CN114058608B - A kind of engineering bacteria and method for producing putrescine - Google Patents

A kind of engineering bacteria and method for producing putrescine Download PDF

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CN114058608B
CN114058608B CN202010757654.4A CN202010757654A CN114058608B CN 114058608 B CN114058608 B CN 114058608B CN 202010757654 A CN202010757654 A CN 202010757654A CN 114058608 B CN114058608 B CN 114058608B
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蔡宇杰
惠红杰
梁鑫鑫
丁彦蕊
白亚军
郑晓晖
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Abstract

本发明公开了一种生产腐胺的工程菌及方法,属于生物工程技术领域。本发明通过对腐胺合成途径中相关的精氨酸脱羧酶和胍基丁胺脲水解酶进行改造,获得了能够解除反馈抑制并提升酶活的两个突变体,利用所述突变体构建了基因工程菌用于腐胺的生产,在全细胞转化条件下,以精氨酸为底物,腐胺产量能达到120g/L及以上。利用两种酶的突变体进行酶转化法,以精氨酸为底物,腐胺产量能达到160g/L。本发明提供的生产腐胺的方法过程简单并且原料容易获得,具有良好的工业化应用前景。

The invention discloses an engineering bacterium and a method for producing putrescine, belonging to the technical field of bioengineering. The present invention obtains two mutants capable of releasing feedback inhibition and improving enzyme activity by transforming the related arginine decarboxylase and agmatine urea hydrolase in the putrescine synthesis pathway, and using the mutants to construct Genetically engineered bacteria are used for the production of putrescine. Under the condition of whole cell transformation, with arginine as the substrate, the output of putrescine can reach 120g/L and above. The mutants of the two enzymes are used for enzymatic conversion, and the putrescine output can reach 160 g/L with arginine as the substrate. The method for producing putrescine provided by the invention is simple in process and easy to obtain raw materials, and has good industrial application prospect.

Description

一种生产腐胺的工程菌及方法A kind of engineering bacteria and method for producing putrescine

技术领域technical field

本发明涉及一种生产腐胺的工程菌及方法,属于生物工程技术领域。The invention relates to an engineering bacterium and a method for producing putrescine, belonging to the technical field of bioengineering.

背景技术Background technique

腐胺(putrescine),又称1,4丁二胺,线性分子式为NH2(CH2)4NH2,广泛分布于微生物、植物和动物体内,是一种重要的生理活性物质。在工业、农业和医药领域具有广泛的应用前景。Putrescine, also known as 1,4-butylene diamine, has a linear molecular formula of NH 2 (CH 2 ) 4 NH 2 . It is widely distributed in microorganisms, plants and animals, and is an important physiologically active substance. It has broad application prospects in the fields of industry, agriculture and medicine.

在目前的报道中,生物体中腐胺主要有两条合成途径,如图1所示:In the current report, there are two main synthetic pathways for putrescine in organisms, as shown in Figure 1:

(1)L-精氨酸在精氨酸脱羧酶的脱羧作用下生成胍基丁胺,同时释放CO2;然后,胍基丁胺在胍基丁胺脲水解酶的作用下脱去一分子尿素,生成产物腐胺。此途径在动物、植物和微生物体内,是最为常见的腐胺合成的途径。在Escherichia coli中,精氨酸脱羧酶共分为两类,一种为诱导型精氨酸脱羧酶(由AidA基因编码),另一种为生物合成型精氨酸脱羧酶(由speA基因编码)。前者在酸性条件下发挥功能,脱羧过程中会消耗一分子质子,从而防止质子在胞内聚集,保证菌体在低pH下生存。在中性pH下,腐胺主要由speA编码的精氨酸脱羧酶来合成腐胺。本文选用speA编码的精氨酸脱羧酶进行催化。(1) L-arginine is decarboxylated by arginine decarboxylase to generate agmatine, and CO 2 is released at the same time; then, agmatine is removed under the action of agmatine urea hydrolase Urea, producing putrescine as a product. This pathway is the most common pathway for putrescine synthesis in animals, plants and microorganisms. In Escherichia coli, arginine decarboxylase is divided into two types, one is inducible arginine decarboxylase (encoded by AidA gene), and the other is biosynthetic arginine decarboxylase (encoded by speA gene ). The former functions under acidic conditions, and a molecule of proton will be consumed during the decarboxylation process, thereby preventing the accumulation of protons in the cell and ensuring the survival of bacteria at low pH. At neutral pH, putrescine is mainly synthesized by arginine decarboxylase encoded by speA. In this paper, arginine decarboxylase encoded by speA was selected for catalysis.

(2)L-精氨酸经精氨酸酶的水解作用生成鸟氨酸,鸟氨酸在鸟氨酸脱羧酶(ODC)的作用下生成产物腐胺,同时释放CO2。研究表明,外源添加L-精氨酸时,E.coli会选择利用ADC途径合成腐胺,不经过鸟氨酸途径。(2) L-arginine is hydrolyzed by arginase to generate ornithine, and ornithine generates putrescine under the action of ornithine decarboxylase (ODC), and releases CO 2 at the same time. Studies have shown that when L-arginine is added exogenously, E.coli will choose to use the ADC pathway to synthesize putrescine instead of the ornithine pathway.

目前,合成途径中的ADC和AUH在菌株体内,受到严格的调控。对于ADC,DavidR.Morris.et al.的研究表明,Mg2+、精氨酸和腐胺对ADC有竞争性抑制关系。数据显示,低浓度的Mg2+将会大大降低ADC的催化活力,同时也会加剧腐胺的产物抑制。精氨酸浓度高于一定浓度时,腐胺的产量反而会下降。精氨酸同样会对AUH产生竞争性抑制(Ki=9×10-3M)。由于双酶在菌体内均收到一定程度的反馈抑制,从而导致利用此途径合成的腐胺量非常低。Currently, ADC and AUH in the synthetic pathway are strictly regulated in the strain. For ADC, research by DavidR.Morris.et al. shows that Mg 2+ , arginine and putrescine have a competitive inhibitory relationship on ADC. The data showed that low concentration of Mg 2+ would greatly reduce the catalytic activity of ADC and also aggravate the product inhibition of putrescine. When the concentration of arginine was higher than a certain concentration, the production of putrescine would decrease instead. Arginine also competitively inhibits AUH (Ki=9×10 -3 M). Since both enzymes are subject to a certain degree of feedback inhibition in the bacteria, the amount of putrescine synthesized by this pathway is very low.

发明内容Contents of the invention

本发明要解决的技术问题是以L-精氨酸为底物高效合成腐胺,尤其是构建双基因共表达工程菌,利用该工程菌催化L-精氨酸合成腐胺,实现腐胺的高效生产。The technical problem to be solved in the present invention is to efficiently synthesize putrescine with L-arginine as a substrate, especially to construct a double-gene co-expression engineering bacterium, and use the engineering bacterium to catalyze L-arginine to synthesize putrescine to realize the production of putrescine. Efficient production.

本发明提供了以廉价L-精氨酸为底物合成腐胺的方法。L-精氨酸被精氨酸脱羧酶脱羧生成胍基丁胺和CO2。随后,胍基丁胺被胍基丁胺脲水解酶催化生成腐胺。本发明选择的野生型精氨酸脱羧酶(ADC)来源于Escherichia coli BL21(DE3),其编码基因为speA,其核苷酸序列如SEQ ID NO.5所示,对应的氨基酸序列如GenBank号QJZ13350.1所示。选择的野生型胍基丁胺脲水解酶(AUH)来源于Escherichia coli BL21(DE3),其编码基因为speB,其核苷酸序列如SEQ ID NO.6所示,对应的氨基酸序列如Genbank号为QJZ13349.1所示。The invention provides a method for synthesizing putrescine by using cheap L-arginine as a substrate. L-arginine is decarboxylated by arginine decarboxylase to generate agmatine and CO 2 . Subsequently, agmatine is catalyzed by agmatine urea hydrolase to produce putrescine. The wild-type arginine decarboxylase (ADC) selected in the present invention is derived from Escherichia coli BL21 (DE3), its coding gene is speA, its nucleotide sequence is shown in SEQ ID NO.5, and the corresponding amino acid sequence is shown in GenBank No. Shown in QJZ13350.1. The selected wild-type agmatine urea hydrolase (AUH) is derived from Escherichia coli BL21 (DE3), its coding gene is speB, its nucleotide sequence is shown in SEQ ID NO.6, and the corresponding amino acid sequence is shown in Genbank No. It is shown in QJZ13349.1.

本发明提供了腐胺合成相关的酶的突变体,所述酶为精氨酸脱羧酶和胍基丁胺脲水解酶;The present invention provides mutants of enzymes related to putrescine synthesis, the enzymes are arginine decarboxylase and agmatine urea hydrolase;

在一种实施方式中,精氨酸脱羧酶的突变体的氨基酸序列如SEQ ID NO.3所示;In one embodiment, the amino acid sequence of the mutant of arginine decarboxylase is shown in SEQ ID NO.3;

在一种实施方式中,胍基丁胺脲水解酶的突变体的氨基酸序列如SEQ ID NO.4所示。In one embodiment, the amino acid sequence of the mutant of agmatine urea hydrolase is shown in SEQ ID NO.4.

本发明提供了一种基因工程菌,所述基因工程菌表达核苷酸序列如SEQ ID NO.1所示的精氨酸脱羧酶基因和核苷酸序列如SEQ ID NO.2所示的胍基丁胺脲水解酶基因。The invention provides a genetically engineered bacterium, which expresses an arginine decarboxylase gene with a nucleotide sequence as shown in SEQ ID NO.1 and a guanidine with a nucleotide sequence as shown in SEQ ID NO.2 butylaminourea hydrolase gene.

在一种实施方式中,以大肠杆菌为宿主。In one embodiment, Escherichia coli is used as the host.

在一种实施方式中,以pACYCDuet、pRSFDuet、pCDFDuet或pCOLADuet为表达载体。In one embodiment, pACYCDuet, pRSFDuet, pCDFDuet or pCOLADuet is used as the expression vector.

在一种实施方式中,以pACYCDuet、pRSFDuet或pCDFDuet为表达载体。In one embodiment, pACYCDuet, pRSFDuet or pCDFDuet is used as the expression vector.

在一种实施方式中,精氨酸脱羧酶基因连接至胍基丁胺脲水解酶基因的后面,或胍基丁胺脲水解酶基因连接至精氨酸脱羧酶基因的后面。In one embodiment, the arginine decarboxylase gene is linked to the agmatine urea hydrolase gene, or the arginine urea hydrolase gene is linked to the arginine decarboxylase gene.

本发明提供一种酶转化生产腐胺的方法,其特征在于,利用所述精氨酸脱羧酶的突变体和胍基丁胺脲水解酶的突变体共同转化精氨酸,生成腐胺。The invention provides a method for producing putrescine by enzymatic conversion, which is characterized in that the mutant of arginine decarboxylase and the mutant of arginine urea hydrolase are used to convert arginine together to generate putrescine.

在一种实施方式中,在40~50℃下反应70~75小时。In one embodiment, the reaction is carried out at 40-50° C. for 70-75 hours.

本发明提供一种生产腐胺的方法,利用所述工程菌全细胞转化生产腐胺。The invention provides a method for producing putrescine, which utilizes whole cells of the engineering bacteria to transform and produce putrescine.

在一种实施方式中,以精氨酸为底物,精氨的浓度不低于300g/L。In one embodiment, arginine is used as a substrate, and the concentration of arginine is not lower than 300 g/L.

在一种实施方式中,反应条件为pH 9.0~10.0、40~50℃、100~200rpm,反应20~30h。In one embodiment, the reaction conditions are pH 9.0-10.0, 40-50° C., 100-200 rpm, and react for 20-30 hours.

本发明还保护所述突变体,或所述工程菌,或所述方法在生产腐胺或者含有腐胺的产品或者以腐胺为前体的物质中的应用。The present invention also protects the mutant, or the engineering bacterium, or the application of the method in the production of putrescine or a product containing putrescine or a substance with putrescine as a precursor.

本发明的有益效果:Beneficial effects of the present invention:

本发明通过对腐胺合成途径中相关的精氨酸脱羧酶和胍基丁胺脲水解酶进行改造,获得了能够解除反馈抑制并提升酶活的两个突变体,利用所述突变体构建了基因工程菌用于腐胺的生产,在全细胞转化条件下,以精氨酸为底物,腐胺产量能达到120g/L及以上。利用两种酶的突变体进行酶转化法,以精氨酸为底物,腐胺产量能达到160g/L。本发明提供的生产腐胺的方法过程简单并且原料容易获得,具有良好的工业化应用前景。The present invention obtains two mutants capable of releasing feedback inhibition and improving enzyme activity by transforming the related arginine decarboxylase and agmatine urea hydrolase in the putrescine synthesis pathway, and using the mutants to construct Genetically engineered bacteria are used for the production of putrescine. Under the condition of whole cell transformation, with arginine as the substrate, the output of putrescine can reach 120g/L and above. The mutants of the two enzymes are used for enzymatic conversion, and the putrescine production can reach 160 g/L with arginine as the substrate. The method for producing putrescine provided by the invention is simple in process and easy to obtain raw materials, and has good industrial application prospect.

附图说明Description of drawings

图1是两种现有的合成腐胺的途径示意图。Figure 1 is a schematic diagram of two existing synthetic putrescine pathways.

具体实施方式Detailed ways

1.本发明所涉及的菌株及质粒1. Bacterial strains and plasmids involved in the present invention

质粒如pCOLADuet-1、pACYCDuet-1、pCDFDuet-1、pETDuet-1、pRSFDuet-1和菌株Escherichia coli JM 109和Escherichia coli BL21(DE3)均购买自购自Novagen公司。Plasmids such as pCOLADuet-1, pACYCDuet-1, pCDFDuet-1, pETDuet-1, pRSFDuet-1 and strains Escherichia coli JM 109 and Escherichia coli BL21 (DE3) were purchased from Novagen.

2.多基因共表达体系的构建及细胞的培养2. Construction of multi-gene co-expression system and cell culture

目前大肠杆菌多基因共表达有多种方法,本发明采用刘向磊(合成生物学技术改造大肠杆菌生产莽草酸及白藜芦醇,2016,上海医药工业研究院,博士论文)所述方法构建,每个基因前均包含T7启动子和RBS结合点。每个质粒上包含两个基因,将构建好的质粒热转导入大肠杆菌感受态细胞中,并涂布于单抗固体平板上,筛选得到阳性转化子,即得到重组大肠杆菌。At present, there are many methods for the co-expression of multiple genes in Escherichia coli. The present invention adopts the method described by Liu Xianglei (Synthetic biology technology transforms E. coli to produce shikimic acid and resveratrol, 2016, Shanghai Pharmaceutical Industry Research Institute, doctoral dissertation). All genes contain T7 promoter and RBS junction. Each plasmid contains two genes, and the constructed plasmid is thermally transfected into E. coli competent cells, and coated on a monoclonal antibody solid plate, and positive transformants are obtained by screening, that is, recombinant E. coli is obtained.

细胞的培养:根据经典的重组大肠杆菌培养及诱导表达方案,将重组大肠杆菌按体积比为2%的量转接到LB发酵培养基(蛋白胨10g/L、酵母粉5g/L、NaCl 10g/L)中,37℃、200rpm培养至细胞OD600达到0.4-0.6后,加入终浓度为0.4mM的IPTG,在15℃诱导表达培养24h。诱导表达结束后,4℃、8000rpm、10min离心收集细胞。Cell culture: According to the classical recombinant Escherichia coli culture and induced expression scheme, the recombinant Escherichia coli was transferred to LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/ In L), culture at 37°C and 200rpm until the OD 600 of the cells reaches 0.4-0.6, then add IPTG with a final concentration of 0.4mM, induce expression and culture at 15°C for 24h. After induction of expression, the cells were collected by centrifugation at 4°C, 8000 rpm, and 10 min.

相应抗生素的浓度分别为:氨苄青霉素为100μg/mL、卡那霉素为40μg/mL、氯霉素为20μg/mL、链霉素为40μg/mL。The concentrations of the corresponding antibiotics were: 100 μg/mL for ampicillin, 40 μg/mL for kanamycin, 20 μg/mL for chloramphenicol, and 40 μg/mL for streptomycin.

3.相关酶的选择3. Selection of relevant enzymes

(1)精氨酸脱羧酶(ADC)(1) Arginine decarboxylase (ADC)

选择来源于Escherichia coli BL21(DE3)的基因speA,其核苷酸序列如SEQ IDNO.5所示,对应的氨基酸序列如GenBank号QJZ13350.1所示。The gene speA derived from Escherichia coli BL21 (DE3) was selected, its nucleotide sequence is shown in SEQ ID NO.5, and the corresponding amino acid sequence is shown in GenBank No. QJZ13350.1.

(2)胍基丁胺脲水解酶(AUH)(2) Agmatine urea hydrolase (AUH)

选择来源于Escherichia coli BL21(DE3)的基因speB,其核苷酸序列如SEQ IDNO.6所示,对应的氨基酸序列如Genbank号为QJZ13349.1所示。The gene speB derived from Escherichia coli BL21 (DE3) was selected, its nucleotide sequence is shown in SEQ ID NO.6, and the corresponding amino acid sequence is shown in Genbank No. QJZ13349.1.

4.样品的检测分析4. Detection and analysis of samples

L-精氨酸、胍基丁胺和腐胺参考并优化了相关文献的方法进行测定(Evaluationof biogenic amines in organic and non-organic wines by HPLC OPAderivatization,Food Technol.Biotechnol.45(1)(2007)62-68)。L-arginine, agmatine and putrescine were determined with reference to and optimized methods in relevant literature (Evaluation of biological amines in organic and non-organic wines by HPLC OPA derivatization, Food Technol.Biotechnol.45(1)(2007) 62-68).

流动相A包含96%的50M乙酸盐缓冲液,4%的四氢呋喃,流动相B为甲醇。Mobile phase A contained 96% 50M acetate buffer, 4% tetrahydrofuran, and mobile phase B was methanol.

梯度洗脱的比例如下:The ratio of gradient elution is as follows:

solvent A:100%(0min),100%(17min),66.7%(22min),0%(30min),0%(35min);Solvent A: 100% (0min), 100% (17min), 66.7% (22min), 0% (30min), 0% (35min);

solvent B:0%(0min),0%(17min),33.3%(22min),100%(30min),100%(35min)。Solvent B: 0% (0min), 0% (17min), 33.3% (22min), 100% (30min), 100% (35min).

色谱柱为Waters Sunfire C18 column,(4.6×250mm,5μm)。The chromatographic column is Waters Sunfire C18 column, (4.6×250mm, 5μm).

液相色谱为:(1260Infinity,Agilent Technologies,Santa Clara,CA)。Liquid chromatography was: (1260 Infinity, Agilent Technologies, Santa Clara, CA).

OPA试剂购买自Agilent。OPA reagent was purchased from Agilent.

5.构建迭代组合突变体文库5. Construction of iterative combinatorial mutant library

根据分子对接的结果,选取ADC的D502、D531、D535及AUH的H151、D153、H163和D232进行第一轮饱和突变。ADC活力提升明显的单点突变体总结在表3中。根据单点突变体,每个位点选取活力提升最明显的两个突变体进行双位点突变,结果如表3所示。最终,根据双位点突变结果,进行三位点组合突变,结果如表4所示。所以,ADC的最优突变株为D502K/D531C/D535P。According to the results of molecular docking, D502, D531, D535 of ADC and H151, D153, H163 and D232 of AUH were selected for the first round of saturation mutation. The single point mutants with significantly improved ADC activity are summarized in Table 3. According to the single-point mutants, two mutants with the most obvious improvement in activity were selected for each site for double-site mutation, and the results are shown in Table 3. Finally, based on the results of double-site mutations, three-site combined mutations were performed, and the results are shown in Table 4. Therefore, the optimal mutant strain of ADC is D502K/D531C/D535P.

按照ADC的改造思路,AUH的单突变及双突变文库筛选结果如表5所示。三位点及四位点突变结果如表6所示。According to the transformation idea of ADC, the screening results of single mutation and double mutation library of AUH are shown in Table 5. The results of three-position and four-position mutations are shown in Table 6.

6.突变体的构建及评价6. Construction and evaluation of mutants

ADC和AUH的饱和突变位点引物设计如表1所示。组合突变体的构建是以上一轮的质粒为模板进行全质粒PCR。组合突变所用到的突变引物如表2所示。The design of primers for saturation mutation sites of ADC and AUH is shown in Table 1. The construction of combinatorial mutants was carried out by PCR of the whole plasmid with the plasmid of the previous round as a template. The mutation primers used for combinatorial mutation are shown in Table 2.

本研究所有突变体均通过全质粒PCR获得。PCR酶选用Mioenzyme公司的2×FastPfu PCR Supermix。扩增体系为:2×FastPfu PCR Supermix 25μL、质粒模板100-200ng、前/后引物各1μL(10μM)、ddH2O补足至50μL。PCR扩增程序为:94℃预变性2min,98℃变性10sec,57℃退火10sec,72℃延伸2min,循环30次,72℃终延伸10min,10℃保温。All mutants in this study were obtained by whole-plasmid PCR. The PCR enzyme was 2×FastPfu PCR Supermix from Mioenzyme Company. The amplification system is: 2×FastPfu PCR Supermix 25μL, plasmid template 100-200ng, front/back primer 1μL (10μM), ddH2O supplemented to 50μL. The PCR amplification program was as follows: pre-denaturation at 94°C for 2 min, denaturation at 98°C for 10 sec, annealing at 57°C for 10 sec, extension at 72°C for 2 min, 30 cycles, final extension at 72°C for 10 min, and incubation at 10°C.

7.蛋白纯化:7. Protein purification:

将得到的粗酶液使用0.22μm纤维素滤膜过滤,采用AKTA avant蛋白纯化系统进行纯化。纯化的具体步骤为:分别用水和对应缓冲液对所需管路进行清洗排气;将5mL Ni-HistrapTM柱连接到仪器上,分别用水和结合液冲洗至少10个柱体积,流速为5mL/min;将粗酶样品装载到镍柱上,用洗脱液进行线性洗脱;分别收集各峰处的蛋白样品,用于酶活测定。所用缓冲液成分如下:20mM Na2HPO4-NaH2PO4缓冲液;结合液包含:20mM磷酸盐缓冲液、50mM NaCl和5mM咪唑;洗脱液包含:20mM磷酸盐缓冲液、50mM NaCl和500mM咪唑。The obtained crude enzyme solution was filtered through a 0.22 μm cellulose filter membrane, and purified using the AKTA avant protein purification system. The specific steps of purification are: clean and exhaust the required pipelines with water and corresponding buffer solution respectively; connect 5mL Ni-HistrapTM column to the instrument, wash at least 10 column volumes with water and binding solution respectively, and the flow rate is 5mL/min ; The crude enzyme sample is loaded on the nickel column, and the eluent is used for linear elution; the protein samples at each peak are collected separately for the determination of enzyme activity. The buffer components used are as follows: 20mM Na 2 HPO 4 -NaH 2 PO 4 buffer; the binding solution contains: 20mM phosphate buffer, 50mM NaCl and 5mM imidazole; the eluent contains: 20mM phosphate buffer, 50mM NaCl and 500mM imidazole.

8.酶活测定:8. Enzyme activity assay:

精氨酸脱羧酶ADC反应体系为:50mM Tris–HCl(pH 7.5),4mM MgSO4,1mM磷酸吡哆醛(PLP),0.1mM和10mM L-精氨酸。反应时间为15min,温度为40℃,反应体积为2mL,最终加入400μL的40%三氯乙酸终止反应。The reaction system of arginine decarboxylase ADC is: 50mM Tris-HCl (pH 7.5), 4mM MgSO 4 , 1mM pyridoxal phosphate (PLP), 0.1mM and 10mM L-arginine. The reaction time was 15 min, the temperature was 40° C., and the reaction volume was 2 mL. Finally, 400 μL of 40% trichloroacetic acid was added to terminate the reaction.

胍基丁胺脲水解酶AUH的反应体系为:50mM Tris–HCl(pH 7.5)、0.1mM二硫苏糖醇和10mM胍基丁胺。反应时间为15min,温度为40℃,反应体积为2mL,最终加入400μL的40%三氯乙酸终止反应。The reaction system of agmatine urea hydrolase AUH is: 50mM Tris-HCl (pH 7.5), 0.1mM dithiothreitol and 10mM agmatine. The reaction time was 15 min, the temperature was 40° C., and the reaction volume was 2 mL. Finally, 400 μL of 40% trichloroacetic acid was added to terminate the reaction.

反应产物胍基丁胺和腐胺的含量由HPLC进行检测。The contents of the reaction products agmatine and putrescine were detected by HPLC.

一个酶活力单位的定义为:每分钟内转化1μmol底物所需要的酶的量。A unit of enzyme activity is defined as the amount of enzyme required to convert 1 μmol of substrate per minute.

表1 ADC和AUH单位点饱和突变引物设计Table 1 Design of primers for single point saturation mutation of ADC and AUH

表2 ADC和AUH组合突变引物设计Table 2 Design of primers for combined mutation of ADC and AUH

实施例1:精氨酸脱羧酶的突变与筛选Example 1: Mutation and screening of arginine decarboxylase

从Escherichia coliBL21(DE3)克隆得到精氨酸脱羧酶基因speA。核苷酸序列如SEQ ID NO.5所示。The arginine decarboxylase gene speA was cloned from Escherichia coli BL21 (DE3). The nucleotide sequence is shown in SEQ ID NO.5.

将克隆得到的基因连接到pETDuet-1载体上,得到含有野生型精氨酸脱羧酶基因的重组载体pETDuet-1-speA,再以pETDuet-1-speA为模板,分别利用表1中的引物对D502-F/R、D531-F/R、D535-F/R引物,进行全质粒PCR,PCR完成后,通过琼脂糖凝胶电泳验证目的片段大小。验证成功后,使用微量分光光度计测定浓度,再利用Quick Cut Dpn I进行母本消化,消化反应体系为:5μL 10×Qucikcut Buffer、1μL Dpn I、4μL DNA、40μL ddH2O,轻轻混匀后瞬时离心,37℃水浴30min。The cloned gene was connected to the pETDuet-1 vector to obtain the recombinant vector pETDuet-1-speA containing the wild-type arginine decarboxylase gene, and then using pETDuet-1-speA as a template, using the primer pairs in Table 1 D502-F/R, D531-F/R, and D535-F/R primers were used for PCR of the whole plasmid. After the PCR was completed, the size of the target fragment was verified by agarose gel electrophoresis. After the verification is successful, use a micro-spectrophotometer to measure the concentration, and then use Quick Cut Dpn I to digest the parent. The digestion reaction system is: 5 μL 10×Qucikcut Buffer, 1 μL Dpn I, 4 μL DNA, 40 μL ddH 2 O, and mix gently Afterwards, it was centrifuged briefly and placed in a water bath at 37°C for 30 minutes.

随后取15μL消化后的PCR产物加入到100μLE.coli JM 1009感受态中,轻混匀,冰浴30min。放入预热的42℃水浴热激处理90s,冰浴2min后加入1mL LB培养基37℃培养1h。最后将菌体均匀涂布在含100μg/mL氨苄青霉素的LB平板上,培养12h,至长出单菌落。挑取单菌落进行测序验证,将测序后突变成功的质粒,取500μL菌液进行菌株保藏,完成突变体库的构建。Then take 15 μL of the digested PCR product and add it to 100 μL of LE.coli JM 1009 competent medium, mix gently, and ice-bath for 30 minutes. Place in a preheated 42°C water bath for heat shock treatment for 90s, ice-bath for 2min, add 1mL LB medium and incubate at 37°C for 1h. Finally, the cells were evenly spread on LB plates containing 100 μg/mL ampicillin, and cultured for 12 hours until a single colony grew. Pick a single colony for sequencing verification, and take 500 μL of the mutated plasmid after sequencing for strain preservation to complete the construction of the mutant library.

将突变成功的质粒热导转入E.coli BL21(DE3)中,用作蛋白的表达并根据蛋白表达量进行筛选。The successfully mutated plasmid was thermally transferred into E.coli BL21(DE3) for protein expression and screened according to the protein expression level.

将转入了含突变体重组质粒的E.coli BL21(DE3)接入96深孔板中进行培养,每孔包含LB培养基450μL,氨苄青霉素为100μg/mL。培养2h后加入终浓度为0.4mM IPTG进行诱导表达,15℃培养5h后离心收集细胞。加入1000U的溶菌酶于37℃、1h进行破壁处理。之后将深孔板于4℃,4000r/min条件下离心15min,取上清200μL并进行纯化、测定酶活。The E.coli BL21(DE3) transformed with the recombinant plasmid containing the mutant was inserted into a 96-deep well plate for culture, each well contained 450 μL of LB medium, and 100 μg/mL of ampicillin. After culturing for 2 hours, IPTG was added at a final concentration of 0.4 mM to induce expression, and cells were collected by centrifugation after culturing at 15°C for 5 hours. Add 1000 U of lysozyme at 37°C for 1 hour to break the wall. Afterwards, the deep-well plate was centrifuged at 4°C and 4000r/min for 15min, and 200μL of the supernatant was taken for purification and determination of enzyme activity.

经HPLC测定酶的活力:如表3所示,单突变体D502C、D502K、D531Y、D531C、D535M、D535P较野生型均有较高的酶活,因此,选取这些位点,组合并进行双突变(所用引物见表2),具体为:The activity of the enzyme was determined by HPLC: as shown in Table 3, the single mutants D502C, D502K, D531Y, D531C, D535M, and D535P all had higher enzyme activities than the wild type. Therefore, these sites were selected, combined and double-mutated (The primers used are shown in Table 2), specifically:

(1)在单突变体D502C的基础上分别对D531Y、D531C、D535M、D535P进行突变;(1) D531Y, D531C, D535M, and D535P were mutated on the basis of the single mutant D502C;

(2)在单突变体D502K的基础上分别对D531Y、D531C、D535M、D535P进行突变;(2) On the basis of single mutant D502K, mutate D531Y, D531C, D535M, and D535P respectively;

(3)在单突变体531Y的基础上分别对D535M、D535P进行突变;(3) On the basis of single mutant 531Y, mutate D535M and D535P respectively;

(4)在单突变体D531C的基础上分别对D535M、D535P进行突变。(4) On the basis of the single mutant D531C, the D535M and D535P were mutated respectively.

利用如单突变相同的筛选和酶活测定方法,得到的双突变体结果如表3所示,D502K/D531Y、D502K/D531C、D502K/D535P、D531C/D535P双突变体的比酶活较高,选取这些双突变体进一步进行突变(所用引物见表2):Using the same screening and enzyme activity assay methods as the single mutation, the results of the double mutants obtained are shown in Table 3. The specific enzyme activities of the D502K/D531Y, D502K/D531C, D502K/D535P, and D531C/D535P double mutants are higher. These double mutants were selected for further mutation (primers used are shown in Table 2):

(1)在双突变体D502K/D531Y的基础上分别对D535M、D535P进行突变;(1) On the basis of the double mutant D502K/D531Y, mutate D535M and D535P respectively;

(2)在双突变体D502K/D531C的基础上分别对D535M、D535P进行突变。(2) On the basis of double mutant D502K/D531C, D535M and D535P were mutated respectively.

利用如单突变相同的筛选和酶活测定方法,测定三突变体的比酶活,结果如表4所示。The specific enzymatic activity of the triple mutant was measured using the same screening and enzymatic activity assay methods as the single mutant, and the results are shown in Table 4.

经多轮突变得到活力最高的突变为D502K/D531C/D535M,序列如SEQ ID NO.1所示。After multiple rounds of mutation, the mutation with the highest activity was D502K/D531C/D535M, the sequence of which is shown in SEQ ID NO.1.

表3 ADC单突变和双突变文库筛选结果Table 3 ADC single mutation and double mutation library screening results

表4 ADC三突变文库筛选结果Table 4 ADC triple mutation library screening results

实施例2:胍基丁胺脲水解酶的突变与筛选表达Example 2: Mutation and screening expression of agmatine urea hydrolase

从Escherichia coliBL21(DE3)克隆得到胍基丁胺脲水解酶基因speB,核苷酸序列如SEQ ID NO.6所示。表达产物用于催化胍基丁胺产生腐胺。The arginidine urea hydrolase gene speB was cloned from Escherichia coliBL21 (DE3), and the nucleotide sequence is shown in SEQ ID NO.6. The expressed product is used to catalyze the production of putrescine from agmatine.

将克隆得到的基因连接到pETDuet-1载体上,得到含有野生型精氨酸脱羧酶基因的重组载体pETDuet-1-speA,再以pETDuet-1-speA为模板,分别利用表1中的引物对H151-F/R、H153-F/R、H163-F/R、D232-F/R,进行全质粒PCR,PCR完成后,通过琼脂糖凝胶电泳验证目的片段大小。验证成功后,使用微量分光光度计测定浓度,再利用Quick Cut Dpn I进行母本消化,消化反应体系为:5μL 10×Qucikcut Buffer、1μL Dpn I、4μL DNA、40μL ddH2O,轻轻混匀后瞬时离心,37℃水浴30min。The cloned gene was connected to the pETDuet-1 vector to obtain the recombinant vector pETDuet-1-speA containing the wild-type arginine decarboxylase gene, and then using pETDuet-1-speA as a template, using the primer pairs in Table 1 For H151-F/R, H153-F/R, H163-F/R, and D232-F/R, carry out the whole plasmid PCR. After the PCR is completed, verify the size of the target fragment by agarose gel electrophoresis. After the verification is successful, use a micro-spectrophotometer to measure the concentration, and then use Quick Cut Dpn I to digest the parent. The digestion reaction system is: 5 μL 10×Qucikcut Buffer, 1 μL Dpn I, 4 μL DNA, 40 μL ddH 2 O, and mix gently Afterwards, it was centrifuged briefly and placed in a water bath at 37°C for 30 minutes.

随后取15μL消化后的PCR产物加入到100μLE.coli JM 109感受态中,轻混匀,冰浴30min。放入预热的42℃水浴热激处理90s,冰浴2min后加入1mL LB培养基37℃培养1h。最后将菌体均匀涂布在含相应抗生素的LB平板上,培养12h,至长出单菌落。挑取单菌落进行测序验证,将测序后突变成功的质粒,取500μL菌液进行菌株保藏,完成突变体库的构建。Then take 15 μL of the digested PCR product and add it to 100 μL E.coli JM 109 competent medium, mix gently, and ice-bath for 30 minutes. Place in a preheated 42°C water bath for heat shock treatment for 90s, ice-bath for 2min, add 1mL LB medium and incubate at 37°C for 1h. Finally, the bacteria were evenly spread on the LB plate containing the corresponding antibiotics, and cultured for 12 hours until a single colony grew. Pick a single colony for sequencing verification, and take 500 μL of the mutated plasmid after sequencing for strain preservation to complete the construction of the mutant library.

将突变成功的质粒热导转入E.coli BL21(DE3)中,用作蛋白的表达并根据蛋白表达量进行筛选。The successfully mutated plasmid was thermally transferred into E.coli BL21(DE3) for protein expression and screened according to the protein expression level.

将转入了突变体序列的E.coli BL21(DE3)接入96深孔板中进行培养,每孔有含100μg/mL氨苄青霉素的LB培养基450μL,。培养2h后加入终浓度为0.4mM IPTG进行诱导表达,15℃培养5h后离心收集细胞。加入1000U的溶菌酶于37℃、1h进行破壁处理。之后将深孔板于4℃,4000r/min条件下离心15min,取上清200μL测定酶活。The E.coli BL21 (DE3) transformed with the mutant sequence was cultured in a 96-deep well plate, and each well contained 450 μL of LB medium containing 100 μg/mL ampicillin. After culturing for 2 hours, IPTG was added at a final concentration of 0.4 mM to induce expression, and cells were collected by centrifugation after culturing at 15°C for 5 hours. Add 1000 U of lysozyme at 37°C for 1 hour to break the wall. Afterwards, the deep-well plate was centrifuged at 4°C and 4000 r/min for 15 min, and 200 μL of the supernatant was taken to measure the enzyme activity.

以胍基丁胺为底物,PLP为辅酶经HPLC测定酶的活力:结果如表5所示,胍基丁胺脲水解酶野生型的比酶活为0.4U/mg,单突变体H151L、D153Y、H163K、H163V、H163A、D232I、D232K,因此,选取这些位点,组合并进行双突变(所用引物见表2),具体为:With agmatine as the substrate, PLP as the coenzyme, the activity of the enzyme was measured by HPLC: the results are shown in Table 5, the specific enzyme activity of the agmatine urea hydrolase wild type is 0.4U/mg, single mutant H151L, D153Y, H163K, H163V, H163A, D232I, D232K, therefore, select these sites, combine and carry out double mutation (primers used are shown in Table 2), specifically:

(1)在单突变体H151L的基础上分别对D153Y、H163V、H163A、D232I、D232K进行突变;(1) On the basis of single mutant H151L, mutate D153Y, H163V, H163A, D232I, and D232K respectively;

(2)在单突变体153Y的基础上分别对H163V、H163A、D232I、D232K进行突变;(2) On the basis of the single mutant 153Y, mutate H163V, H163A, D232I, and D232K respectively;

(3)在单突变体H163V的基础上分别对D232I、D232K进行突变;(3) On the basis of the single mutant H163V, mutate D232I and D232K respectively;

(4)在单突变体H163A的基础上分别对D232I、D232K进行突变。(4) On the basis of single mutant H163A, D232I and D232K were mutated respectively.

利用如单突变相同的筛选和酶活测定方法,测定双突变体的比酶活,结果如表5显示。The specific enzymatic activity of the double mutants was measured using the same screening and enzymatic activity assay methods as the single mutants, and the results are shown in Table 5.

表5 AUH单突变和双突变文库筛选结果Table 5 AUH single mutation and double mutation library screening results

根据双突变体的比酶活,在双突变体的基础上,构建H151L/D153Y/H163A、H151L/H163A/D232I、H151L/H163A/D232K、D153Y/H163A/D232I、D153Y/H163A/D232K(所用引物见表2),利用上述相同的筛选和酶活测定方法,测定三突变体的比酶活,结果如表6所示。According to the specific enzyme activity of double mutants, on the basis of double mutants, construct H151L/D153Y/H163A, H151L/H163A/D232I, H151L/H163A/D232K, D153Y/H163A/D232I, D153Y/H163A/D232K (primers used See Table 2), using the above-mentioned same screening and enzyme activity determination method, the specific enzyme activity of the triple mutant was determined, and the results are shown in Table 6.

选取比酶活较高的D153Y/H163A/D232I、H151L/H163A/D232K,在其基础上,分别在D153Y/H163A/D232I上对H151L突变,在H151L/H163A/D232K基础上对D153Y进行突变(所用引物见表2),结果如表6所示。Select D153Y/H163A/D232I and H151L/H163A/D232K with higher specific enzyme activity, and on the basis of them, mutate H151L on D153Y/H163A/D232I, and mutate D153Y on the basis of H151L/H163A/D232K (used Primers are shown in Table 2), and the results are shown in Table 6.

经多轮突变得到活力最高的突变为H151L/D153Y/H163A/D232K,序列如SEQ IDNO.2所示。组合突变后表达的酶的比酶活为:57U/mg。结合实验结果及三维结构分析,突变株所受到的腐胺和亚精胺等反馈抑制已解除。After multiple rounds of mutation, the mutation with the highest activity was H151L/D153Y/H163A/D232K, the sequence of which is shown in SEQ ID NO.2. The specific enzyme activity of the expressed enzyme after combined mutation is: 57U/mg. Combined with the experimental results and three-dimensional structure analysis, the feedback inhibition of putrescine and spermidine in the mutant strain has been relieved.

表6 AUH三突变和四突变文库筛选结果Table 6 AUH triple mutation and quadruple mutation library screening results

实施例3:同时表达2种酶的重组大肠杆菌的构建Example 3: Construction of recombinant Escherichia coli expressing two enzymes simultaneously

重组大肠杆菌构建:选择突变后的speA和speB基因(核苷酸序列分别如SEQ IDNO.1和2所示),分别连接到pCOLADuet-1(卡那抗性)、pACYCDuet-1(氯霉素抗性)、pCDFduet-1(链霉素抗性)、pETDuet-1(氨苄青霉素抗性)和pRSFDuet-1(卡那抗性)质粒上,每个质粒上表达2个基因,每个基因前均包含T7启动子和RBS结合点,基因后带有T7终止子。Recombinant Escherichia coli construction: select mutated speA and speB genes (nucleotide sequences shown in SEQ ID NO.1 and 2 respectively), and connect to pCOLADuet-1 (Kana resistance), pACYCDuet-1 (chloramphenicol resistance), pCDFduet-1 (streptomycin resistance), pETDuet-1 (ampicillin resistance) and pRSFDuet-1 (canna resistance) plasmids, each expressing 2 genes, each gene before Both contain T7 promoter and RBS junction, with T7 terminator behind the gene.

将质粒转入大肠杆菌Escherichia coli BL21中,利用相对应的抗生平板筛选得到阳性转化子,即得到可强化表达双基因的重组大肠杆菌,如表7所示。The plasmid was transformed into E. coli Escherichia coli BL21, and positive transformants were obtained by screening with corresponding antibiotic plates, that is, recombinant E. coli capable of enhanced expression of double genes was obtained, as shown in Table 7.

全细胞转化生产腐胺:Whole-cell transformation to produce putrescine:

初始反应时,反应体系中细胞湿重20g/L,L-精氨酸为300g/L、PLP为5g/L,MgSO4为9.6g/L、DTT为0.02g/L、pH 9.5,150rpm、于45℃反应,时间为24小时。得到最适的组合为pACYCDuet-speB-speA。全细胞催化结果如表7所示。During the initial reaction, the wet weight of cells in the reaction system was 20g/L, L-arginine was 300g/L, PLP was 5g/L, MgSO4 was 9.6g/L, DTT was 0.02g/L, pH 9.5, 150rpm, React at 45°C for 24 hours. The most suitable combination was obtained as pACYCDuet-speB-speA. The whole-cell catalytic results are shown in Table 7.

表7不同菌株的转化结果Transformation results of different bacterial strains in table 7

实施例4:应用两种酶体外合成腐胺Embodiment 4: Application of two kinds of enzymes to synthesize putrescine in vitro

将双基因speA和speB及其突变体分别连接到pETDuet-1载体上,采用与实施例1同样的方法表达纯化后得到精氨酸脱羧酶和胍基丁胺脲水解酶。然后于50mL反应体系中加入这两种纯酶,精氨酸脱羧酶和胍基丁胺脲水解酶的添加量分别为6.3mg和4mg,反应体系中含有300g/L L-精氨酸,5g/LPLP为,9.6g/L MgSO4,0.02g/L DTT,pH 9.5,150rpm、于45℃反应,转化72小时。最终液相色谱测得腐胺产量为160g/L。The double genes speA and speB and their mutants were respectively connected to the pETDuet-1 vector, expressed and purified by the same method as in Example 1 to obtain arginine decarboxylase and agmatine urea hydrolase. Then add these two pure enzymes in 50mL reaction system, the addition amount of arginine decarboxylase and agmatine urea hydrolase is respectively 6.3mg and 4mg, contains 300g/L L-arginine in the reaction system, 5g /LPLP is, 9.6g/L MgSO 4 , 0.02g/L DTT, pH 9.5, 150rpm, reaction at 45°C, conversion for 72 hours. The output of putrescine measured by final liquid chromatography was 160g/L.

虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

SEQUENCE LISTINGSEQUENCE LISTING

<110> 江南大学<110> Jiangnan University

<120> 一种生产腐胺的工程菌及方法<120> A kind of engineering bacteria and method for producing putrescine

<160> 6<160> 6

<170> PatentIn version 3.3<170> PatentIn version 3.3

<210> 1<210> 1

<211> 1977<211> 1977

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<400> 1<400> 1

atgtctgacg acatgtctat gggtttgcct tcgtcagcgg gcgaacacgg tgtactacgc 60atgtctgacg acatgtctat gggtttgcct tcgtcagcgg gcgaacacgg tgtactacgc 60

tccatgcagg aggttgcaat gagctcccag gaagccagca agatgctgcg tacttacaat 120tccatgcagg aggttgcaat gagctcccag gaagccagca agatgctgcg tacttacaat 120

attgcctggt ggggcaataa ctactatgac gttaacgagc tgggccacat tagcgtgtgc 180attgcctggt ggggcaataa ctactatgac gttaacgagc tgggccacat tagcgtgtgc 180

ccggacccgg acgtcccgga agctcgcgtc gatctcgcgc agttagtgaa aactcgtgaa 240ccggacccgg acgtcccgga agctcgcgtc gatctcgcgc agttagtgaa aactcgtgaa 240

gcacagggcc agcgtctgcc tgcactgttc tgtttcccac agatcctgca gcaccgtttg 300gcacagggcc agcgtctgcc tgcactgttc tgtttcccac agatcctgca gcaccgtttg 300

cgttccatta acgccgcgtt caaacgtgcg agggaatcct acggctataa cggcgattac 360cgttccatta acgccgcgtt caaacgtgcg agggaatcct acggctataa cggcgattac 360

ttccttgttt atccgatcaa agttaaccag caccgccgcg tgattgagtc cctgattcat 420ttccttgttt atccgatcaa agttaaccag caccgccgcg tgattgagtc cctgattcat 420

tcgggcgaac cgctgggtct ggaagccggt tccaaagccg agttgatggc agtactggca 480tcgggcgaac cgctgggtct ggaagccggt tccaaagccg agttgatggc agtactggca 480

catgctggca tgacccgtag cgtcatcgtc tgcaacggtt ataaagaccg cgaatatatc 540catgctggca tgacccgtag cgtcatcgtc tgcaacggtt ataaagaccg cgaatatatc 540

cgcctggcat taattggcga gaagatgggg cacaaggtct atctggtcat tgagaagatg 600cgcctggcat taattggcga gaagatgggg cacaaggtct atctggtcat tgagaagatg 600

tcagaaatcg ccattgtgct ggatgaagca gaacgtctga atgtcgttcc tcgtctgggc 660tcagaaatcg ccattgtgct ggatgaagca gaacgtctga atgtcgttcc tcgtctgggc 660

gtgcgtgcac gtctggcttc gcagggttcg ggtaaatggc agtcctccgg cggggaaaaa 720gtgcgtgcac gtctggcttc gcagggttcg ggtaaatggc agtcctccgg cggggaaaaa 720

tcgaagttcg gcctggctgc gactcaggta ctgcaactgg ttgaaaccct gcgtgaagcc 780tcgaagttcg gcctggctgc gactcaggta ctgcaactgg ttgaaaccct gcgtgaagcc 780

gggcgtctcg acagcctgca actactgcac ttccacctcg gttcgcagat ggcgaatatt 840gggcgtctcg acagcctgca actactgcac ttccacctcg gttcgcagat ggcgaatatt 840

cgcgatatcg cgacaggcgt tcgtgaatcc gcgcgtttct atgtggaact gcacaagctg 900cgcgatatcg cgacaggcgt tcgtgaatcc gcgcgtttct atgtggaact gcacaagctg 900

ggcgtcaata ttcagtgctt cgacgtcggc ggcggtctgg gcgtggatta tgaaggtact 960ggcgtcaata ttcagtgctt cgacgtcggc ggcggtctgg gcgtggatta tgaaggtact 960

cgttcgcagt ccgactgttc ggtgaactac ggcctcaatg aatacgccaa caacattatc 1020cgttcgcagt ccgactgttc ggtgaactac ggcctcaatg aatacgccaa caacattatc 1020

tgggcgattg gcgatgcgtg tgaagaaaac ggtctgccgc atccgacggt aatcaccgaa 1080tgggcgattg gcgatgcgtg tgaagaaaac ggtctgccgc atccgacggt aatcaccgaa 1080

tcgggtcgtg cggtgactgc gcatcacacc gtgctggtgt ctaatatcat cggcgtggaa 1140tcgggtcgtg cggtgactgc gcatcacacc gtgctggtgt ctaatatcat cggcgtggaa 1140

cgtaacgaat acacggtgcc gaccgcgcct gcagaagatg cgccgcgcgc gctgcaaagc 1200cgtaacgaat acacggtgcc gaccgcgcct gcagaagatg cgccgcgcgc gctgcaaagc 1200

atgtgggaaa cctggcagga gatgcacgaa ccgggaactc gccgttctct gcgtgaatgg 1260atgtgggaaa cctggcagga gatgcacgaa ccgggaactc gccgttctct gcgtgaatgg 1260

ttacacgaca gtcagatgga tctgcacgac attcatatcg gctactcttc cggcatcttt 1320ttacacgaca gtcagatgga tctgcacgac attcatatcg gctactcttc cggcatcttt 1320

agcctgcaag aacgtgcatg ggctgagcag ctttatttga gcatgtgcca tgaagtgcaa 1380agcctgcaag aacgtgcatg ggctgagcag ctttatttga gcatgtgcca tgaagtgcaa 1380

aagcagctgg atccgcaaaa ccgtgctcat cgtccgatta tcgacgagct gcaggaacgt 1440aagcagctgg atccgcaaaa ccgtgctcat cgtccgatta tcgacgagct gcaggaacgt 1440

atggcggaca aaatgtacgt caacttctcg ctgttccagt cgatgccgga cgcatggggg 1500atggcggaca aaatgtacgt caacttctcg ctgttccagt cgatgccgga cgcatggggg 1500

atcaaacagt tgttcccggt tctgccgctg gaagggctgg atcaagtgcc ggaacgtcgc 1560atcaaacagt tgttcccggt tctgccgctg gaagggctgg atcaagtgcc ggaacgtcgc 1560

gctgtgctgc tggatattac ctgtgactct tgcggtgcta tcatgcacta tattgatggt 1620gctgtgctgc tggatattac ctgtgactct tgcggtgcta tcatgcacta tattgatggt 1620

gacggtattg ccacgacaat gccaatgccg gagtacgatc cagagaatcc gccgatgctc 1680gacggtattg ccacgacaat gccaatgccg gagtacgatc cagagaatcc gccgatgctc 1680

ggtttcttta tggtcggcgc atatcaggag atcctcggca acatgcacaa cctgttcggt 1740ggtttcttta tggtcggcgc atatcaggag atcctcggca acatgcacaa cctgttcggt 1740

gataccgaag cggttgacgt gttcgtcttc cctgacggta gcgtagaagt agaactgtct 1800gataccgaag cggttgacgt gttcgtcttc cctgacggta gcgtagaagt agaactgtct 1800

gacgaaggcg ataccgtggc ggacatgctg caatatgtac agctcgatcc gaaaacgctg 1860gacgaaggcg ataccgtggc ggacatgctg caatatgtac agctcgatcc gaaaacgctg 1860

ttaacccagt tccgcgatca agtgaagaaa accgatcttg atgctgaact gcaacaacag 1920ttaacccagt tccgcgatca agtgaagaaa accgatcttg atgctgaact gcaacaacag 1920

ttccttgaag agttcgaggc aggtttgtac ggttatactt atcttgaaga tgagtaa 1977ttccttgaag agttcgaggc aggtttgtac ggttatactt atcttgaaga tgagtaa 1977

<210> 2<210> 2

<211> 921<211> 921

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<400> 2<400> 2

atgagcacct taggtcatca atacgataac tcactggttt ccaatgcctt tggtttttta 60atgagcacct taggtcatca atacgataac tcactggttt ccaatgcctt tggtttttta 60

cgcctgccga tgaacttcca gccgtatgac agcgatgcag actgggtgat tactggcgtg 120cgcctgccga tgaacttcca gccgtatgac agcgatgcag actgggtgat tactggcgtg 120

ccgttcgata tggccacttc tggtcgtgcg ggtggtcgcc acggtccggc agcgatccgt 180ccgttcgata tggccacttc tggtcgtgcg ggtggtcgcc acggtccggc agcgatccgt 180

caggtttcga cgaatctggc ctgggaacac aaccgcttcc cgtggaattt cgacatgcgt 240caggtttcga cgaatctggc ctgggaacac aaccgcttcc cgtggaattt cgacatgcgt 240

gagcgtctga acgtcgtgga ctgcggcgat ctggtatatg cctttggcga tgcccgtgag 300gagcgtctga acgtcgtgga ctgcggcgat ctggtatatg cctttggcga tgcccgtgag 300

atgagcgaaa agctgcaggc gcacgccgag aagctgctgg ctgccggtaa gcgtatgctc 360atgagcgaaa agctgcaggc gcacgccgag aagctgctgg ctgccggtaa gcgtatgctc 360

tctttcggtg gtgaccactt tgttacgctg ccgctgctgc gtgctcatgc gaagcatttc 420tctttcggtg gtgaccactt tgttacgctg ccgctgctgc gtgctcatgc gaagcatttc 420

ggcaaaatgg cgctggtaca ctttgacgcc ctaacctaca cctatgcgaa cggttgtgaa 480ggcaaaatgg cgctggtaca ctttgacgcc ctaacctaca cctatgcgaa cggttgtgaa 480

tttgacgcag gcactatgtt ctataccgcg ccgaaagaag gtctgatcga cccgaatcat 540tttgacgcag gcactatgtt ctataccgcg ccgaaagaag gtctgatcga cccgaatcat 540

tccgtgcaga ttggtattcg taccgagttt gataaagaca acggctttac cgtgctggac 600tccgtgcaga ttggtattcg taccgagttt gataaagaca acggctttac cgtgctggac 600

gcctgccagg tgaacgatcg cagcgtggat gacgttatcg cccaagtgaa acagattgtg 660gcctgccagg tgaacgatcg cagcgtggat gacgttatcg cccaagtgaa acagattgtg 660

ggtgatatgc cggtttacct gacttttgat atcaaatgcc tggatcctgc ttttgcacca 720ggtgatatgc cggtttacct gacttttgat atcaaatgcc tggatcctgc ttttgcacca 720

ggcaccggta cgccagtgat tggcggcctg acctccgatc gcgctattaa actggtacgc 780ggcaccggta cgccagtgat tggcggcctg acctccgatc gcgctattaa actggtacgc 780

ggcctgaaag atctcaacat tgttgggatg gacgtagtgg aagtggctcc ggcatacgat 840ggcctgaaag atctcaacat tgttgggatg gacgtagtgg aagtggctcc ggcatacgat 840

cagtcggaaa tcactgctct ggcagcggca acgctggcgc tggaaatgct gtatattcag 900cagtcggaaa tcactgctct ggcagcggca acgctggcgc tggaaatgct gtatattcag 900

gcggcgaaaa agggcgagta a 921gcggcgaaaa agggcgagta a 921

<210> 3<210> 3

<211> 658<211> 658

<212> PRT<212> PRT

<213> 人工序列<213> Artificial sequence

<400> 3<400> 3

Met Ser Asp Asp Met Ser Met Gly Leu Pro Ser Ser Ala Gly Glu HisMet Ser Asp Asp Met Ser Met Gly Leu Pro Ser Ser Ala Gly Glu His

1 5 10 151 5 10 15

Gly Val Leu Arg Ser Met Gln Glu Val Ala Met Ser Ser Gln Glu AlaGly Val Leu Arg Ser Met Gln Glu Val Ala Met Ser Ser Gln Glu Ala

20 25 30 20 25 30

Ser Lys Met Leu Arg Thr Tyr Asn Ile Ala Trp Trp Gly Asn Asn TyrSer Lys Met Leu Arg Thr Tyr Asn Ile Ala Trp Trp Gly Asn Asn Tyr

35 40 45 35 40 45

Tyr Asp Val Asn Glu Leu Gly His Ile Ser Val Cys Pro Asp Pro AspTyr Asp Val Asn Glu Leu Gly His Ile Ser Val Cys Pro Asp Pro Asp

50 55 60 50 55 60

Val Pro Glu Ala Arg Val Asp Leu Ala Gln Leu Val Lys Thr Arg GluVal Pro Glu Ala Arg Val Asp Leu Ala Gln Leu Val Lys Thr Arg Glu

65 70 75 8065 70 75 80

Ala Gln Gly Gln Arg Leu Pro Ala Leu Phe Cys Phe Pro Gln Ile LeuAla Gln Gly Gln Arg Leu Pro Ala Leu Phe Cys Phe Pro Gln Ile Leu

85 90 95 85 90 95

Gln His Arg Leu Arg Ser Ile Asn Ala Ala Phe Lys Arg Ala Arg GluGln His Arg Leu Arg Ser Ile Asn Ala Ala Phe Lys Arg Ala Arg Glu

100 105 110 100 105 110

Ser Tyr Gly Tyr Asn Gly Asp Tyr Phe Leu Val Tyr Pro Ile Lys ValSer Tyr Gly Tyr Asn Gly Asp Tyr Phe Leu Val Tyr Pro Ile Lys Val

115 120 125 115 120 125

Asn Gln His Arg Arg Val Ile Glu Ser Leu Ile His Ser Gly Glu ProAsn Gln His Arg Arg Val Ile Glu Ser Leu Ile His Ser Gly Glu Pro

130 135 140 130 135 140

Leu Gly Leu Glu Ala Gly Ser Lys Ala Glu Leu Met Ala Val Leu AlaLeu Gly Leu Glu Ala Gly Ser Lys Ala Glu Leu Met Ala Val Leu Ala

145 150 155 160145 150 155 160

His Ala Gly Met Thr Arg Ser Val Ile Val Cys Asn Gly Tyr Lys AspHis Ala Gly Met Thr Arg Ser Val Ile Val Cys Asn Gly Tyr Lys Asp

165 170 175 165 170 175

Arg Glu Tyr Ile Arg Leu Ala Leu Ile Gly Glu Lys Met Gly His LysArg Glu Tyr Ile Arg Leu Ala Leu Ile Gly Glu Lys Met Gly His Lys

180 185 190 180 185 190

Val Tyr Leu Val Ile Glu Lys Met Ser Glu Ile Ala Ile Val Leu AspVal Tyr Leu Val Ile Glu Lys Met Ser Glu Ile Ala Ile Val Leu Asp

195 200 205 195 200 205

Glu Ala Glu Arg Leu Asn Val Val Pro Arg Leu Gly Val Arg Ala ArgGlu Ala Glu Arg Leu Asn Val Val Pro Arg Leu Gly Val Arg Ala Arg

210 215 220 210 215 220

Leu Ala Ser Gln Gly Ser Gly Lys Trp Gln Ser Ser Gly Gly Glu LysLeu Ala Ser Gln Gly Ser Gly Lys Trp Gln Ser Ser Gly Gly Glu Lys

225 230 235 240225 230 235 240

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

245 250 255 245 250 255

Leu Arg Glu Ala Gly Arg Leu Asp Ser Leu Gln Leu Leu His Phe HisLeu Arg Glu Ala Gly Arg Leu Asp Ser Leu Gln Leu Leu His Phe His

260 265 270 260 265 270

Leu Gly Ser Gln Met Ala Asn Ile Arg Asp Ile Ala Thr Gly Val ArgLeu Gly Ser Gln Met Ala Asn Ile Arg Asp Ile Ala Thr Gly Val Arg

275 280 285 275 280 285

Glu Ser Ala Arg Phe Tyr Val Glu Leu His Lys Leu Gly Val Asn IleGlu Ser Ala Arg Phe Tyr Val Glu Leu His Lys Leu Gly Val Asn Ile

290 295 300 290 295 300

Gln Cys Phe Asp Val Gly Gly Gly Leu Gly Val Asp Tyr Glu Gly ThrGln Cys Phe Asp Val Gly Gly Gly Leu Gly Val Asp Tyr Glu Gly Thr

305 310 315 320305 310 315 320

Arg Ser Gln Ser Asp Cys Ser Val Asn Tyr Gly Leu Asn Glu Tyr AlaArg Ser Gln Ser Asp Cys Ser Val Asn Tyr Gly Leu Asn Glu Tyr Ala

325 330 335 325 330 335

Asn Asn Ile Ile Trp Ala Ile Gly Asp Ala Cys Glu Glu Asn Gly LeuAsn Asn Ile Ile Trp Ala Ile Gly Asp Ala Cys Glu Glu Asn Gly Leu

340 345 350 340 345 350

Pro His Pro Thr Val Ile Thr Glu Ser Gly Arg Ala Val Thr Ala HisPro His Pro Thr Val Ile Thr Glu Ser Gly Arg Ala Val Thr Ala His

355 360 365 355 360 365

His Thr Val Leu Val Ser Asn Ile Ile Gly Val Glu Arg Asn Glu TyrHis Thr Val Leu Val Ser Asn Ile Ile Gly Val Glu Arg Asn Glu Tyr

370 375 380 370 375 380

Thr Val Pro Thr Ala Pro Ala Glu Asp Ala Pro Arg Ala Leu Gln SerThr Val Pro Thr Ala Pro Ala Glu Asp Ala Pro Arg Ala Leu Gln Ser

385 390 395 400385 390 395 400

Met Trp Glu Thr Trp Gln Glu Met His Glu Pro Gly Thr Arg Arg SerMet Trp Glu Thr Trp Gln Glu Met His Glu Pro Gly Thr Arg Arg Ser

405 410 415 405 410 415

Leu Arg Glu Trp Leu His Asp Ser Gln Met Asp Leu His Asp Ile HisLeu Arg Glu Trp Leu His Asp Ser Gln Met Asp Leu His Asp Ile His

420 425 430 420 425 430

Ile Gly Tyr Ser Ser Gly Ile Phe Ser Leu Gln Glu Arg Ala Trp AlaIle Gly Tyr Ser Ser Gly Ile Phe Ser Leu Gln Glu Arg Ala Trp Ala

435 440 445 435 440 445

Glu Gln Leu Tyr Leu Ser Met Cys His Glu Val Gln Lys Gln Leu AspGlu Gln Leu Tyr Leu Ser Met Cys His Glu Val Gln Lys Gln Leu Asp

450 455 460 450 455 460

Pro Gln Asn Arg Ala His Arg Pro Ile Ile Asp Glu Leu Gln Glu ArgPro Gln Asn Arg Ala His Arg Pro Ile Ile Asp Glu Leu Gln Glu Arg

465 470 475 480465 470 475 480

Met Ala Asp Lys Met Tyr Val Asn Phe Ser Leu Phe Gln Ser Met ProMet Ala Asp Lys Met Tyr Val Asn Phe Ser Leu Phe Gln Ser Met Pro

485 490 495 485 490 495

Asp Ala Trp Gly Ile Lys Gln Leu Phe Pro Val Leu Pro Leu Glu GlyAsp Ala Trp Gly Ile Lys Gln Leu Phe Pro Val Leu Pro Leu Glu Gly

500 505 510 500 505 510

Leu Asp Gln Val Pro Glu Arg Arg Ala Val Leu Leu Asp Ile Thr CysLeu Asp Gln Val Pro Glu Arg Arg Ala Val Leu Leu Asp Ile Thr Cys

515 520 525 515 520 525

Asp Ser Cys Gly Ala Ile Met His Tyr Ile Asp Gly Asp Gly Ile AlaAsp Ser Cys Gly Ala Ile Met His Tyr Ile Asp Gly Asp Gly Ile Ala

530 535 540 530 535 540

Thr Thr Met Pro Met Pro Glu Tyr Asp Pro Glu Asn Pro Pro Met LeuThr Thr Met Pro Met Pro Glu Tyr Asp Pro Glu Asn Pro Pro Met Leu

545 550 555 560545 550 555 560

Gly Phe Phe Met Val Gly Ala Tyr Gln Glu Ile Leu Gly Asn Met HisGly Phe Phe Met Val Gly Ala Tyr Gln Glu Ile Leu Gly Asn Met His

565 570 575 565 570 575

Asn Leu Phe Gly Asp Thr Glu Ala Val Asp Val Phe Val Phe Pro AspAsn Leu Phe Gly Asp Thr Glu Ala Val Asp Val Phe Val Phe Pro Asp

580 585 590 580 585 590

Gly Ser Val Glu Val Glu Leu Ser Asp Glu Gly Asp Thr Val Ala AspGly Ser Val Glu Val Glu Leu Ser Asp Glu Gly Asp Thr Val Ala Asp

595 600 605 595 600 605

Met Leu Gln Tyr Val Gln Leu Asp Pro Lys Thr Leu Leu Thr Gln PheMet Leu Gln Tyr Val Gln Leu Asp Pro Lys Thr Leu Leu Thr Gln Phe

610 615 620 610 615 620

Arg Asp Gln Val Lys Lys Thr Asp Leu Asp Ala Glu Leu Gln Gln GlnArg Asp Gln Val Lys Lys Thr Asp Leu Asp Ala Glu Leu Gln Gln Gln

625 630 635 640625 630 635 640

Phe Leu Glu Glu Phe Glu Ala Gly Leu Tyr Gly Tyr Thr Tyr Leu GluPhe Leu Glu Glu Phe Glu Ala Gly Leu Tyr Gly Tyr Thr Tyr Leu Glu

645 650 655 645 650 655

Asp GluAsp Glu

<210> 4<210> 4

<211> 306<211> 306

<212> PRT<212> PRT

<213> 人工序列<213> Artificial sequence

<400> 4<400> 4

Met Ser Thr Leu Gly His Gln Tyr Asp Asn Ser Leu Val Ser Asn AlaMet Ser Thr Leu Gly His Gln Tyr Asp Asn Ser Leu Val Ser Asn Ala

1 5 10 151 5 10 15

Phe Gly Phe Leu Arg Leu Pro Met Asn Phe Gln Pro Tyr Asp Ser AspPhe Gly Phe Leu Arg Leu Pro Met Asn Phe Gln Pro Tyr Asp Ser Asp

20 25 30 20 25 30

Ala Asp Trp Val Ile Thr Gly Val Pro Phe Asp Met Ala Thr Ser GlyAla Asp Trp Val Ile Thr Gly Val Pro Phe Asp Met Ala Thr Ser Gly

35 40 45 35 40 45

Arg Ala Gly Gly Arg His Gly Pro Ala Ala Ile Arg Gln Val Ser ThrArg Ala Gly Gly Arg His Gly Pro Ala Ala Ile Arg Gln Val Ser Thr

50 55 60 50 55 60

Asn Leu Ala Trp Glu His Asn Arg Phe Pro Trp Asn Phe Asp Met ArgAsn Leu Ala Trp Glu His Asn Arg Phe Pro Trp Asn Phe Asp Met Arg

65 70 75 8065 70 75 80

Glu Arg Leu Asn Val Val Asp Cys Gly Asp Leu Val Tyr Ala Phe GlyGlu Arg Leu Asn Val Val Asp Cys Gly Asp Leu Val Tyr Ala Phe Gly

85 90 95 85 90 95

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

100 105 110 100 105 110

Leu Ala Ala Gly Lys Arg Met Leu Ser Phe Gly Gly Asp His Phe ValLeu Ala Ala Gly Lys Arg Met Leu Ser Phe Gly Gly Asp His Phe Val

115 120 125 115 120 125

Thr Leu Pro Leu Leu Arg Ala His Ala Lys His Phe Gly Lys Met AlaThr Leu Pro Leu Leu Arg Ala His Ala Lys His Phe Gly Lys Met Ala

130 135 140 130 135 140

Leu Val His Phe Asp Ala Leu Thr Tyr Thr Tyr Ala Asn Gly Cys GluLeu Val His Phe Asp Ala Leu Thr Tyr Thr Tyr Ala Asn Gly Cys Glu

145 150 155 160145 150 155 160

Phe Asp Ala Gly Thr Met Phe Tyr Thr Ala Pro Lys Glu Gly Leu IlePhe Asp Ala Gly Thr Met Phe Tyr Thr Ala Pro Lys Glu Gly Leu Ile

165 170 175 165 170 175

Asp Pro Asn His Ser Val Gln Ile Gly Ile Arg Thr Glu Phe Asp LysAsp Pro Asn His Ser Val Gln Ile Gly Ile Arg Thr Glu Phe Asp Lys

180 185 190 180 185 190

Asp Asn Gly Phe Thr Val Leu Asp Ala Cys Gln Val Asn Asp Arg SerAsp Asn Gly Phe Thr Val Leu Asp Ala Cys Gln Val Asn Asp Arg Ser

195 200 205 195 200 205

Val Asp Asp Val Ile Ala Gln Val Lys Gln Ile Val Gly Asp Met ProVal Asp Asp Val Ile Ala Gln Val Lys Gln Ile Val Gly Asp Met Pro

210 215 220 210 215 220

Val Tyr Leu Thr Phe Asp Ile Lys Cys Leu Asp Pro Ala Phe Ala ProVal Tyr Leu Thr Phe Asp Ile Lys Cys Leu Asp Pro Ala Phe Ala Pro

225 230 235 240225 230 235 240

Gly Thr Gly Thr Pro Val Ile Gly Gly Leu Thr Ser Asp Arg Ala IleGly Thr Gly Thr Pro Val Ile Gly Gly Leu Thr Ser Ser Asp Arg Ala Ile

245 250 255 245 250 255

Lys Leu Val Arg Gly Leu Lys Asp Leu Asn Ile Val Gly Met Asp ValLys Leu Val Arg Gly Leu Lys Asp Leu Asn Ile Val Gly Met Asp Val

260 265 270 260 265 270

Val Glu Val Ala Pro Ala Tyr Asp Gln Ser Glu Ile Thr Ala Leu AlaVal Glu Val Ala Pro Ala Tyr Asp Gln Ser Glu Ile Thr Ala Leu Ala

275 280 285 275 280 285

Ala Ala Thr Leu Ala Leu Glu Met Leu Tyr Ile Gln Ala Ala Lys LysAla Ala Thr Leu Ala Leu Glu Met Leu Tyr Ile Gln Ala Ala Lys Lys

290 295 300 290 295 300

Gly GluGly Glu

305305

<210> 5<210> 5

<211> 1977<211> 1977

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<400> 5<400> 5

atgtctgacg acatgtctat gggtttgcct tcgtcagcgg gcgaacacgg tgtactacgc 60atgtctgacg acatgtctat gggtttgcct tcgtcagcgg gcgaacacgg tgtactacgc 60

tccatgcagg aggttgcaat gagctcccag gaagccagca agatgctgcg tacttacaat 120tccatgcagg aggttgcaat gagctcccag gaagccagca agatgctgcg tacttacaat 120

attgcctggt ggggcaataa ctactatgac gttaacgagc tgggccacat tagcgtgtgc 180attgcctggt ggggcaataa ctactatgac gttaacgagc tgggccacat tagcgtgtgc 180

ccggacccgg acgtcccgga agctcgcgtc gatctcgcgc agttagtgaa aactcgtgaa 240ccggacccgg acgtcccgga agctcgcgtc gatctcgcgc agttagtgaa aactcgtgaa 240

gcacagggcc agcgtctgcc tgcactgttc tgtttcccac agatcctgca gcaccgtttg 300gcacagggcc agcgtctgcc tgcactgttc tgtttcccac agatcctgca gcaccgtttg 300

cgttccatta acgccgcgtt caaacgtgcg agggaatcct acggctataa cggcgattac 360cgttccatta acgccgcgtt caaacgtgcg agggaatcct acggctataa cggcgattac 360

ttccttgttt atccgatcaa agttaaccag caccgccgcg tgattgagtc cctgattcat 420ttccttgttt atccgatcaa agttaaccag caccgccgcg tgattgagtc cctgattcat 420

tcgggcgaac cgctgggtct ggaagccggt tccaaagccg agttgatggc agtactggca 480tcgggcgaac cgctgggtct ggaagccggt tccaaagccg agttgatggc agtactggca 480

catgctggca tgacccgtag cgtcatcgtc tgcaacggtt ataaagaccg cgaatatatc 540catgctggca tgacccgtag cgtcatcgtc tgcaacggtt ataaagaccg cgaatatatc 540

cgcctggcat taattggcga gaagatgggg cacaaggtct atctggtcat tgagaagatg 600cgcctggcat taattggcga gaagatgggg cacaaggtct atctggtcat tgagaagatg 600

tcagaaatcg ccattgtgct ggatgaagca gaacgtctga atgtcgttcc tcgtctgggc 660tcagaaatcg ccattgtgct ggatgaagca gaacgtctga atgtcgttcc tcgtctgggc 660

gtgcgtgcac gtctggcttc gcagggttcg ggtaaatggc agtcctccgg cggggaaaaa 720gtgcgtgcac gtctggcttc gcagggttcg ggtaaatggc agtcctccgg cggggaaaaa 720

tcgaagttcg gcctggctgc gactcaggta ctgcaactgg ttgaaaccct gcgtgaagcc 780tcgaagttcg gcctggctgc gactcaggta ctgcaactgg ttgaaaccct gcgtgaagcc 780

gggcgtctcg acagcctgca actactgcac ttccacctcg gttcgcagat ggcgaatatt 840gggcgtctcg acagcctgca actactgcac ttccacctcg gttcgcagat ggcgaatatt 840

cgcgatatcg cgacaggcgt tcgtgaatcc gcgcgtttct atgtggaact gcacaagctg 900cgcgatatcg cgacaggcgt tcgtgaatcc gcgcgtttct atgtggaact gcacaagctg 900

ggcgtcaata ttcagtgctt cgacgtcggc ggcggtctgg gcgtggatta tgaaggtact 960ggcgtcaata ttcagtgctt cgacgtcggc ggcggtctgg gcgtggatta tgaaggtact 960

cgttcgcagt ccgactgttc ggtgaactac ggcctcaatg aatacgccaa caacattatc 1020cgttcgcagt ccgactgttc ggtgaactac ggcctcaatg aatacgccaa caacattatc 1020

tgggcgattg gcgatgcgtg tgaagaaaac ggtctgccgc atccgacggt aatcaccgaa 1080tgggcgattg gcgatgcgtg tgaagaaaac ggtctgccgc atccgacggt aatcaccgaa 1080

tcgggtcgtg cggtgactgc gcatcacacc gtgctggtgt ctaatatcat cggcgtggaa 1140tcgggtcgtg cggtgactgc gcatcacacc gtgctggtgt ctaatatcat cggcgtggaa 1140

cgtaacgaat acacggtgcc gaccgcgcct gcagaagatg cgccgcgcgc gctgcaaagc 1200cgtaacgaat acacggtgcc gaccgcgcct gcagaagatg cgccgcgcgc gctgcaaagc 1200

atgtgggaaa cctggcagga gatgcacgaa ccgggaactc gccgttctct gcgtgaatgg 1260atgtgggaaa cctggcagga gatgcacgaa ccgggaactc gccgttctct gcgtgaatgg 1260

ttacacgaca gtcagatgga tctgcacgac attcatatcg gctactcttc cggcatcttt 1320ttacacgaca gtcagatgga tctgcacgac attcatatcg gctactcttc cggcatcttt 1320

agcctgcaag aacgtgcatg ggctgagcag ctttatttga gcatgtgcca tgaagtgcaa 1380agcctgcaag aacgtgcatg ggctgagcag ctttatttga gcatgtgcca tgaagtgcaa 1380

aagcagctgg atccgcaaaa ccgtgctcat cgtccgatta tcgacgagct gcaggaacgt 1440aagcagctgg atccgcaaaa ccgtgctcat cgtccgatta tcgacgagct gcaggaacgt 1440

atggcggaca aaatgtacgt caacttctcg ctgttccagt cgatgccgga cgcatggggg 1500atggcggaca aaatgtacgt caacttctcg ctgttccagt cgatgccgga cgcatggggg 1500

atcgaccagt tgttcccggt tctgccgctg gaagggctgg atcaagtgcc ggaacgtcgc 1560atcgaccagt tgttcccggt tctgccgctg gaagggctgg atcaagtgcc ggaacgtcgc 1560

gctgtgctgc tggatattac ctgtgactct gacggtgcta tcgaccacta tattgatggt 1620gctgtgctgc tggatattac ctgtgactct gacggtgcta tcgaccacta tattgatggt 1620

gacggtattg ccacgacaat gccaatgccg gagtacgatc cagagaatcc gccgatgctc 1680gacggtattg ccacgacaat gccaatgccg gagtacgatc cagagaatcc gccgatgctc 1680

ggtttcttta tggtcggcgc atatcaggag atcctcggca acatgcacaa cctgttcggt 1740ggtttcttta tggtcggcgc atatcaggag atcctcggca acatgcacaa cctgttcggt 1740

gataccgaag cggttgacgt gttcgtcttc cctgacggta gcgtagaagt agaactgtct 1800gataccgaag cggttgacgt gttcgtcttc cctgacggta gcgtagaagt agaactgtct 1800

gacgaaggcg ataccgtggc ggacatgctg caatatgtac agctcgatcc gaaaacgctg 1860gacgaaggcg ataccgtggc ggacatgctg caatatgtac agctcgatcc gaaaacgctg 1860

ttaacccagt tccgcgatca agtgaagaaa accgatcttg atgctgaact gcaacaacag 1920ttaacccagt tccgcgatca agtgaagaaa accgatcttg atgctgaact gcaacaacag 1920

ttccttgaag agttcgaggc aggtttgtac ggttatactt atcttgaaga tgagtaa 1977ttccttgaag agttcgaggc aggtttgtac ggttatactt atcttgaaga tgagtaa 1977

<210> 6<210> 6

<211> 921<211> 921

<212> DNA<212>DNA

<213> 人工序列<213> Artificial sequence

<400> 6<400> 6

atgagcacct taggtcatca atacgataac tcactggttt ccaatgcctt tggtttttta 60atgagcacct taggtcatca atacgataac tcactggttt ccaatgcctt tggtttttta 60

cgcctgccga tgaacttcca gccgtatgac agcgatgcag actgggtgat tactggcgtg 120cgcctgccga tgaacttcca gccgtatgac agcgatgcag actgggtgat tactggcgtg 120

ccgttcgata tggccacttc tggtcgtgcg ggtggtcgcc acggtccggc agcgatccgt 180ccgttcgata tggccacttc tggtcgtgcg ggtggtcgcc acggtccggc agcgatccgt 180

caggtttcga cgaatctggc ctgggaacac aaccgcttcc cgtggaattt cgacatgcgt 240caggtttcga cgaatctggc ctgggaacac aaccgcttcc cgtggaattt cgacatgcgt 240

gagcgtctga acgtcgtgga ctgcggcgat ctggtatatg cctttggcga tgcccgtgag 300gagcgtctga acgtcgtgga ctgcggcgat ctggtatatg cctttggcga tgcccgtgag 300

atgagcgaaa agctgcaggc gcacgccgag aagctgctgg ctgccggtaa gcgtatgctc 360atgagcgaaa agctgcaggc gcacgccgag aagctgctgg ctgccggtaa gcgtatgctc 360

tctttcggtg gtgaccactt tgttacgctg ccgctgctgc gtgctcatgc gaagcatttc 420tctttcggtg gtgaccactt tgttacgctg ccgctgctgc gtgctcatgc gaagcatttc 420

ggcaaaatgg cgctggtaca ctttgacgcc cacaccgata cctatgcgaa cggttgtgaa 480ggcaaaatgg cgctggtaca ctttgacgcc cacaccgata cctatgcgaa cggttgtgaa 480

tttgaccacg gcactatgtt ctataccgcg ccgaaagaag gtctgatcga cccgaatcat 540tttgaccacg gcactatgtt ctataccgcg ccgaaagaag gtctgatcga cccgaatcat 540

tccgtgcaga ttggtattcg taccgagttt gataaagaca acggctttac cgtgctggac 600tccgtgcaga ttggtattcg taccgagttt gataaagaca acggctttac cgtgctggac 600

gcctgccagg tgaacgatcg cagcgtggat gacgttatcg cccaagtgaa acagattgtg 660gcctgccagg tgaacgatcg cagcgtggat gacgttatcg cccaagtgaa acagattgtg 660

ggtgatatgc cggtttacct gacttttgat atcgactgcc tggatcctgc ttttgcacca 720ggtgatatgc cggtttacct gacttttgat atcgactgcc tggatcctgc ttttgcacca 720

ggcaccggta cgccagtgat tggcggcctg acctccgatc gcgctattaa actggtacgc 780ggcaccggta cgccagtgat tggcggcctg acctccgatc gcgctattaa actggtacgc 780

ggcctgaaag atctcaacat tgttgggatg gacgtagtgg aagtggctcc ggcatacgat 840ggcctgaaag atctcaacat tgttgggatg gacgtagtgg aagtggctcc ggcatacgat 840

cagtcggaaa tcactgctct ggcagcggca acgctggcgc tggaaatgct gtatattcag 900cagtcggaaa tcactgctct ggcagcggca acgctggcgc tggaaatgct gtatattcag 900

gcggcgaaaa agggcgagta a 921gcggcgaaaa agggcgagta a 921

Claims (9)

1. Mutants of enzymes associated with putrescine synthesis, characterized in that the enzymes are arginine decarboxylase and agmatine urea hydrolase;
the amino acid sequence of the mutant of the arginine decarboxylase is shown as SEQ ID NO. 3;
the amino acid sequence of the mutant of agmatine urea hydrolase is shown as SEQ ID NO. 4.
2. A genetically engineered bacterium is characterized by taking escherichia coli as a host and expressing arginine decarboxylase with a nucleotide sequence shown as SEQ ID NO.1 and agmatine urea hydrolase gene with a nucleotide sequence shown as SEQ ID NO. 2.
3. The engineering bacterium according to claim 2, wherein pACYCDuet, pRSFDuet, pCDFDuet or pcoladat is used as an expression vector.
4. A method for producing putrescine by enzymatic conversion, characterized in that arginine is converted to putrescine by using the mutant of claim 1.
5. The method of claim 4, wherein the reaction is carried out at 40-50 ℃ for 70-75 hours.
6. A method for producing putrescine, which is characterized in that arginine is used as a substrate, and the putrescine is produced by whole cell transformation of the genetically engineered bacterium of claim 2 or 3.
7. The method of claim 6, wherein the concentration of arginine is not less than 300g/L.
8. The method according to claim 6, wherein the reaction conditions are pH 9.0 to 10.0, 40 to 50 ℃, 100 to 200rpm, and the reaction is carried out for 20 to 30 hours.
9. Use of a mutant according to claim 1, or a genetically engineered bacterium according to any one of claims 2 to 3, or a method according to any one of claims 4 to 8, for the production of putrescine or a putrescine-containing product or a putrescine-precursor-containing substance.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN110257448A (en) * 2019-07-02 2019-09-20 山东国力生物科技有限公司 A method of using thallus whole-cell catalytic conversion of Arginine be gamatine

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110257448A (en) * 2019-07-02 2019-09-20 山东国力生物科技有限公司 A method of using thallus whole-cell catalytic conversion of Arginine be gamatine

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