CN113355300B - 芳香族异戊烯基转移酶突变体、用于其表达的重组菌的构建方法及由其构建的重组菌 - Google Patents

Abstract

本发明涉及芳香族异戊烯基转移酶突变体、用于其表达的重组菌的构建方法及由其构建的重组菌。具体地，本发明的芳香族异戊烯基转移酶突变体的氨基酸序列相对于由SEQ ID No.28所示的氨基酸序列，具有以下突变中的一种或多种：i)序列SEQ ID No.28的第161位Q替换为A、N、S或者V：ii)序列SEQ ID No.28的第286位的G替换为N、S或者V：iii)序列SEQ ID No.28的第288位Y替换为A、N、S或者V。本发明构建的重组菌通过高效表达所述芳香族异戊烯基转移酶突变体而具有强大的大麻萜酚酸及其衍生物工业化生产的潜力。

Description

芳香族异戊烯基转移酶突变体、用于其表达的重组菌的构建 方法及由其构建的重组菌

技术领域

本申请属于生物工程领域，具体涉及芳香族异戊烯基转移酶突变体、用于其表达的重组菌的构建方法及由其构建的重组菌。

背景技术

大麻素(cannabinoids)，指来源于植物大麻(Cannabis sativa)的一大类化学分子，种类超过150种。目前国际上使用较多的大麻素有大麻二酚(cannabidiol，缩写为CBD)、四氢大麻酚(tetrahydrocannabinol，缩写为THC)、大麻萜酚(cannabigerol，缩写为CBG)、大麻色烯(cannabichromene，缩写为CBC)、大麻酚(cannabinol，缩写为CBN)。CBD是植物大麻中的主要化学成分之一，是大麻素中的非成瘾性成分。THC是大麻中的主要精神活性物质，可成瘾，是世界各国被严格管制的物质。CBG因其在植物大麻中含量较低，通常被归类为微量大麻素或稀有大麻素，因其为其它大麻素的共同前体，被称为是“大麻素之母”，随着大麻植株的生长，大部分CBG会转化为CBD和THC，只会残留微量的CBG在植株体内，供应严重受限，使得CBG应用的发展受到了较大的限制。

大麻素可用于临床、食品和化妆品等产品中。其中CBD在部分国家已经用于临床，主要针对的适应症有：儿童癫痫、痉挛、焦虑、恐慌症(Panic Disorder)、广场恐惧症(Agoraphobia)等。THC主要针对的适应症有：过敏，发炎，抑郁，偏头痛，焦虑等临床症状。CBG药理作用包括：抗真菌，抗昆虫，抗炎镇痛，降眼压，神经保护，抗抑郁，刺激食欲，加强癌细胞凋亡等等。机制上，CBG可以阻断瞬时受体电位M8(TRPM8)，激活TRPA1、TRPV1、TRPV2通道并抑制内源性大麻素的再摄取。因此，CBG会影响生理的诸多部分，具有多种多样的效果，这些影响与CBG和内源性大麻素系统的相互作用，与充当温和的CB1受体拮抗剂有关，而且都没有THC的精神活性。

在大麻素的生物合成途径中有一个共同的前体：大麻萜酚酸(cannabigerolicacid,缩写为CBGA)。CBGA的合成来源于两个前体：牻牛儿基二磷酸盐(geranyldiphosphate，缩写为GPP)与橄榄酸(olivetolic acid，缩写为OA)。GPP来源于2-甲基-D-赤藓糖醇-4-磷酸生物合成途径(MEP途径)或者甲羟戊酸生物合成途径(mevalonate pathway，MVA途径)，OA的合成从己酰辅酶A(hexanoyl-CoA)开始经聚酮合酶(PKS)生物合成途径引入3分子丙二酰辅酶A(malonyl-CoA)得到；己酰辅酶A的来源是脂肪酸从头合成的己酸(hexanoic acid)，也可以来源于脂肪酸代谢的中间产物。CBGA可以转化为CBDA、THCA、CBCA等。THCA可以转化为CBNA。CBGA、CBDA、THCA、CBCA和CBNA通过加热可以脱羧基转化为CBG、CBD、THC、CBC和CBN。

GPP是大麻素重要前体之一，GPP的生物合成是以棕榈酸异丙酯(Isopropylpalmitate，缩写为IPP)和二甲基烯丙基二磷酸三铵盐(Dimethylallyl Diphosphate，缩写为DMAPP)作为单元结构组成。IPP和DMAPP的生物合成途径有明确路线，分为MVA途径和MEP途径。MEP途径：由丙酮酸(pyruvate)和3-磷酸甘油醛(3-phosphoglyceraldehyde，缩写为G3P)缩合时掉一个CO2形成C5化合物，再经一系列反应最终形成IPP或DMAPP。本发明主要围绕MVA途径展开，MEP途径不做赘述。

MVA途径：该途径可以描述为3个乙酰辅酶A(acetyl-CoA)形成一个C6化合物甲羟戊酸(mevalonate)，再接两个磷酸基团后，脱羧去掉一个CO2，形成IPP，IPP和DMAPP之间可以通过异构酶相互转化。生物合成过程中涉及以下酶：

AACT家族的酶(乙酰乙酰-CoA硫解酶，acetoacetyl-CoA thiolase，)以乙酰辅酶A为底物合成乙酰乙酰辅酶A(acetoacetyl-CoA)；

HMGS家族的酶(HMG合酶，HMG synthase)以乙酰乙酰辅酶A为底物合成3-羟基-3-甲基戊二酰辅酶A(3-Hydroxy-3-methylglutaryl-CoA，HMG-CoA)；

HMGR家族的酶(3-羟基-3-甲基戊二酰-CoA还原酶，3-Hydroxy-3-methylglutaryl-CoA reductase)以HMG-CoA为底物合成甲羟戊酸(mevalonate，MVA)；

MK家族的酶(MVA激酶，MVA kinase)以MVA为底物合成甲羟戊酸-5-磷酸(mevalonate-5-phosphate)；

PMK家族的酶(磷酸MVA激酶，phospho MVA kinase)以甲羟戊酸-5-磷酸为底物合成甲羟戊酸-5-焦磷酸(mevalonate-5-diphosphate)；

PMD家族的酶(MVA焦磷酸脱羧酶，MVA diphosphate decarboxylase)以甲羟戊酸-5-焦磷酸为底物合成异戊烯焦磷酸(isopentenyl diphosphate，IPP)；

IDI家族的酶(IPP异构酶)可以将IPP转化为DMAPP；

GPPS家族的酶(GPP合酶，GPP synthase)以1分子IPP和1分子DMAPP合成单萜前体GPP(参考文献：VranováE,Coman D,Gruissem W.,2013,Network analysis of the MVAand MEP pathways for isoprenoid synthesis,Annu Rev Plant Biol.,2013；64:665-700)。

Olivetolic acid/Olivetol生物合成途径如下：

乙酰辅酶A(acetyl-CoA)通过羧化生成丙二酰辅酶A(malonyl-CoA)，参与该反应的酶有乙酰辅酶A羧化酶(acetyl-CoA carboxylase)，或者一分子丙二酸(malonate)通过丙二酰辅酶A合酶(malonyl-CoA synthetase)催化合成一分子丙二酰辅酶A(malonyl-CoA)。己酸(Hexanoic acid)通过酰化生成己酰辅酶A(Hexanoyl-CoA)，参与该反应的酶有FadD、FadK等。一分子丙二酰辅酶A(malonyl-CoA)和一分子己酰辅酶A(malonyl-CoA)缩合形成3-氧代辛酰基辅酶A(3-oxooctanoyl-CoA)，一分子3-氧代辛酰基辅酶A与丙二酰辅酶A(malonyl-CoA)缩合生成3,5-二氧代十碳酰辅酶A(3,5-dioxodocanoyl-CoA)，一分子3,5-二氧代十碳酰辅酶A与一分子丙二酰辅酶A缩合生成3,5,7-三氧代十二碳酰辅酶A(3,5,7-trioxododecanoyl-CoA)，参与以上三步反应的酶均为橄榄酸合酶(olivetolic acidsynthase，OLS)。3,5,7-三氧十二碳酰辅酶A可以环化生产橄榄醇(olivetol)或者橄榄酸(olivetolic acid)，参与该反应的酶为橄榄酸环化酶(olivetolic acid cyclase，OAC)。参考文献：Zaigao Tan,James Clomburg,and Ramon Gonzalez,2018,Synthetic pathwayfor the production of olivetolic acid in Escherichia coli,ACS Synth Biol.,7(8):1886-1896)。

鉴于CBGA的重要性，现有技术中开发了用于其生成的多种重组菌。已有文献报道在底盘菌Escherichia coli strain JST06(DE3)atoBCT5(ΔldhA,ΔpoxB,Δpta,ΔadhE,ΔfrdA,ΔyciA,ΔybgC,ΔydiI,ΔtesA,ΔfadM,ΔtesB)的基础上导入MVA途径所需要酶的表达基因(HMGS，HMGR，MK，PMK，PMD，IDI，GPPS)，然后再导入一个芳香族异戊烯基转移酶(aromatic prenyltransferas)表达基因(nphB)，CBGA的产量为0.5mg/L。将NphB的第286位氨基酸G突变位S，CBGA的产率为1mg/L。这个产量离产业化还有较大差距。(参考文献：ShuaiQian,James M.Clomburg,Ramon Gonzalez,2019,Engineering Escherichia coli as aplatform for the in vivo synthesis of prenylated aromatics,Biotechnol Bioeng,116(5):1116-1127)。另有文献报道在底盘菌Saccharomyces cerevisiae yCAN10中导入MVA途径需要的基因(mvaE，mvaS，ERG12，ERG8，ERG19，idi，ERG20)，然后再导入一个来源于植物大麻的基因CsPT4，CBGA的产率为5.67mg/L/h。但是酵母的培养时间长(通常需要72小时以上)，并且需要低温(30℃)，发酵过程的能耗比大肠杆菌更高。(参考文献：XiaozhouLuo,et al.,2019,Complete biosynthesis of cannabinoids and their unnaturalanalogues in yeast,Nature,567(7746):123-126)。

因此，目前现有技术的缺点主要在于以下几个方面：(1)大麻素主要来源于植物大麻，但诸多因素使得大麻素的成本持续处于高位；例如，相比于生物合成更难获得高纯度产品，种植管制严格，产能有限，需要高昂的植物种植和下游提取的投入，生产稳定性较低，稀有大麻素含量低。(2)大麻素的化学合成法的成本高昂，并且存在环境污染和条件苛刻等问题。(3)以大肠杆菌为底盘菌生物合成大麻素的产率还达不到产业化的标准。

有鉴于此，仍需要开发可以以高产量、低成本生产CBGA的重组菌。

发明内容

本发明的一个技术目的是提供一种突变体，其为芳香族异戊烯基转移酶(aromatic prenyltransferase，NphB)突变体。

本发明的另一个技术目的是提供一种质粒，其包含芳香族异戊烯基转移酶(aromatic prenyltransferase，NphB)突变体的编码序列。

本发明的又一个技术目的是提供一种重组菌的构建方法。

本发明的又一个技术目的是提供一种由上述方法构建的重组菌。

本发明的又一个技术目的是提供所述重组菌用于发酵生产CBGA的用途。

本发明的又一个技术目的是提供一种生产CBGA的方法。

因此，第一方面，本发明提供了一种芳香族异戊烯基转移酶(NphB)突变体，其氨基酸序列相对于由SEQ ID No.28所示的氨基酸序列，具有以下突变的一种或多种：

i)序列SEQ ID No.28的第161位Q替换为A、N、S或者V：

ii)序列SEQ ID No.28的第286位的G替换为N、S或者V：

iii)序列SEQ ID No.28的第288位Y替换为A、N、S或者V。

优选地，本发明的NphB突变体的氨基酸序列为SEQ ID No.30、SEQ ID No.32或SEQID No.34。

优选地，本发明的芳香族异戊烯基转移酶突变体的核苷酸序列为SEQ ID No.29、SEQ ID No.31或SEQ ID No.33。

第二方面，本发明提供一种质粒，其包含上述NphB突变体的编码序列。

第三方面，本发明提供一种构建重组菌的方法，所述方法包括以下步骤：

1)以大肠杆菌BW25113为出发菌株，(例如，通过λ-RED同源重组的方法)将MVA途径所需的基因导入其基因组中以获得底盘菌；

2)构建包含上述NphB突变体的编码序列的质粒；

3)将步骤2)中构建的质粒导入步骤1)获得的底盘菌中即得到所述重组菌。

在具体实施方式中，所述MVA途径所需的基因包括：AtoB、HMGS、HMGR、MK、PMK、PMD、IDI和ERG20或它们各自的同功酶的编码基因，其中，PMK表示磷酸MVA激酶(phospho MVAkinase)，PMD表示MVA二磷酸脱羧酶(MVA diphosphate decarboxylase)，MK表示MVA激酶(MVA kinase)，IDI表示IPP异构酶，AtoB表示乙酰乙酰-CoA硫解酶(acetoacetyl-CoAthiolase)，HMGS表示HMG合酶(HMG synthase)，HMGR表示3-羟基-3-甲基戊二酰-CoA还原酶(3-Hydroxy-3-methylglutaryl-CoA reductase)，ERG20表示GPP合酶(GPP synthase)。

在具体实施方式中，在所述重组菌中额外导入橄榄酸/橄榄醇(olivetolic acid/olivetol)生物合成途径相关的酶。

在具体实施方式中，所述重组菌通过在其基因组中额外包含用于编码橄榄酸/橄榄醇生物合成途径的相关蛋白的基因序列，或者通过包含含有用于编码橄榄酸/橄榄醇生物合成途径的相关蛋白的基因序列的质粒，而额外导入橄榄酸/橄榄醇生物合成途径相关的酶。

如果所述重组菌中额外导入的酶为：乙酰辅酶A羧化酶/丙二酰辅酶A合酶、FadD/FadK、OLS、OAC，则底盘菌培养时可以添加己酸作为前体合成CBGA或者CBG。上述组合中如果使用丙二酰辅酶A合酶，则需要在培养时额外添加丙二酸。

在具体实施方式中，所述重组菌进一步额外导入大麻二酸合酶(CBDAS)。

第四方面，本发明提供一种根据上述方法构建的重组菌。

第五方面，本发明提供上述重组菌用于发酵生产大麻萜酚酸(CBGA)的用途。

第六方面，本发明提供一种生产大麻萜酚酸的方法，其中，所述方法包括采用上述重组菌发酵生产大麻萜酚酸。

第七方面，本发明提供一种生产大麻素类化合物的方法，包括使用采用上述重组菌发酵生产的大麻萜酚酸作为原料进一步合成大麻素类化合物的步骤。

在具体实施方式中，所述大麻素类化合物包括CBDA(大麻二酸)、THCA(四氢大麻酚酸)、CBCA(大麻色素羧酸)、CBNA(大麻酸)、CBG(大麻萜酚)、CBD(大麻二酚)、THC(四氢大麻酚)、CBC(大麻环萜酚)、CBN(大麻酚)。

有益效果

本发明的重组菌包含用以表达NphB突变体的质粒，可以用于高效生产CBGA，相较于现有技术，CBGA的产率得到显著提高。因此，所述重组菌具有强大的CBGA工业化生产的潜力。

附图说明

图1为本申请实施例1中序列3包含的基因元件示意图。

图2为本申请实施例1中序列4包含的基因元件示意图。

图3为本申请实施例1中序列3和4整合到基因组上后的基因元件示意图。

图4为本申请实施例1中序列1包含的基因元件示意图。

图5为本申请实施例1中序列5包含的基因元件示意图。

图6为本申请实施例1中序列6包含的基因元件示意图。

图7为本申请实施例1中序列2包含的基因元件示意图。

图8为本申请实施例2中p15A-BsaI质粒的示意图。

图9为本申请实施例2中构建的CBGA合成酶(CBGAS)表达质粒的示意图。

图10示出本申请实施例3中用HPLC检测方法检测发酵液萃取物中的CBGA含量，其中CZ-1、CZ-2、CZ-3和CZ-4分别表示将p15A-CBGAS-1、p15A-CBGAS-2、p15A-CBGAS-3、p15A-CBGAS-4质粒转化到底盘菌中(插入了序列1和序列2的BW25113)获得用于CBGA合成的各重组菌。

具体实施方式

在下文中，将通过实施例详细描述本发明。然而，在此提供的实施例仅用于说明目的，并不用于限制本发明。

下述实施例所使用的实验方法如无特殊说明，均为常规方法。

下述实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。

所用酶试剂采购自Thermo Fisher公司和New England Biolabs(NEB)公司，所用的小分子标准品采购自Sigma公司，提取质粒所用的试剂盒购自天根生化科技(北京)有限公司，回收DNA片段的试剂盒购自美国omega公司，相应的操作步骤严格按照产品说明书进行。所有培养基如无特殊说明均用去离子水配制，培养基中酵母提取物和蛋白胨购自英国OXID公司，其它试剂购自国药集团化学试剂公司。基因合成服务由华大基因研究院提供。

在下文中采用的大肠杆菌培养基如下：

LB培养基：5g/L酵母提取物，10g/L蛋白胨,10g/L NaCl。调pH值至7.0-7.2，高压蒸汽灭菌30分钟SOB培养基：5g/L酵母提取物，20g/L蛋白胨,0.5g/L NaCl，浓度为1M的KCl2.5ml。调pH值至7.0-7.2，高压蒸汽灭菌。

ZY培养基：蛋白胨10g/L，酵母提取物5g/L，蒸馏水溶解后，调pH至7.0。高压蒸汽灭菌30分钟。

50×M：1.25mol/L Na₂HPO₄，1.25mol/L KH₂PO₄，2.5mol/L NH4Cl，0.25mol/LNa₂SO₄，高压蒸汽灭菌30分钟。

50×5052：25％甘油，2.5％葡萄糖，高压蒸汽灭菌30分钟。

1M MgSO₄：称取24.6g MgSO₄·7H₂O加H₂O溶解，定容至100mL，然后高压蒸汽灭菌30分钟。

1000×微量元素：50mmol/L FeCl₃，20mmol/L CaCl₂，10mmol/L MnCl₂，10mmol/LZnSO₄，CoCl₂，NiCl₂，Na₂MO₄，Na₂SeO₃，H₃BO₃各2mmol/L。

ZYM培养基：在ZY培养基中加入2mL 50×5052，2mL 50×M，200μL浓度1M的MgSO4，100μL 1000×微量元素。

大肠杆菌BW25113(ATCC编号:购自美国菌种保藏中心(American Type CultureCollection))。BW25113菌株来源于E.coli K-12W1485，是K12的衍生菌株，与MG1655类似，也是一种经过较少改造，比较接近于“野生型”的大肠杆菌重组菌株。

实施例1：通过λ-RED同源重组的方法将MVA途径所需的基因导入大肠杆菌BW25113基因组中得到具有高产GPP能力的底盘菌。

为构建高效合成CBGA等大麻素的底盘微生物，首先合成序列3(SEQ ID No.:1)和序列4(SEQ ID No.:2)。

图1为序列3包含的基因元件示意图。如图1所示，序列3包含CmR、PMD、MK和idi，其中CmR为筛选标记，PMD：MVA二磷酸脱羧酶(MVA diphosphate decarboxylase)，MK：MVA激酶(MAV kinase)，IDI：IPP异构酶，arm1和arm2为BW25113基因组序列；

图2为序列4包含的基因元件示意图。如图2所示，序列4包含aadA、ERG20、PMK和PMD，aadA为筛选标记两端带有FRT位点，ERG20：GPP合成酶(GPP synthase)，PMK：磷酸MVA激酶(phospho MVA kinas e)，PMD：MVA二磷酸脱羧酶(MVA diphosphate decarboxylase)，arm1为BW25113基因组同源序列；

通过λ-RED同源重组的方法将上述序列分别插入到大肠杆菌BW25113的基因组特定位置，λ-RED同源双交换为线环同源重组(linear-plus-circular homologousrecombination)，即线性片段两端各有一个同源臂与基因组目标序列两端同源。

根据文献(Tomoya Baba,Hsuan-Cheng Huan,Kirill Datsenko,Barry L.Wanner,and Hirotada Mori,2008,The Applications of Systematic In-Frame,Single-GeneKnockout Mutant Collection of Escherichia coli K-12.Methods Mol.Biol.416:183–194)提供的方法将序列3整合到BW25113基因组上。步骤如下：

1.制备BW25113的感受态；

2.导入质粒pKD46；

3.将BW25113(pKD46)接种到3ml含有氨苄青霉素的LB，氨苄青霉素的浓度为100μg/L，30℃摇床过夜培养；

4.取过夜培养的BW25113(pKD46)细胞悬液100μl，加入到10ml SOB培养基中，再加入浓度为1M的阿拉伯糖100μl和10μl浓度为100mg/L的氨苄青霉素。30℃摇床培养至OD600＝0.4-0.6；

5.4℃条件下，离心收集菌体，并用10ml预冷的超纯水重悬菌体，同样的方法再洗两次菌体，最后用50μl浓度为10％的甘油溶液重悬菌体；

6.加入50ng的序列3基因片段，混匀后将混合液加入电击杯；

7.将电击杯放进电击仪，进行一次电击，电击条件为200Ω、25μF、2.5KV；

8.加入1ml预冷的SOB培养基，混合液转入无菌的EP管中，30℃摇床培养1小时；

9.将菌液均匀涂布于含有氨苄青霉素和氯霉素的LB平板培养基上，30℃静置培养16-20小时；

10.验证平板上长出的转化子：

在整合了序列3的菌株中将序列4整合到基因组上，方法同上，其中第9步放置37℃静置培养16小时。

序列3和4整合到基因组上后，基因组上的序列如图3所示。在图3中，包含aadA、Erg20、PMK、PMD、MK和idi，Erg20：GPP合酶(GPP synthase)，PMK：磷酸MVA激酶(phospho MVAkinase)，PMD：MVA二磷酸脱羧酶(MVA diphosphate decarboxylase)，MK：MVA激酶(MVAkinase)，IDI：IPP异构酶，aadA为筛选标记，两端带有FRT位点，arm1和arm2为BW25113基因组序列。

根据文献(Datsenko,K.A.,Wanner,B.L.,2000,One-step inactivation ofchromosomal genes in Escherichia coli K-12using PCR products.Proc.Natl.Acad.Sci.U.S.A.97:6640–6645)提供的方法将aadA抗性基因删除，具体步骤如下：

1.制备上述菌株的感受态；

2.转化pCP20进入感受态，培养温度为30℃；

3.挑取单克隆至3ml SOB培养基，30℃过夜培养；

4.取100μl转接至10ml SOB，30℃摇床培养3-4小时，OD600＝0.4；

5.4℃条件下离心收集菌体，用冰浴的无菌水洗菌体两次；

6.重悬于50μl-100μl无菌水中，用于电击转化；

7.加入100ng的pCP20，电击转化(电击仪设置：1.8KV，5.5ms)，30℃复苏1小时；

8.均匀涂布于含有氨苄青霉素的LB平板上，30℃培养2-3天；

9.挑取单克隆在没有抗生素的LB平板上划线，42℃培养过夜；

10.挑20-30个克隆分别至壮观霉素抗性平板和无抗平板，壮观霉素平板上不能生长，同时无抗平板上长起的克隆为目标克隆，进行PCR验证。

通过上述方法将筛选标记aadA删除，aadA删除后得到序列1(如图4所示)。序列1包含ERG20、PMK、PMD、MK和idi，GPP合酶(GPP synthase)，PMK：磷酸MVA激酶(phosphoMVAkinase)，PMD：MVA二磷酸脱羧酶(MVA diphosphate decarboxylase)，MK：MVA激酶(MVAkinase)，IDI：IPP异构酶，arm1和arm2为BW25113基因组序列，这些基因共用一个启动子p119(SEQ ID NO.:8)，基因前分别含有RBS序列(SEQ ID NO.:7)。

MK的基因序列为SEQ ID NO.:10，对应的氨基酸序列为SEQ ID NO.:11(来源于酵母Saccharomyces cerevisiae S288C)；PMK的基因序列为SEQ ID NO.:12，对应的氨基酸序列为SEQ ID NO.:13(来源于酵母Saccharomyces cerevisiae S288C)；PMD的基因序列为SEQ ID NO.:14，对应的氨基酸序列为SEQ ID NO.:15(来源于酵母Saccharomycescerevisiae S288C)；idi的基因序列为SEQ ID NO.:16，对应的氨基酸序列为SEQ ID NO.:17(来源于大肠杆菌Escherichia coli)；ERG20的基因序列为SEQ ID NO.:18，对应的氨基酸序列为SEQ ID NO.:19(来源于酵母Saccharomyces cerevisiae S288C)。

如下文所述，在插入序列1的菌株的基因组上继续插入atoB、HMGR、HMGS和idi基因。

首先合成序列5(SEQ ID No.:4)和序列6(SEQ ID No.:5)。

图5为序列5包含的基因元件示意图。如图5所示，序列5包含CmR、HMGS、HMGR和idi；HMGS：HMG合酶(HMG synthase)，HMGR：3-羟基-3-甲基戊二酰-CoA还原酶(3-Hydroxy-3-methylglutaryl-CoAreductase)，IDI：IPP异构酶，CmR为筛选标记，使宿主具有氯霉素抗性，arm3和arm4为BW25113基因组同源序列；

图6为序列6包含的基因元件示意图。如图6所示，序列6包含aadA、araC、atoB、HMGS；atoB：乙酰乙酰-CoA硫解酶(acetoacetyl-CoAthiolase)，HMGS：HMG合酶(HMGsynthase)，aadA：aadA为筛选标记，使得宿主具有壮观霉素抗性，两端带有FRT位点，araC：为阿拉伯糖启动子元件。

序列5和6插入基因组的方法同上文中序列3、序列4的方法，此时在基因组上插入了如图7所示的序列2(SEQ ID No.:6)。

如图7所示，序列2包含araC、atoB、HMGS、HMGR和idi；AtoB：acetoacetyl-CoAthiolase，HMGS：HMG synthase，HMGR：3-Hydroxy-3-methylglutaryl-CoAreductase，IDI：IPP异构酶，arm3和arm4为BW25113基因组同源序列，araC：为阿拉伯糖启动子元件。

序列2(SEQ ID NO.:6)包括atoB，HMGS，HMGR和idi的编码基因，这4个基因共用一个启动子pBAD(SEQ ID NO.:9)，pBAD为诱导性启动子，在阿拉伯糖存在下工作。基因前分别含有RBS序列(SEQ ID NO.:7)。

atoB基因序列为SEQ ID NO.:20，对应的氨基酸序列为SEQ ID NO.:21(序列来源于大肠杆菌Escherichia coli)；HMGS的基因序列为SEQ ID NO.:22，对应的氨基酸序列为SEQ ID NO.:23(序列来源于Staphylococcus aureus)；HMGR的基因序列为SEQ ID NO.:24，对应的氨基酸序列为SEQ ID NO.:25(来源于Staphylococcus aureus)。

通过在BW25113中插入序列1和序列2，基因序列包括了MVA途径需要各个基因，使得该底盘菌具有高产GPP的能力。

实施例2：包含芳香族异戊烯基转移酶(NphB)突变体的编码序列的质粒的构建

首先构建骨架质粒：p15A-BsaI(SEQ ID NO.:26)(参见图7)。

然后，使用golden gate连接法，分别将合成CBGAS编码基因(下文合成的CBGAS-1、CBGAS-2、CBGAS-3和CBGAS-4序列)替换p15A-BsaI质粒中BsaI位点之间的序列得到p15A-CBGAS-1、p15A-CBGAS-2、p15A-CBGAS-3、p15A-CBGAS-4质粒。其中，反应体系如下表1所示：

表1

反应条件如下表2：

表2

反应完成后取10μl反应液转化大肠杆菌DH5α(购自南京市金斯瑞生物科技有限公司)，完成质粒的构建。构建的质粒如图9所示。

分别合成CBGAS-1、CBGAS-2、CBGAS-3和CBGAS-4(对应nphB编码序列或者其不同的突变体的编码序列)，序列前端包含BsaI酶切位点和RBS序列(SEQ ID NO.:35)，序列尾端包含另一个BsaI酶切位点，nphB序列来源于Streptomyces coelicolor CL190，序列针对大肠杆菌MG1655进行密码子优化。

其中，CBGAS-1、CBGAS-2、CBGAS-3、CBGAS-4序列分别为SEQ ID NO.:27，SEQ IDNO.:29，SEQ ID NO.:31，SEQ ID NO.:33；CBGAS-1、CBGAS-2、CBGAS-3、CBGAS-4中nphB编码的氨基酸序列分别为SEQ ID NO.:28，SEQ ID NO.:30，SEQ ID NO.:32，SEQ ID NO.:34。

其中，CBGAS-2中nphB编码的氨基酸序列与CBGAS-1中nphB编码的氨基酸序列相比，第161位氨基酸由Q突变为A；CBGAS-3中nphB编码的氨基酸序列与CBGAS-1中nphB编码的氨基酸序列相比，第286位氨基酸由G突变为S，第288位氨基酸由Y突变为A；CBGAS-4中nphB编码的氨基酸序列与CBGAS-1中nphB编码的氨基酸序列相比，第161位氨基酸由Q突变为A，第286位氨基酸由G突变为S，第288位氨基酸由Y突变为A。

实施例3：质粒转化与重组菌发酵生产CBGA

将实施例2中构建的p15A-CBGAS-1、p15A-CBGAS-2、p15A-CBGAS-3、p15A-CBGAS-4质粒分别转化到实施例1中得到的底盘菌中(插入了序列1和序列2的BW25113)获得用于CBGA合成的重组菌，重组菌分别命名为：CZ-1、CZ-2、CZ-3和CZ-4。重组菌按照下面的方法分别处理，每个重组菌做三个平行样品。

发酵和制样：

1.重组菌接种至3mLLB液体培养基中，37℃，220转/分钟，过夜培养，约14小时，终OD600值达到2-3；

2.在24深孔板中加入ZYM培养基，每个孔加入2ml；

3.将步骤1中菌液转接至步骤2中的ZYM培养基，转接后OD600为0.01；

4.待菌液生长至OD600为0.2，加入诱导剂(添加量：鼠李糖0.2％，阿拉伯糖0.2％)和前体olivetolic acid(200mg/L)，从接种至发酵结束总共24小时；

5.取3mL发酵液，离心弃上清，沉淀中加入1mL甲醇；

6.再进行一次离心，用0.22μM滤器过滤上清，然后转移至HPLC样品瓶中。

停止发酵时发酵液中的细胞浓度如下表3所示：

表3

用HPLC检测方法分析发酵液萃取物中的CBGA含量。HPLC检测方法如下表4所示：

表4

经检测，重组菌CZ-1、CZ-2、CZ-3和CZ-4发酵液样品中CBGA产量如下表5中所示，

表5

从以上表5中的数据可以看出，重组菌CZ-4的CBGA的平均产量为83.91mg/L，远高于现有的技术(文献：Shuai Qian,James M.Clomburg,Ramon Gonzalez,2019,EngineeringEscherichia coli as a platform for the in vivo synthesis of prenylatedaromatics,Biotechnol Bioeng,116(5):1116-1127)所公布的1mg/L。

综上，本发明开发出一种高产CBGA的重组菌，培养过程中通过阿拉伯糖和鼠李糖诱导，获得高浓度的CBGA，平均浓度为83.91mg/L，由于深孔板培养时发酵液的细胞浓度(OD600)仅在4-5之间，而工业化培养中细胞浓度能到100以上，因此本发明开发的重组菌具有强大的CBGA工业化潜力。

序列表

<110> 深圳蓝晶生物科技有限公司

<120> 芳香族异戊烯基转移酶突变体、用于其表达的重组菌的构建方法及由其构建的重组菌

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<400> 2

gaattcaaaa aaaacctgcg catctgcgca ggctggtgta attcatgtgc tcaacccgaa 60

gttgacttca cctatcaata cctctgggat caccacttta gcaacctgaa gccaaacgcc 120

accagcggtc aatcgcaaca gcgtttcgca aagtgtaacc aaaggtttgc aatctctttt 180

tttgtcttac ttatttgcgt ttggctcaca ttcccacgat gaaaacacgc ctgtaggctg 240

gagctgcttc gaagttccta tactttctag agaataggaa cttcggaata ggaacttcat 300

gagctcagcc aatcgactgg cgagcggcat cttatttgcc gactaccttg gtgatctcgc 360

ctttcacgta gtggacaaat tcttccaact gatctgcgcg cgaggccaag cgatcttctt 420

cttgtccaag ataagcctgt ctagcttcaa gtatgacggg ctgatactgg gccggcaggc 480

gctccattgc ccagtcggca gcgacatcct tcggcgcgat tttgccggtt actgcgctgt 540

accaaatgcg ggacaacgta agcactacat ttcgctcatc gccagcccag tcgggcggcg 600

agttccatag cgttaaggtt tcatttagcg cctcaaatag atcctgttca ggaaccggat 660

caaagagttc ctccgccgct ggacctacca aggcaacgct atgttctctt gcttttgtca 720

gcaagatagc cagatcaatg tcgatcgtgg ctggctcgaa gatacctgca agaatgtcat 780

tgcgctgcca ttctccaaat tgcagttcgc gcttagctgg ataacgccac ggaatgatgt 840

cgtcgtgcac aacaatggtg acttctacag cgcggagaat ctcgctctct ccaggggaag 900

ccgaagtttc caaaaggtcg ttgatcaaag ctcgccgcgt tgtttcatca agccttacgg 960

tcaccgtaac cagcaaatca atatcactgt gtggcttcag gccgccatcc actgcggagc 1020

cgtacaaatg tacggccagc aacgtcggtt cgagatggcg ctcgatgacg ccaactacct 1080

ctgatagttg agtcgatact tcggcgatca ccgcttccct catgacattg cactccaccg 1140

ctgatgacat cagtcgatca tagcacgatc aacggcactg ttgcaaatag tcggtggtga 1200

taaacttatc atcccctttt gctgatggag ctgcacatga acccattcaa aggccggcat 1260

tttcagcgtg acatcattct gtgggccgta cgctggtact gcaaatacgg catcagttac 1320

cgtgagctgc attttccgct gcataaccct gcttcggggt cattatagcg attttttcgg 1380

tatatccatc ctttttcgca cgatatacag gattttgcca aagggttcgt gtagactttc 1440

cttggtgtat ccaacggcgt cagccgggca ggataggtga agtaggccca cccgcgagcg 1500

ggtgttcctt cttcactgtc ccttattcgc acctggcggt gctcaacggg aatcctgctc 1560

tgcgaggctg gcgggaactt cgaagttcct atactttcta gagaatagga acttcgaact 1620

gcaggtcgac ggatccccgg aatcacagct aacaccacgt cgtccctatc tgctgcccta 1680

ggtctatgag tggttgctgg ataacttgac agctagctca gtcctaggta taatgctagc 1740

agggagacca caacggtttc cctctacaaa taattttgtt taacttttct agagaaagag 1800

gagaaatact agatggccag tgaaaaagaa atccgtcgcg agcgctttct gaacgtgttc 1860

ccgaaactgg tggaagaact caacgccagt ctgctggcct atggtatgcc gaaagaagcg 1920

tgcgactggt acgcgcacag tctgaattac aacaccccgg gcggcaagct gaatcgtggt 1980

ctgagcgttg tggacacgta cgcgattctg agcaacaaaa ccgtggagca gctgggccaa 2040

gaagaatatg agaaggtggc cattctgggc tggtgtatcg aactgctgca agcctactgg 2100

ctggttgcgg acgacatgat ggacaagagc attacccgcc gcggccagcc gtgttggtac 2160

aaagttccgg aggttggcga gatcgccatc tgggatgcgt ttatgctgga agcggccatc 2220

tacaagctgc tcaagagcca ctttcgcaac gagaagtact acatcgacat cacggagctg 2280

ttccatgagg tgacgtttca gaccgagctc ggccagctca tggatctgat caccgccccg 2340

gaggacaaag ttgacctcag caagttcagt ctgaaaaaac acagctttat cgtgaccttc 2400

aagacggcct actacagctt ttatctgcca gttgcgctcg cgatgtatgt tgccggcatc 2460

accgacgaga aggatctgaa acaagcccgc gatgtgctga ttccgctggg tgagtacttc 2520

cagatccaag atgactatct ggactgtttc ggcaccccgg aacagattgg caagattggc 2580

accgacatcc aagataacaa atgcagctgg gtgatcaaca aggcgctgga actcgccagt 2640

gcggaacagc gcaaaacgct cgacgagaat tacggcaaaa aagacagtgt ggccgaagcg 2700

aagtgcaaga aaatctttaa cgatctgaag attgagcagc tctaccacga atacgaggag 2760

agcattgcca aggatctcaa ggccaagatc agtcaagttg acgagagtcg cggcttcaaa 2820

gccgacgtgc tcacggcctt tctgaacaag gtttacaaac gcagcaaata aaaagaggag 2880

aaaatgagcg aattacgtgc attcagcgcg ccaggtaagg cactgctggc cggtggctac 2940

ctggtgttag acaccaagta cgaggcgttc gtcgtcggct tatctgcccg tatgcatgca 3000

gttgcccacc cgtatggtag cctgcagggc tctgacaagt tcgaagtgcg tgtgaagagc 3060

aagcagttca aggacggcga gtggctgtac cacattagcc caaagagcgg cttcatcccg 3120

gttagcattg gtggcagcaa gaacccattt atcgagaagg tcattgccaa cgtcttcagc 3180

tacttcaagc cgaatatgga cgattactgc aaccgcaacc tgttcgtcat cgacattttc 3240

agcgacgacg cgtaccacag ccaagaggac tctgttacgg agcatcgtgg taaccgccgc 3300

ctgagcttcc acagccatcg cattgaggag gtgccgaaga cgggtctggg ttctagcgcc 3360

ggtttagtta ccgtcttaac gacggcgtta gcgagcttct tcgtgagcga cctggagaac 3420

aacgtggaca agtaccgcga agtgattcat aacctggcgc aggtggcaca ttgtcaggcc 3480

caaggtaaga ttggctctgg ttttgatgtg gcagcggccg cctatggctc tatccgctat 3540

cgccgctttc cgccggccct gatcagcaat ctgccggaca tcggctctgc gacgtatggt 3600

agcaaactgg cgcatctggt ggacgaagaa gactggaaca tcaccattaa gtctaatcac 3660

ctgccgagcg gcttaacgtt atggatgggc gatatcaaga acggcagcga aacggttaag 3720

ctggtgcaga aagtgaaaaa ctggtacgac agccacatgc cggaaagcct gaagatttac 3780

acggagctgg accacgccaa tagccgtttc atggatggtc tgagcaagct ggaccgcctg 3840

cacgaaaccc acgacgacta cagcgaccaa atcttcgaga gcctggagcg caatgactgc 3900

acctgccaga agtacccgga gatcacggag gtccgcgatg ccgtggcaac gattcgccgt 3960

agcttccgca aaattacgaa ggagagcggc gcggatatcg aaccaccggt ccagacgtct 4020

ctgctggacg actgtcaaac cttaaagggc gtgttaacgt gcctgattcc gggcgcgggt 4080

ggttacgacg ccattgccgt catcacgaaa caggacgtcg atctgcgcgc acaaacggcc 4140

aacgacaaac gtttcagcaa agtccaatgg ctggatgtta cgcaggccga ctggggtgtt 4200

cgcaaggaga aggacccgga aacgtatctg gataagtgaa aagaggagaa aatgaccgtt 4260

tacacagcat ccgttaccgc acccgtcaac atcgcaaccc ttaagtattg ggggaaaagg 4320

gacacgaagt tgaatctgcc caccaattcg tccatatcag tgactttatc gcaagatgac 4380

ctcagaacgt tgacctctgc ggctactgca cctgagtttg aacgcgacac tttgtggtta 4440

aatggagaac cacacagcat cgacaatgaa agaactcaaa attgtctgcg cgacctacgc 4500

caattaagaa aggaaatgga atcgaaggac gcctcattgc ccacattatc tcaatggaaa 4560

ctccacattg tctccgaaaa taactttcct acagcagctg gtttagcttc ctccgctgct 4620

ggctttgctg cattggtctc tgcaattgct aagttatacc aattaccaca gtcaacttca 4680

gaaatatcta gaatagcaag aaaggggtct ggttcagctt gtagatcgtt gtttggcgga 4740

tacgtggcct ggaattc 4757

<210> 3

<211> 5943

<212> DNA

<213> 人工序列

<220>

<223> 序列 1

<400> 3

attccgggga tccgtcgacc tgcagttcga agttcctatt ctctagaaag tataggaact 60

tcgaagcagc tccagcctac acacagctaa caccacgtcg tccctatctg ctgccctagg 120

tctatgagtg gttgctggat aacttgacag ctagctcagt cctaggtata atgctagcag 180

ggagaccaca acggtttccc tctacaaata attttgttta acttttctag agaaagagga 240

gaaatactag atggccagtg aaaaagaaat ccgtcgcgag cgctttctga acgtgttccc 300

gaaactggtg gaagaactca acgccagtct gctggcctat ggtatgccga aagaagcgtg 360

cgactggtac gcgcacagtc tgaattacaa caccccgggc ggcaagctga atcgtggtct 420

gagcgttgtg gacacgtacg cgattctgag caacaaaacc gtggagcagc tgggccaaga 480

agaatatgag aaggtggcca ttctgggctg gtgtatcgaa ctgctgcaag cctactggct 540

ggttgcggac gacatgatgg acaagagcat tacccgccgc ggccagccgt gttggtacaa 600

agttccggag gttggcgaga tcgccatctg ggatgcgttt atgctggaag cggccatcta 660

caagctgctc aagagccact ttcgcaacga gaagtactac atcgacatca cggagctgtt 720

ccatgaggtg acgtttcaga ccgagctcgg ccagctcatg gatctgatca ccgccccgga 780

ggacaaagtt gacctcagca agttcagtct gaaaaaacac agctttatcg tgaccttcaa 840

gacggcctac tacagctttt atctgccagt tgcgctcgcg atgtatgttg ccggcatcac 900

cgacgagaag gatctgaaac aagcccgcga tgtgctgatt ccgctgggtg agtacttcca 960

gatccaagat gactatctgg actgtttcgg caccccggaa cagattggca agattggcac 1020

cgacatccaa gataacaaat gcagctgggt gatcaacaag gcgctggaac tcgccagtgc 1080

ggaacagcgc aaaacgctcg acgagaatta cggcaaaaaa gacagtgtgg ccgaagcgaa 1140

gtgcaagaaa atctttaacg atctgaagat tgagcagctc taccacgaat acgaggagag 1200

cattgccaag gatctcaagg ccaagatcag tcaagttgac gagagtcgcg gcttcaaagc 1260

cgacgtgctc acggcctttc tgaacaaggt ttacaaacgc agcaaataaa aagaggagaa 1320

aatgagcgaa ttacgtgcat tcagcgcgcc aggtaaggca ctgctggccg gtggctacct 1380

ggtgttagac accaagtacg aggcgttcgt cgtcggctta tctgcccgta tgcatgcagt 1440

tgcccacccg tatggtagcc tgcagggctc tgacaagttc gaagtgcgtg tgaagagcaa 1500

gcagttcaag gacggcgagt ggctgtacca cattagccca aagagcggct tcatcccggt 1560

tagcattggt ggcagcaaga acccatttat cgagaaggtc attgccaacg tcttcagcta 1620

cttcaagccg aatatggacg attactgcaa ccgcaacctg ttcgtcatcg acattttcag 1680

cgacgacgcg taccacagcc aagaggactc tgttacggag catcgtggta accgccgcct 1740

gagcttccac agccatcgca ttgaggaggt gccgaagacg ggtctgggtt ctagcgccgg 1800

tttagttacc gtcttaacga cggcgttagc gagcttcttc gtgagcgacc tggagaacaa 1860

cgtggacaag taccgcgaag tgattcataa cctggcgcag gtggcacatt gtcaggccca 1920

aggtaagatt ggctctggtt ttgatgtggc agcggccgcc tatggctcta tccgctatcg 1980

ccgctttccg ccggccctga tcagcaatct gccggacatc ggctctgcga cgtatggtag 2040

caaactggcg catctggtgg acgaagaaga ctggaacatc accattaagt ctaatcacct 2100

gccgagcggc ttaacgttat ggatgggcga tatcaagaac ggcagcgaaa cggttaagct 2160

ggtgcagaaa gtgaaaaact ggtacgacag ccacatgccg gaaagcctga agatttacac 2220

ggagctggac cacgccaata gccgtttcat ggatggtctg agcaagctgg accgcctgca 2280

cgaaacccac gacgactaca gcgaccaaat cttcgagagc ctggagcgca atgactgcac 2340

ctgccagaag tacccggaga tcacggaggt ccgcgatgcc gtggcaacga ttcgccgtag 2400

cttccgcaaa attacgaagg agagcggcgc ggatatcgaa ccaccggtcc agacgtctct 2460

gctggacgac tgtcaaacct taaagggcgt gttaacgtgc ctgattccgg gcgcgggtgg 2520

ttacgacgcc attgccgtca tcacgaaaca ggacgtcgat ctgcgcgcac aaacggccaa 2580

cgacaaacgt ttcagcaaag tccaatggct ggatgttacg caggccgact ggggtgttcg 2640

caaggagaag gacccggaaa cgtatctgga taagtgaaaa gaggagaaaa tgaccgttta 2700

cacagcatcc gttaccgcac ccgtcaacat cgcaaccctt aagtattggg ggaaaaggga 2760

cacgaagttg aatctgccca ccaattcgtc catatcagtg actttatcgc aagatgacct 2820

cagaacgttg acctctgcgg ctactgcacc tgagtttgaa cgcgacactt tgtggttaaa 2880

tggagaacca cacagcatcg acaatgaaag aactcaaaat tgtctgcgcg acctacgcca 2940

attaagaaag gaaatggaat cgaaggacgc ctcattgccc acattatctc aatggaaact 3000

ccacattgtc tccgaaaata actttcctac agcagctggt ttagcttcct ccgctgctgg 3060

ctttgctgca ttggtctctg caattgctaa gttataccaa ttaccacagt caacttcaga 3120

aatatctaga atagcaagaa aggggtctgg ttcagcttgt agatcgttgt ttggcggata 3180

cgtggcctgg gaaatgggaa aagctgaaga tggtcatgat tccatggcag tacaaatcgc 3240

agacagctct gactggcctc agatgaaagc ttgtgtccta gttgtcagcg atattaaaaa 3300

ggatgtgagt tccactcagg gtatgcaatt gaccgtggca acctccgaac tatttaaaga 3360

aagaattgaa catgtcgtac caaagagatt tgaagtcatg cgtaaagcca ttgttgaaaa 3420

agatttcgcc acctttgcaa aggaaacaat gatggattcc aactctttcc atgccacatg 3480

tttggactct ttccctccaa tattctacat gaatgacact tccaagcgta tcatcagttg 3540

gtgccacacc attaatcagt tttacggaga aacaatcgtt gcatacacgt ttgatgcagg 3600

tccaaatgct gtgttgtact acttagctga aaatgagtcg aaactctttg catttatcta 3660

taaattgttt ggctctgttc ctggatggga caagaaattt actactgagc agcttgaggc 3720

tttcaaccat caatttgaat catctaactt tactgcacgt gaattggatc ttgagttgca 3780

aaaggatgtt gccagagtga ttttaactca agtcggttca ggcccacaag aaacaaacga 3840

atctttgatt gacgcaaaga ctggtctacc aaaggaataa aaagaggaga aaatgtctct 3900

gccattcctg acgtctgcgc caggtaaggt gatcatcttc ggcgagcact ctgcggtgta 3960

caataagccg gccgtcgccg cctctgtgtc tgcgttacgc acctacctgc tgatcagcga 4020

atcttctgca ccggacacga tcgagctgga ctttccggac atcagcttca accacaagtg 4080

gagcatcaac gacttcaacg cgatcacgga ggaccaggtg aacagccaaa agctggccaa 4140

agcccagcaa gcaaccgacg gtctgtctca ggagctggtg tctctgctgg acccgctgtt 4200

agcgcagtta agcgagagct tccattacca cgccgcgttc tgcttcctgt acatgttcgt 4260

ttgcctgtgc ccgcacgcaa agaacatcaa gttcagcctg aagagcacgc tgccgattgg 4320

cgcaggctta ggctctagcg catctatcag cgtgagcctg gcgctggcga tggcctatct 4380

gggtggcctg attggcagca acgacctgga gaaactgagc gaaaacgaca agcacatcgt 4440

gaaccagtgg gcctttatcg gcgagaagtg cattcatggc accccgagcg gcattgacaa 4500

cgcagttgcc acgtatggca acgccctgct gttcgagaaa gacagccaca acggcacgat 4560

caacacgaac aacttcaagt tcctggacga cttcccggcg atcccgatga ttctgaccta 4620

cacccgtatc ccacgcagca ccaaggattt agtcgcccgc gtgcgtgttt tagtcaccga 4680

aaagttcccg gaggtgatga agccgatcct ggacgcgatg ggcgagtgcg cgctgcaggg 4740

tctggagatc atgaccaagc tgagcaagtg caagggcacc gacgatgagg cggtggagac 4800

caacaatgag ctgtacgagc agctgctgga gctgatccgt atcaatcacg gcctgctggt 4860

ctctatcggt gtgtctcacc cgggcctgga actgatcaaa aacctgagcg acgacctgcg 4920

cattggctct acgaaattaa cgggtgcagg tggcggtggc tgctctttaa cgctgctgcg 4980

ccgtgacatt acgcaggagc aaatcgacag cttcaagaag aagctgcagg acgacttcag 5040

ctacgagacg ttcgagacgg acctgggcgg cacgggctgt tgcctgctga gcgccaaaaa 5100

tctgaacaag gacctgaaga tcaaaagcct ggtgttccag ctgttcgaaa acaagacgac 5160

cacgaagcag cagatcgacg acctgttact gccgggtaac accaatctgc cgtggacgtc 5220

ttaaaaagag gagaaaatgc aaacggaaca cgtcatttta ttgaatgcac agggagttcc 5280

cacgggtacg ctggaaaagt atgccgcaca cacggcagac acccgcttac atctcgcgtt 5340

ctccagttgg ctgtttaatg ccaaaggaca attattagtt acccgccgcg cactgagcaa 5400

aaaagcatgg cctggcgtgt ggactaactc ggtttgtggg cacccacaac tgggagaaag 5460

caacgaagac gcagtgatcc gccgttgccg ttatgagctt ggcgtggaaa ttacgcctcc 5520

tgaatctatc tatcctgact ttcgctaccg cgccaccgat ccgagtggca ttgtggaaaa 5580

tgaagtgtgt ccggtatttg ccgcacgcac cactagtgcg ttacagatca atgatgatga 5640

agtgatggat tatcaatggt gtgatttagc agatgtatta cacggtattg atgccacgcc 5700

gtgggcgttc agtccgtgga tggtgatgca ggcgacaaat cgcgaagcca gaaaacgatt 5760

atctgcattt acccagctta aataatgcct ggcggcagta gcgcggtggt cccacctgac 5820

cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat 5880

gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc 5940

ctt 5943

<210> 4

<211> 5216

<212> DNA

<213> 人工序列

<220>

<223> 序列 5

<400> 4

gatatcgtaa cggtactggt ctggcgaaag gattcttgcg attcccgacg gcgaaacagt 60

tccgtatctc gtctaatttt aacccgcgtc gtactaatcc ggtgaccggt cgcgttgcac 120

cacacagagg tgttgatttc gccatgccgc agggtacgcc agtgctttca gtgggtgacg 180

gtgaagtggt ggttgccaaa cgcagtggcg cagcaggtta ttatgtggct attcgtcatg 240

gtcgcagcta caccacgcgt tatatgcact tgcgcaagat tctggtgaaa ccgggacaga 300

aggtgaaacg tggcgaccgt atcgcgcttt ccggtaatac cggacgttca accgggccgc 360

atctgcacta tgaagtatgg ataaaccagc aggccgtaaa cccgctgacg gcaaaactgc 420

cgcgtaccga agggctgacc ggctccgatc gtcgcgaatt cctggcgcag gccaaagaga 480

ttgtgccgca gctacggttt gattaattac gccccgccct gccactcatc gcagtactgt 540

tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga 600

atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg 660

ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag 720

ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt 780

tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg 840

tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg 900

tgaacactat cccatatcac cagctcaccg tctttcattg ccatacgaaa ttccggatga 960

gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc 1020

tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga 1080

gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg 1140

gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac 1200

tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg 1260

tgccgatcaa aagaggagaa aatgacaata ggtatcgata aaataaactt ttacgttcca 1320

aagtactatg tagacatggc taaattagca gaagcacgcc aagtagaccc aaacaaattt 1380

ttaattggaa ttggtcaaac tgaaatggct gttagtcctg taaaccaaga catcgtttca 1440

atgggcgcta acgctgctaa ggacattata acagacgaag acaaaaagaa aattggtatg 1500

gtaattgtgg caactgaatc agcagttgat gctgctaaag cagccgctgt tcaaattcac 1560

aacttattag gtattcaacc ttttgcacgc tgctttgaaa tgaaagaagc ttgttatgct 1620

gcaacaccag caattcaatt agctaaagat tatttagcaa ctagaccgaa tgaaaaagta 1680

ttagttattg ctacagatac agcacgttat ggattgaact caggcggcga gccaacacaa 1740

ggtgctggcg cagttgcgat ggttattgca cataatccaa gcattttggc attaaatgaa 1800

gatgctgttg cttacactga agacgtttat gatttctggc gtccaactgg acataaatat 1860

ccattagttg atggtgcatt atctaaagat gcttatatcc gctcattcca acaaagctgg 1920

aatgaatacg caaaacgtca aggtaagtcg ctagctgact tcgcatctct atgcttccat 1980

gttccattta caaaaatggg taaaaaggca ttagagtcaa tcattgataa cgctgatgaa 2040

acaactcaag agcgtttacg ttcaggatat gaagatgctg tagattataa ccgttatgtc 2100

ggtaatattt atactggatc attatattta agcctaatat cattacttga aaatcgagat 2160

ttacaagctg gtgaaacaat cggtttattc agttatggct caggttcagt tggtgaattt 2220

tatagtgcga cattagttga aggctacaaa gatcatttag atcaagctgc acataaagca 2280

ttattaaata accgtactga agtatctgtt gatgcatatg aaacattctt caaacgtttt 2340

gatgacgttg aatttgacga agaacaagat gctgttcatg aagatcgtca tattttctac 2400

ttatcaaata ttgaaaataa cgttcgcgaa tatcacagac cagagtaaaa agaggagaaa 2460

atgtccatgc aaagtttaga taagaatttt cgacatttat ctcgtaaaga aaagttacaa 2520

caattggttg ataagcaatg gttatcagaa gaacaattcg acattttact gaatcatcca 2580

ttaatcgatg aagaagtagc caatagttta attgaaaatg tcatcgcgca aggtgcatta 2640

cccgttggat tattaccgaa tatcattgtg gacgataagg catatgttgt acctatgatg 2700

gtggaagagc cttcagttgt cgctgcagct agttatggtg caaagctagt gaatcagact 2760

ggcggattta aaacggtatc ttctgaacgt attatgatag gtcaaatcgt ctttgatggc 2820

gttgacgata ctgaaaaatt atcagcagac attaaagctt tagaaaagca aattcataaa 2880

attgcggatg aggcatatcc ttctattaaa gcgcgtggtg gtggttacca acgtatagcg 2940

attgatacat ttcctgagca acagttacta tctttaaaag tatttgttga tacgaaagat 3000

gctatgggcg ctaatatgct taatacgatt ttagaggcca taactgcatt tttaaaaaat 3060

gaatttccgc aaagcgacat tttaatgagt attttatcca atcatgcaac agcgtccgtt 3120

gttaaagttc aaggcgaaat tgatgttaaa gatttagcaa ggggcgagag aactggagaa 3180

gaggttgcca aacgaatgga acgtgcttct gtattggccc aagtagatat tcatcgtgca 3240

gcaacacata ataaaggtgt tatgaatggc atacatgctg ttgttttagc aacaggaaat 3300

gatacgcgtg gtgcagaagc aagtgcgcat gcatacgcga gtcgtgacgg acagtatcgt 3360

ggtattgcta catggcgtta cgatcaagat cgtcaacgat tgattggtac aattgaagtg 3420

cctatgacat tggcaatcgt tggcggtggt acaaaagtat taccaattgc taaagcttca 3480

ttagagctac taaatgtaga gtcagcacaa gaattaggtc atgtagttgc tgccgttggt 3540

ttagcgcaaa actttgcagc atgtcgcgcg cttgtgtcag aaggtattca acaaggtcat 3600

atgagtttac aatataaatc attagctatc gttgtagggg caaaaggtga tgaaattgct 3660

aaagtagctg aagctttgaa aaaagaaccc cgtgcaaata cacaagcagc ggaacatatt 3720

ttacaagaaa ttagacaaca ataaaaagag gagaaaatgc aaacggaaca cgtcatttta 3780

ttgaatgcac agggagttcc cacgggtacg ctggaaaagt atgccgcaca cacggcagac 3840

acccgcttac atctcgcgtt ctccagttgg ctgtttaatg ccaaaggaca attattagtt 3900

acccgccgcg cactgagcaa aaaagcatgg cctggcgtgt ggactaactc ggtttgtggg 3960

cacccacaac tgggagaaag caacgaagac gcagtgatcc gccgttgccg ttatgagctt 4020

ggcgtggaaa ttacgcctcc tgaatctatc tatcctgact ttcgctaccg cgccaccgat 4080

ccgagtggca ttgtggaaaa tgaagtgtgt ccggtatttg ccgcacgcac cactagtgcg 4140

ttacagatca atgatgatga agtgatggat tatcaatggt gtgatttagc agatgtatta 4200

cacggtattg atgccacgcc gtgggcgttc agtccgtgga tggtgatgca ggcgacaaat 4260

cgcgaagcca gaaaacgatt atctgcattt acccagctta aataatcggt ctgcagctgg 4320

tgccgcgcgg cagccaccac caccaccacc actaatacag attaaatcag aacgcagaag 4380

cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat 4440

gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag 4500

agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggccttct 4560

atacgaagtt attcgtcgac ctaattctct cgcgaggaga ggccttcgcc tgatgataag 4620

ttcaagtttg cttcagaata ttcgaaatct gttgaactat cattgaactg taggccggat 4680

gtggcgtttt cgccgcatcc ggcaacgtac ttactctacc gttaaaatac gcgtggtatt 4740

agtagaaccc acggtactca tcacgtcgcc ctgggtgaca atcaccaggt caccagacat 4800

caagtaacct ttatcgcgca gcagattaac cgcttcgctg gcagctgcta cgccgtcatt 4860

agcgctatca aagtgcaccg gcgtaacgcc acgatagaga gcagtcaggt tcagcgtacg 4920

ttcatggcgc gacatggcga aaattggcag accagagctg atacgggagg tcatcagcgc 4980

ggtacgaccc gattcggtca tggtgatgat cgccgtaacg cctttcaggt ggttagctgc 5040

gtacattgct gacatggcaa tagcttcttc cacattgtcg aactgaacgt ccagacggtg 5100

tttagaaacg ttgatgctcg ggattttttc cgcacccagg caaacgcgcg ccatggctgc 5160

aacggtttct gacggatact gcccagcggc agtttctgca gacagcatca gatatc 5216

<210> 5

<211> 4956

<212> DNA

<213> 人工序列

<220>

<223> 序列 6

<400> 5

actagtgtaa cggtactggt ctggcgaaag gattcttgcg attcccgacg gcgaaacagt 60

tccgtatctc gtctaatttt aacccgcgtc gtactaatcc ggtgaccggt cgcgttgcac 120

cacacagagg tgttgatttc gccatgccgc agggtacgcc agtgctttca gtgggtgacg 180

gtgaagtggt ggttgccaaa cgcagtggcg cagcaggtta ttatgtggct attcgtcatg 240

gtcgcagcta caccacgcgt tatatgcact tgcgcaagat tctggtgaaa ccgggacaga 300

aggtgaaacg tggcgaccgt atcgcgcttt ccggtaatac cggacgttca accgggccgc 360

atctgcacta tgaagtatgg ataaaccagc aggccgtaaa cccgctgacg gcaaaactgc 420

cgcgtaccga agggctgacc ggctccgatc gtcgcgaatt cctggcgcag gccaaagaga 480

ttgtgccgca gctacggttt gattaattaa catccattcg cagccggtac gcaattccgg 540

ggatccgtcg acctgcagtt cgaagttcct attctctaga aagtatagga acttcgaagt 600

tcccgccagc ctcgcagagc aggattcccg ttgagcaccg ccaggtgcga ataagggaca 660

gtgaagaagg aacacccgct cgcgggtggg cctacttcac ctatcctgcc cggctgacgc 720

cgttggatac accaaggaaa gtctacacga accctttggc aaaatcctgt atatcgtgcg 780

aaaaaggatg gatataccga aaaaatcgct ataatgaccc cgaagcaggg ttatgcagcg 840

gaaaatgcag ctcacggtaa ctgatgccgt atttgcagta ccagcgtacg gcccacagaa 900

tgatgtcacg ctgaaaatgc cggcctttga atgggttcat gtgcagctcc atcagcaaaa 960

ggggatgata agtttatcac caccgactat ttgcaacagt gccgttgatc gtgctatgat 1020

cgactgatgt catcagcggt ggagtgcaat gtcatgaggg aagcggtgat cgccgaagta 1080

tcgactcaac tatcagaggt agttggcgtc atcgagcgcc atctcgaacc gacgttgctg 1140

gccgtacatt tgtacggctc cgcagtggat ggcggcctga agccacacag tgatattgat 1200

ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac 1260

cttttggaaa cttcggcttc ccctggagag agcgagattc tccgcgctgt agaagtcacc 1320

attgttgtgc acgacgacat cattccgtgg cgttatccag ctaagcgcga actgcaattt 1380

ggagaatggc agcgcaatga cattcttgca ggtatcttcg agccagccac gatcgacatt 1440

gatctggcta tcttgctgac aaaagcaaga gaacatagcg ttgccttggt aggtccagcg 1500

gcggaggaac tctttgatcc ggttcctgaa caggatctat ttgaggcgct aaatgaaacc 1560

ttaacgctat ggaactcgcc gcccgactgg gctggcgatg agcgaaatgt agtgcttacg 1620

ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc 1680

gactgggcaa tggagcgcct gccggcccag tatcagcccg tcatacttga agctagacag 1740

gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt 1800

gtccactacg tgaaaggcga gatcaccaag gtagtcggca aataagatgc cgctcgccag 1860

tcgattggct gagctcatga agttcctatt ccgaagttcc tattctctag aaagtatagg 1920

aacttcgaag cagctccagc ctacaaatgt gcctgtcaaa tggacgaagc agggattctg 1980

caaaccctat gctactccgt caagccgtca attgtctgat tcgttaccaa ttatgacaac 2040

ttgacggcta catcattcac tttttcttca caaccggcac ggaactcgct cgggctggcc 2100

ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc aacattgcga 2160

ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg gctgatacgt 2220

tggtcctcgc gccagcttaa gacgctaatc cctaactgct ggcggaaaag atgtgacaga 2280

cgcgacggcg acaagcaaac atgctgtgcg acgctggcga tatcaaaatt gctgtctgcc 2340

aggtgatcgc tgatgtactg acaagcctcg cgtacccgat tatccatcgg tggatggagc 2400

gactcgttaa tcgcttccat gcgccgcagt aacaattgct caagcagatt tatcgccagc 2460

agctccgaat agcgcccttc cccttgcccg gcgttaatga tttgcccaaa caggtcgctg 2520

aaatgcggct ggtgcgcttc atccgggcga aagaaccccg tattggcaaa tattgacggc 2580

cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt aaacccactg gtgataccat 2640

tcgcgagcct ccggatgacg accgtagtga tgaatctctc ctggcgggaa cagcaaaata 2700

tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg accgcgaatg 2760

gtgagattga gaatataacc tttcattccc agcggtcggt cgataaaaaa atcgagataa 2820

ccgttggcct caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc 2880

agcaggggat cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa 2940

accaattgtc catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg 3000

ctaaccaaac cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc 3060

atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat 3120

ttgcacggcg tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct 3180

acctgacgct ttttatcgca actctctact gtttctccat acccgttttt tgggctaaca 3240

ggaggaatta accatgaaaa attgtgtcat cgtcagtgcg gtacgtactg ctatcggtag 3300

ttttaacggt tcactcgctt ccaccagcgc catcgacctg ggggcgacag taattaaagc 3360

cgccattgaa cgtgcaaaaa tcgattcaca acacgttgat gaagtgatta tgggtaacgt 3420

gttacaagcc gggctggggc aaaatccggc gcgtcaggca ctgttaaaaa gcgggctggc 3480

agaaacggtg tgcggattca cggtcaataa agtatgtggt tcgggtctta aaagtgtggc 3540

gcttgccgcc caggccattc aggcaggtca ggcgcagagc attgtggcgg ggggtatgga 3600

aaatatgagt ttagccccct acttactcga tgcaaaagca cgctctggtt atcgtcttgg 3660

agacggacag gtttatgacg taatcctgcg cgatggcctg atgtgcgcca cccatggtta 3720

tcatatgggg attaccgccg aaaacgtggc taaagagtac ggaattaccc gtgaaatgca 3780

ggatgaactg gcgctacatt cacagcgtaa agcggcagcc gcaattgagt ccggtgcttt 3840

tacagccgaa atcgtcccgg taaatgttgt cactcgaaag aaaaccttcg tcttcagtca 3900

agacgagttc ccgaaagcga actcaacggc tgaagcgtta ggtgcattgc gcccggcctt 3960

cgataaagca ggaacagtca ccgctgggaa cgcgtctggt attaacgacg gtgctgccgc 4020

tctggtgatt atggaagaat ctgcggcgct ggcagcaggc cttacccccc tggctcgcat 4080

taaaagttat gccagcggtg gcgtgccccc cgcattgatg ggtatggggc cagtacctgc 4140

cacgcaaaaa gcgttacaac tggcggggct gcaactggcg gatattgatc tcattgaggc 4200

taatgaagca tttgctgcac agttccttgc cgttgggaaa aacctgggct ttgattctga 4260

gaaagtgaat gtcaacggcg gggccatcgc gctcgggcat cctatcggtg ccagtggtgc 4320

tcgtattctg gtcacactat tacatgccat gcaggcacgc gataaaacgc tggggctggc 4380

aacactgtgc attggcggcg gtcagggaat tgcgatggtg attgaacggt tgaattgaaa 4440

agaggagaaa atgacaatag gtatcgataa aataaacttt tacgttccaa agtactatgt 4500

agacatggct aaattagcag aagcacgcca agtagaccca aacaaatttt taattggaat 4560

tggtcaaact gaaatggctg ttagtcctgt aaaccaagac atcgtttcaa tgggcgctaa 4620

cgctgctaag gacattataa cagacgaaga caaaaagaaa attggtatgg taattgtggc 4680

aactgaatca gcagttgatg ctgctaaagc agccgctgtt caaattcaca acttattagg 4740

tattcaacct tttgcacgct gctttgaaat gaaagaagct tgttatgctg caacaccagc 4800

aattcaatta gctaaagatt atttagcaac tagaccgaat gaaaaagtat tagttattgc 4860

tacagataca gcacgttatg gattgaactc aggcggcgag ccaacacaag gtgctggcgc 4920

agttgcgatg gttattgcac ataatccaag actagt 4956

<210> 6

<211> 6042

<212> DNA

<213> 人工序列

<220>

<223> 序列 2

<400> 6

ttaacatcca ttcgcagccg gtacgcatgt aggctggagc tgcttcgaag ttcctatact 60

ttctagagaa taggaacttc gaactgcagg tcgacggatc cccggaataa tgtgcctgtc 120

aaatggacga agcagggatt ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct 180

gattcgttac caattatgac aacttgacgg ctacatcatt cactttttct tcacaaccgg 240

cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag aaatagagtt 300

gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa 360

gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta atccctaact 420

gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt gcgacgctgg 480

cgatatcaaa attgctgtct gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc 540

gattatccat cggtggatgg agcgactcgt taatcgcttc catgcgccgc agtaacaatt 600

gctcaagcag atttatcgcc agcagctccg aatagcgccc ttccccttgc ccggcgttaa 660

tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc 720

ccgtattggc aaatattgac ggccagttaa gccattcatg ccagtaggcg cgcggacgaa 780

agtaaaccca ctggtgatac cattcgcgag cctccggatg acgaccgtag tgatgaatct 840

ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac aaattctcgt ccctgatttt 900

tcaccacccc ctgaccgcga atggtgagat tgagaatata acctttcatt cccagcggtc 960

ggtcgataaa aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc gccaccagat 1020

gggcattaaa cgagtatccc ggcagcaggg gatcattttg cgcttcagcc atacttttca 1080

tactcccgcc attcagagaa gaaaccaatt gtccatattg catcagacat tgccgtcact 1140

gcgtctttta ctggctcttc tcgctaacca aaccggtaac cccgcttatt aaaagcattc 1200

tgtaacaaag cgggaccaaa gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg 1260

gcagaaaagt ccacattgat tatttgcacg gcgtcacact ttgctatgcc atagcatttt 1320

tatccataag attagcggat cctacctgac gctttttatc gcaactctct actgtttctc 1380

catacccgtt ttttgggcta acaggaggaa ttaaccatga aaaattgtgt catcgtcagt 1440

gcggtacgta ctgctatcgg tagttttaac ggttcactcg cttccaccag cgccatcgac 1500

ctgggggcga cagtaattaa agccgccatt gaacgtgcaa aaatcgattc acaacacgtt 1560

gatgaagtga ttatgggtaa cgtgttacaa gccgggctgg ggcaaaatcc ggcgcgtcag 1620

gcactgttaa aaagcgggct ggcagaaacg gtgtgcggat tcacggtcaa taaagtatgt 1680

ggttcgggtc ttaaaagtgt ggcgcttgcc gcccaggcca ttcaggcagg tcaggcgcag 1740

agcattgtgg cggggggtat ggaaaatatg agtttagccc cctacttact cgatgcaaaa 1800

gcacgctctg gttatcgtct tggagacgga caggtttatg acgtaatcct gcgcgatggc 1860

ctgatgtgcg ccacccatgg ttatcatatg gggattaccg ccgaaaacgt ggctaaagag 1920

tacggaatta cccgtgaaat gcaggatgaa ctggcgctac attcacagcg taaagcggca 1980

gccgcaattg agtccggtgc ttttacagcc gaaatcgtcc cggtaaatgt tgtcactcga 2040

aagaaaacct tcgtcttcag tcaagacgag ttcccgaaag cgaactcaac ggctgaagcg 2100

ttaggtgcat tgcgcccggc cttcgataaa gcaggaacag tcaccgctgg gaacgcgtct 2160

ggtattaacg acggtgctgc cgctctggtg attatggaag aatctgcggc gctggcagca 2220

ggccttaccc ccctggctcg cattaaaagt tatgccagcg gtggcgtgcc ccccgcattg 2280

atgggtatgg ggccagtacc tgccacgcaa aaagcgttac aactggcggg gctgcaactg 2340

gcggatattg atctcattga ggctaatgaa gcatttgctg cacagttcct tgccgttggg 2400

aaaaacctgg gctttgattc tgagaaagtg aatgtcaacg gcggggccat cgcgctcggg 2460

catcctatcg gtgccagtgg tgctcgtatt ctggtcacac tattacatgc catgcaggca 2520

cgcgataaaa cgctggggct ggcaacactg tgcattggcg gcggtcaggg aattgcgatg 2580

gtgattgaac ggttgaattg aaaagaggag aaaatgacaa taggtatcga taaaataaac 2640

ttttacgttc caaagtacta tgtagacatg gctaaattag cagaagcacg ccaagtagac 2700

ccaaacaaat ttttaattgg aattggtcaa actgaaatgg ctgttagtcc tgtaaaccaa 2760

gacatcgttt caatgggcgc taacgctgct aaggacatta taacagacga agacaaaaag 2820

aaaattggta tggtaattgt ggcaactgaa tcagcagttg atgctgctaa agcagccgct 2880

gttcaaattc acaacttatt aggtattcaa ccttttgcac gctgctttga aatgaaagaa 2940

gcttgttatg ctgcaacacc agcaattcaa ttagctaaag attatttagc aactagaccg 3000

aatgaaaaag tattagttat tgctacagat acagcacgtt atggattgaa ctcaggcggc 3060

gagccaacac aaggtgctgg cgcagttgcg atggttattg cacataatcc aagcattttg 3120

gcattaaatg aagatgctgt tgcttacact gaagacgttt atgatttctg gcgtccaact 3180

ggacataaat atccattagt tgatggtgca ttatctaaag atgcttatat ccgctcattc 3240

caacaaagct ggaatgaata cgcaaaacgt caaggtaagt cgctagctga cttcgcatct 3300

ctatgcttcc atgttccatt tacaaaaatg ggtaaaaagg cattagagtc aatcattgat 3360

aacgctgatg aaacaactca agagcgttta cgttcaggat atgaagatgc tgtagattat 3420

aaccgttatg tcggtaatat ttatactgga tcattatatt taagcctaat atcattactt 3480

gaaaatcgag atttacaagc tggtgaaaca atcggtttat tcagttatgg ctcaggttca 3540

gttggtgaat tttatagtgc gacattagtt gaaggctaca aagatcattt agatcaagct 3600

gcacataaag cattattaaa taaccgtact gaagtatctg ttgatgcata tgaaacattc 3660

ttcaaacgtt ttgatgacgt tgaatttgac gaagaacaag atgctgttca tgaagatcgt 3720

catattttct acttatcaaa tattgaaaat aacgttcgcg aatatcacag accagagtaa 3780

aaagaggaga aaatgtccat gcaaagttta gataagaatt ttcgacattt atctcgtaaa 3840

gaaaagttac aacaattggt tgataagcaa tggttatcag aagaacaatt cgacatttta 3900

ctgaatcatc cattaatcga tgaagaagta gccaatagtt taattgaaaa tgtcatcgcg 3960

caaggtgcat tacccgttgg attattaccg aatatcattg tggacgataa ggcatatgtt 4020

gtacctatga tggtggaaga gccttcagtt gtcgctgcag ctagttatgg tgcaaagcta 4080

gtgaatcaga ctggcggatt taaaacggta tcttctgaac gtattatgat aggtcaaatc 4140

gtctttgatg gcgttgacga tactgaaaaa ttatcagcag acattaaagc tttagaaaag 4200

caaattcata aaattgcgga tgaggcatat ccttctatta aagcgcgtgg tggtggttac 4260

caacgtatag cgattgatac atttcctgag caacagttac tatctttaaa agtatttgtt 4320

gatacgaaag atgctatggg cgctaatatg cttaatacga ttttagaggc cataactgca 4380

tttttaaaaa atgaatttcc gcaaagcgac attttaatga gtattttatc caatcatgca 4440

acagcgtccg ttgttaaagt tcaaggcgaa attgatgtta aagatttagc aaggggcgag 4500

agaactggag aagaggttgc caaacgaatg gaacgtgctt ctgtattggc ccaagtagat 4560

attcatcgtg cagcaacaca taataaaggt gttatgaatg gcatacatgc tgttgtttta 4620

gcaacaggaa atgatacgcg tggtgcagaa gcaagtgcgc atgcatacgc gagtcgtgac 4680

ggacagtatc gtggtattgc tacatggcgt tacgatcaag atcgtcaacg attgattggt 4740

acaattgaag tgcctatgac attggcaatc gttggcggtg gtacaaaagt attaccaatt 4800

gctaaagctt cattagagct actaaatgta gagtcagcac aagaattagg tcatgtagtt 4860

gctgccgttg gtttagcgca aaactttgca gcatgtcgcg cgcttgtgtc agaaggtatt 4920

caacaaggtc atatgagttt acaatataaa tcattagcta tcgttgtagg ggcaaaaggt 4980

gatgaaattg ctaaagtagc tgaagctttg aaaaaagaac cccgtgcaaa tacacaagca 5040

gcggaacata ttttacaaga aattagacaa caataaaaag aggagaaaat gcaaacggaa 5100

cacgtcattt tattgaatgc acagggagtt cccacgggta cgctggaaaa gtatgccgca 5160

cacacggcag acacccgctt acatctcgcg ttctccagtt ggctgtttaa tgccaaagga 5220

caattattag ttacccgccg cgcactgagc aaaaaagcat ggcctggcgt gtggactaac 5280

tcggtttgtg ggcacccaca actgggagaa agcaacgaag acgcagtgat ccgccgttgc 5340

cgttatgagc ttggcgtgga aattacgcct cctgaatcta tctatcctga ctttcgctac 5400

cgcgccaccg atccgagtgg cattgtggaa aatgaagtgt gtccggtatt tgccgcacgc 5460

accactagtg cgttacagat caatgatgat gaagtgatgg attatcaatg gtgtgattta 5520

gcagatgtat tacacggtat tgatgccacg ccgtgggcgt tcagtccgtg gatggtgatg 5580

caggcgacaa atcgcgaagc cagaaaacga ttatctgcat ttacccagct taaataatcg 5640

gtctgcagct ggtgccgcgc ggcagccacc accaccacca ccactaatac agattaaatc 5700

agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc 5760

acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc 5820

tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag 5880

actgggcctt ctatacgaag ttattcgtcg acctaattct ctcgcgagga gaggccttcg 5940

cctgatgata agttcaagtt tgcttcagaa tattcgaaat ctgttgaact atcattgaac 6000

tgtaggccgg atgtggcgtt ttcgccgcat ccggcaacgt ac 6042

<210> 7

<211> 25

<212> DNA

<213> 人工序列

<220>

<223> RBS 序列

<400> 7

actagagaaa gaggagaaat actag 25

<210> 8

<211> 35

<212> DNA

<213> 人工序列

<220>

<223> p119 启动子

<400> 8

ttgacagcta gctcagtcct aggtataatg ctagc 35

<210> 9

<211> 273

<212> DNA

<213> 人工序列

<220>

<223> pBAD 启动子

<400> 9

aaaccaattg tccatattgc atcagacatt gccgtcactg cgtcttttac tggctcttct 60

cgctaaccaa accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag 120

ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg cagaaaagtc cacattgatt 180

atttgcacgg cgtcacactt tgctatgcca tagcattttt atccataaga ttagcggatc 240

ctacctgacg ctttttatcg caactctcta ctg 273

<210> 10

<211> 1332

<212> DNA

<213> 人工序列

<220>

<223> MK 基因

<400> 10

atgtctctgc cattcctgac gtctgcgcca ggtaaggtga tcatcttcgg cgagcactct 60

gcggtgtaca ataagccggc cgtcgccgcc tctgtgtctg cgttacgcac ctacctgctg 120

atcagcgaat cttctgcacc ggacacgatc gagctggact ttccggacat cagcttcaac 180

cacaagtgga gcatcaacga cttcaacgcg atcacggagg accaggtgaa cagccaaaag 240

ctggccaaag cccagcaagc aaccgacggt ctgtctcagg agctggtgtc tctgctggac 300

ccgctgttag cgcagttaag cgagagcttc cattaccacg ccgcgttctg cttcctgtac 360

atgttcgttt gcctgtgccc gcacgcaaag aacatcaagt tcagcctgaa gagcacgctg 420

ccgattggcg caggcttagg ctctagcgca tctatcagcg tgagcctggc gctggcgatg 480

gcctatctgg gtggcctgat tggcagcaac gacctggaga aactgagcga aaacgacaag 540

cacatcgtga accagtgggc ctttatcggc gagaagtgca ttcatggcac cccgagcggc 600

attgacaacg cagttgccac gtatggcaac gccctgctgt tcgagaaaga cagccacaac 660

ggcacgatca acacgaacaa cttcaagttc ctggacgact tcccggcgat cccgatgatt 720

ctgacctaca cccgtatccc acgcagcacc aaggatttag tcgcccgcgt gcgtgtttta 780

gtcaccgaaa agttcccgga ggtgatgaag ccgatcctgg acgcgatggg cgagtgcgcg 840

ctgcagggtc tggagatcat gaccaagctg agcaagtgca agggcaccga cgatgaggcg 900

gtggagacca acaatgagct gtacgagcag ctgctggagc tgatccgtat caatcacggc 960

ctgctggtct ctatcggtgt gtctcacccg ggcctggaac tgatcaaaaa cctgagcgac 1020

gacctgcgca ttggctctac gaaattaacg ggtgcaggtg gcggtggctg ctctttaacg 1080

ctgctgcgcc gtgacattac gcaggagcaa atcgacagct tcaagaagaa gctgcaggac 1140

gacttcagct acgagacgtt cgagacggac ctgggcggca cgggctgttg cctgctgagc 1200

gccaaaaatc tgaacaagga cctgaagatc aaaagcctgg tgttccagct gttcgaaaac 1260

aagacgacca cgaagcagca gatcgacgac ctgttactgc cgggtaacac caatctgccg 1320

tggacgtctt aa 1332

<210> 11

<211> 443

<212> PRT

<213> 人工序列

<220>

<223> MK

<400> 11

Met Ser Leu Pro Phe Leu Thr Ser Ala Pro Gly Lys Val Ile Ile Phe

1 5 10 15

Gly Glu His Ser Ala Val Tyr Asn Lys Pro Ala Val Ala Ala Ser Val

20 25 30

Ser Ala Leu Arg Thr Tyr Leu Leu Ile Ser Glu Ser Ser Ala Pro Asp

35 40 45

Thr Ile Glu Leu Asp Phe Pro Asp Ile Ser Phe Asn His Lys Trp Ser

50 55 60

Ile Asn Asp Phe Asn Ala Ile Thr Glu Asp Gln Val Asn Ser Gln Lys

65 70 75 80

Leu Ala Lys Ala Gln Gln Ala Thr Asp Gly Leu Ser Gln Glu Leu Val

85 90 95

Ser Leu Leu Asp Pro Leu Leu Ala Gln Leu Ser Glu Ser Phe His Tyr

100 105 110

His Ala Ala Phe Cys Phe Leu Tyr Met Phe Val Cys Leu Cys Pro His

115 120 125

Ala Lys Asn Ile Lys Phe Ser Leu Lys Ser Thr Leu Pro Ile Gly Ala

130 135 140

Gly Leu Gly Ser Ser Ala Ser Ile Ser Val Ser Leu Ala Leu Ala Met

145 150 155 160

Ala Tyr Leu Gly Gly Leu Ile Gly Ser Asn Asp Leu Glu Lys Leu Ser

165 170 175

Glu Asn Asp Lys His Ile Val Asn Gln Trp Ala Phe Ile Gly Glu Lys

180 185 190

Cys Ile His Gly Thr Pro Ser Gly Ile Asp Asn Ala Val Ala Thr Tyr

195 200 205

Gly Asn Ala Leu Leu Phe Glu Lys Asp Ser His Asn Gly Thr Ile Asn

210 215 220

Thr Asn Asn Phe Lys Phe Leu Asp Asp Phe Pro Ala Ile Pro Met Ile

225 230 235 240

Leu Thr Tyr Thr Arg Ile Pro Arg Ser Thr Lys Asp Leu Val Ala Arg

245 250 255

Val Arg Val Leu Val Thr Glu Lys Phe Pro Glu Val Met Lys Pro Ile

260 265 270

Leu Asp Ala Met Gly Glu Cys Ala Leu Gln Gly Leu Glu Ile Met Thr

275 280 285

Lys Leu Ser Lys Cys Lys Gly Thr Asp Asp Glu Ala Val Glu Thr Asn

290 295 300

Asn Glu Leu Tyr Glu Gln Leu Leu Glu Leu Ile Arg Ile Asn His Gly

305 310 315 320

Leu Leu Val Ser Ile Gly Val Ser His Pro Gly Leu Glu Leu Ile Lys

325 330 335

Asn Leu Ser Asp Asp Leu Arg Ile Gly Ser Thr Lys Leu Thr Gly Ala

340 345 350

Gly Gly Gly Gly Cys Ser Leu Thr Leu Leu Arg Arg Asp Ile Thr Gln

355 360 365

Glu Gln Ile Asp Ser Phe Lys Lys Lys Leu Gln Asp Asp Phe Ser Tyr

370 375 380

Glu Thr Phe Glu Thr Asp Leu Gly Gly Thr Gly Cys Cys Leu Leu Ser

385 390 395 400

Ala Lys Asn Leu Asn Lys Asp Leu Lys Ile Lys Ser Leu Val Phe Gln

405 410 415

Leu Phe Glu Asn Lys Thr Thr Thr Lys Gln Gln Ile Asp Asp Leu Leu

420 425 430

Leu Pro Gly Asn Thr Asn Leu Pro Trp Thr Ser

435 440

<210> 12

<211> 1356

<212> DNA

<213> 人工序列

<220>

<223> PMK 基因

<400> 12

atgagcgaat tacgtgcatt cagcgcgcca ggtaaggcac tgctggccgg tggctacctg 60

gtgttagaca ccaagtacga ggcgttcgtc gtcggcttat ctgcccgtat gcatgcagtt 120

gcccacccgt atggtagcct gcagggctct gacaagttcg aagtgcgtgt gaagagcaag 180

cagttcaagg acggcgagtg gctgtaccac attagcccaa agagcggctt catcccggtt 240

agcattggtg gcagcaagaa cccatttatc gagaaggtca ttgccaacgt cttcagctac 300

ttcaagccga atatggacga ttactgcaac cgcaacctgt tcgtcatcga cattttcagc 360

gacgacgcgt accacagcca agaggactct gttacggagc atcgtggtaa ccgccgcctg 420

agcttccaca gccatcgcat tgaggaggtg ccgaagacgg gtctgggttc tagcgccggt 480

ttagttaccg tcttaacgac ggcgttagcg agcttcttcg tgagcgacct ggagaacaac 540

gtggacaagt accgcgaagt gattcataac ctggcgcagg tggcacattg tcaggcccaa 600

ggtaagattg gctctggttt tgatgtggca gcggccgcct atggctctat ccgctatcgc 660

cgctttccgc cggccctgat cagcaatctg ccggacatcg gctctgcgac gtatggtagc 720

aaactggcgc atctggtgga cgaagaagac tggaacatca ccattaagtc taatcacctg 780

ccgagcggct taacgttatg gatgggcgat atcaagaacg gcagcgaaac ggttaagctg 840

gtgcagaaag tgaaaaactg gtacgacagc cacatgccgg aaagcctgaa gatttacacg 900

gagctggacc acgccaatag ccgtttcatg gatggtctga gcaagctgga ccgcctgcac 960

gaaacccacg acgactacag cgaccaaatc ttcgagagcc tggagcgcaa tgactgcacc 1020

tgccagaagt acccggagat cacggaggtc cgcgatgccg tggcaacgat tcgccgtagc 1080

ttccgcaaaa ttacgaagga gagcggcgcg gatatcgaac caccggtcca gacgtctctg 1140

ctggacgact gtcaaacctt aaagggcgtg ttaacgtgcc tgattccggg cgcgggtggt 1200

tacgacgcca ttgccgtcat cacgaaacag gacgtcgatc tgcgcgcaca aacggccaac 1260

gacaaacgtt tcagcaaagt ccaatggctg gatgttacgc aggccgactg gggtgttcgc 1320

aaggagaagg acccggaaac gtatctggat aagtga 1356

<210> 13

<211> 451

<212> PRT

<213> 人工序列

<220>

<223> PMK

<400> 13

Met Ser Glu Leu Arg Ala Phe Ser Ala Pro Gly Lys Ala Leu Leu Ala

1 5 10 15

Gly Gly Tyr Leu Val Leu Asp Thr Lys Tyr Glu Ala Phe Val Val Gly

20 25 30

Leu Ser Ala Arg Met His Ala Val Ala His Pro Tyr Gly Ser Leu Gln

35 40 45

Gly Ser Asp Lys Phe Glu Val Arg Val Lys Ser Lys Gln Phe Lys Asp

50 55 60

Gly Glu Trp Leu Tyr His Ile Ser Pro Lys Ser Gly Phe Ile Pro Val

65 70 75 80

Ser Ile Gly Gly Ser Lys Asn Pro Phe Ile Glu Lys Val Ile Ala Asn

85 90 95

Val Phe Ser Tyr Phe Lys Pro Asn Met Asp Asp Tyr Cys Asn Arg Asn

100 105 110

Leu Phe Val Ile Asp Ile Phe Ser Asp Asp Ala Tyr His Ser Gln Glu

115 120 125

Asp Ser Val Thr Glu His Arg Gly Asn Arg Arg Leu Ser Phe His Ser

130 135 140

His Arg Ile Glu Glu Val Pro Lys Thr Gly Leu Gly Ser Ser Ala Gly

145 150 155 160

Leu Val Thr Val Leu Thr Thr Ala Leu Ala Ser Phe Phe Val Ser Asp

165 170 175

Leu Glu Asn Asn Val Asp Lys Tyr Arg Glu Val Ile His Asn Leu Ala

180 185 190

Gln Val Ala His Cys Gln Ala Gln Gly Lys Ile Gly Ser Gly Phe Asp

195 200 205

Val Ala Ala Ala Ala Tyr Gly Ser Ile Arg Tyr Arg Arg Phe Pro Pro

210 215 220

Ala Leu Ile Ser Asn Leu Pro Asp Ile Gly Ser Ala Thr Tyr Gly Ser

225 230 235 240

Lys Leu Ala His Leu Val Asp Glu Glu Asp Trp Asn Ile Thr Ile Lys

245 250 255

Ser Asn His Leu Pro Ser Gly Leu Thr Leu Trp Met Gly Asp Ile Lys

260 265 270

Asn Gly Ser Glu Thr Val Lys Leu Val Gln Lys Val Lys Asn Trp Tyr

275 280 285

Asp Ser His Met Pro Glu Ser Leu Lys Ile Tyr Thr Glu Leu Asp His

290 295 300

Ala Asn Ser Arg Phe Met Asp Gly Leu Ser Lys Leu Asp Arg Leu His

305 310 315 320

Glu Thr His Asp Asp Tyr Ser Asp Gln Ile Phe Glu Ser Leu Glu Arg

325 330 335

Asn Asp Cys Thr Cys Gln Lys Tyr Pro Glu Ile Thr Glu Val Arg Asp

340 345 350

Ala Val Ala Thr Ile Arg Arg Ser Phe Arg Lys Ile Thr Lys Glu Ser

355 360 365

Gly Ala Asp Ile Glu Pro Pro Val Gln Thr Ser Leu Leu Asp Asp Cys

370 375 380

Gln Thr Leu Lys Gly Val Leu Thr Cys Leu Ile Pro Gly Ala Gly Gly

385 390 395 400

Tyr Asp Ala Ile Ala Val Ile Thr Lys Gln Asp Val Asp Leu Arg Ala

405 410 415

Gln Thr Ala Asn Asp Lys Arg Phe Ser Lys Val Gln Trp Leu Asp Val

420 425 430

Thr Gln Ala Asp Trp Gly Val Arg Lys Glu Lys Asp Pro Glu Thr Tyr

435 440 445

Leu Asp Lys

450

<210> 14

<211> 1191

<212> DNA

<213> 人工序列

<220>

<223> PMD 基因

<400> 14

atgaccgttt acacagcatc cgttaccgca cccgtcaaca tcgcaaccct taagtattgg 60

gggaaaaggg acacgaagtt gaatctgccc accaattcgt ccatatcagt gactttatcg 120

caagatgacc tcagaacgtt gacctctgcg gctactgcac ctgagtttga acgcgacact 180

ttgtggttaa atggagaacc acacagcatc gacaatgaaa gaactcaaaa ttgtctgcgc 240

gacctacgcc aattaagaaa ggaaatggaa tcgaaggacg cctcattgcc cacattatct 300

caatggaaac tccacattgt ctccgaaaat aactttccta cagcagctgg tttagcttcc 360

tccgctgctg gctttgctgc attggtctct gcaattgcta agttatacca attaccacag 420

tcaacttcag aaatatctag aatagcaaga aaggggtctg gttcagcttg tagatcgttg 480

tttggcggat acgtggcctg ggaaatggga aaagctgaag atggtcatga ttccatggca 540

gtacaaatcg cagacagctc tgactggcct cagatgaaag cttgtgtcct agttgtcagc 600

gatattaaaa aggatgtgag ttccactcag ggtatgcaat tgaccgtggc aacctccgaa 660

ctatttaaag aaagaattga acatgtcgta ccaaagagat ttgaagtcat gcgtaaagcc 720

attgttgaaa aagatttcgc cacctttgca aaggaaacaa tgatggattc caactctttc 780

catgccacat gtttggactc tttccctcca atattctaca tgaatgacac ttccaagcgt 840

atcatcagtt ggtgccacac cattaatcag ttttacggag aaacaatcgt tgcatacacg 900

tttgatgcag gtccaaatgc tgtgttgtac tacttagctg aaaatgagtc gaaactcttt 960

gcatttatct ataaattgtt tggctctgtt cctggatggg acaagaaatt tactactgag 1020

cagcttgagg ctttcaacca tcaatttgaa tcatctaact ttactgcacg tgaattggat 1080

cttgagttgc aaaaggatgt tgccagagtg attttaactc aagtcggttc aggcccacaa 1140

gaaacaaacg aatctttgat tgacgcaaag actggtctac caaaggaata a 1191

<210> 15

<211> 396

<212> PRT

<213> 人工序列

<220>

<223> PMD

<400> 15

Met Thr Val Tyr Thr Ala Ser Val Thr Ala Pro Val Asn Ile Ala Thr

1 5 10 15

Leu Lys Tyr Trp Gly Lys Arg Asp Thr Lys Leu Asn Leu Pro Thr Asn

20 25 30

Ser Ser Ile Ser Val Thr Leu Ser Gln Asp Asp Leu Arg Thr Leu Thr

35 40 45

Ser Ala Ala Thr Ala Pro Glu Phe Glu Arg Asp Thr Leu Trp Leu Asn

50 55 60

Gly Glu Pro His Ser Ile Asp Asn Glu Arg Thr Gln Asn Cys Leu Arg

65 70 75 80

Asp Leu Arg Gln Leu Arg Lys Glu Met Glu Ser Lys Asp Ala Ser Leu

85 90 95

Pro Thr Leu Ser Gln Trp Lys Leu His Ile Val Ser Glu Asn Asn Phe

100 105 110

Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ala Gly Phe Ala Ala Leu

115 120 125

Val Ser Ala Ile Ala Lys Leu Tyr Gln Leu Pro Gln Ser Thr Ser Glu

130 135 140

Ile Ser Arg Ile Ala Arg Lys Gly Ser Gly Ser Ala Cys Arg Ser Leu

145 150 155 160

Phe Gly Gly Tyr Val Ala Trp Glu Met Gly Lys Ala Glu Asp Gly His

165 170 175

Asp Ser Met Ala Val Gln Ile Ala Asp Ser Ser Asp Trp Pro Gln Met

180 185 190

Lys Ala Cys Val Leu Val Val Ser Asp Ile Lys Lys Asp Val Ser Ser

195 200 205

Thr Gln Gly Met Gln Leu Thr Val Ala Thr Ser Glu Leu Phe Lys Glu

210 215 220

Arg Ile Glu His Val Val Pro Lys Arg Phe Glu Val Met Arg Lys Ala

225 230 235 240

Ile Val Glu Lys Asp Phe Ala Thr Phe Ala Lys Glu Thr Met Met Asp

245 250 255

Ser Asn Ser Phe His Ala Thr Cys Leu Asp Ser Phe Pro Pro Ile Phe

260 265 270

Tyr Met Asn Asp Thr Ser Lys Arg Ile Ile Ser Trp Cys His Thr Ile

275 280 285

Asn Gln Phe Tyr Gly Glu Thr Ile Val Ala Tyr Thr Phe Asp Ala Gly

290 295 300

Pro Asn Ala Val Leu Tyr Tyr Leu Ala Glu Asn Glu Ser Lys Leu Phe

305 310 315 320

Ala Phe Ile Tyr Lys Leu Phe Gly Ser Val Pro Gly Trp Asp Lys Lys

325 330 335

Phe Thr Thr Glu Gln Leu Glu Ala Phe Asn His Gln Phe Glu Ser Ser

340 345 350

Asn Phe Thr Ala Arg Glu Leu Asp Leu Glu Leu Gln Lys Asp Val Ala

355 360 365

Arg Val Ile Leu Thr Gln Val Gly Ser Gly Pro Gln Glu Thr Asn Glu

370 375 380

Ser Leu Ile Asp Ala Lys Thr Gly Leu Pro Lys Glu

385 390 395

<210> 16

<211> 549

<212> DNA

<213> 人工序列

<220>

<223> idi 基因

<400> 16

atgcaaacgg aacacgtcat tttattgaat gcacagggag ttcccacggg tacgctggaa 60

aagtatgccg cacacacggc agacacccgc ttacatctcg cgttctccag ttggctgttt 120

aatgccaaag gacaattatt agttacccgc cgcgcactga gcaaaaaagc atggcctggc 180

gtgtggacta actcggtttg tgggcaccca caactgggag aaagcaacga agacgcagtg 240

atccgccgtt gccgttatga gcttggcgtg gaaattacgc ctcctgaatc tatctatcct 300

gactttcgct accgcgccac cgatccgagt ggcattgtgg aaaatgaagt gtgtccggta 360

tttgccgcac gcaccactag tgcgttacag atcaatgatg atgaagtgat ggattatcaa 420

tggtgtgatt tagcagatgt attacacggt attgatgcca cgccgtgggc gttcagtccg 480

tggatggtga tgcaggcgac aaatcgcgaa gccagaaaac gattatctgc atttacccag 540

cttaaataa 549

<210> 17

<211> 182

<212> PRT

<213> 人工序列

<220>

<223> idi

<400> 17

Met Gln Thr Glu His Val Ile Leu Leu Asn Ala Gln Gly Val Pro Thr

1 5 10 15

Gly Thr Leu Glu Lys Tyr Ala Ala His Thr Ala Asp Thr Arg Leu His

20 25 30

Leu Ala Phe Ser Ser Trp Leu Phe Asn Ala Lys Gly Gln Leu Leu Val

35 40 45

Thr Arg Arg Ala Leu Ser Lys Lys Ala Trp Pro Gly Val Trp Thr Asn

50 55 60

Ser Val Cys Gly His Pro Gln Leu Gly Glu Ser Asn Glu Asp Ala Val

65 70 75 80

Ile Arg Arg Cys Arg Tyr Glu Leu Gly Val Glu Ile Thr Pro Pro Glu

85 90 95

Ser Ile Tyr Pro Asp Phe Arg Tyr Arg Ala Thr Asp Pro Ser Gly Ile

100 105 110

Val Glu Asn Glu Val Cys Pro Val Phe Ala Ala Arg Thr Thr Ser Ala

115 120 125

Leu Gln Ile Asn Asp Asp Glu Val Met Asp Tyr Gln Trp Cys Asp Leu

130 135 140

Ala Asp Val Leu His Gly Ile Asp Ala Thr Pro Trp Ala Phe Ser Pro

145 150 155 160

Trp Met Val Met Gln Ala Thr Asn Arg Glu Ala Arg Lys Arg Leu Ser

165 170 175

Ala Phe Thr Gln Leu Lys

180

<210> 18

<211> 1059

<212> DNA

<213> 人工序列

<220>

<223> ERG20 基因

<400> 18

atggccagtg aaaaagaaat ccgtcgcgag cgctttctga acgtgttccc gaaactggtg 60

gaagaactca acgccagtct gctggcctat ggtatgccga aagaagcgtg cgactggtac 120

gcgcacagtc tgaattacaa caccccgggc ggcaagctga atcgtggtct gagcgttgtg 180

gacacgtacg cgattctgag caacaaaacc gtggagcagc tgggccaaga agaatatgag 240

aaggtggcca ttctgggctg gtgtatcgaa ctgctgcaag cctactggct ggttgcggac 300

gacatgatgg acaagagcat tacccgccgc ggccagccgt gttggtacaa agttccggag 360

gttggcgaga tcgccatctg ggatgcgttt atgctggaag cggccatcta caagctgctc 420

aagagccact ttcgcaacga gaagtactac atcgacatca cggagctgtt ccatgaggtg 480

acgtttcaga ccgagctcgg ccagctcatg gatctgatca ccgccccgga ggacaaagtt 540

gacctcagca agttcagtct gaaaaaacac agctttatcg tgaccttcaa gacggcctac 600

tacagctttt atctgccagt tgcgctcgcg atgtatgttg ccggcatcac cgacgagaag 660

gatctgaaac aagcccgcga tgtgctgatt ccgctgggtg agtacttcca gatccaagat 720

gactatctgg actgtttcgg caccccggaa cagattggca agattggcac cgacatccaa 780

gataacaaat gcagctgggt gatcaacaag gcgctggaac tcgccagtgc ggaacagcgc 840

aaaacgctcg acgagaatta cggcaaaaaa gacagtgtgg ccgaagcgaa gtgcaagaaa 900

atctttaacg atctgaagat tgagcagctc taccacgaat acgaggagag cattgccaag 960

gatctcaagg ccaagatcag tcaagttgac gagagtcgcg gcttcaaagc cgacgtgctc 1020

acggcctttc tgaacaaggt ttacaaacgc agcaaataa 1059

<210> 19

<211> 352

<212> PRT

<213> 人工序列

<220>

<223> ERG20

<400> 19

Met Ala Ser Glu Lys Glu Ile Arg Arg Glu Arg Phe Leu Asn Val Phe

1 5 10 15

Pro Lys Leu Val Glu Glu Leu Asn Ala Ser Leu Leu Ala Tyr Gly Met

20 25 30

Pro Lys Glu Ala Cys Asp Trp Tyr Ala His Ser Leu Asn Tyr Asn Thr

35 40 45

Pro Gly Gly Lys Leu Asn Arg Gly Leu Ser Val Val Asp Thr Tyr Ala

50 55 60

Ile Leu Ser Asn Lys Thr Val Glu Gln Leu Gly Gln Glu Glu Tyr Glu

65 70 75 80

Lys Val Ala Ile Leu Gly Trp Cys Ile Glu Leu Leu Gln Ala Tyr Trp

85 90 95

Leu Val Ala Asp Asp Met Met Asp Lys Ser Ile Thr Arg Arg Gly Gln

100 105 110

Pro Cys Trp Tyr Lys Val Pro Glu Val Gly Glu Ile Ala Ile Trp Asp

115 120 125

Ala Phe Met Leu Glu Ala Ala Ile Tyr Lys Leu Leu Lys Ser His Phe

130 135 140

Arg Asn Glu Lys Tyr Tyr Ile Asp Ile Thr Glu Leu Phe His Glu Val

145 150 155 160

Thr Phe Gln Thr Glu Leu Gly Gln Leu Met Asp Leu Ile Thr Ala Pro

165 170 175

Glu Asp Lys Val Asp Leu Ser Lys Phe Ser Leu Lys Lys His Ser Phe

180 185 190

Ile Val Thr Phe Lys Thr Ala Tyr Tyr Ser Phe Tyr Leu Pro Val Ala

195 200 205

Leu Ala Met Tyr Val Ala Gly Ile Thr Asp Glu Lys Asp Leu Lys Gln

210 215 220

Ala Arg Asp Val Leu Ile Pro Leu Gly Glu Tyr Phe Gln Ile Gln Asp

225 230 235 240

Asp Tyr Leu Asp Cys Phe Gly Thr Pro Glu Gln Ile Gly Lys Ile Gly

245 250 255

Thr Asp Ile Gln Asp Asn Lys Cys Ser Trp Val Ile Asn Lys Ala Leu

260 265 270

Glu Leu Ala Ser Ala Glu Gln Arg Lys Thr Leu Asp Glu Asn Tyr Gly

275 280 285

Lys Lys Asp Ser Val Ala Glu Ala Lys Cys Lys Lys Ile Phe Asn Asp

290 295 300

Leu Lys Ile Glu Gln Leu Tyr His Glu Tyr Glu Glu Ser Ile Ala Lys

305 310 315 320

Asp Leu Lys Ala Lys Ile Ser Gln Val Asp Glu Ser Arg Gly Phe Lys

325 330 335

Ala Asp Val Leu Thr Ala Phe Leu Asn Lys Val Tyr Lys Arg Ser Lys

340 345 350

<210> 20

<211> 1185

<212> DNA

<213> 人工序列

<220>

<223> AtoB 基因

<400> 20

atgaaaaatt gtgtcatcgt cagtgcggta cgtactgcta tcggtagttt taacggttca 60

ctcgcttcca ccagcgccat cgacctgggg gcgacagtaa ttaaagccgc cattgaacgt 120

gcaaaaatcg attcacaaca cgttgatgaa gtgattatgg gtaacgtgtt acaagccggg 180

ctggggcaaa atccggcgcg tcaggcactg ttaaaaagcg ggctggcaga aacggtgtgc 240

ggattcacgg tcaataaagt atgtggttcg ggtcttaaaa gtgtggcgct tgccgcccag 300

gccattcagg caggtcaggc gcagagcatt gtggcggggg gtatggaaaa tatgagttta 360

gccccctact tactcgatgc aaaagcacgc tctggttatc gtcttggaga cggacaggtt 420

tatgacgtaa tcctgcgcga tggcctgatg tgcgccaccc atggttatca tatggggatt 480

accgccgaaa acgtggctaa agagtacgga attacccgtg aaatgcagga tgaactggcg 540

ctacattcac agcgtaaagc ggcagccgca attgagtccg gtgcttttac agccgaaatc 600

gtcccggtaa atgttgtcac tcgaaagaaa accttcgtct tcagtcaaga cgagttcccg 660

aaagcgaact caacggctga agcgttaggt gcattgcgcc cggccttcga taaagcagga 720

acagtcaccg ctgggaacgc gtctggtatt aacgacggtg ctgccgctct ggtgattatg 780

gaagaatctg cggcgctggc agcaggcctt acccccctgg ctcgcattaa aagttatgcc 840

agcggtggcg tgccccccgc attgatgggt atggggccag tacctgccac gcaaaaagcg 900

ttacaactgg cggggctgca actggcggat attgatctca ttgaggctaa tgaagcattt 960

gctgcacagt tccttgccgt tgggaaaaac ctgggctttg attctgagaa agtgaatgtc 1020

aacggcgggg ccatcgcgct cgggcatcct atcggtgcca gtggtgctcg tattctggtc 1080

acactattac atgccatgca ggcacgcgat aaaacgctgg ggctggcaac actgtgcatt 1140

ggcggcggtc agggaattgc gatggtgatt gaacggttga attga 1185

<210> 21

<211> 394

<212> PRT

<213> 人工序列

<220>

<223> AtoB

<400> 21

Met Lys Asn Cys Val Ile Val Ser Ala Val Arg Thr Ala Ile Gly Ser

1 5 10 15

Phe Asn Gly Ser Leu Ala Ser Thr Ser Ala Ile Asp Leu Gly Ala Thr

20 25 30

Val Ile Lys Ala Ala Ile Glu Arg Ala Lys Ile Asp Ser Gln His Val

35 40 45

Asp Glu Val Ile Met Gly Asn Val Leu Gln Ala Gly Leu Gly Gln Asn

50 55 60

Pro Ala Arg Gln Ala Leu Leu Lys Ser Gly Leu Ala Glu Thr Val Cys

65 70 75 80

Gly Phe Thr Val Asn Lys Val Cys Gly Ser Gly Leu Lys Ser Val Ala

85 90 95

Leu Ala Ala Gln Ala Ile Gln Ala Gly Gln Ala Gln Ser Ile Val Ala

100 105 110

Gly Gly Met Glu Asn Met Ser Leu Ala Pro Tyr Leu Leu Asp Ala Lys

115 120 125

Ala Arg Ser Gly Tyr Arg Leu Gly Asp Gly Gln Val Tyr Asp Val Ile

130 135 140

Leu Arg Asp Gly Leu Met Cys Ala Thr His Gly Tyr His Met Gly Ile

145 150 155 160

Thr Ala Glu Asn Val Ala Lys Glu Tyr Gly Ile Thr Arg Glu Met Gln

165 170 175

Asp Glu Leu Ala Leu His Ser Gln Arg Lys Ala Ala Ala Ala Ile Glu

180 185 190

Ser Gly Ala Phe Thr Ala Glu Ile Val Pro Val Asn Val Val Thr Arg

195 200 205

Lys Lys Thr Phe Val Phe Ser Gln Asp Glu Phe Pro Lys Ala Asn Ser

210 215 220

Thr Ala Glu Ala Leu Gly Ala Leu Arg Pro Ala Phe Asp Lys Ala Gly

225 230 235 240

Thr Val Thr Ala Gly Asn Ala Ser Gly Ile Asn Asp Gly Ala Ala Ala

245 250 255

Leu Val Ile Met Glu Glu Ser Ala Ala Leu Ala Ala Gly Leu Thr Pro

260 265 270

Leu Ala Arg Ile Lys Ser Tyr Ala Ser Gly Gly Val Pro Pro Ala Leu

275 280 285

Met Gly Met Gly Pro Val Pro Ala Thr Gln Lys Ala Leu Gln Leu Ala

290 295 300

Gly Leu Gln Leu Ala Asp Ile Asp Leu Ile Glu Ala Asn Glu Ala Phe

305 310 315 320

Ala Ala Gln Phe Leu Ala Val Gly Lys Asn Leu Gly Phe Asp Ser Glu

325 330 335

Lys Val Asn Val Asn Gly Gly Ala Ile Ala Leu Gly His Pro Ile Gly

340 345 350

Ala Ser Gly Ala Arg Ile Leu Val Thr Leu Leu His Ala Met Gln Ala

355 360 365

Arg Asp Lys Thr Leu Gly Leu Ala Thr Leu Cys Ile Gly Gly Gly Gln

370 375 380

Gly Ile Ala Met Val Ile Glu Arg Leu Asn

385 390

<210> 22

<211> 1167

<212> DNA

<213> 人工序列

<220>

<223> HMGS 基因

<400> 22

atgacaatag gtatcgataa aataaacttt tacgttccaa agtactatgt agacatggct 60

aaattagcag aagcacgcca agtagaccca aacaaatttt taattggaat tggtcaaact 120

gaaatggctg ttagtcctgt aaaccaagac atcgtttcaa tgggcgctaa cgctgctaag 180

gacattataa cagacgaaga caaaaagaaa attggtatgg taattgtggc aactgaatca 240

gcagttgatg ctgctaaagc agccgctgtt caaattcaca acttattagg tattcaacct 300

tttgcacgct gctttgaaat gaaagaagct tgttatgctg caacaccagc aattcaatta 360

gctaaagatt atttagcaac tagaccgaat gaaaaagtat tagttattgc tacagataca 420

gcacgttatg gattgaactc aggcggcgag ccaacacaag gtgctggcgc agttgcgatg 480

gttattgcac ataatccaag cattttggca ttaaatgaag atgctgttgc ttacactgaa 540

gacgtttatg atttctggcg tccaactgga cataaatatc cattagttga tggtgcatta 600

tctaaagatg cttatatccg ctcattccaa caaagctgga atgaatacgc aaaacgtcaa 660

ggtaagtcgc tagctgactt cgcatctcta tgcttccatg ttccatttac aaaaatgggt 720

aaaaaggcat tagagtcaat cattgataac gctgatgaaa caactcaaga gcgtttacgt 780

tcaggatatg aagatgctgt agattataac cgttatgtcg gtaatattta tactggatca 840

ttatatttaa gcctaatatc attacttgaa aatcgagatt tacaagctgg tgaaacaatc 900

ggtttattca gttatggctc aggttcagtt ggtgaatttt atagtgcgac attagttgaa 960

ggctacaaag atcatttaga tcaagctgca cataaagcat tattaaataa ccgtactgaa 1020

gtatctgttg atgcatatga aacattcttc aaacgttttg atgacgttga atttgacgaa 1080

gaacaagatg ctgttcatga agatcgtcat attttctact tatcaaatat tgaaaataac 1140

gttcgcgaat atcacagacc agagtaa 1167

<210> 23

<211> 388

<212> PRT

<213> 人工序列

<220>

<223> HMGS

<400> 23

Met Thr Ile Gly Ile Asp Lys Ile Asn Phe Tyr Val Pro Lys Tyr Tyr

1 5 10 15

Val Asp Met Ala Lys Leu Ala Glu Ala Arg Gln Val Asp Pro Asn Lys

20 25 30

Phe Leu Ile Gly Ile Gly Gln Thr Glu Met Ala Val Ser Pro Val Asn

35 40 45

Gln Asp Ile Val Ser Met Gly Ala Asn Ala Ala Lys Asp Ile Ile Thr

50 55 60

Asp Glu Asp Lys Lys Lys Ile Gly Met Val Ile Val Ala Thr Glu Ser

65 70 75 80

Ala Val Asp Ala Ala Lys Ala Ala Ala Val Gln Ile His Asn Leu Leu

85 90 95

Gly Ile Gln Pro Phe Ala Arg Cys Phe Glu Met Lys Glu Ala Cys Tyr

100 105 110

Ala Ala Thr Pro Ala Ile Gln Leu Ala Lys Asp Tyr Leu Ala Thr Arg

115 120 125

Pro Asn Glu Lys Val Leu Val Ile Ala Thr Asp Thr Ala Arg Tyr Gly

130 135 140

Leu Asn Ser Gly Gly Glu Pro Thr Gln Gly Ala Gly Ala Val Ala Met

145 150 155 160

Val Ile Ala His Asn Pro Ser Ile Leu Ala Leu Asn Glu Asp Ala Val

165 170 175

Ala Tyr Thr Glu Asp Val Tyr Asp Phe Trp Arg Pro Thr Gly His Lys

180 185 190

Tyr Pro Leu Val Asp Gly Ala Leu Ser Lys Asp Ala Tyr Ile Arg Ser

195 200 205

Phe Gln Gln Ser Trp Asn Glu Tyr Ala Lys Arg Gln Gly Lys Ser Leu

210 215 220

Ala Asp Phe Ala Ser Leu Cys Phe His Val Pro Phe Thr Lys Met Gly

225 230 235 240

Lys Lys Ala Leu Glu Ser Ile Ile Asp Asn Ala Asp Glu Thr Thr Gln

245 250 255

Glu Arg Leu Arg Ser Gly Tyr Glu Asp Ala Val Asp Tyr Asn Arg Tyr

260 265 270

Val Gly Asn Ile Tyr Thr Gly Ser Leu Tyr Leu Ser Leu Ile Ser Leu

275 280 285

Leu Glu Asn Arg Asp Leu Gln Ala Gly Glu Thr Ile Gly Leu Phe Ser

290 295 300

Tyr Gly Ser Gly Ser Val Gly Glu Phe Tyr Ser Ala Thr Leu Val Glu

305 310 315 320

Gly Tyr Lys Asp His Leu Asp Gln Ala Ala His Lys Ala Leu Leu Asn

325 330 335

Asn Arg Thr Glu Val Ser Val Asp Ala Tyr Glu Thr Phe Phe Lys Arg

340 345 350

Phe Asp Asp Val Glu Phe Asp Glu Glu Gln Asp Ala Val His Glu Asp

355 360 365

Arg His Ile Phe Tyr Leu Ser Asn Ile Glu Asn Asn Val Arg Glu Tyr

370 375 380

His Arg Pro Glu

385

<210> 24

<211> 1284

<212> DNA

<213> 人工序列

<220>

<223> HMGR 基因

<400> 24

atgtccatgc aaagtttaga taagaatttt cgacatttat ctcgtaaaga aaagttacaa 60

caattggttg ataagcaatg gttatcagaa gaacaattcg acattttact gaatcatcca 120

ttaatcgatg aagaagtagc caatagttta attgaaaatg tcatcgcgca aggtgcatta 180

cccgttggat tattaccgaa tatcattgtg gacgataagg catatgttgt acctatgatg 240

gtggaagagc cttcagttgt cgctgcagct agttatggtg caaagctagt gaatcagact 300

ggcggattta aaacggtatc ttctgaacgt attatgatag gtcaaatcgt ctttgatggc 360

gttgacgata ctgaaaaatt atcagcagac attaaagctt tagaaaagca aattcataaa 420

attgcggatg aggcatatcc ttctattaaa gcgcgtggtg gtggttacca acgtatagcg 480

attgatacat ttcctgagca acagttacta tctttaaaag tatttgttga tacgaaagat 540

gctatgggcg ctaatatgct taatacgatt ttagaggcca taactgcatt tttaaaaaat 600

gaatttccgc aaagcgacat tttaatgagt attttatcca atcatgcaac agcgtccgtt 660

gttaaagttc aaggcgaaat tgatgttaaa gatttagcaa ggggcgagag aactggagaa 720

gaggttgcca aacgaatgga acgtgcttct gtattggccc aagtagatat tcatcgtgca 780

gcaacacata ataaaggtgt tatgaatggc atacatgctg ttgttttagc aacaggaaat 840

gatacgcgtg gtgcagaagc aagtgcgcat gcatacgcga gtcgtgacgg acagtatcgt 900

ggtattgcta catggcgtta cgatcaagat cgtcaacgat tgattggtac aattgaagtg 960

cctatgacat tggcaatcgt tggcggtggt acaaaagtat taccaattgc taaagcttca 1020

ttagagctac taaatgtaga gtcagcacaa gaattaggtc atgtagttgc tgccgttggt 1080

ttagcgcaaa actttgcagc atgtcgcgcg cttgtgtcag aaggtattca acaaggtcat 1140

atgagtttac aatataaatc attagctatc gttgtagggg caaaaggtga tgaaattgct 1200

aaagtagctg aagctttgaa aaaagaaccc cgtgcaaata cacaagcagc ggaacatatt 1260

ttacaagaaa ttagacaaca ataa 1284

<210> 25

<211> 427

<212> PRT

<213> 人工序列

<220>

<223> HMGR

<400> 25

Met Ser Met Gln Ser Leu Asp Lys Asn Phe Arg His Leu Ser Arg Lys

1 5 10 15

Glu Lys Leu Gln Gln Leu Val Asp Lys Gln Trp Leu Ser Glu Glu Gln

20 25 30

Phe Asp Ile Leu Leu Asn His Pro Leu Ile Asp Glu Glu Val Ala Asn

35 40 45

Ser Leu Ile Glu Asn Val Ile Ala Gln Gly Ala Leu Pro Val Gly Leu

50 55 60

Leu Pro Asn Ile Ile Val Asp Asp Lys Ala Tyr Val Val Pro Met Met

65 70 75 80

Val Glu Glu Pro Ser Val Val Ala Ala Ala Ser Tyr Gly Ala Lys Leu

85 90 95

Val Asn Gln Thr Gly Gly Phe Lys Thr Val Ser Ser Glu Arg Ile Met

100 105 110

Ile Gly Gln Ile Val Phe Asp Gly Val Asp Asp Thr Glu Lys Leu Ser

115 120 125

Ala Asp Ile Lys Ala Leu Glu Lys Gln Ile His Lys Ile Ala Asp Glu

130 135 140

Ala Tyr Pro Ser Ile Lys Ala Arg Gly Gly Gly Tyr Gln Arg Ile Ala

145 150 155 160

Ile Asp Thr Phe Pro Glu Gln Gln Leu Leu Ser Leu Lys Val Phe Val

165 170 175

Asp Thr Lys Asp Ala Met Gly Ala Asn Met Leu Asn Thr Ile Leu Glu

180 185 190

Ala Ile Thr Ala Phe Leu Lys Asn Glu Phe Pro Gln Ser Asp Ile Leu

195 200 205

Met Ser Ile Leu Ser Asn His Ala Thr Ala Ser Val Val Lys Val Gln

210 215 220

Gly Glu Ile Asp Val Lys Asp Leu Ala Arg Gly Glu Arg Thr Gly Glu

225 230 235 240

Glu Val Ala Lys Arg Met Glu Arg Ala Ser Val Leu Ala Gln Val Asp

245 250 255

Ile His Arg Ala Ala Thr His Asn Lys Gly Val Met Asn Gly Ile His

260 265 270

Ala Val Val Leu Ala Thr Gly Asn Asp Thr Arg Gly Ala Glu Ala Ser

275 280 285

Ala His Ala Tyr Ala Ser Arg Asp Gly Gln Tyr Arg Gly Ile Ala Thr

290 295 300

Trp Arg Tyr Asp Gln Asp Arg Gln Arg Leu Ile Gly Thr Ile Glu Val

305 310 315 320

Pro Met Thr Leu Ala Ile Val Gly Gly Gly Thr Lys Val Leu Pro Ile

325 330 335

Ala Lys Ala Ser Leu Glu Leu Leu Asn Val Glu Ser Ala Gln Glu Leu

340 345 350

Gly His Val Val Ala Ala Val Gly Leu Ala Gln Asn Phe Ala Ala Cys

355 360 365

Arg Ala Leu Val Ser Glu Gly Ile Gln Gln Gly His Met Ser Leu Gln

370 375 380

Tyr Lys Ser Leu Ala Ile Val Val Gly Ala Lys Gly Asp Glu Ile Ala

385 390 395 400

Lys Val Ala Glu Ala Leu Lys Lys Glu Pro Arg Ala Asn Thr Gln Ala

405 410 415

Ala Glu His Ile Leu Gln Glu Ile Arg Gln Gln

420 425

<210> 26

<211> 6026

<212> DNA

<213> 人工序列

<220>

<223> p15A-BsaI 质粒

<400> 26

agcggataac aatttcacac aggagagctc aaagagtgga acaatgcagg acgccgtccg 60

gtaacggtgt tgagcctcgg ttgtgtggtg attattgtcg ccgctaacat cgtcggcatc 120

ggcatggcga attaatcttt ctgcgaattg agatgacgcc actggctggg cgtcatcccg 180

gtttcccggg taaacaccac cgaaaaatag ttactatctt caaagccaca ttcggtcgaa 240

atatcactga ttaacaggcg gctatgctgg agaagatatt gcgcatgaca cactctgacc 300

tgtcgcagat attgattgat ggtcattcca gtctgctggc gaaattgctg acgcaaaacg 360

cgctcactgc acgatgcctc atcacaaaat ttatccagcg caaagggact tttcaggcta 420

gccgccagcc gggtaatcag cttatccagc aacgtttcgc tggatgttgg cggcaacgaa 480

tcactggtgt aacgatggcg attcagcaac atcaccaact gcccgaacag caactcagcc 540

atttcgttag caaacggcac atgctgacta ctttcatgct caagctgacc gataacctgc 600

cgcgcctgcg ccatccccat gctacctaag cgccagtgtg gttgccctgc gctggcgtta 660

aatcccggaa tcgccccctg ccagtcaaga ttcagcttca gacgctccgg gcaataaata 720

atattctgca aaaccagatc gttaacggaa gcgtaggagt gtttatcgtc agcatgaatg 780

taaaagagat cgccacgggt aatgcgataa gggcgatcgt tgagtacatg caggccatta 840

ccgcgccaga caatcaccag ctcacaaaaa tcatgtgtat gttcagcaaa gacatcttgc 900

ggataacggt cagccacagc gactgcctgc tggtcgctgg caaaaaaatc atctttgaga 960

agttttaact gatgcgccac cgtggctacc tcggccagag aacgaagttg attattcgca 1020

atatggcgta caaatacgtt gagaagattc gcgttattgc agaaagccat cccgtccctg 1080

gcgaatatca cgcggtgacc agttaaactc tcggcgaaaa agcgtcgaaa agtggttact 1140

gtcgctgaat ccacagcgat aggcgatgtc agtaacgctg gcctcgctgt ggcgtagcag 1200

atgtcgggct ttcatcagtc gcaggcggtt caggtatcgc tgaggcgtca gtcccgtttg 1260

ctgcttaagc tgccgatgta gcgtacgcag tgaaagagaa aattgatccg ccacggcatc 1320

ccaattcacc tcatcggcaa aatggtcctc cagccaggcc agaagcaagt tgagacgtga 1380

tgcgctgttt tccaggttct cctgcaaact gcttttacgc agcaagagca gtaattgcat 1440

aaacaagatc tcgcgactgg cggtcgaggg taaatcattt tccccttcct gctgttccat 1500

ctgtgcaacc agctgtcgca cctgctgcaa tacgctgtgg ttaacgcgcc agtgagacgg 1560

atactgccca tccagctctt gtggcagcaa ctgattcagc ccggcgagaa actgaaatcg 1620

atccggcgag cgatacagca cattggtcag acacagatta tcggtatgtt catacagatg 1680

ccgatcatga tcgcgtacga aacagaccgt gccaccggtg atggtatagg gctgcccatt 1740

aaacacatga atacccgtgc catgttcgac aatcacaatt tcatgaaaat catgatgatg 1800

ttcaggaaaa tccgcctgcg ggagccgggg ttctatcgcc acggacgcgt taccagacgg 1860

aaaaaaatcc acactatgta atacggtcat actggcctcc tgatgtcgtc aacacggcga 1920

aatagtaatc acgaggtcag gttcttacct taaattttcg acggaaaacc acgtaaaaaa 1980

cgtcgatttt tcaagataca gcgtgaattt tcaggaaatg cggtgagcat cacatcacca 2040

caattcagca aattgtgaac atcatcacgt tcatctttcc ctggttgcca atggcccatt 2100

ttcctgtcag taacgagaag gtcgcgaatt caggcgcttt ttagactggt cgtaatgaaa 2160

ttcaactagt gctctgcagg agctgtcacc ggatgtgctt tccggtctga tgagtccgtg 2220

aggacgaaac agcctctaca aataattttg tttaagagtc gagacccatt cctgatcctt 2280

gacagctagc tcagtcctag gtataatact aggagttact agagaaagag gagaaatact 2340

agatgcgtaa aggcgaagag ctgttcactg gtgtcgtccc tattctggtg gaactggatg 2400

gtgatgtcaa cggtcataag ttttccgtgc gtggcgaggg tgaaggtgac gcaactaatg 2460

gtaaactgac gctgaagttc atctgtacta ctggtaaact gccggtacct tggccgactc 2520

tggtaacgac gctgacttat ggtgttcagt gctttgctcg ttatccggac catatgaagc 2580

agcatgactt cttcaagtcc gccatgccgg aaggctatgt gcaggaacgc acgatttcct 2640

ttaaggatga cggcacgtac aaaacgcgtg cggaagtgaa atttgaaggc gataccctgg 2700

taaaccgcat tgagctgaaa ggcattgact ttaaagaaga cggcaatatc ctgggccata 2760

agctggaata caattttaac agccacaatg tttacatcac cgccgataaa caaaaaaatg 2820

gcattaaagc gaattttaaa attcgccaca acgtggagga tggcagcgtg cagctggctg 2880

atcactacca gcaaaacact ccaatcggtg atggtcctgt tctgctgcca gacaatcact 2940

atctgagcac gcaaagcgtt ctgtctaaag atccgaacga gaaacgcgat catatggttc 3000

tgctggagtt cgtaaccgca gcgggcatca cgcatggtat ggatgaactg tacaaataat 3060

cgtcactcca ccggtgctta ataaccaggc atcaaataaa acgaaaggct cagtcgaaag 3120

actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag agtcacactg 3180

gctcaccttc gggtgggcct ttctgcgttt atatactaga gctgctaaca aagcccgaaa 3240

ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc 3300

taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tactagaggt 3360

catgcttgcc atctgttttc ttgcaagatg ctcactcaaa ggcggtaatg gtctcactcg 3420

tcgtgactgg gaaaaccctg gcgactagtc ttggactcct gttgatagat ccagtaatga 3480

cctcagaact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg 3540

gtgagaatcc aggggtcccc aataattacg atttaaattg gcgaaaatga gacgtgggtc 3600

tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 3660

gatcttcacc tagatccttt tggttcatgt gcagctccat cagcaaaagg ggatgataag 3720

tttatcacca ccgactattt gcaacagcgc cggtgatcgt gctatgatcg actgatgtca 3780

tcagcggtgg agtgcaatgt cgtgcaatac gaatggcgaa aagccgagct catcggtcag 3840

cttctcaacc ttggggttac ccccggcggt gtgctgctgg tccacagctc cttccgtagc 3900

gtccggcccc tcgaagatgg gccacttgga ctgatcgagg ccctgcgtgc tgcgctgggt 3960

ccgggaggga cgctcgtcat gccctcgtgg tcaggtctgg acgacgagcc gttcgatcct 4020

gccacgtcgc ccgttacacc ggaccttgga gttgtctctg acacattctg gcgcctgcca 4080

aatgtaaagc gcagcgccca tccatttgcc tttgcggcag cggggccaca ggcagagcag 4140

atcatctctg atccattgcc cctgccacct cactcgcctg caagcccggt cgcccgtgtc 4200

catgaactcg atgggcaggt acttctcctc ggcgtgggac acgatgccaa cacgacgctg 4260

catcttgccg agttgatggc aaaggttccc tatggggtgc cgagacactg caccattctt 4320

caggatggca agttggtacg cgtcgattat ctcgagaatg accactgctg tgagcgcttt 4380

gccttggcgg acaggtggct caaggagaag agccttcaga aggaaggtcc agtcggtcat 4440

gcctttgctc ggttgatccg ctcccgcgac attgtggcga cagccctggg tcaactgggc 4500

cgagatccgt tgatcttcct gcatccgcca gaggcgggat gcgaagaatg cgatgccgct 4560

cgccagtcga ttggctgagc tcatgagcgg agaacgagat gacgttggag gggcaaggtc 4620

gcgctgattg ctggggcaac acgtggagcg gatcggtttg acttttgtcc ttttccgctg 4680

cataaccctg cttcggggtc attatagcga ttttttcggt atatccatcc tttttcgcac 4740

gatatacagg attttgccaa agggttcgtg tagactttcc ttggtgtatc caacggcgtc 4800

agccgggcag gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc ttcactgtcc 4860

cttattcgca cctggcggtg ctcaacggga atcctgctct gcgaggctgg ccgtaggccg 4920

gccgcgatgc aggtggctgc tgaaccccca gccggaactg accccacaag gccctagcgg 4980

agtgtatact ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg 5040

caggagaaaa aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc 5100

ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta 5160

cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg 5220

gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga 5280

cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc gtttcccctg 5340

gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt 5400

tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc 5460

aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac 5520

tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt 5580

aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac 5640

aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca 5700

gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac 5760

gcgcagacca aaacgatctc aagaagatca tcttattaac tacatggctc tgctgtagtg 5820

agtgggttgc gctccggcag cggtcctgat cccccgcaga aaaaaaggat ctcaagaaga 5880

tcctttgatc ttttctacgg cgcgcccagc tgtctagggc ggcggatttg tcctactcag 5940

gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt 6000

tcgttttatt tgatgccttt aattaa 6026

<210> 27

<211> 972

<212> DNA

<213> 人工序列

<220>

<223> CBGAS-1 基因

<400> 27

ggtctcagag ttactagaga aagaggagaa atactagatg agcgaagcgg cggatgtgga 60

acgcgtgtat gcggcgatgg aagaagcggc gggtctgtta ggtgttgcgt gtgcgcgtga 120

taaaatttat ccgctgctga gcacctttca ggataccctg gtggaaggcg gcagcgtggt 180

tgtgtttagc atggcgagcg gccgccatag caccgaatta gattttagca ttagcgtgcc 240

gaccagccat ggcgatccgt atgcgaccgt tgtggaaaaa ggcctgtttc cggcgaccgg 300

ccatccggtt gatgatctgt tagcggatac ccagaaacat ctgccggtga gcatgtttgc 360

gattgatggc gaagtgaccg gcggctttaa gaaaacctat gcgttctttc cgaccgacaa 420

catgccgggc gtggcggaat taagcgcgat tcctagcatg ccgccggcgg tggcagaaaa 480

tgcggaatta tttgcgcgct atggcctgga taaagtgcag atgaccagca tggattataa 540

gaaacgccag gtgaacctgt attttagcga actgagcgcg cagaccctgg aagcggaaag 600

cgttttagcg ctggttcgcg aattaggcct gcatgtgccg aacgaactgg gcctgaaatt 660

ttgcaaacgc agctttagcg tgtatccgac cctgaactgg gaaaccggca aaattgatcg 720

cctgtgcttt gcggtgatta gcaacgatcc gaccctggtg ccgagcagcg atgaaggcga 780

tattgaaaaa tttcataact acgcgaccaa ggcgccgtat gcgtatgtgg gcgaaaaacg 840

caccctggtg tatggcctga ccctgagccc taaagaagaa tattataaac tgggcgcgta 900

ctatcacatt accgatgtgc agcgcggcct gctgaaagcg tttgatagcc tggaagatta 960

actcgagaga cc 972

<210> 28

<211> 307

<212> PRT

<213> 人工序列

<220>

<223> CBGAS-1

<400> 28

Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu

1 5 10 15

Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro

20 25 30

Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val

35 40 45

Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser

50 55 60

Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu

65 70 75 80

Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala

85 90 95

Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu

100 105 110

Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn

115 120 125

Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala

130 135 140

Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val

145 150 155 160

Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe

165 170 175

Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu

180 185 190

Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe

195 200 205

Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly

210 215 220

Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu

225 230 235 240

Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala

245 250 255

Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr

260 265 270

Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Gly Ala Tyr

275 280 285

Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser

290 295 300

Leu Glu Asp

305

<210> 29

<211> 972

<212> DNA

<213> 人工序列

<220>

<223> CBGAS-2 基因

<400> 29

ggtctcagag ttactagaga aagaggagaa atactagatg agcgaagcgg cggatgtgga 60

acgcgtgtat gcggcgatgg aagaagcggc gggtctgtta ggtgttgcgt gtgcgcgtga 120

taaaatttat ccgctgctga gcacctttca ggataccctg gtggaaggcg gcagcgtggt 180

tgtgtttagc atggcgagcg gccgccatag caccgaatta gattttagca ttagcgtgcc 240

gaccagccat ggcgatccgt atgcgaccgt tgtggaaaaa ggcctgtttc cggcgaccgg 300

ccatccggtt gatgatctgt tagcggatac ccagaaacat ctgccggtga gcatgtttgc 360

gattgatggc gaagtgaccg gcggctttaa gaaaacctat gcgttctttc cgaccgacaa 420

catgccgggc gtggcggaat taagcgcgat tcctagcatg ccgccggcgg tggcagaaaa 480

tgcggaatta tttgcgcgct atggcctgga taaagtggcg atgaccagca tggattataa 540

gaaacgccag gtgaacctgt attttagcga actgagcgcg cagaccctgg aagcggaaag 600

cgttttagcg ctggttcgcg aattaggcct gcatgtgccg aacgaactgg gcctgaaatt 660

ttgcaaacgc agctttagcg tgtatccgac cctgaactgg gaaaccggca aaattgatcg 720

cctgtgcttt gcggtgatta gcaacgatcc gaccctggtg ccgagcagcg atgaaggcga 780

tattgaaaaa tttcataact acgcgaccaa ggcgccgtat gcgtatgtgg gcgaaaaacg 840

caccctggtg tatggcctga ccctgagccc taaagaagaa tattataaac tgggcgcgta 900

ctatcacatt accgatgtgc agcgcggcct gctgaaagcg tttgatagcc tggaagatta 960

actcgagaga cc 972

<210> 30

<211> 307

<212> PRT

<213> 人工序列

<220>

<223> CBGAS-2

<400> 30

Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu

1 5 10 15

Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro

20 25 30

Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val

35 40 45

Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser

50 55 60

Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu

65 70 75 80

Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala

85 90 95

Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu

100 105 110

Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn

115 120 125

Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala

130 135 140

Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val

145 150 155 160

Ala Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe

165 170 175

Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu

180 185 190

Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe

195 200 205

Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly

210 215 220

Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu

225 230 235 240

Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala

245 250 255

Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr

260 265 270

Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Gly Ala Tyr

275 280 285

Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser

290 295 300

Leu Glu Asp

305

<210> 31

<211> 972

<212> DNA

<213> 人工序列

<220>

<223> CBGAS-3 基因

<400> 31

ggtctcagag ttactagaga aagaggagaa atactagatg agcgaagcgg cggatgtgga 60

acgcgtgtat gcggcgatgg aagaagcggc gggtctgtta ggtgttgcgt gtgcgcgtga 120

taaaatttat ccgctgctga gcacctttca ggataccctg gtggaaggcg gcagcgtggt 180

tgtgtttagc atggcgagcg gccgccatag caccgaatta gattttagca ttagcgtgcc 240

gaccagccat ggcgatccgt atgcgaccgt tgtggaaaaa ggcctgtttc cggcgaccgg 300

ccatccggtt gatgatctgt tagcggatac ccagaaacat ctgccggtga gcatgtttgc 360

gattgatggc gaagtgaccg gcggctttaa gaaaacctat gcgttctttc cgaccgacaa 420

catgccgggc gtggcggaat taagcgcgat tcctagcatg ccgccggcgg tggcagaaaa 480

tgcggaatta tttgcgcgct atggcctgga taaagtgcag atgaccagca tggattataa 540

gaaacgccag gtgaacctgt attttagcga actgagcgcg cagaccctgg aagcggaaag 600

cgttttagcg ctggttcgcg aattaggcct gcatgtgccg aacgaactgg gcctgaaatt 660

ttgcaaacgc agctttagcg tgtatccgac cctgaactgg gaaaccggca aaattgatcg 720

cctgtgcttt gcggtgatta gcaacgatcc gaccctggtg ccgagcagcg atgaaggcga 780

tattgaaaaa tttcataact acgcgaccaa ggcgccgtat gcgtatgtgg gcgaaaaacg 840

caccctggtg tatggcctga ccctgagccc taaagaagaa tattataaac tgagcgcggc 900

atatcacatt accgatgtgc agcgcggcct gctgaaagcg tttgatagcc tggaagatta 960

actcgagaga cc 972

<210> 32

<211> 307

<212> PRT

<213> 人工序列

<220>

<223> CBGAS-3

<400> 32

Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu

1 5 10 15

Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro

20 25 30

Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val

35 40 45

Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser

50 55 60

Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu

65 70 75 80

Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala

85 90 95

Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu

100 105 110

Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn

115 120 125

Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala

130 135 140

Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val

145 150 155 160

Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe

165 170 175

Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu

180 185 190

Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe

195 200 205

Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly

210 215 220

Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu

225 230 235 240

Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala

245 250 255

Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr

260 265 270

Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Ser Ala Ala

275 280 285

Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser

290 295 300

Leu Glu Asp

305

<210> 33

<211> 972

<212> DNA

<213> 人工序列

<220>

<223> CBGAS-4 基因

<400> 33

ggtctcagag ttactagaga aagaggagaa atactagatg agcgaagcgg cggatgtgga 60

acgcgtgtat gcggcgatgg aagaagcggc gggtctgtta ggtgttgcgt gtgcgcgtga 120

taaaatttat ccgctgctga gcacctttca ggataccctg gtggaaggcg gcagcgtggt 180

tgtgtttagc atggcgagcg gccgccatag caccgaatta gattttagca ttagcgtgcc 240

gaccagccat ggcgatccgt atgcgaccgt tgtggaaaaa ggcctgtttc cggcgaccgg 300

ccatccggtt gatgatctgt tagcggatac ccagaaacat ctgccggtga gcatgtttgc 360

gattgatggc gaagtgaccg gcggctttaa gaaaacctat gcgttctttc cgaccgacaa 420

catgccgggc gtggcggaat taagcgcgat tcctagcatg ccgccggcgg tggcagaaaa 480

tgcggaatta tttgcgcgct atggcctgga taaagtggcg atgaccagca tggattataa 540

gaaacgccag gtgaacctgt attttagcga actgagcgcg cagaccctgg aagcggaaag 600

cgttttagcg ctggttcgcg aattaggcct gcatgtgccg aacgaactgg gcctgaaatt 660

ttgcaaacgc agctttagcg tgtatccgac cctgaactgg gaaaccggca aaattgatcg 720

cctgtgcttt gcggtgatta gcaacgatcc gaccctggtg ccgagcagcg atgaaggcga 780

tattgaaaaa tttcataact acgcgaccaa ggcgccgtat gcgtatgtgg gcgaaaaacg 840

caccctggtg tatggcctga ccctgagccc taaagaagaa tattataaac tgagcgcggc 900

atatcacatt accgatgtgc agcgcggcct gctgaaagcg tttgatagcc tggaagatta 960

actcgagaga cc 972

<210> 34

<211> 307

<212> PRT

<213> 人工序列

<220>

<223> CBGAS-4

<400> 34

Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu

1 5 10 15

Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro

20 25 30

Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val

35 40 45

Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser

50 55 60

Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu

65 70 75 80

Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala

85 90 95

Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu

100 105 110

Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn

115 120 125

Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala

130 135 140

Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val

145 150 155 160

Ala Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe

165 170 175

Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu

180 185 190

Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe

195 200 205

Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly

210 215 220

Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu

225 230 235 240

Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala

245 250 255

Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr

260 265 270

Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Ser Ala Ala

275 280 285

Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser

290 295 300

Leu Glu Asp

305

<210> 35

<211> 25

<212> DNA

<213> 人工序列

<220>

<223> 在CBGASs中的RBS

<400> 35

actagagaaa gaggagaaat actag 25

Claims (11)

1.一种芳香族异戊烯基转移酶突变体，其氨基酸序列为SEQ ID No.34。

2.一种质粒，其包含如权利要求1所述的芳香族异戊烯基转移酶突变体的编码序列。

3.一种构建重组菌的方法，所述方法包括以下步骤：

1）以大肠杆菌BW25113为出发菌株将MVA途径所需的基因导入其基因组中以获得底盘菌；

2）构建包含如权利要求1所述的芳香族异戊烯基转移酶突变体的编码序列的质粒；

3）将步骤2）中构建的质粒导入步骤1）获得的底盘菌中即得到所述重组菌。

4.根据权利要求3所述的方法，其中，所述MVA途径所需的基因包括：乙酰乙酰-CoA硫解酶、HMG合酶、3-羟基-3-甲基戊二酰-CoA还原酶、MVA激酶、磷酸MVA激酶、MVA二磷酸脱羧酶、IPP异构酶和ERG20或它们各自的同功酶的编码基因。

5.根据权利要求3所述的方法，进一步包括向所述重组菌中额外导入大麻二酸合酶。

6.一种根据权利要求3或4所述的方法构建的重组菌。

7.如权利要求6所述的重组菌用于发酵生产大麻萜酚酸的用途。

8.一种生产大麻萜酚酸的方法，其中，所述方法包括采用如权利要求6所述的重组菌发酵生产大麻萜酚酸。

9.一种生产大麻素类化合物的方法，包括使用大麻萜酚酸作为原料合成所述大麻素类化合物的步骤，其中，所述大麻萜酚酸采用如权利要求6所述的重组菌发酵生产。

10.根据权利要求9所的方法，其中，所述大麻素类化合物包括大麻二酸、四氢大麻酚酸、大麻色素羧酸、大麻酸、大麻萜酚、大麻二酚、四氢大麻酚、大麻环萜酚或大麻酚。

11.一种根据权利要求5所述的方法构建的重组菌。

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