCGGTCTGGTAGCAATGGTTGGTCACACCCGCCGGTTTAATCCGAGCCATCAATATGTTCACAAACAAATAGAAAAGGGTGATTTTCATGTGCAGCAGATGGATGTCCAAACATATTTTTTTCGTCGTACCAATACCAATGCACTGGGTCAACCGCGCAGCTGGACCGATCATTTACTGTGGCATCACGCAGCACATACCGTTGATTTATTTGCATATCAGGCAGGTAAAATTGTTCAGGCACACGCAATGCAAGGGCCGATTCATCCAGATTTAGGTATTGCCATGGATATGAGCATTCAGTTAAAAAGTGAAACCGGTGCAATTTGTACCCTGAGCCTGAGCTTCAACAATAATGGACCGCTGGGGACATTTTTTCGTTACATTGGTGATACAGAAACCTACATTGCGCGCTATGATGATCTGGTTAATGGAAGAGATGAAAAAATAGATGTGAGCAAAGTTGACGTGAGCATGAATGGTATTGAACTGCAGGATCGGGAATTTTTTGCAGCAATTCGCGAGGGTCGTGAGCCGAATAGCAGTGTTGCACAGGTGCTGCCGTGTTATGAAGTTCTGCATCAATTAGAACAGCAGCTGGCCCGTGATAATTAA 960
<210>20
<211>1319
<212> DNA
<213>人工序列9AB
<400>20
ATGGCACTGCAGAAACCGTACTTAGACGTTCCGGGGACGACGATTTTTGACGCGGAACAGAGCCGCAAAGGGTACCACCTGAACCAGTTTTGTATGAGCTTAATGAAAGCACAGAATCGCGCACGTTTTAAAGCAGATGAACGGGCATATCTGGACGAATGGGCGATGACCGAAGAACAGAAACAGGCTGTTTTAGCACGGGATCTTAATCGGTGTATTGCCCTGGGAGGAAATATTTATTTTCTGGCAAAAATCGGAGCAACAGACGGTAAAAGCTTCCAGCAGATGGCAGGAAGTATGACAGGTATGACCGAAGATGAATACCGCAATATGATGATTGGTGGGGGACGTAGCGCAGAGGGAAATCGCTATATAGGTGAAGATGGGGATGCACAGGCACATCACCAGCCGCAGGGAGCAGCAGGAAAAAAAGGAACCTAAGGATCCGAAGGAGATATACCATGGCAAAAATCACCGCGAGCGTTTTCACCAGCCACGTTCCGGCGATTGGGGCGGCGATTGACTTAGGCAAAACCACCGAACCGTACTGGGTTCCGCTGTTCGCAGGCTACGAACCGTCCAAGCAGTGGATGAAAGATAATAAACCAGATGTTATCTTCCTGGTTTATAATGATCACGCAACCGCATTCAGCCTGGAAATGATTCCGACCTTTGCAATTGGTTGTGCCGCAGAATTCCAACCAGCGGATGAAGGATATGGCGCACGTCCGGTTCCGACTGTTATTGGTCATCCTGATCTGGCATCTCATATCGCACAGAGCGTTATTCAGGATGATTTTGATTTAACCATCGTTAACAAGATGGATGTGGATCATGGTTTAACCGTTCCGTTAAGCCTGATGTGTGGTGAACCGGATGCATGGCCGTGTCCGGTAATTCCGTTTGCAGTTAATGTTGTTCAGTATCCGGTTCCGAGCGGTCAGCGTTGTTTTAAGCTGGGCCAGGCAATTAGACGTGCCATTGAGAGCTATGATCAGCCGCTTAACGTTCAGATTTGGGGTACCGGTGGCATGAGTCATCAGCTGCAGGGTCCGCGGGCAGGTCTGATTAATCGTGAATGGGATAACGCATGGTTAGACAAACTGATTGAAGATCCTGAAGCAGCAGCAGCAACGCCTCATATTGATTACGTTCGTGAAGCAGGTAGTGAAGGTATAGAACTTGTTATGTGGCTGATTGCACGCGGAGCAATGGCAGACGTTGCGGGTGGGCCTAAACCGACAGTACGTCACCGTTTTTATCATGTTCCTGCAAGCAATACAGCAGTTGGGCACGTGATTCTGGAGAATAATGTTTAA 1319
<210>21
<211>957
<212> DNA
<213>人工序列9C
<400>21
ATGACCAAAACCATTAAGGTGGCCCTGGCCGGGGCGGGGGCATTTGGTATTAAACATCTGGACGGCATTAAAAATATCGACGGCGTTGAAGTTGTTAGCCTGATTAGCCGCGACATTGAAAAAACCAGAGAAACGGCAGCACAATATGGTATAGGGCATGTGACAACCGAACTGGCTCATAGTCTGGCACTGCCGGAAGTGGATGCAGTTATCCTGTGTACCCCGACCCAAATGCACGCAAGCCAGAGCATAGCATGTTTAAAGGCAGGCAAACACGTACAAGTTGAAATTCCGCTGGCAGATAGTTGGACCGATGCACAGGCAGTTGCAGATCTGGCACGTAGCTCAGGTCTGGTAGCAATGTGTGGTCATACCCGGAGATTTAATCCGAGCCATCAGGCAGTGCACAATAAAATAGTTGCAGGGGAATTTAATATCCAGCAGATGGATGTTCAGACCTATTTTTTTCGTCGTACCAATATGAATGCACTGGGACAGGCCCGCAGCTGGACCGATCATCTGCTTTGGCATCACGCAGCACATACCGTTGACTTATTTGCATATCAGTGTGGTAGCCCTATTGTTAAAGCAAACGCAATTCAGGGACCTATTCATCCGACACTGGGTATTGCAATGGATATGAGCATCCAACTGCAGGCAGCAAATGGGGCAATTTGTACCTTAAGCCTGAGCTTTAATAATGACGGTCCGCTGGGCACATTTTTTCGCTATATTGGAGATAGTGCAACCTACATTGCGCGCTATGATGATTTATTTACCGGTAAGGAAGAGAAGATTGATGTTTCTCGGGTGGCAGTGAGCATGAATGGTATTGAACTGCAGGATCGGGAATTTTTTGCAGCAATTCGCGAAGGTCGTGAGCCGAATAGCAGCGTTGCACAGGTTTTACCGTGCTATCAGGTTCTGCATCAGCTGGAACAGCAGCTGTTAGCCTAA 957
<210>22
<211>1337
<212> DNA
<213>人工序列10AB
<400>22
ATGAGCCTGGAAAAACCGTACCTGGACGTGCCGGGCACCATTATTTTTGACGCGGAACAGAGCCGCAAAGGGTACTGGCTGAACCAGTTTTGTATGAGCCTGATGAAAGCACAGAACCGCGAACGTTTTAAAACCGATGAACGGGCGTATCTGGATGAGTGGCCGATGACCGAAGAACAGAAGCAGGCGGTTCTGGCCCGCGACCTTAATTGGTGTATGCGCACAGGTGGTAATATTTATTTTCTGGCAAAGATTGGAGCAACAGATGGTAAAAGCTTTCAGCAGATGGCAGGTAGTATGACCGGTATGACCGAAACCGAATATCGTGATATGATGATTCAAGGTGGGCGTAGCGTTGAGGGAAATAGATATGTTGGTGAAGATGGGGATGCACAGGCACATCGTCAGCCGCAGGGGGCGGCAGGAAAAAAAGTGTAAGGATCCGAAGGAGATATACCATGGCAAAAATCACCGCGAGCGTTTTCACCAGCCACGTTCCGGCGATTGGGGCGGCGATGGATCTGGGCAAGACCCAGGAAGACTACTGGAAACCGGTTTTTGCGGGGTACGACTTTAGCAAACAGTGGATGAAAGATAATAAGCCAGATGTAATTTTCCTGGTGTTTAATGATCACGCAACCGCATTTAGCTTAGAAATGATTCCGACCTTTGCTATTGGGACAGCAGCAGAGTTTCAGCCGGCAGACGAAGGATGGGGTCCGCGCCCTGTTCCTAAAGTTATTGGTCATCCAGATCTGGCATCTCACATTGCACAGAGCGTTATTCAGCAGGACTTTGATTTAACAATTGTTAACAAGATGGACGTGGATCATGGTCTGACTGTTCCGCTTAGCCTGATGTGTGGTGAACTGGATCCGAAAACCGATGCATGGCCGTGTCCGGTTATACCATTTGCAGTTAATGTTGTGCAGTATCCGGTTCCGAGCGGTAAACGTTGTTTTATGTTAGGACAAGCCATTCGTAAAGCAGTTGAAAGCTACGATGAAGATCTGAATGTTCATATTTGGGGTACAGGTGGGATGAGTCATCAGCTTCAGGGTGCACGTGCGGGTCTGATTAATCGTGAATGGGATAATGCATGGCTGGATCAGATGATTGCAGATCCGGTTGGCTGTGCAAACACACCGCACATTGATTATGTTCGTGAAGCAGGTAGTGAAGGTATTGAACTGGTTATGTGGCTGATTGCAAGAGGTGCGATGAGCGATATTGTGGATGGTAAAGTGCAGGGTCCGGCGCCGACCGTTAAACATCGTTTTTATCATGTTCCGGCGAGCAATACCGCAGTTGGTCATCTGATTCTGGAAAATAATTAA 1337
<210>23
<211>951
<212> DNA
<213>人工序列10C
<400>23
ATGACCATTAAGGTGGCCCTGGCCGGGGCGGGGGCATTTGGTATTAAACATCTGGACGGGATTAAAAACATTGACGGGGTGGAAGTGGTGAGCCTGATCAGCCGGGACCTGGAAAAAACCAAAGAAGTGGCGGATAAATATGGTATTCAGCATGTGACAACCGATTTAGCAGATAGCCTGGCGCTGAAAGAAGTGGATGCAGTTATACTGTGTACGCCGACCCAGATGCATGCAGAACAGACACTTGCATGTCTGAAGGCAGGTAAACATGTTCAGGTTGAAATTCCGCTGGCAGACAGTCTGAAAGGAGCAGAAGATGTGGTTGCACTGCAAAAACAAACCGGCCTGGTTGCAATGTGCGGCCATACCCGTAGATTTAATCCAAGCCATCAATATGTTCACAATAAAATTCAGGCAGGTGAATTTAACATCCAGCAGATGGATGTTCAAACCTACTTTTTCCGTCGTACCAACACCAATGCACTGGGTCAGGCCCGCTCCTGGACCGATCATCTGTTATGGCATCACGCAGCACATACCGTTGACCTGTTTGCCTACCAGTGTAATTCACCGATTGTTATGGCAAATGCAATCCAGGGTCCTATTCATCCGGTTCTTGGGATTGCAATGGATATGAGCATCCAGTTAAAAGCAGCCAATGGGGCAATTTGTACCTTAAGCCTGTCCTTTAACAATGACGGACCATTAGGCACATTTTTCCGTTACATTGGGGATACAGCAACCTACCTGGCGCGCTACGATGATCTGTTTACAGGTAAAGAAGAGAAAATCGATGTTAGCAAAGTTGCAGTGAGCATGAACGGCATTGAACTGCAGGATCGCGAGTTTTTTGCAGCAATAAAGGAAGGTAGAGAACCGAATAGCAGCGTTGGTAAAGTGTTTAACTGTTATCAGGTTTTACACAATCTGGAACAGCAGCTGAAAGGGTAA 951
<210>24
<211>1319
<212> DNA
<213>人工序列11AB
<400>24
ATGAGCCTGAACAAACCGTACAAAGACATTCCGGGTACCACCATTTTTGATGCAGAAGAAAGCCGCAAGGGTTATTGGCTGAACCAGTTTTGCAGCAGCCTGATGAAAGCAGAAAATCGCACCCGTTTCAAAGCAGATGAAAGAGCCTATCTGAACGAATGGAAGATGACCGAAGCACAGAAAGATGCAGTGATTTCCCGCGATCTGAATGAATGTATTCGCCTGGGAGGTAATATCTACTTTCTGGCACGTATTGGAGCAACCGATGGTCTGACCTTTCAACAGATGGCAGGAAGCATGACCGGTATGAGCGAAGAAGAATATCGCAATATGATGGTTGGTGGTGGTCGGAGCCCGGAAGGTAATCGTTATCTGGGTGAAGATGGTGATGCACAGCCGCATCGGCAGCCGCAGGGAAATCATAATAAAGAAGAAGCATAAGGATCCGAAGGAGATATACCATGGCACGGATTACGGCGAGCGTTTACACCAGCCACATTCCGGCGGTTGGTGTTGCGATGGATTTAGGGAAGACACAGGAGCCGTACTGGCAGCCGGTTTTTGAAGGATACGAATATAGCAAAGAATGGCTGAAAGAAAATAAGCCGGATGTTGTGTTCCTGGTTTATAATGATCACGGTACCGCATTCAGCTTAGAAATGATTCCGACCTTTGCGCTGGGTACAGGTGCAGAATATCCGCCGGCCGATGAAGGATGGGGTCCGCGTCCGGTTCCGATGGTTCATGGCCATCCGGAATTATCAGCACACATTGCACAGAGCGTTATTCAGGATGATTTTGACTTAACCCTGATTAATGAAATGCCAGTAGATCATGGTCTTACCGTTCCGCTGTCGGTTATGTGTGGCCAACCGGAGGCCTGGCCGTTTAAAGTTATTCCGTTTCATGTTAATGTGGTTCAGTATCCTGTTCCGAGTGGGAAACGTTGTTTTGAATTAGGACAGGCAATTCGTCGTGCGGTTGAAAGCTTTGATGAAGATTTAAATGTGCAGATTTGGGGTACCGGTGGTATGAGTCATCAGCTGCAGGGTGCACGGGCGGGTCTGATTAATCGGGAATGGGATAACGCATTTCTGGATCGTCTGATTGCAGATCCGGCCGACCTTAGCAACATGCCGCACATTGATTACGTTCGTGAAGCAGGTAGCGAAGGCGTTGAGCTTGTTATGTGGTTAATTGCACGCGGTGCCATGGCAGATGTTAACGGTGGTGCAGCACCGACCGTTAAACATCGTTTTTATCATGTTCCTGCAAGCAATACAGCGGTTGGTCATCTGATTCTGGAAAATAATATTTAA 1319
<210>25
<211>963
<212> DNA
<213>人工序列11C
<400>25
ATGAGCACCACCATTAAAGTGGCGCTGGCCGGCGCGGGCGCATTCGGTATTAAACACCTGGACGGCATTAAAAATATTGACGGTGTGGAAGTGGTTAGCCTGATTAGCCGGGACCTGGAAAATACCAAAAAAGTTGCTGAACAATACGGTATTGGACATGTTACGACCGAACTGAGCGAAGCACTGGCACTGCCGGAAGTGGATGCAGTTATTTTATGTACCCCGACCCAGATGCATGCAGAACAGGCAATTGCATGTCTGAAAGCAGGGAAACACGTTCAAGTTGAAATTCCGATGGCAGATAATTTAGCAGATGCGGAAGAAATTGTTCGCCTGCAAAAAGAAACCGAATTAGTTGCAATGGTAGGTCATACGCGTCGGTTTAATCCGAGCCATCAGTATGTGCACAATCAGATAACAGCAGGTCAGTTTAACATACAGCAGATGGATGTCCAAACCTATTTCTTTCGTCGTACCAACACCAATGCACTGGGTCAAGCCCGCAGCTGGACAGATCATTTACTTTGGCATCACGCAGCACATACCGTCGACCTGTTTGCCTACCAGGCAGGTTCACCTATTGTTCAAGCAAACGCAATTCAGGGTCCTATTCATCCGACACTGGGTATTGCAATGGATATGAGCATCCAACTGAAAGCAGCAAATGGAGCAATTTGTACCTTAAGTCTGAGTTTTAATAACGATGGTCCGCTGGGCACATATTTTCGTTATATTGGTGATACAGCCACCTACCTGGCACGTTATGATGATCTGTTTACCGGGAAAGATGAACAGATAGATGTTTCCAAGGTTGCAGTGAGCATGAATGGTATAGAACTGCAGGATCGTGAATTTTTTGCAGCAATCAAAGAAGGTCGTGAACCGAATAGCAGCGTGGAAGGAGTTTTTGCATGTTATAAGGTTTTACATGACCTGGAACAGCAGCTGAACGCAGCAGGTTAA 963
<210>26
<211>1322
<212> DNA
<213>人工序列12AB
<400>26
ATGGCACTGGACAAACCGTACTTAGACGTGCCGGGGACGATTATTTTTGACGCGGAACAGAGCCGCCGCGGTTACTGGCTGAACCAGTTTTGTATGAGCTTAATGAAAGCAGAAAACCGCGCACGTTTTAAAGCAGATGAGAGAGCATATTTAGATGAGTGGGCCATGAGTGAAGAACAGAAACAAGCAGTTCTTGCAAGAGATCTGAATTGGTGTATGAGAACAGGTGGAAATATTTATTTCCTGGCAAAAATTGGTGCAACAGATGGAAAAAGTTTTCAGCAGATGGCAGGAAGTATGACCGGAATGACCGAAGAAGAATATCGTAATATGATGATAAACGGTGGACGGAGCGTGGAGGGTAATCGGGTTGTGGGTGAAGATGGTGACGCACAGGCACATCGGCAGCCGCAGGGAGCAGCAGGAAAAAAAGCATAAGGATCCGAAGGAGATATACCATGGCACGGATTTCGGCGAGCGTTTACACCAGCCACGTTCCGGCGATTGGTGCGGCAATGGATTTAGGGAAAGATCACGAACCGTACTGGCAGCCGCTGTTTGCGGGATACGAACCGAGCAAACAATGGCTGAAAGATAATAAACCCGATGTTATTTTCCTGGTGTTTAATGATCACGCAACCGCATTTAGCCTGGATATGATTCCGACCTTTGCAATTGGAACAGCAGCAGAATATCAGCCAGCCGATGAAGGTTGGGGCCCGCGGCCTGTTCCTAAAGTTGTTGGCCATCCCGAACTGGCAAGCCATATTGCACAGAGCGTTATTCAGCAGGACTTTGACTTAACCATTGTTAACAAAATGGACGTGGATCATGGTCTGACTGTTCCGCTTAGCCTGATGTGTGGCAAACTGGATCCGAAAGAAGGCGCATGGCCGTGTCCGGTTATTCCGTTTGCAGTTAATGTGGTGCAATACCCGGTTCCGAGTGGTAAACGTTGTTTTCAGCTTGGGCAAGCGATTCGTCGTGCTGTTGAAAGTTTTGATCAGCCGCTGAATGTTCAGATTTGGGGTACAGGTGGAATGAGTCATCAGCTTCAGGGTGCGCGTGCGGGTCTGATTAATAAAGAATGGGATAATCGTTTCCTGGATCGTTTAATTGCAGATCCTGCTGGCCTGGCGGAGGTTAGTCATATTGAATACGTTCGTGAAGCAGGTAGTGAAGGTATTGAGCTGGTTATGTGGTTAATTGCACGCGGTGCAATGGCAGATGTTGCGGGTGGGCCTGCACCGACTTTAAAGCATCGTTTTTTTCATGTGCCGGCCAGCAATACGGCAGTGGGACACCTGATTTTAGAAAATTAA 1322
<210>27
<211>951
<212> DNA
<213>人工序列12C
<400>27
ATGACCATTAAGGTGGCCCTGGCCGGGGCGGGGGCATTTGGTATTAAACATCTGGACGGGATTAAAAACATTGACGGGGTGGAAGTGGTGAGCCTGATCAGCCGGGACCTGGACAAAACGAAAGAAGTGGCGGATAAATATGGTATTCAGCATGTTACAACCAATCTGGAAGATAGCCTGGCACTGAAAGAAGTGGATGCAGTGATTTTATGTACCCCGACCCAGATGCATGCAAGCCAAACATTAGCATGTCTGAAGGCTGGTAAACACGTTCAGGTTGAAATTCCGCTGTGTGATGTTCTTCAGGATGGTGAAGAAGTTGTGCAGGTTGCAAAAACAAGCGGTCTGGTAGCAATGTGTGGCCATACGCGTCGGTTTAATCCATCCCATCAATATGTTCACCAGAAAATTGTTGCAGGTGAATTTAATATCCAGCAGATGGATGTTCAGACCTACTTTTTTCGTCGTACCAACATGAATGCACTGGGTCAGGCCCGTTCCTGGACAGATCACCTGTTATGGCATCACGCGGCACATACCGTTGACCTGTTTGCATACCAGGCGGGTAGCCCTATTGTTAAAGCAAATGCAGTTCAGGGTCCGATTCACAAAGATCTGGGTATTGCAATGGATATGAGCATTCAGTTAAAAGCCGCAAATGGTGCAATTTGTACCTTAAGTCTGTCATTTAATAACGACGGACCGTTAGGGACATTTTTTCGGTATATTGGGGATACAGCAACCTACATTGCGCGCTATGATGATCTGGTTAATGGAAAAGAAGAGAAGATAGATGTTAGCAAAGTTGATGTGAGCATGAATGGGATTGAGCTGCAGGATCGGGAGTTTTTTGCAGCAATTCGCGAGGGTAGAGAACCGAATAGCAGTGTTGCACAGGTTCTGCCGTGTTATCAGGTTCTGCATCAGCTGGAACAGCAGCTGAACGGTTAA 951
<210>28
<211>1253
<212> DNA
<213>人工序列13AB
<400>28
ATGAGCCACAAACCGTACGACGACATTCCGGGTACCACCATTTTCGACCCCGACATGGCATTCAAAGGCTACCACCTGAACCAGTTCGCACTGAGCCTGATGAAAGCAGAGAACCGCGAACAGTTCAAAGTTAATGAAAGAGCATATCTGGACACCTGGGCCATGACCGAAGAACAGAAACAGGCAGTCCTGGCCCGCGATTATAATTGGATGATGCGCCTGGGTGGAAATGTATATTTTCTGGCAAAAATCTTCAGCACCGACGGTCTGAGCTTTCAAGTTGCAGCAGCAAGCATGACCGGTATGACCCAAGAAGATTACGCCCAGATGATGTTAGATGGTGGACGTAGTCCGGAGGGTAATCTGTACAAAAAAGAACAGGCATAAGGATCCGAAGGAGATATACCATGGCAAAAATCACCGCGGGCGTTGCGACGAGCCACGTTCCCGCAATTGGCGCCGCCATTGACCTGAGCAAAACCGAACAGCCGTACTGGCAGCCGGTTTTTAAAGGCTACGAATACGTTAAAGCATGGATTAAAGAACAGAAACCGGATGTTATTTTCCTTGTTTATAACGATCACGCAACCGCATTCGACATGAATATGATTCCGACCTTTGCAATTGGTTGTGCCCCGGAATTTAAACCGGCGGATGAGGGTTGGGGCGCACGCCCTGTTCCTACTGTTAAAGGCCATCCGCAGCTGGCAGCACATATCACCCAGAGCTTAATTCAGGATAACTTTGACCTGACAATAATTAACAAGATGGATGTTGATCACGGTTTAACCGTTCCGCTGAGCCTGGTGTTTGGTCAGGTTGAAGAATGGCCGTGCCTGATTATTCCGTTTGCAGTTAATGTTGTTCTTTATCCGCCTCCTAGCGGTCAGCGTTGTTATGATCTGGGCAAGGCCTTACGTAAAGCGATTGAAAGCTTTCCGGAAGATTTAAAAGTTCAGGTTTGGGGTACCGGTGGTATGAGTCATCAGTTACAGGGTGCACGTGCAGGGCTGATTAATAAAGCATTTGATAATGATTTCCTGGACCGTATTATTGATAAAGCAGATGAGCTGGCGCAGGTTCCGCATATTAATTATGTTCGTGAGGCCGGTAGTGAAGGTATTGAGCTGGTTATGTGGTTAATTATGAGAGGGGCGTTAAATGATGAAGTTGAACTGAAACATCGTTTTTATCATGTGCCGGCGAGCAATACCGCAGTTGGGCATCTGGTTTTAGAGAATAAACCTAGCTAA 1253
<210>29
<211>945
<212> DNA
<213>人工序列13C
<400>29
ATGAAGATCTGTATGGTTGGGCAGGGCGCATTTGGGCAGAAACATCTGGACGCCCTGAAAAATATTCCGGACGTTGAAGTTGTGAGCCTGGTGGGGGGTACGGAAGAAAGCACCCGCGAGGTGGCAGAGAAGTATGGTATCCCGTTTTGGACATTAGATTTAGCAGAGGGTCTTGCACAGCCGGGTGTTGATGCAGCAATTATAACCAGTCCGACACCTATTCATGCAGCCCAAGCATTACAGGTTATGGATGCAGGCAAACATGTTATGATTGAAATTCCGATGACAGATAGCTTAGCCGATGCGGAAGCAGTATGTGCAAAACAGCAAGAAACCGGCTTAGTTGCAATGGCAGGTCACACCCGCCGTTTCAATCCGTCCCATCAGTGGATACACAAAAAAATAGTGGCAGGTGAATTAAAAATCCAGCAGATGGATGTCCAGACCTATTTTTTTCGTCGTCAGAATTTAAACGCACTGGGTCAAGCCCGCAGCTGGACCGATCATCTGCTGTGGCATCACGCGTGTCATACTGTGGACCTGTTTGCATACCAGACCGGTGAAAAAATTGTGCAAGTTCAAGCCCTGCAAGGTCCTAAACATCCGGAATTAGGTATCGCAATGGATATGAGCATTGGCCTGAAAAGCGAAAGCGGAGCAATTTGTACCTTAAGCCTGAGCTTCAACAATAACGGTCCTCTGGGTAGCTTTTTCCGTTACATTTGTGATAATGGCACCTACCTGGCGCGGTACGATGATCTGGTTGATGGGTATGAAAAACCGATTGATGTCTCTCAGGTGGACGTGAGCATGAACGGTATTGAACTGCAGGATCGTGAGTTTATTGCAGCAATAAAAGAAAAACGTGAGCCGAATGCAAGTGTGCAGCAGGCGCTGGCAGCAATGCGGGTTTTAGATCTGCTGGAACAGCAGCTGGAAAATTAA 945
<210>30
<211>1259
<212> DNA
<213>人工序列14AB
<400>30
ATGAGCCTGGACAAACCGTACACCAACATTCCGGGCACCACCATTTTTGATGCAGACATGGCGCGCATTGGCTACCACCTGAACCAGTTTTGCATGAGCCTGATGAAGGCGGAAAACCGCGAACGCTTTAAAGCAGACGAACGCGCATATTTAAATGAATGGCCTATGACAGAAGAACAGAAACTGGCTGTTTTAGATCGCGATTATAATAGAATGATGGACCAGGGAGGCAACATCTATTTTCTGGCAAAAATTTTTAGCAGCGACGGTCTGAGCTTTCAGCACGCAGCAGCAACCATGACTGGAATGAGCCAGGAAGAATATGCACAGATGATGCTGAATGGAGGTCGGTCACCGCAGGGTAATCGGTATCTGGGAGAAGAAAAAGGTAAATAAGGATCCGAAGGAGATATACCATGGCAAAAATCACCGCGGGCGTTGCGACGAGCCACGTTCCCGCAATTGGCGCCGCCATTGACCTGGGCAAAACCCAGGAACCCTACTGGGCCCCCCTGTTCGAAGGCTACGAATTCGCGAAACAGTGGATTGCAGAAGAAAAACCAGATGTTATTCTGCTGGTGTATAATGATCATGCAAGCGCATTTTCCATGGATTTTGTTCCGACCTTTGCCATTGGTTGTGCCGAGAAATTTGAGCCGGCGGATGAAGGATGGGGCCCGCGTCCTGTTCCTACCGTTGAGGGGCATCCGAGACTGGCATCTCACCTGGCGCAGAGCGTTATTCAGCAAGATTTTGATCTGACAATCGTTAATAAGATGGATGTTGATCATGGGTTAACCGTTCCGCTTAGCCTGATGTTTGGTCAGCCAGATGCATGGCCCTGTAAAGTTATTCCGATCGCAGTTAATGTTGTTTTATTTCCCCCGCCGAGCGGTAGACGGTGTTATGAACTGGGCAAAGCATTAAGAAAAGCAGTTGATAGCTTTGATGAAGATCTGAATGTGCAGATTTGGGGTACCGGTGGTATGAGCCACCAGCTTCAAGGTCCGCGTGCAGGTCTGATTAATCAAGAATTTGATAAGGAGTTCCTGGAAAAAATTGTGAATAATCCGGAAGAACTGAGCCGTAAACCGCACATTGATTACGTACGTGAAGCAGGTAGTGAAGGCATCGAATTAGTTATGTGGCTGATTATGCGGGGAGCACTGAATCCGGAGGTTATCGAAAAAAAACGGTTTTATCATGTGCCTGCCAGCAATACCGCAGTTGGTCATCTTGTGCTGGAAAACAAAGAGTAA 1259
<210>31
<211>951
<212> DNA
<213>人工序列14C
<400>31
ATGAAGGTTGCCCTGGCCGGCCCGGGGGCATTTGGTATTAAACACCTGGACGCGATTGAAAAAATCGACGGCGTGGAAGTTGTTTCGCTGATTGGGCGCGACCTGGAAAAAACGAAAAAAGTGGCGGAAAAATATCATATTGGTCATACGAGTACCGAACTGGCAGACGCACTGGCACTGCCGGAAGTGGATGCAGTTATACTGTGTACCCCGACCCAAATGCATGCAGCACAGAGCATAGAATGTATGCGTGCAGGGAAGCACGTTGAAGTTGAAATTCCTCTGGCAGATAGTTGGGAAGAAGCAGAAGAAGTTCTGAAAGTTCAAAAAGAAACCGGCAAAGTGTGTATGGTTGGTCATACCCGGCGTTTTAATCCGAGCCATCAATATGTTAACAAAAAAATTCGCGCAGGTGAACTGAATATACAGCAGATGGATGTTCAGACATATTTTTTTCGCCGTACCAACACCAATGCACTGGGTGAAGCACGCTCATGGACCGATCACCTGCTGTGGCACCACGCAGCACATACCGTTGACCTGTTTCGTTACCAGGCAGGTGCAGAGATTGTTAGTGCAAATGCGCTGGAAGGTCCTAAACATCCGGAACTGGGTATAGCAATGGATATGAGCATTCAAATGAAAGCAGCAAATGGAGCAATTTGTACCTTAAGTCTGAGCTTTAACAATGATGGTCCGTTAGGTACATTTTTCCGTTATATTTGTGACAATGGTACCTACATTGCGAGATACGATGATCTGGTTAATGGTAAGGAAGAGCCGATTGATGTTAGCAAAGTTGATGTTAGCATGAATGGTATTGAACTGCAGGATCGTGAATTTTTTGCAGCAATAGCCGAAGGGCGGGAACCGAATAGCAGTGTTGCACAGGTGCTGCCTTGTTATAAAGTTCTGCATGATCTGGAACAGCAACTGAACGCAGCACAATAA 951
<210>32
<211>1317
<212> DNA
<213>人工序列15AB
<400>32
ATGACCCTGGACAAACCGTACAAAAACGTGCCGGGGACCACCATTTTCGACGCCGATCAGGCCCGTAAGGGTTACCACCTGAATCAGTTCAGCATGAGCCTGATGAAACCGGAAAACAGAGAACGTTATCTGGCAGACCGCGAAGCGTATCTGGACGAGTGGCCGCTGACCCCGGCACAGCGTCAGGGTGTTTTAGATCTGGATCTTAATGCGTGTATTCGCGAAGGTGGAAATATTTATTTTCTGTCTAAGATCGGTGCAACACACGGTCTGAGCTTTCAGCAAATGGCAGGTAGTATGACCGGAATGAGCGAGGCAGCATATCGTGATATGATGATTGGAGGTGGTCGTCGTCCGGAGGGTAATAGACTGAAAGATCTGGATGGTTGGGCACCGCCAGCAACCGAAAAAAGCGAAGTTGTGCGGCCAGATGCACCGGCGAAATTTACCAGCGCGCTGTTTACAAGCCATGTTCCGGCAATCGGTGCAGCAATGGATTTAGGGAAAACAGAAGAACCGTACTGGAAAAAAGTGTTTGATGGTTATGAATGGACAAGACGGTGGGCAAAAGAAAATACCCCAGATGTTGTGATCTTAGTTTATAATGATCACGCAACCGCATTTGACAGCAATATTATTCCGACCTTCGTTCTGGGTACGGGTGCGCATTATCCGGTTGCGGATGAGGGGTACGGCCCGCGTCCGGTTCCGGATGTTAAAGGTTATCCGGAATTAGCAGCGCATATAGCACAGAGCGTTATTCAGGATGATTTTGATTTAACTCTGGTTAATGAGATGGTTGTTGATCATGGACTGACCGTTCCGTTAAGCCTGGTGTTTGGCGATGTTGATGAGTGGCCATGTCGTGTTATTCCGCTGCCCGTTAATGTTGTTCAGTATCCTGTTCCGAGTGGTCGTCGTTGTTATGAGCTTGGGCGTGCAATTCGCCGTGCGCTGGATAAGTGGGATGGTCCGAAACTGAATGTGCAGATTTGGGGTACCGGCGGGATGTCACATCAATTACAGGGTCCGCGTGCAGGTCTGATCAATGAAGAATGGGACAATGCTTTTTTAGATCATCTGATTGCGGATCCGCTGGGCCTGACAGAGTGGCCGCACATGGAGTATGTTGACGAAGCAGGTAGTGAAGGTATTGAATTAGTTGATTGGTTAATCGCACGCGGTGCAATGGATGATCAATTTGGTGGGGAAGCACCGGAAATGAATCACCGTTTTTATCATGTTCCGGCCAGCAATACAGCAGTGGGACACCTGGTTATGACCAATCCGACGGCATCACAGGAAGATCCGAGCTAA 1317
<210>33
<211>957
<212> DNA
<213>人工序列15C
<400>33
ATGACCGCCGGTAAAGTTCGCATTGCCGTTGTGGGGGCCGCCGGGGCATTTGGTATGAAACATCTGGATGGGCTGCGTAATATCGCAGAAGCCGAAGTGACCGTTGTGAGCGGGACCCGTCCAGAAAGCGTGCAGGCAGTTGCAGAACAGTATGGTATACCGAATGCCGTTGTGGGCTTAGATGCAGTTCTGGCAAGAGATGATGTTGATGCAGTTATTCTGGCGACCCCGACCCAACAGCATGCAGCACAGACCCAGGCAGTGTTAGCGGGTCGCAAACATGTTCAGGTGGAAATCCCGTTAGCAGATAGTCTGGCAGATGCAGAAGCAACCCTGGCCGCAGCAGAAGCAAGCGGTCGTATTGCAATGGTTGGTCATACCCGGAGATTTAATCCAAGCCATCAACTTATACATAACCGTATAGCAGCAGGTGAATTTGCAGTGCAGCAGATGGATGTTCAAACATATTTCTTTCGTCGTAGCAATACCAATGCAAAAGGTGAAGCCCGTAGCTGGACCGATCATCTGTTATGGCATCACGCAGCACATACCGTTGATTTATTCGCCTACCAGGCAGGTCGCATTGTGCAGGCAAACGCAATCCAAGGGCCGATCCATCCGGAACTGGGAATTGCCATGGACATGAGTATTCAATTAAAAGCAGAAAGCGGTGCGATCTGTACCCTGAGCTTAAGCTTTAACAATAATGGTCCGTTTGGCAGCTTTTTTCGCTATATTGGTGATAGTGAGACCTACATAGCACGGTACGATGATCTGGTTAATGGACGTGAAGAACCGATTGATGTTAGCGACGTTGCAGTCAGCACAAATGGTATTGAACTGCAGGATCGGGAGTTTGTTGCAGCAATCCTGGAAGGTCGTGAACCGAATAGCAGTATAAGACAGGTGATTGATTGTTATAGAGTATTAGGTGCACTGGAAGAGCAGCTGAGCTAA 957
<210>34
<211>1305
<212> DNA
<213>人工序列16AB
<400>34
ATGAGCCTGGACAAAACGTACAAACTGGTGCCGGGGACGACCATTTTTGACGCGGAACAGAGCGCCAAAGGGTACCACCTGAACCAGTTCTGTATGAGCCTGATGACCGCAGAGAACAGAGCAGCATTTCTGGCAGATGAACGTGCGTACTTAGATGCATGGCCACTGAGAGAAGAACAAAAACAAGCACTGCTGGATCGCGATCTGAATGCAGCAATGAGAGAAGGAGGAAACATATATTTTCTGGCAAAATGGGGGGCAACCCTGGGGTTTAGTTTTCAACAAATGGCAGGAAGCATGACCGGTATGACCGAAGAAGAATATAGAGCAATGATGGTGGGTGGAGGAAGAAGCGTGGAAGGTAATCGGATTGATCATGCGGTTCTGGATGCAGCATATACAAACCCGGAACCGAGCGCAGAACACGCAACAATAACCGGCGCAGTATTTACGAGTCATGTGCCGGCGATCGGTGCAGCACTGGATCACGGTAAAACAGAAGAACCGTATTGGCAACCGGTGTTTGAAGGTTATGCATATTCAAAAACATGGGAACGTGAAAATGTTCCAGATGTTATTTTTCTTGTGTACAATGATCACGCAAGCGCATTTGATCAGAGTATGATTCCGACCTTTGTTCTGGGGACAGGTGCAAGCTATCCGATTGCGGATGAAGGTTACGGTCCGCGGCCGGTTCCGGGTATTGAAGGGGATCCGGATTTAGCAGCGCATATAGCACACAGCCTGATTCGGGATGATTTTGATTTAACCCTGGTTAATGAGATGGCAGTGGATCATGGTTTAACAGTTCCTCTTAGTCTGATGTTTGGTGATGTTGAGAAATGGCCTTGTAAAGTTATTCCTTTTCATGTTAACGTGGTGCAGTATCCTGTTCCGAGCGGGGCACGTTGTTTTAAGCTGGGCCAGGCATTACGGAAAGCCATTGAGAGTTATGATCGGCCGCTGAAGGTTCATGTTTGGGGTACTGGTGGCATGAGTCATCAGCTTCAGGGTCCGCGTGCAGGTCTGATTAATCGTGAATGGGATAATGCATTCCTGGATCGTTTAATTGCAGATCCAGCCGGACTGGCAGAAGTTCCGCACTTAGAGTACGTTGAAGAAGCAGGTAGTGAAGGTATAGAACTGGTGATGTGGCTGATTGCACGTGGTGCATTATCAGATGTTGACGGTGCAGTTGAAGTTAAACATCGTTTTTATCATGTGCCGGCCAGCAATACCGCAGTGGGCCATCTGATTTTAGAAAATCATCCGCGTGCCGAAGCCCCGGCAGAGGGTGAAAATTAA 1305
<210>35
<211>975
<212> DNA
<213>人工序列16C
<400>35
ATGACCGACGACCGTACCCTGCGTATTGCACTGGCCGGTGGTGGGGCCTTTGGCGCAAAACACGCAGCCGCCCTGCGTCGCATTGAAGGTGTTGAAGTGGCCGCCGTGGTGAGCGGCAGCCTGGAAGGAGCACGCAAATTTGCAGCAGAACAGGGTGTTGGTAGAGCGGTTGCAAGCTTAGATGAAGTGCTGGCAATGGATGATATTGATGCAGTTATCCTGGCGACCCCGACCCAGATGCATGCAGCACAGACGCTGGCATGTTTAGATGCAGGTAAACATGTTCAGACAGAAATTCCGCTGGCAGATAGCTTAGCAGATGCCGAAGCATGTCAGTCAGCACAAGAAAGAACCGGTTTAGTTGCAATGGCAGGTCATACGCGTCGTTTTAATCCGTCCCATCAATGGGTGCACCGTCGTATTGAAGCACGTGATTATAATATTCAACAGATGGATGTTCAGACCTATTTTTTTCGCCGTGAAAATTTAAACGCATTAGGTGAACGCCGCAGCTGGACAGATCATCTTTTATGGCATCACGCAGCACATACCGTTGACCTGTTTGCATATCAGACCGGTAGCCCTATTGTTCAAGCAAATGCAATTCAGGGCCCGATTCATCCCGAACTGGGTATCGCAATGGATATGAGTATTCAGTTAAGAGCAGCAAATGGAGCAATTTGTACCCTTAGCCTGTCATTTAACAATGATGGTCCGCTGGGCACCTTTTTTCGTTATATTGGAAATACAGGCACCTACATTGCCCGGTATGATGATTTATTTACCGGTAAAGATGAGCCGATTGATGTGAGCGGTGTTGATGTGAGCATGGATGGTATTGAACTTCAGGATCGTGAATTTGTGGCAGCAATTCGCGAAGGTCGTGAACCGAATGCCAGCATTGCACAGGTGTTACCGTGTTATAGAACCCTGGCAGCACTGGAACGGCAGCTGACCGCAGCAGCAGGGAGCTAA 975
Claims (10)
1.一种用于全细胞催化生产2-吡喃酮-4,6-二羧酸的基因工程重组菌,其特征在于,所述基因工程重组菌是通过在宿主细胞中共表达原儿茶酸-4,5-二氧化酶复合体的AB基因和4-羧基-2-羟基粘康酸-6-半醛脱氢酶的C基因而获得,优选地,所述宿主细胞为大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、需钠弧菌(Vibrio natriegens)。
2.根据权利要求1所述的基因工程重组菌,其特征在于,所述原儿茶酸-4,5-二氧化酶复合体αβ亚基的AB基因和4-羧基-2-羟基粘康酸-6-半醛脱氢酶的C基因来源于变形菌门(Proteobacteria)或放线菌门(Actinobacteria)的细菌;
优选地,原儿茶酸-4,5-二氧化酶复合体α亚基来源于NCBI登录号为TIX48797.1、MBO9517659.1、MBB5734100.1、QWT16175.1、MBE1527979.1、RIV77917.1、TCU95342.1、KAB0542660.1、ACB35890.1、MBQ0919761.1、AXF85167.1、KQP37453.1、SKA71457.1、NHO66815.1;原儿茶酸-4,5-二氧化酶复合体β亚基来源于NCBI登录号为TIX48798.1、MBO9517658.1、MBB5734099.1、QWT16174.1、MBE1527978.1、RIV77918.1、TCU95343.1、KAB0542661.1、ACB35891.1、MBQ0919762.1、AXF85168.1、KQP37452.1、SKA71463.1、NHO66816.1;单亚基原儿茶酸-4,5-二氧化酶的NCBI登录号分别为EZP27614.1和AYG79827.1;4-羧基-2-羟基粘康酸-6-半醛脱氢酶的NCBI登录号为TIX48799.1、MBO9517657.1、MBB5734098.1、QWT16173.1、MBE1527977.1、RIV77919.1、TCU95344.1、KAB0542662.1、ACB35892.1、MBQ0919763.1、AXF85169.1、KQP37451.1、SKA71468.1、NHO66817.1、EZP27613.1、AYG79828.1。
3.根据权利要求2所述的基因工程重组菌,其特征在于,原儿茶酸-4,5-二氧化酶复合体αβ亚基编码基因AB和4-羧基-2-羟基粘康酸-6-半醛脱氢酶编码基因C在基因组上以基因簇的形式排列,分别来源于NCBI中登录号为SSHH01000004.1、JAGIBN010000003.1、JACIJL010000010.1、CP076557.1、JADBDT010000001.1、QXFK01000016.1、SMBU01000015.1、VZPC01000007.1、CP001013.1、JAGPWB010000023.1、CP031124.1、LMNA01000027.1、FUYB01000003.1、JAAONZ010000012.1、JFYO01000005.1、CP032698.1基因组上对应基因簇内的核苷酸序列。
4.根据权利要求1所述的基因工程重组菌,其特征在于,所述原儿茶酸-4,5-二氧化酶复合体αβ亚基编码基因AB和4-羧基-2-羟基粘康酸-6-半醛脱氢酶编码基因C分别连接至pETDuet-1或pRSFDuet-1或pACYCDue-1或pCOLADuet-1或pCDFDuet-1中任意一种共表达载体上T7启动子后面得到ABC基因共表达载体;
优选地,在共表达载体上的连接顺序是AB基因在前,C基因在后,或者C基因在前,AB基因在后;
优选地,其中AB基因串联在一起进行表达,两个基因编码框中间包含一段RBS序列,如SEQ ID NO:1所示的核苷酸序列GGATCCGAAGGAGATATACC;
优选地,将所述基因序列按照大肠杆菌密码子偏爱性进行密码子优化,更优选的优化后的AB基因的核苷酸序列分别如SEQ ID NO:4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34所示;优化后的C基因的核苷酸序列分别如SEQ ID NO:5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35所示。
5.根据权利要求1所述的基因工程重组菌,其特征在于,将所述ABC基因表达载体转化至原核生物细胞内进行蛋白共表达;优选地,以大肠杆菌,例如大肠杆菌BL21(DE3)或BL21(DE3)pLysS或Rosetta(DE3)或Rosetta (DE3)pLysS中的任意一种菌株作为宿主细胞,得到基因工程重组大肠杆菌。
6.一种催化生产2-吡喃酮-4,6-二羧酸的全细胞催化剂,其特征在于,培养如权利要求1至5任一项所述基因工程重组菌并表达所述AB基因和所述C基因,得到全细胞催化剂。
7.根据权利要求6所述的全细胞催化剂,其特征在于,利用异丙基-β-D-硫代半乳糖苷或乳糖诱导ABC基因共表达,获得全细胞催化剂。
8.一种全细胞催化生产2-吡喃酮-4,6-二羧酸的方法,其特征在于,采用如权利要求1至5任一项所述的重组菌,或权利要求6或7所述的全细胞催化剂,以原儿茶酸作为底物,催化合成2-吡喃酮-4,6-二羧酸。
9.根据权利要求8所述的方法,其特征在于,所述全细胞催化的体系的原儿茶酸浓度为1-100 g/L,或者采用分批补加策略,每次补加浓度小于20 g/L;催化底物为原儿茶酸纯品或微生物发酵获得的原儿茶酸发酵液;全细胞催化剂用量为1-100 OD600。
10.根据权利要求8所述的方法,其特征在于,所述全细胞催化体系的反应体系为M9培养基、磷酸盐缓冲液、Tris-HCl缓冲液、乙酸盐缓冲液的任意一种;反应pH为5-8;反应温度为30-43℃;溶氧水平为1%-100%;反应时间为0.5-24 h。
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