CN110511874B - 基于杜氏藻代谢途径的链孢红素高产工程菌及其构建方法与应用 - Google Patents
基于杜氏藻代谢途径的链孢红素高产工程菌及其构建方法与应用 Download PDFInfo
- Publication number
- CN110511874B CN110511874B CN201910665236.XA CN201910665236A CN110511874B CN 110511874 B CN110511874 B CN 110511874B CN 201910665236 A CN201910665236 A CN 201910665236A CN 110511874 B CN110511874 B CN 110511874B
- Authority
- CN
- China
- Prior art keywords
- leu
- ala
- gly
- val
- ser
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
- C12Y103/99—Oxidoreductases acting on the CH-CH group of donors (1.3) with other acceptors (1.3.99)
- C12Y103/99026—All-trans-zeta-carotene desaturase (1.3.99.26)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
- C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
- C12Y205/01029—Geranylgeranyl diphosphate synthase (2.5.1.29)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y502/00—Cis-trans-isomerases (5.2)
- C12Y502/01—Cis-trans-Isomerases (5.2.1)
- C12Y502/01012—Zeta-carotene isomerase (5.2.1.12)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开一种基于杜氏藻代谢途径的链孢红素高产工程菌及其构建方法与应用,涉及基因工程菌领域。本发明第一次实现全部利用来自杜氏藻(如杜氏巴氏藻或杜氏盐藻)的类胡萝卜素途径的基因来构建工程菌。当杜氏藻为杜氏巴氏藻时,所述链孢红素高产工程菌的链孢红素的产率为2.4mg/g(细胞干重);当杜氏藻为杜氏盐藻时,所述链孢红素高产工程菌的链孢红素的产率为3.1mg/g(细胞干重)。本发明采用的载体的启动子都是T7启动子,包含lacZ调控元件,其在IPTG作用的大肠杆菌BL21(DE3)中能够高表达,这有利于链孢红素的大量积累,可用于实际应用生产。
Description
技术领域
本发明涉及基因工程菌领域,涉及植物类胡萝卜素代谢途径载体的构建克隆系统,特别涉及一种基于杜氏藻代谢途径的链孢红素高产工程菌及其构建方法与应用;具体涉及一种杜氏藻(如杜氏巴氏藻(Dunaliella bardawil)、杜氏盐藻(Dunaliella saline))中牦牛儿基牻牛儿基焦磷酸合成酶(Ggps)、八氢番茄红素合成酶(Psy)、八氢番茄红素脱氢酶(Pds)、15-顺式-ζ-胡萝卜素异构酶(Ziso)、ζ-胡萝卜素脱氢酶(Zds)基因的cDNA并构建出高产链孢红素的工程菌。
背景技术
类胡萝卜素分子是含8个异戊二烯单位的萜类化合物,其作为有机色素广泛存在于植物的叶绿体和有色体中,以及其它的一些光合组织中,比如藻类,部分细菌和真菌中。在特定条件下,杜氏藻细胞中类胡萝卜素含量可达到细胞干重的14%。其高含量可能跟酶的转化效率有很大的关系。
发明内容
为了克服现有技术的缺点与不足,本发明的首要目的在于提供一种基于杜氏藻代谢途径的链孢红素高产工程菌。
本发明的另一目的在于提供上述基于杜氏藻代谢途径的链孢红素高产工程菌的构建方法。
本发明的再一目的在于提供上述基于杜氏藻代谢途径的链孢红素高产工程菌的应用。
本发明的目的通过下述技术方案实现:
一种基于杜氏藻代谢途径的链孢红素高产工程菌,基于杜氏巴氏藻(Dunaliellabardawil)代谢途径时,所述链孢红素高产工程菌含有编码GenBank:APW83740.1所示氨基酸序列的牦牛儿基牻牛儿基焦磷酸合成酶(Ggps)基因、编码SEQ ID NO:2所示氨基酸序列的八氢番茄红素合成酶(Psy)基因、编码GenBank:ADD52599.1所示氨基酸序列的八氢番茄红素脱氢酶(Pds)基因、编码SEQ ID NO:4所示氨基酸序列的15-顺式-ζ-胡萝卜素异构酶(Ziso)基因和编码SEQ ID NO:6所示氨基酸序列的ζ-胡萝卜素脱氢酶(Zds)基因;
基于杜氏盐藻(Dunaliella saline)代谢途径时,所述链孢红素高产工程菌含有编码SEQ ID NO:8所示氨基酸序列的牦牛儿基牻牛儿基焦磷酸合成酶(Ggps)基因、编码GenBank:AAB51287.1所示氨基酸序列的八氢番茄红素合成酶(Psy)基因、编码GenBank:CAA75094.1所示氨基酸序列的八氢番茄红素脱氢酶(Pds)基因、编码SEQ ID NO:10所示氨基酸序列的15-顺式-ζ-胡萝卜素异构酶(Ziso)基因和编码SEQ ID NO:12所示氨基酸序列的ζ-胡萝卜素脱氢酶(Zds)基因;
优选的,基于杜氏巴氏藻(Dunaliella bardawil)代谢途径时,编码GenBank:APW83740.1所示氨基酸序列的牦牛儿基牻牛儿基焦磷酸合成酶(Ggps)基因的核苷酸序列为如GenBank:KX231795.1所示的核苷酸序列;其中,GenBank:APW83740.1与GenBank:KX231795.1中的来源Dunaliella salina记载有误,实际来源应该是Dunaliellabardawil;
编码SEQ ID NO:2所示氨基酸序列的八氢番茄红素合成酶(Psy)基因的核苷酸序列为如SEQ ID NO:1所示的核苷酸序列;
编码GenBank:ADD52599.1所示氨基酸序列的八氢番茄红素脱氢酶(Pds)基因的核苷酸序列为如GenBank:GQ923693.1所示的核苷酸序列;其中,GenBank:ADD52599.1与GenBank:GQ923693.1中的来源Dunaliella salina记载有误,实际来源应该是Dunaliellabardawil;
编码SEQ ID NO:4所示氨基酸序列的15-顺式-ζ-胡萝卜素异构酶(Ziso)基因的核苷酸序列为如SEQ ID NO:3所示的核苷酸序列;
编码SEQ ID NO:6所示氨基酸序列的ζ-胡萝卜素脱氢酶(Zds)基因的核苷酸序列为如SEQ ID NO:5所示的核苷酸序列;
优选的,基于杜氏盐藻(Dunaliella saline)代谢途径时,编码SEQ ID NO:8所示氨基酸序列的牦牛儿基牻牛儿基焦磷酸合成酶(Ggps)基因的核苷酸序列为如SEQ ID NO:7所示的核苷酸序列;
编码GenBank:AAB51287.1所示氨基酸序列的八氢番茄红素合成酶(Psy)基因的核苷酸序列为如GenBank:U91900.1所示的核苷酸序列;其中,GenBank:U91900.1中的来源Dunaliella bardawil记载有误,实际来源应该是Dunaliella saline;
编码GenBank:CAA75094.1所示氨基酸序列的八氢番茄红素脱氢酶(Pds)基因的核苷酸序列为如GenBank:Y14807.1所示的核苷酸序列;其中,GenBank:Y14807.1中的来源Dunaliella bardawil记载有误,实际来源应该是Dunaliella saline;
编码SEQ ID NO:10所示氨基酸序列的15-顺式-ζ-胡萝卜素异构酶(Ziso)基因的核苷酸序列为如SEQ ID NO:9所示的核苷酸序列;
编码SEQ ID NO:12所示氨基酸序列的ζ-胡萝卜素脱氢酶(Zds)基因的核苷酸序列为如SEQ ID NO:11所示的核苷酸序列;
所述的基于杜氏藻代谢途径的链孢红素高产工程菌的构建方法,包括如下步骤:
(1)利用相关基因工程手段从杜氏藻中克隆到牦牛儿基牻牛儿基焦磷酸合成酶(Ggps)、八氢番茄红素合成酶(Psy)、八氢番茄红素脱氢酶(Pds)、15-顺式-ζ-胡萝卜素异构酶(Ziso)、ζ-胡萝卜素脱氢酶(Zds);
(2)将Ggps和Psy构建在pACYduet-1载体上,氯霉素抗性,得到重组载体pACYduet-ggps-psy;将Pds和Zds构建在pCDFduet-1载体上,链霉素抗性,得到重组载体pCDFduet-pds-zds;将Ziso构建在pETduet-1载体上,氨苄抗性,得到重组载体pETduet-ziso;
(3)然后将步骤(2)构建的三个重组载体共转化到大肠杆菌BL21(DE3)中,获得基于杜氏藻代谢途径的链孢红素高产工程菌。
优选的,所述的杜氏藻为杜氏巴氏藻(Dunaliella bardawil)或杜氏盐藻(Dunaliella saline)。
优选的,重组载体构建时,在5个目的基因的N端(5′端)均连接有rbs(核糖体结合位点)序列,或者,均连接有T7启动子(T7_promoter)序列和rbs(核糖体结合位点)序列。
所述的基于杜氏藻代谢途径的链孢红素高产工程菌在生产链孢红素中的应用。
当杜氏藻为杜氏巴氏藻(Dunaliella bardawil)时,所述链孢红素高产工程菌的链孢红素的产率为2.4mg/g(细胞干重)。
当杜氏藻为杜氏盐藻(Dunaliella saline)时,所述链孢红素高产工程菌的链孢红素的产率为3.1mg/g(细胞干重)。
本发明相对于现有技术具有如下的优点及效果:
本发明第一次实现全部利用来自杜氏藻(如杜氏巴氏藻或杜氏盐藻)的类胡萝卜素途径的基因来构建工程菌。而杜氏藻(如杜氏巴氏藻或杜氏盐藻)是已知的产链孢红素最高的植物之一。本发明采用的载体的启动子都是T7启动子,包含lacZ调控元件,其在IPTG作用的大肠杆菌BL21(DE3)中能够高表达,这有利于链孢红素的大量积累,可用于实际应用生产。
附图说明
图1是本发明pACYduet-Dbggps-Dbpsy(A)和pCDFduet-Dbpds-Dbzds(B)构建图。
图2是本发明pETduet-Dbziso构建图。
图3是本发明基于杜氏藻代谢途径的链孢红素高产工程菌与普通大肠杆菌BL21(DE3)颜色比较;其中,A是基于杜氏巴氏藻代谢途径的链孢红素高产工程菌;B是基于杜氏盐藻代谢途径的链孢红素高产工程菌。
图4是链孢红素液相图;其中,A是实施例1;B是实施例2。
图5是本发明pACYduet1-Dsggps-Dspsy构建图。
图6是本发明pCDFduet1-Dspds-Dszds构建图。
图7是本发明pETduet1-Dsziso构建图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
下列实施例中未注明具体实验条件的试验方法,通常按照常规实验条件或按照制造厂所建议的实验条件。所使用的材料、试剂等,如无特殊说明,为从商业途径得到的试剂和材料。
实施例1
1.制备线性化载体
(1)用NcoI和HindIII(购自Thermo Fisher Scientific)双酶切质粒pACYduet-1,pCDFduet-1和pETduet-1(购自EMD Biosciences)。37℃水浴3h。酶切反应体系如下:
10×Buffer 5μL、两种酶各1μL、质粒10μL、Add dH2O To 50μL。
(2)1%琼脂糖凝胶电泳检测酶切产物。
(3)酶切之后,采用PCR纯化试剂盒纯化线性化载体。
2.PCR扩增目的片段
(1)以杜氏巴氏藻(Dunaliella bardawil)(购自中科院水生生物研究所淡水藻种库,编号FACHB-847)反转录的cDNA为模板,使用Takara的PrimeSTAR HS DNA Polymerase(高保真酶,购自Takara),进行PCR反应。
(2)1%琼脂糖凝胶电泳检测PCR产物。
(3)用OMEGA公司的胶回收试剂盒E.Z.N.A.TM Gel Extraction Kit回收目的片段,纯化PCR产物。测序结果如GenBank:KX231795.1、SEQ ID NO:1、GenBank:GQ923693.1、SEQ ID NO:3、5所示,对应编码的氨基酸序列如GenBank:APW83740.1、SEQ ID NO:2、GenBank:ADD52599.1、SEQ ID NO:4、6所示。
3.In-Fusion克隆
用NcoI和HindIII双酶切质粒pACYduet-1并插入用引物Pac_dbggps_F和Pac_dbggps_R扩增得到的Dbggps,得到pACYduet-Dbggps;再用NdeI和EcoRV双酶切pACYduet-Dbggps并插入用引物Pac_dbpsy_F和Pac_dbpsy_R扩增得到的Dbpsy,得到pACYduet-Dbggps-Dbpsy。引物序列如表1所示。
用NcoI和HindIII双酶切质粒pCDFduet-1并插入用引物Pcd_dbpds_F和Pcd_dbpds_R扩增得到的Dbpds,得到pCDFduet-Dbpds;再用NdeI和EcoRV双酶切pCDFduet-Dbpds并插入用引物Pcd_dbzds_F和Pcd_dbzds_R扩增得到的Dbzds,得到pCDFduet-Dbpds-Dbzds。引物序列如表1所示。
用NcoI和HindIII双酶切质粒pETduet-1并插入用引物Pet_dbziso_F和Pet_dbziso_R扩增得到的Dbziso,得到pETduet-Dbziso。引物序列如表1所示。
(1)建立In-Fusion(购自Takara)克隆反应体系:
5X In-Fusion HD Enzyme Premix | 2μL |
线性化载体 | 1μL |
纯化的PCR片段 | 2μL |
dH<sub>2</sub>O | 5μL |
Total | 10μL |
(2)50℃孵育15min,然后置于冰上。
(3)连接产物分别转化大肠杆菌DH5α,涂布于含有氯霉素(CmR或CamR)、链霉素(Sm)或氨苄青霉素(Ap或Amp)的相应平板上。在37℃生化培养箱中分别倒置培养12~16h。注意培养时间不宜过长,否则抗性失效,生长出卫星菌落,给筛选阳性克隆造成困难。
表1构建重组质粒的引物
4.质粒的提取
参照OMEGA公司的E.Z.N.A.TM Plasmid Mini Kit试剂盒的说明书,提取重组质粒pACYduet-Dbggps-Dbpsy(图1A)、pCDFduet-Dbpds-Dbzds(图1B)、pETduet-Dbziso(图2)。
(1)10000g,1min离心,收集菌体。(2)加入250μL SolutionⅠ,涡旋十几秒,混匀。(3)加入250μL SolutionⅡ,上下颠倒4~6次,室温放置2min。(4)加入350μL SolutionⅢ,轻轻摇匀,10000g,10min离心。(5)转上清至Hibind柱,10000g,1min离心。(6)弃掉滤液,加入500μL Buffer HB,10000g,1min离心。(7)弃掉滤液,加入700μL DNA Wash Buffer,10000g,1min离心。(8)重复洗一次(步骤(7))。(9)10000g,空管离心2min。(10)弃掉收集管将HiBind柱套进干净的1.5mL EP管,加入50~100μL灭菌水(60℃预热)洗脱DNA,静置1~2min后,10000g,1min离心。
5.转化大肠杆菌BL21(DE3)
将提取的重组质粒pACYduet-Dbggps-Dbpsy、pCDFduet-Dbpds-Dbzds和pETduet-Dbziso共转化到大肠杆菌BL21(DE3),涂布于含有CmR、Sm和Ap抗生素的平板上,在37℃生化培养箱中倒置培养12~16h。经过筛选获得目的菌株,即基于杜氏巴氏藻代谢途径的链孢红素高产工程菌,如图3A所示,白色为BL21(DE3)对照菌株,土黄色的为高产链孢红素菌株。
6.转化子的培养
将目的菌株接种于50mL已灭菌的LB液体培养基中(加入氯霉素、氨苄青霉素、链霉素抗性),于200rpm,37℃下振荡培养6h。加入IPTG进行诱导,培养6小时。
7.提取转化子的类胡萝卜素
(1)将24mL菌液于4℃,8000rpm下离心2min,弃上清。(2)用去离子水重新悬浮大肠杆菌细胞,4℃,8000rpm离心2min,弃上清。(3)重复步骤(2)。(4)加6mL丙酮至大肠杆菌沉淀,涡旋振荡分散。(5)55℃水浴20min,每隔5min振荡一次。(6)将混合物于4℃,8000rpm下离心20min,收集上清。(7)用丙酮定容至6mL,-20℃保存。
8.HPLC定性分析转化子的类胡萝卜素成分
(1)将流动相用0.22μm孔径滤膜进行抽滤处理,接着超声处理60min去除液体中的气泡。
(2)将类胡萝卜素提取物以及链孢红素标准品溶液,用0.2μm孔径的聚碳酸酯滤膜进行过滤处理。
(3)调节色谱柱(C30YMC carotenoid column,5μm,250*4.6mm)温度在25℃,以1.0mL/min的流速用甲醇冲洗至平衡(约2h)。
(4)改为用流动相甲基叔丁基醚(A相)/甲醇(B相)(梯度洗脱:0min,90%A相和10%B相;10min,60%A相和40%B相;20min 50%A相和50%B相;25min,10%A相和90%B相;29.5min,90%A相和10%B相),以1mL/min的流速进行洗柱,直至平衡。
(5)将浓度为100μg/mL的链孢红素标准品存储液稀释为0.625μg/mL、1.25μg/mL、2.5μg/mL、5μg/mL、10μg/mL和20μg/mL等不同浓度的工作液,用HPLC仪测定473nm波长下工作液的峰面积,根据结果绘制标准曲线。
(6)上样20μL,在波长473nm下检测40min。
结果如图4A所示,表明在18.2min左右出现了链孢红素的特征峰,这与标准品的出峰时间是一致的,另外根据该峰面积,结合得到的标准曲线方程:y=63.124x-0.4755(x为峰面积,y为链孢红素的含量),再根据细胞的干重,我们可以计算出链孢红素的产率为2.4mg/g(细胞干重)。
实施例2
1.制备线性化载体
(1)用Asc I和Sal I(购自Thermo Fisher Scientific)双酶切质粒pACYduet-1,pCDFduet-1和pETduet-1(购自EMD Biosciences)。37℃水浴3h。酶切反应体系如下:
10×Buffer 5μL、两种酶各1μL、质粒10μL、Add dH2O To 50μL。
(2)1%琼脂糖凝胶电泳检测酶切产物。
(3)酶切之后,采用PCR纯化试剂盒纯化线性化载体。
2.PCR扩增目的片段
(1)以杜氏盐藻(Dunaliella saline)(购自Culture Collection of Algae andProtozoa,英国;编号CCAP 19/18)反转录的cDNA为模板,使用Takara的PrimeSTAR HS DNAPolymerase(高保真酶,购自Takara),进行PCR反应。
(2)1%琼脂糖凝胶电泳检测PCR产物。
(3)用OMEGA公司的胶回收试剂盒E.Z.N.A.TM Gel Extraction Kit回收目的片段,纯化PCR产物。测序结果如SEQ ID NO:7、GenBank:U91900.1、GenBank:Y14807.1、SEQ IDNO:9、11所示,对应编码的氨基酸序列如SEQ ID NO:8、GenBank:AAB51287.1、GenBank:CAA75094.1、SEQ ID NO:10、12所示。
3.In-Fusion克隆
用Asc I和Sal I双酶切质粒pACYduet-1并插入用引物Pac_dsggps_F和Pac_dsggps_R扩增得到的Dsggps,得到pACYduet1-Dsggps;再用NdeI和EcoRV双酶切pACYduet1-Dsggps并插入用引物Pac_dspsy_F和Pac_dspsy_R扩增得到的Dspsy,得到pACYduet1-Dsggps-Dspsy。引物序列如表2所示。
用Asc I和Sal I双酶切质粒pCDFduet-1并插入用引物Pcd_dspds_F和Pcd_dspds_R扩增得到的Dspds,得到pCDFduet1-Dspds;再用NdeI和EcoRV双酶切pCDFduet1-Dspds并插入用引物Pcd_dszds_F和Pcd_dszds_R扩增得到的Dszds,得到pCDFduet1-Dspds-Dszds。引物序列如表2所示。
用Asc I和Sal I双酶切质粒pETduet-1并插入用引物Pet_dsziso_F和Pet_dsziso_R扩增得到的Dsziso,得到pETduet1-Dsziso。引物序列如表2所示。
(1)建立In-Fusion(购自Takara)克隆反应体系:
5X In-Fusion HD Enzyme Premix | 2μL |
线性化载体 | 1μL |
纯化的PCR片段 | 2μL |
dH<sub>2</sub>O | 5μL |
Total | 10μL |
(2)50℃孵育15min,然后置于冰上。
(3)连接产物分别转化大肠杆菌DH5α,涂布于含有氯霉素(CmR或CamR)、链霉素(Sm)或氨苄青霉素(Ap或Amp)的相应的平板上。在37℃生化培养箱中分别倒置培养12~16h。注意培养时间不宜过长,否则抗性失效,生长出卫星菌落,给筛选阳性克隆造成困难。
表2构建重组质粒的引物
4.质粒的提取
参照OMEGA公司的E.Z.N.A.TM Plasmid Mini Kit试剂盒的说明书,提取重组质粒pACYduet1-Dsggps-Dspsy(图5)、pCDFduet1-Dspds-Dszds(图6)、pETduet1-Dsziso(图7)。
(1)10000g,1min离心,收集菌体。(2)加入250μL SolutionⅠ,涡旋十几秒,混匀。(3)加入250μL SolutionⅡ,上下颠倒4~6次,室温放置2min。(4)加入350μL SolutionⅢ,轻轻摇匀,10000g,10min离心。(5)转上清至Hibind柱,10000g,1min离心。(6)弃掉滤液,加入500μL Buffer HB,10000g,1min离心。(7)弃掉滤液,加入700μL DNA Wash Buffer,10000g,1min离心。(8)重复洗一次(步骤(7))。(9)10000g,空管离心2min。(10)弃掉收集管将HiBind柱套进干净的1.5mL EP管,加入50~100μL灭菌水(60℃预热)洗脱DNA,静置1~2min后,10000g,1min离心。
5.转化大肠杆菌BL21(DE3)
将提取的重组质粒pACYduet1-Dsggps-Dspsy、pCDFduet1-Dspds-Dszds和pETduet1-Dsziso共转化到大肠杆菌BL21(DE3),涂布于含有CamR、Sm和Amp抗生素的平板上,在37℃生化培养箱中倒置培养12~16h。经过筛选获得目的菌株,即基于杜氏盐藻代谢途径的链孢红素高产工程菌,如图3B所示,白色为BL21(DE3)对照菌株,土黄色的为高产链孢红素菌株。
6.转化子的培养
将目的菌株接种于50mL已灭菌的LB液体培养基中(加入氯霉素、氨苄青霉素、链霉素抗性),于200rpm,37℃下振荡培养6h。加入IPTG进行诱导,培养6小时。
7.提取转化子的类胡萝卜素
(1)将24mL菌液于4℃,8000rpm下离心2min,弃上清。(2)用去离子水重新悬浮大肠杆菌细胞,4℃,8000rpm离心2min,弃上清。(3)重复步骤(2)。(4)加6mL丙酮至大肠杆菌沉淀,涡旋振荡分散。(5)55℃水浴20min,每隔5min振荡一次。(6)将混合物于4℃,8000rpm下离心20min,收集上清。(7)用丙酮定容至6mL,-20℃保存。
8.HPLC定性分析转化子的类胡萝卜素成分
(1)将流动相用0.22μm孔径滤膜进行抽滤处理,接着超声处理60min去除液体中的气泡。
(2)将类胡萝卜素提取物以及链孢红素标准品溶液,用0.2μm孔径的聚碳酸酯滤膜进行过滤处理。
(3)调节色谱柱(C30YMC carotenoid column,5μm,250*4.6mm)温度在25℃,以1.0mL/min的流速用甲醇冲洗至平衡(约2h)。
(4)改为用流动相甲基叔丁基醚(A相)/甲醇(B相)(梯度洗脱:0min,90%A相和10%B相;10min,60%A相和40%B相;20min 50%A相和50%B相;25min,10%A相和90%B相;29.5min,90%A相和10%B相),以1mL/min的流速进行洗柱,直至平衡。
(5)链孢红素的标准曲线参照实施例1步骤8(5)。
(6)上样20μL,在波长473nm下检测40min。
结果如图4B所示,结果表明在18.2min左右出现了链孢红素的特征峰,这与标准品的出峰时间是一致的,另外根据该峰面积,结合得到的标准曲线方程:y=63.124x-0.4755(x为峰面积,y为链孢红素的含量),再根据细胞的干重,可以计算出链孢红素的产率为3.1mg/g(细胞干重)。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 华南理工大学
<120> 基于杜氏藻代谢途径的链孢红素高产工程菌及其构建方法与应用
<160> 32
<170> SIPOSequenceListing 1.0
<211> 1305
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 杜氏巴氏藻(Dunaliella bardawil):Psy
<400> 1
atgacgctgt caatgttgga cgcgcgaagg atggcacagc gaacagcaac ttcctcctcc 60
tcctctccta gcatcatata tgccccatcg cccataagca atcgcagcgg caggcgcgca 120
gcagcgaatc acggcatcag gaatggtagt cgcagagcag caggccggat gggactctgc 180
agcactgtgc aagtgaactg cacgctcgcg atgccccagc ccaaccacgg ccagaagatg 240
cgattgcagc agcagcagca gcaacaactg cagcagcagc agcaacagca actatcggga 300
aagcaggtgg aggagcaggc gatgctgcag tgcataaaga ccgctcagtc agtgcccccc 360
tccaccggac tcctcaatcc tcgcggcctg cgatggcagg gcagcagctt ggaagcagcg 420
tacgagcgat gtggggcggt gtgcagcgag tacgccaaga ccttctacct cggtactcag 480
ctcatgacac cagtgcaggc caggtgcatc tgggccatct atgtgtggtg ccgccgcaca 540
gatgagctgg tggatggccc caatgcatca aagatcacgc ctcaggccct agacagatgg 600
gaggagcgcc ttgaaagcat gttccaaggc aagccctatg atgtgctgga cgcggcgctc 660
acagacacca tctccaaatt ccctctggag gtgcaaccct tcagagacat gatcgagggc 720
atgcgaatgg acctcttcaa gtcgcggtat cacacctttg atgagctgta cgagtactgc 780
tatcgtgtgg cgggcacagt ggggctgatg accatgccag tgatggggat tgatcccaac 840
tacaagggtc caattgacaa ggtctacaag gccgcccttg cgctgggtac ggcaaaccag 900
ctcaccaaca ttctgcgaga tgtgggagag gacatcagag agcgtgaccg tatctacttg 960
cccctggatg agctcaagca gttcggcatc tccgaagagg aggtaaaagc aggtatccac 1020
aagccatcgc aaggcaaggt ggatgagcgg tggcgagcgt tcatgaagtt ccagatcaag 1080
cgtgcgcgag agtacttcca ggaagcagag gatggggtag actacttgga cgtgaaggcg 1140
cggtggccag tgtggtcagc gctgatcctt taccgccaaa tcttggatgt cattgagaag 1200
aatgactacg acaacttctc catgcgcgca tacgtgccta agtccaagaa gtttgcatcg 1260
ttgccgatgg ccttgttccg ggccttggtg cccaagaaca aataa 1305
<211> 434
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 杜氏巴氏藻(Dunaliella bardawil):Psy
<400> 2
Met Thr Leu Ser Met Leu Asp Ala Arg Arg Met Ala Gln Arg Thr Ala
1 5 10 15
Thr Ser Ser Ser Ser Ser Pro Ser Ile Ile Tyr Ala Pro Ser Pro Ile
20 25 30
Ser Asn Arg Ser Gly Arg Arg Ala Ala Ala Asn His Gly Ile Arg Asn
35 40 45
Gly Ser Arg Arg Ala Ala Gly Arg Met Gly Leu Cys Ser Thr Val Gln
50 55 60
Val Asn Cys Thr Leu Ala Met Pro Gln Pro Asn His Gly Gln Lys Met
65 70 75 80
Arg Leu Gln Gln Gln Gln Gln Gln Gln Leu Gln Gln Gln Gln Gln Gln
85 90 95
Gln Leu Ser Gly Lys Gln Val Glu Glu Gln Ala Met Leu Gln Cys Ile
100 105 110
Lys Thr Ala Gln Ser Val Pro Pro Ser Thr Gly Leu Leu Asn Pro Arg
115 120 125
Gly Leu Arg Trp Gln Gly Ser Ser Leu Glu Ala Ala Tyr Glu Arg Cys
130 135 140
Gly Ala Val Cys Ser Glu Tyr Ala Lys Thr Phe Tyr Leu Gly Thr Gln
145 150 155 160
Leu Met Thr Pro Val Gln Ala Arg Cys Ile Trp Ala Ile Tyr Val Trp
165 170 175
Cys Arg Arg Thr Asp Glu Leu Val Asp Gly Pro Asn Ala Ser Lys Ile
180 185 190
Thr Pro Gln Ala Leu Asp Arg Trp Glu Glu Arg Leu Glu Ser Met Phe
195 200 205
Gln Gly Lys Pro Tyr Asp Val Leu Asp Ala Ala Leu Thr Asp Thr Ile
210 215 220
Ser Lys Phe Pro Leu Glu Val Gln Pro Phe Arg Asp Met Ile Glu Gly
225 230 235 240
Met Arg Met Asp Leu Phe Lys Ser Arg Tyr His Thr Phe Asp Glu Leu
245 250 255
Tyr Glu Tyr Cys Tyr Arg Val Ala Gly Thr Val Gly Leu Met Thr Met
260 265 270
Pro Val Met Gly Ile Asp Pro Asn Tyr Lys Gly Pro Ile Asp Lys Val
275 280 285
Tyr Lys Ala Ala Leu Ala Leu Gly Thr Ala Asn Gln Leu Thr Asn Ile
290 295 300
Leu Arg Asp Val Gly Glu Asp Ile Arg Glu Arg Asp Arg Ile Tyr Leu
305 310 315 320
Pro Leu Asp Glu Leu Lys Gln Phe Gly Ile Ser Glu Glu Glu Val Lys
325 330 335
Ala Gly Ile His Lys Pro Ser Gln Gly Lys Val Asp Glu Arg Trp Arg
340 345 350
Ala Phe Met Lys Phe Gln Ile Lys Arg Ala Arg Glu Tyr Phe Gln Glu
355 360 365
Ala Glu Asp Gly Val Asp Tyr Leu Asp Val Lys Ala Arg Trp Pro Val
370 375 380
Trp Ser Ala Leu Ile Leu Tyr Arg Gln Ile Leu Asp Val Ile Glu Lys
385 390 395 400
Asn Asp Tyr Asp Asn Phe Ser Met Arg Ala Tyr Val Pro Lys Ser Lys
405 410 415
Lys Phe Ala Ser Leu Pro Met Ala Leu Phe Arg Ala Leu Val Pro Lys
420 425 430
Asn Lys
<211> 1104
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 杜氏巴氏藻(Dunaliella bardawil):Ziso
<400> 3
atggcgagct tgtgtagagc tgcccttggg caggccagtg cgaaggggct gagtggctta 60
caaacttcct ccaagcctct catctccaag agccctcttg tagcaagacc accatgcatc 120
agaattagcg aacgctgccc agtcttagaa aattccgtat ggagcagaag acgagcggag 180
gtgtgtgtgc gggctgctag cgacgaggag tcgccccgac ctgcggggct tgttggcgag 240
gatgcggcag ccttcgacgt ttcccagcag agtaccaagt cctgggcgct gtttactggg 300
cttctgactg gcgtgctggg cctcatttac ctggtatgga tccagccggg agcaggtctg 360
gcagatgact tcttggggtt ggtcgagggc ttcagcaaca acaacccgga ggcaacaatc 420
ctccttatcc tctttgtgtt tgctgttgta cacagtgggc tggcaggcct gcgccccaaa 480
ggggagcagc tgatcggcgc acgagcatac cgggtgattt ttgcccttgt cagcctgccc 540
ttggccatcg tggccatcgt gtacttcatc aaccatagat acgatggcat gcccttatgg 600
gatctcaggg gtgtgacggg agtgcatgag ctggtgtggc tcctcaactt tgtgtccttc 660
tacttccttt acccttccac ctttaacatc cttgaggtgg ctgcagtgga cgagcccaag 720
ctgcatatgt gggaaacggg aatcatgcga atcactcgtc acccacaaat ggtagggcag 780
ctaatatggt gcgcagcaca cacgttgtgg atcgggaaca gcttcatgct cgtgacctca 840
gcaggcctca tggctcatca tctctttggt tgctggcatg gtgaccggcg gttgtctgcc 900
aagtatggcg aggcctttga aatcgtcaag gcgcggacaa gtacctttcc attgcaagct 960
atttgggagg gccgccaggt cttgccagct gattactaca aggagttcct gcgggcgccc 1020
tacttcgctg tgactgcctt caccctgggc gcctacttcg cacaccccat catgcagtct 1080
gccagcttct atttgagatg gtag 1104
<211> 367
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 杜氏巴氏藻(Dunaliella bardawil):Ziso
<400> 4
Met Ala Ser Leu Cys Arg Ala Ala Leu Gly Gln Ala Ser Ala Lys Gly
1 5 10 15
Leu Ser Gly Leu Gln Thr Ser Ser Lys Pro Leu Ile Ser Lys Ser Pro
20 25 30
Leu Val Ala Arg Pro Pro Cys Ile Arg Ile Ser Glu Arg Cys Pro Val
35 40 45
Leu Glu Asn Ser Val Trp Ser Arg Arg Arg Ala Glu Val Cys Val Arg
50 55 60
Ala Ala Ser Asp Glu Glu Ser Pro Arg Pro Ala Gly Leu Val Gly Glu
65 70 75 80
Asp Ala Ala Ala Phe Asp Val Ser Gln Gln Ser Thr Lys Ser Trp Ala
85 90 95
Leu Phe Thr Gly Leu Leu Thr Gly Val Leu Gly Leu Ile Tyr Leu Val
100 105 110
Trp Ile Gln Pro Gly Ala Gly Leu Ala Asp Asp Phe Leu Gly Leu Val
115 120 125
Glu Gly Phe Ser Asn Asn Asn Pro Glu Ala Thr Ile Leu Leu Ile Leu
130 135 140
Phe Val Phe Ala Val Val His Ser Gly Leu Ala Gly Leu Arg Pro Lys
145 150 155 160
Gly Glu Gln Leu Ile Gly Ala Arg Ala Tyr Arg Val Ile Phe Ala Leu
165 170 175
Val Ser Leu Pro Leu Ala Ile Val Ala Ile Val Tyr Phe Ile Asn His
180 185 190
Arg Tyr Asp Gly Met Pro Leu Trp Asp Leu Arg Gly Val Thr Gly Val
195 200 205
His Glu Leu Val Trp Leu Leu Asn Phe Val Ser Phe Tyr Phe Leu Tyr
210 215 220
Pro Ser Thr Phe Asn Ile Leu Glu Val Ala Ala Val Asp Glu Pro Lys
225 230 235 240
Leu His Met Trp Glu Thr Gly Ile Met Arg Ile Thr Arg His Pro Gln
245 250 255
Met Val Gly Gln Leu Ile Trp Cys Ala Ala His Thr Leu Trp Ile Gly
260 265 270
Asn Ser Phe Met Leu Val Thr Ser Ala Gly Leu Met Ala His His Leu
275 280 285
Phe Gly Cys Trp His Gly Asp Arg Arg Leu Ser Ala Lys Tyr Gly Glu
290 295 300
Ala Phe Glu Ile Val Lys Ala Arg Thr Ser Thr Phe Pro Leu Gln Ala
305 310 315 320
Ile Trp Glu Gly Arg Gln Val Leu Pro Ala Asp Tyr Tyr Lys Glu Phe
325 330 335
Leu Arg Ala Pro Tyr Phe Ala Val Thr Ala Phe Thr Leu Gly Ala Tyr
340 345 350
Phe Ala His Pro Ile Met Gln Ser Ala Ser Phe Tyr Leu Arg Trp
355 360 365
<211> 1749
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 杜氏巴氏藻(Dunaliella bardawil):Zds
<400> 5
atgttggggc tgcagagcaa ggaatcgcaa ctgtgcacca caaatgtgcc agccaggcgt 60
ggctatgcgc agtgcaccag tacccgcacc cgcagacgga cgcgctgcac cacccaagcc 120
attgccaccc cccctgctcc tccaaagacc acacccaggg agtggaccac ccaggatgtg 180
agcaaggtgg ccttgaagga tgtgcctttg aagtccttgt acccggatga gcctgcccct 240
ccaaagccag gtgcccccaa gatgcgtgtg gccattgtgg gcagtggact ggctggcctg 300
tcgacagcag tggagctgct agaccaaggg catgaggtgg acatctatga ccagcgcagc 360
tgggttggag gcaaggtggc ctcatggcaa gacaaggatg gcaaccacat tgagatgggc 420
ttgcacgtct tcttcggctg ctaccacaac cttttccgtc tgatggccaa gtgtggagta 480
ctggagaact tgctggtgaa ggagcatact cacacctttt gcaacaatga tggggatgtc 540
agggagcttg acttccgttt tgaggtcgga ggacagaaaa tcggggctcc cttccatggc 600
ctgaaagcct tcttcacaac cccccagctt tctgtgggag acaaggcagc caatgcgctg 660
gcgctgggca ccagccctat tgtgcgctcc ttgatagacc cagagggggg catgagtgat 720
gtgcgaaacc tggacaacat cagcttctgg gactggttca agagccatgg cgggtctgag 780
caatccatga agcgcatgtg ggatcccatt gcgtatgctt tgggtttctt ggactgcaaa 840
gacatcagtg cgcgctgcat gctgaccatc ttccagttct ttgccaccaa gaccgacgcc 900
tccgtcctgc gcatgctgaa cggatctcca gctgagaggc tcctgaagcc catcaccgac 960
tacattgagg ccaagggagg ccgcatccac ttgcgtcagg gttgcaagga ggttctattt 1020
gaggatggcc ctgacggcaa gcctgtagtg accggcatgt gcatgggccg ggatggccaa 1080
gttgtgaagg ctgatgccta tgttgcagcc ctggacgtcc ctggggcgaa gcagctcttg 1140
ccgcaggcat ggagaaagta cccccagttt gacaaaatct acaagctcaa tggcgtgcct 1200
gtgatcactg tacagctgcg ctacaatggt tgggtgacgg agatgcagga cccagagaag 1260
gtcaagcagc tgacccagcc ccaaggcatc aacaacctgt tgtacagccc tgatgcgttc 1320
ttctcctgct tcgccgacct tgcccttgtg agccctgtgg agtacttcca tgagggcaag 1380
ggctccctca tgcaagtcgt gatcacgcct gctgccccgt acatgccatg gaccaacgag 1440
gccattgctg aggaggctga ccggcaggtc cgccagctct tccccagcgc ccgtaagctg 1500
gacatgatct ggcatagtgt tgtgaagatt ggccagtcct tgtaccagga ggccccaggc 1560
atggaccctt acaggcccga gcaggccacg cctgtgccca acttcttcct ggcaggcagc 1620
tacaccaagc aagactacat cgactcaatg gagggcgcca ccttgtcagg tcggcagtgt 1680
gctggtgaga ttatgaaggc tgtgcccttg atccaaagcc tgtcaaaggc gccacttccg 1740
agcatgtaa 1749
<211> 582
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 杜氏巴氏藻(Dunaliella bardawil):Zds
<400> 6
Met Leu Gly Leu Gln Ser Lys Glu Ser Gln Leu Cys Thr Thr Asn Val
1 5 10 15
Pro Ala Arg Arg Gly Tyr Ala Gln Cys Thr Ser Thr Arg Thr Arg Arg
20 25 30
Arg Thr Arg Cys Thr Thr Gln Ala Ile Ala Thr Pro Pro Ala Pro Pro
35 40 45
Lys Thr Thr Pro Arg Glu Trp Thr Thr Gln Asp Val Ser Lys Val Ala
50 55 60
Leu Lys Asp Val Pro Leu Lys Ser Leu Tyr Pro Asp Glu Pro Ala Pro
65 70 75 80
Pro Lys Pro Gly Ala Pro Lys Met Arg Val Ala Ile Val Gly Ser Gly
85 90 95
Leu Ala Gly Leu Ser Thr Ala Val Glu Leu Leu Asp Gln Gly His Glu
100 105 110
Val Asp Ile Tyr Asp Gln Arg Ser Trp Val Gly Gly Lys Val Ala Ser
115 120 125
Trp Gln Asp Lys Asp Gly Asn His Ile Glu Met Gly Leu His Val Phe
130 135 140
Phe Gly Cys Tyr His Asn Leu Phe Arg Leu Met Ala Lys Cys Gly Val
145 150 155 160
Leu Glu Asn Leu Leu Val Lys Glu His Thr His Thr Phe Cys Asn Asn
165 170 175
Asp Gly Asp Val Arg Glu Leu Asp Phe Arg Phe Glu Val Gly Gly Gln
180 185 190
Lys Ile Gly Ala Pro Phe His Gly Leu Lys Ala Phe Phe Thr Thr Pro
195 200 205
Gln Leu Ser Val Gly Asp Lys Ala Ala Asn Ala Leu Ala Leu Gly Thr
210 215 220
Ser Pro Ile Val Arg Ser Leu Ile Asp Pro Glu Gly Gly Met Ser Asp
225 230 235 240
Val Arg Asn Leu Asp Asn Ile Ser Phe Trp Asp Trp Phe Lys Ser His
245 250 255
Gly Gly Ser Glu Gln Ser Met Lys Arg Met Trp Asp Pro Ile Ala Tyr
260 265 270
Ala Leu Gly Phe Leu Asp Cys Lys Asp Ile Ser Ala Arg Cys Met Leu
275 280 285
Thr Ile Phe Gln Phe Phe Ala Thr Lys Thr Asp Ala Ser Val Leu Arg
290 295 300
Met Leu Asn Gly Ser Pro Ala Glu Arg Leu Leu Lys Pro Ile Thr Asp
305 310 315 320
Tyr Ile Glu Ala Lys Gly Gly Arg Ile His Leu Arg Gln Gly Cys Lys
325 330 335
Glu Val Leu Phe Glu Asp Gly Pro Asp Gly Lys Pro Val Val Thr Gly
340 345 350
Met Cys Met Gly Arg Asp Gly Gln Val Val Lys Ala Asp Ala Tyr Val
355 360 365
Ala Ala Leu Asp Val Pro Gly Ala Lys Gln Leu Leu Pro Gln Ala Trp
370 375 380
Arg Lys Tyr Pro Gln Phe Asp Lys Ile Tyr Lys Leu Asn Gly Val Pro
385 390 395 400
Val Ile Thr Val Gln Leu Arg Tyr Asn Gly Trp Val Thr Glu Met Gln
405 410 415
Asp Pro Glu Lys Val Lys Gln Leu Thr Gln Pro Gln Gly Ile Asn Asn
420 425 430
Leu Leu Tyr Ser Pro Asp Ala Phe Phe Ser Cys Phe Ala Asp Leu Ala
435 440 445
Leu Val Ser Pro Val Glu Tyr Phe His Glu Gly Lys Gly Ser Leu Met
450 455 460
Gln Val Val Ile Thr Pro Ala Ala Pro Tyr Met Pro Trp Thr Asn Glu
465 470 475 480
Ala Ile Ala Glu Glu Ala Asp Arg Gln Val Arg Gln Leu Phe Pro Ser
485 490 495
Ala Arg Lys Leu Asp Met Ile Trp His Ser Val Val Lys Ile Gly Gln
500 505 510
Ser Leu Tyr Gln Glu Ala Pro Gly Met Asp Pro Tyr Arg Pro Glu Gln
515 520 525
Ala Thr Pro Val Pro Asn Phe Phe Leu Ala Gly Ser Tyr Thr Lys Gln
530 535 540
Asp Tyr Ile Asp Ser Met Glu Gly Ala Thr Leu Ser Gly Arg Gln Cys
545 550 555 560
Ala Gly Glu Ile Met Lys Ala Val Pro Leu Ile Gln Ser Leu Ser Lys
565 570 575
Ala Pro Leu Pro Ser Met
580
<211> 1077
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 杜氏盐藻(Dunaliella saline):Ggps
<400> 7
atggccgccc atcagatgca gctctttagt agccaacgga tgtgctcaac gtcctcaagg 60
agcatgaggc cagcagtctg cagccggccg caagtgccac gaattcgccc tgcaaacgtt 120
agacaaggtc gccatcaggc cttcaggaca atggccattg caactgcaga cgaggcccag 180
cagtccactt cttcctttga cttcaagagc tatatgaagg agcgtgcagt catggtgaat 240
gatgcgttgg acaaggccct gccgcagcgc tatccagagg tgctgctaga gtctatgagg 300
tactcactcc tagctggtgg caagcgcgtg cgcccatgcc tgaccttggc tgcctgcgaa 360
tgcgttggag gggacattgc gcacgcaatg cccactgcct gtgcaatgga ggtggttcac 420
accatgagcc tgatccacga cgacctaccc tccatggaca atgatgattt ccgcagaggg 480
tctcccacca accacaagaa atacggagag gacattgcca ttcttgccgg agacgccctg 540
ctttctttcg ccttcgagca cgtcgcgcgc gccaccactg gcacctcgcc tgagcgcgtg 600
ttgcgtgtga tcattgagct gggcaaggct gtgggtgcag atgggctaac aggaggacag 660
gttgtggaca tcaagagcga aaaccaggag gtgggcctgg aagttctgca gtacatccat 720
gagcacaaga cagcggccct gctagaggca gctgtggtgt gtggcgcgct ggtgggcggc 780
gcggatgatg tgacagtgga gaagatgcgc aagtttgcac tcaacatcgg ccttgcattc 840
caggtggtgg acgacatcct ggattgtacc cagaccacag agcagctggg caagactgca 900
ggcaaggaca tgggcgtgaa caagaccaca taccccaaac tgcttggcct ggagaagagt 960
aagcagaccg cggaggacct gatcacagag gccatccagc agctagatgg cttccccccg 1020
gagaagcgcg ccccccttgt agctctggcc aagtacattg gctaccgtca aaactaa 1077
<211> 358
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 杜氏盐藻(Dunaliella saline):Ggps
<400> 8
Met Ala Ala His Gln Met Gln Leu Phe Ser Ser Gln Arg Met Cys Ser
1 5 10 15
Thr Ser Ser Arg Ser Met Arg Pro Ala Val Cys Ser Arg Pro Gln Val
20 25 30
Pro Arg Ile Arg Pro Ala Asn Val Arg Gln Gly Arg His Gln Ala Phe
35 40 45
Arg Thr Met Ala Ile Ala Thr Ala Asp Glu Ala Gln Gln Ser Thr Ser
50 55 60
Ser Phe Asp Phe Lys Ser Tyr Met Lys Glu Arg Ala Val Met Val Asn
65 70 75 80
Asp Ala Leu Asp Lys Ala Leu Pro Gln Arg Tyr Pro Glu Val Leu Leu
85 90 95
Glu Ser Met Arg Tyr Ser Leu Leu Ala Gly Gly Lys Arg Val Arg Pro
100 105 110
Cys Leu Thr Leu Ala Ala Cys Glu Cys Val Gly Gly Asp Ile Ala His
115 120 125
Ala Met Pro Thr Ala Cys Ala Met Glu Val Val His Thr Met Ser Leu
130 135 140
Ile His Asp Asp Leu Pro Ser Met Asp Asn Asp Asp Phe Arg Arg Gly
145 150 155 160
Ser Pro Thr Asn His Lys Lys Tyr Gly Glu Asp Ile Ala Ile Leu Ala
165 170 175
Gly Asp Ala Leu Leu Ser Phe Ala Phe Glu His Val Ala Arg Ala Thr
180 185 190
Thr Gly Thr Ser Pro Glu Arg Val Leu Arg Val Ile Ile Glu Leu Gly
195 200 205
Lys Ala Val Gly Ala Asp Gly Leu Thr Gly Gly Gln Val Val Asp Ile
210 215 220
Lys Ser Glu Asn Gln Glu Val Gly Leu Glu Val Leu Gln Tyr Ile His
225 230 235 240
Glu His Lys Thr Ala Ala Leu Leu Glu Ala Ala Val Val Cys Gly Ala
245 250 255
Leu Val Gly Gly Ala Asp Asp Val Thr Val Glu Lys Met Arg Lys Phe
260 265 270
Ala Leu Asn Ile Gly Leu Ala Phe Gln Val Val Asp Asp Ile Leu Asp
275 280 285
Cys Thr Gln Thr Thr Glu Gln Leu Gly Lys Thr Ala Gly Lys Asp Met
290 295 300
Gly Val Asn Lys Thr Thr Tyr Pro Lys Leu Leu Gly Leu Glu Lys Ser
305 310 315 320
Lys Gln Thr Ala Glu Asp Leu Ile Thr Glu Ala Ile Gln Gln Leu Asp
325 330 335
Gly Phe Pro Pro Glu Lys Arg Ala Pro Leu Val Ala Leu Ala Lys Tyr
340 345 350
Ile Gly Tyr Arg Gln Asn
355
<211> 1122
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 杜氏盐藻(Dunaliella saline):Ziso
<400> 9
atggcaagct tgtgtgcagg tcgggccctt gggctggctg gtcaggggct gactggctca 60
catgcctcct ctcgaggcct cccagccaag cctctcgtct ccaggagccc gatccttgcc 120
aagacgccac catgcatcag aaatagggat ctccagcaag ccttaggaat ttccgtaccc 180
cacagaagac gatcggaagt gtgtgtgcgg gctgccagcg aggaggagtc gtccccacct 240
gcagggcttg tgggcgagga tgcggctgcc ttcgacgttt ctcaacaaag caccaagtct 300
tgggcgatat tcactgggct tctgactggc gtgctgggcc tcatttacct ggtttggatc 360
cagccgggag cagggctggc agatgacttc ctgagcactg tcgagagctt cagcaacaac 420
aaccccgagg caacaatcct cctcatcctg tttgtgtttg cggtcgcgca cagtgggcta 480
gcagccctgc gcccaaaagg cgagcagctg atcggtgctc gagcattccg cgtgattttt 540
gcccttgtca gcctgcccct ggccatcgtg gcggtggtgt atttcatcaa ccacagatat 600
gatggcatac ccttatggga tctcaggggt gtgacgggag tgcatgagct ggtgtggacc 660
ctcaacttca tttccttcta cttcctgtac ccgtccacct tcaacatcct tgaggtggct 720
gcagtggatg agcccaagct ccacatgtgg gaaaccggca tcatgcgaat cactcgtcac 780
ccacaaatgg tagggcaggc catctggtgt gcagcgcaca cgctgtggat cgggaacagc 840
ttcatgctgg tgacttcggc aggcctcatg gctcaccacc ttttcgggtg ctggcatggt 900
gacaagcgcc tgtcaaacaa gtatggcgag gcctttgaaa tcgtgaagac gcgcaccagc 960
actttcccga tgcaggctgt ttgggagggc cgccagaaat tgccagctga cttctacaag 1020
gagttcctgc gagcccccta ctttgctgtg actgcgttca ccctgggcgc ctattatgca 1080
caccccatca tgcagtctgc tagcttctac ctggggtggt ga 1122
<211> 373
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 杜氏盐藻(Dunaliella saline):Ziso
<400> 10
Met Ala Ser Leu Cys Ala Gly Arg Ala Leu Gly Leu Ala Gly Gln Gly
1 5 10 15
Leu Thr Gly Ser His Ala Ser Ser Arg Gly Leu Pro Ala Lys Pro Leu
20 25 30
Val Ser Arg Ser Pro Ile Leu Ala Lys Thr Pro Pro Cys Ile Arg Asn
35 40 45
Arg Asp Leu Gln Gln Ala Leu Gly Ile Ser Val Pro His Arg Arg Arg
50 55 60
Ser Glu Val Cys Val Arg Ala Ala Ser Glu Glu Glu Ser Ser Pro Pro
65 70 75 80
Ala Gly Leu Val Gly Glu Asp Ala Ala Ala Phe Asp Val Ser Gln Gln
85 90 95
Ser Thr Lys Ser Trp Ala Ile Phe Thr Gly Leu Leu Thr Gly Val Leu
100 105 110
Gly Leu Ile Tyr Leu Val Trp Ile Gln Pro Gly Ala Gly Leu Ala Asp
115 120 125
Asp Phe Leu Ser Thr Val Glu Ser Phe Ser Asn Asn Asn Pro Glu Ala
130 135 140
Thr Ile Leu Leu Ile Leu Phe Val Phe Ala Val Ala His Ser Gly Leu
145 150 155 160
Ala Ala Leu Arg Pro Lys Gly Glu Gln Leu Ile Gly Ala Arg Ala Phe
165 170 175
Arg Val Ile Phe Ala Leu Val Ser Leu Pro Leu Ala Ile Val Ala Val
180 185 190
Val Tyr Phe Ile Asn His Arg Tyr Asp Gly Ile Pro Leu Trp Asp Leu
195 200 205
Arg Gly Val Thr Gly Val His Glu Leu Val Trp Thr Leu Asn Phe Ile
210 215 220
Ser Phe Tyr Phe Leu Tyr Pro Ser Thr Phe Asn Ile Leu Glu Val Ala
225 230 235 240
Ala Val Asp Glu Pro Lys Leu His Met Trp Glu Thr Gly Ile Met Arg
245 250 255
Ile Thr Arg His Pro Gln Met Val Gly Gln Ala Ile Trp Cys Ala Ala
260 265 270
His Thr Leu Trp Ile Gly Asn Ser Phe Met Leu Val Thr Ser Ala Gly
275 280 285
Leu Met Ala His His Leu Phe Gly Cys Trp His Gly Asp Lys Arg Leu
290 295 300
Ser Asn Lys Tyr Gly Glu Ala Phe Glu Ile Val Lys Thr Arg Thr Ser
305 310 315 320
Thr Phe Pro Met Gln Ala Val Trp Glu Gly Arg Gln Lys Leu Pro Ala
325 330 335
Asp Phe Tyr Lys Glu Phe Leu Arg Ala Pro Tyr Phe Ala Val Thr Ala
340 345 350
Phe Thr Leu Gly Ala Tyr Tyr Ala His Pro Ile Met Gln Ser Ala Ser
355 360 365
Phe Tyr Leu Gly Trp
370
<211> 1752
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 杜氏盐藻(Dunaliella saline):Zds
<400> 11
atgctgggac tgcacggtaa ggatgccctc caggctggcc gcaccaacca gggcccgcca 60
gtcaggacta gctttgtgca gtctacccgt accaggagaa ggacgcgctg caccacccat 120
gccatcgctg cgccccctgc acctccaaag accacaccca aggagtggac cacccaggat 180
cttagcaagg tggccctgaa agacaggcct ctgaagtccc tgtacccgga tgagcccgcc 240
ccaccaaagc caggtgatcc caagctacgc gtggccatcg tgggcagtgg actggctgga 300
ctgtccacag cagtggagct gctagaccag gggcatgagg tggacatcta tgaccagcga 360
ccctttctag gaggcaaagt ggcttcatgg gtagacaagg atggcaacca catagagatg 420
ggcttgcatg tcttcttcgg gtgctaccac aaccttttcc gactgatggc caagtgcggt 480
gtactggaaa acttgctggt caaggatcac acccacacct tttgcaacgc tgatggggac 540
gtcagggagc ttgacttccg ctttgaagct ggaggacaga agattggggc cccattccat 600
ggcctgaaag ccttctttac cacccctcag ctcactgtgg ccgacaaggc acaaaatgca 660
ttggcactgg gcacgagccc tatcgtgcgc gccttgatag atcctgaggg gggtatgcaa 720
gatgtgagga acctagacaa cattagcttc tgggactggt tcaagagcca tggcggatca 780
gagctctcca tgaagcgtat gtgggacccc attgcctacg ctttgggctt cttggactgc 840
aaggacatca gtgcgcgctg catgttgaca atcttccaat tttttgccac caagaccgac 900
gcttctgtcc tccgcatgct taacggatct cctggggaga ggctgttgaa gcccatcgtt 960
aactacattg agtccaaggg tggccgcatc cacctgcgcc agggttgcaa ggaagtactc 1020
tacgaggatg gccctgacgg cacacctgtg gtgactggca tgcgcatggg ccgggatggc 1080
caaattgtga aagctgatgc ctatgtggct gccttggatg ttcctggggc caagcagctc 1140
ttaccgcagg cgtggaggaa gtaccctcaa tttgacaaca tctacagcct gattggcgtg 1200
cctgtgatca ccgttcagct gcgatacaat ggatgggtga ccgagatgca ggacccagag 1260
aaggtcaagc agctgaccca gccccaaggc atcaacaact tgctatacag ccctgatgcg 1320
ttctactcct gctttgctga cctcgctctt gtgagccctg tggagtactt caaggagggc 1380
caggggtcgc tcatgcaagt cgtgattaca ccagcggccc cctacatgcc atgggaaaac 1440
aaggccattg ctgaggaggc tgaccgccag acccgccggc tgttccccag cgcgcgcaac 1500
ttggacatga tctggcacag tgttgtgaag atcggccagt ccctgtacca ggaggcccca 1560
ggcatggacc ctttcaggcc tgagcaggcc acgcctgtgc ccaacttctt cttggcaggc 1620
agttacacca agcaagacta catcgactcc atggagggtg ccaccctgtc aggccgacaa 1680
tgtgctggcg aggtcgtgaa agccgtgccc cggatccaga gcctatcggt ggcgccactg 1740
gcaagcattt ga 1752
<211> 583
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 杜氏盐藻(Dunaliella saline):Zds
<400> 12
Met Leu Gly Leu His Gly Lys Asp Ala Leu Gln Ala Gly Arg Thr Asn
1 5 10 15
Gln Gly Pro Pro Val Arg Thr Ser Phe Val Gln Ser Thr Arg Thr Arg
20 25 30
Arg Arg Thr Arg Cys Thr Thr His Ala Ile Ala Ala Pro Pro Ala Pro
35 40 45
Pro Lys Thr Thr Pro Lys Glu Trp Thr Thr Gln Asp Leu Ser Lys Val
50 55 60
Ala Leu Lys Asp Arg Pro Leu Lys Ser Leu Tyr Pro Asp Glu Pro Ala
65 70 75 80
Pro Pro Lys Pro Gly Asp Pro Lys Leu Arg Val Ala Ile Val Gly Ser
85 90 95
Gly Leu Ala Gly Leu Ser Thr Ala Val Glu Leu Leu Asp Gln Gly His
100 105 110
Glu Val Asp Ile Tyr Asp Gln Arg Pro Phe Leu Gly Gly Lys Val Ala
115 120 125
Ser Trp Val Asp Lys Asp Gly Asn His Ile Glu Met Gly Leu His Val
130 135 140
Phe Phe Gly Cys Tyr His Asn Leu Phe Arg Leu Met Ala Lys Cys Gly
145 150 155 160
Val Leu Glu Asn Leu Leu Val Lys Asp His Thr His Thr Phe Cys Asn
165 170 175
Ala Asp Gly Asp Val Arg Glu Leu Asp Phe Arg Phe Glu Ala Gly Gly
180 185 190
Gln Lys Ile Gly Ala Pro Phe His Gly Leu Lys Ala Phe Phe Thr Thr
195 200 205
Pro Gln Leu Thr Val Ala Asp Lys Ala Gln Asn Ala Leu Ala Leu Gly
210 215 220
Thr Ser Pro Ile Val Arg Ala Leu Ile Asp Pro Glu Gly Gly Met Gln
225 230 235 240
Asp Val Arg Asn Leu Asp Asn Ile Ser Phe Trp Asp Trp Phe Lys Ser
245 250 255
His Gly Gly Ser Glu Leu Ser Met Lys Arg Met Trp Asp Pro Ile Ala
260 265 270
Tyr Ala Leu Gly Phe Leu Asp Cys Lys Asp Ile Ser Ala Arg Cys Met
275 280 285
Leu Thr Ile Phe Gln Phe Phe Ala Thr Lys Thr Asp Ala Ser Val Leu
290 295 300
Arg Met Leu Asn Gly Ser Pro Gly Glu Arg Leu Leu Lys Pro Ile Val
305 310 315 320
Asn Tyr Ile Glu Ser Lys Gly Gly Arg Ile His Leu Arg Gln Gly Cys
325 330 335
Lys Glu Val Leu Tyr Glu Asp Gly Pro Asp Gly Thr Pro Val Val Thr
340 345 350
Gly Met Arg Met Gly Arg Asp Gly Gln Ile Val Lys Ala Asp Ala Tyr
355 360 365
Val Ala Ala Leu Asp Val Pro Gly Ala Lys Gln Leu Leu Pro Gln Ala
370 375 380
Trp Arg Lys Tyr Pro Gln Phe Asp Asn Ile Tyr Ser Leu Ile Gly Val
385 390 395 400
Pro Val Ile Thr Val Gln Leu Arg Tyr Asn Gly Trp Val Thr Glu Met
405 410 415
Gln Asp Pro Glu Lys Val Lys Gln Leu Thr Gln Pro Gln Gly Ile Asn
420 425 430
Asn Leu Leu Tyr Ser Pro Asp Ala Phe Tyr Ser Cys Phe Ala Asp Leu
435 440 445
Ala Leu Val Ser Pro Val Glu Tyr Phe Lys Glu Gly Gln Gly Ser Leu
450 455 460
Met Gln Val Val Ile Thr Pro Ala Ala Pro Tyr Met Pro Trp Glu Asn
465 470 475 480
Lys Ala Ile Ala Glu Glu Ala Asp Arg Gln Thr Arg Arg Leu Phe Pro
485 490 495
Ser Ala Arg Asn Leu Asp Met Ile Trp His Ser Val Val Lys Ile Gly
500 505 510
Gln Ser Leu Tyr Gln Glu Ala Pro Gly Met Asp Pro Phe Arg Pro Glu
515 520 525
Gln Ala Thr Pro Val Pro Asn Phe Phe Leu Ala Gly Ser Tyr Thr Lys
530 535 540
Gln Asp Tyr Ile Asp Ser Met Glu Gly Ala Thr Leu Ser Gly Arg Gln
545 550 555 560
Cys Ala Gly Glu Val Val Lys Ala Val Pro Arg Ile Gln Ser Leu Ser
565 570 575
Val Ala Pro Leu Ala Ser Ile
580
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
tttaacttta ataaggagat ataccatggc cgcccatcaa atgca 45
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gacttaagca ttatgcggcc gcaagcttct agttttggcg gtagc 45
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
agttaagtat aagaaggaga tatacatatg acgctgtcaa tgttg 45
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gtttctttac cagactcgag ttatttgttc ttgggcacca a 41
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ttaactttaa taaggagata taccatgcag gttatgcagg gca 43
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
acttaagcat tatgcggccg caagctttta gaaccaaggg aag 43
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
agtataagaa ggagatatac atatgttggg gctgca 36
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gcgatcgcgt ggccggccga tatcttacat gctcgga 37
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
cacagccagg atccgaattc gatggcgagc ttgtgtagag ctg 43
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atgcggccgc aagcttctac catctcaaat agaagctg 38
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tccgaattcg agctcggcga tggccgccca tcagatgc 38
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
cggccgcaag cttgtcgatt agttttgacg gtagccaatg tac 43
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gtataagaag gagatataca tatggcaatg accctgtcta tgctggacg 49
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
atcgcgtggc cggccgattt acttgttctg gttctggggc 40
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
tccgaattcg agctcggcga tgcagaccat gcagggcag 39
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
cggccgcaag cttgtcgatt agaaccaagg gaaggtgact g 41
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
gtataagaag gagatataca tatggcaatg ctgggactgc acggtaa 47
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
atcgcgtggc cggccgattc aaatgcttgc cagtggcg 38
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
tccgaattcg agctcggcga tggcaagctt gtgtgcagg 39
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
ttgtcgacct gcaggcgcgt caccacccca ggtagaagct 40
Claims (6)
1.一种基于杜氏盐藻代谢途径的链孢红素高产工程菌,其特征在于:
所述链孢红素高产工程菌含有编码SEQ ID NO:8所示氨基酸序列的牦牛儿基牻牛儿基焦磷酸合成酶Ggps基因、编码GenBank:AAB51287.1所示氨基酸序列的八氢番茄红素合成酶Psy基因、编码GenBank:CAA75094.1所示氨基酸序列的八氢番茄红素脱氢酶Pds基因、编码SEQ ID NO:10所示氨基酸序列的15-顺式-ζ-胡萝卜素异构酶Ziso基因和编码SEQ ID NO:12所示氨基酸序列的ζ-胡萝卜素脱氢酶Zds基因。
2.根据权利要求1所述的基于杜氏盐藻代谢途径的链孢红素高产工程菌,其特征在于:
编码SEQ ID NO:8所示氨基酸序列的牦牛儿基牻牛儿基焦磷酸合成酶Ggps基因的核苷酸序列为如SEQ ID NO:7所示的核苷酸序列;
编码GenBank:AAB51287.1所示氨基酸序列的八氢番茄红素合成酶Psy基因的核苷酸序列为如GenBank:U91900.1所示的核苷酸序列;
编码GenBank:CAA75094.1所示氨基酸序列的八氢番茄红素脱氢酶Pds基因的核苷酸序列为如GenBank:Y14807.1所示的核苷酸序列;
编码SEQ ID NO:10所示氨基酸序列的15-顺式-ζ-胡萝卜素异构酶Ziso基因的核苷酸序列为如SEQ ID NO:9所示的核苷酸序列;
编码SEQ ID NO:12所示氨基酸序列的ζ-胡萝卜素脱氢酶Zds基因的核苷酸序列为如SEQ ID NO:11所示的核苷酸序列。
3.权利要求1~2任一项所述的基于杜氏盐藻代谢途径的链孢红素高产工程菌的构建方法,其特征在于,包括如下步骤:
(1)利用相关基因工程手段从杜氏盐藻中克隆到牦牛儿基牻牛儿基焦磷酸合成酶Ggps、八氢番茄红素合成酶Psy、八氢番茄红素脱氢酶Pds、15-顺式-ζ-胡萝卜素异构酶Ziso、ζ-胡萝卜素脱氢酶Zds;
(2)将Ggps和Psy构建在pACYduet-1载体上,氯霉素抗性,得到重组载体pACYduet-ggps-psy;将Pds和Zds构建在pCDFduet-1载体上,链霉素抗性,得到重组载体pCDFduet-pds-zds;将Ziso构建在pETduet-1载体上,氨苄抗性,得到重组载体pETduet-ziso;
(3)然后将步骤(2)构建的三个重组载体共转化到大肠杆菌BL21(DE3)中,获得基于杜氏盐藻代谢途径的链孢红素高产工程菌。
4.根据权利要求3所述的基于杜氏盐藻代谢途径的链孢红素高产工程菌的构建方法,其特征在于:
重组载体构建时,在5个目的基因的5′端均连接有核糖体结合位点rbs序列,或者,均连接有T7_promoter序列和核糖体结合位点rbs序列。
5.权利要求1~2任一项所述的基于杜氏盐藻代谢途径的链孢红素高产工程菌在生产链孢红素中的应用。
6.根据权利要求5所述的应用,特征在于:
所述链孢红素高产工程菌的链孢红素的产率为3.1 mg/g细胞干重。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910665236.XA CN110511874B (zh) | 2019-07-23 | 2019-07-23 | 基于杜氏藻代谢途径的链孢红素高产工程菌及其构建方法与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910665236.XA CN110511874B (zh) | 2019-07-23 | 2019-07-23 | 基于杜氏藻代谢途径的链孢红素高产工程菌及其构建方法与应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110511874A CN110511874A (zh) | 2019-11-29 |
CN110511874B true CN110511874B (zh) | 2021-03-30 |
Family
ID=68623931
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910665236.XA Active CN110511874B (zh) | 2019-07-23 | 2019-07-23 | 基于杜氏藻代谢途径的链孢红素高产工程菌及其构建方法与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110511874B (zh) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108165048A (zh) * | 2017-12-20 | 2018-06-15 | 河南科技大学 | 一种天然食用色素盐藻黄及其制备方法和应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102154330B (zh) * | 2011-01-14 | 2012-12-05 | 华南理工大学 | 基于巴氏杜氏藻代谢途径的β-胡萝卜素工程菌及构建方法 |
CN104531755B (zh) * | 2014-12-19 | 2017-06-06 | 西南大学 | 干扰lcyb、lcye表达同时异位表达tt8在制备红色花瓣芸薹属植物中的应用 |
-
2019
- 2019-07-23 CN CN201910665236.XA patent/CN110511874B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108165048A (zh) * | 2017-12-20 | 2018-06-15 | 河南科技大学 | 一种天然食用色素盐藻黄及其制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN110511874A (zh) | 2019-11-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109666683B (zh) | 乙酰辅酶A乙酰转移酶基因RKAcaT2及其应用 | |
CN109777815B (zh) | HMG-CoA合成酶基因RKHMGCS及其应用 | |
CN112410355B (zh) | 一种酰基辅酶a氧化酶2基因rkacox2及其应用 | |
CN111454854B (zh) | 一株产虾青素的红冬孢酵母基因工程菌株 | |
CN109536518A (zh) | 一种八氢番茄红素脱氢酶基因RKcrtI及其应用 | |
CN110499272B (zh) | 基于杜氏藻代谢途径的β-胡萝卜素高产工程菌及其构建方法与应用 | |
CN106459949A (zh) | 补身醇合酶及生产补身醇的方法 | |
CN112342178B (zh) | 重组微生物、其制备方法及在生产塔格糖中的应用 | |
CN106102454B (zh) | 使用o-甲基转移酶生物合成生成紫檀芪的方法 | |
CN110540983B (zh) | 基于杜氏藻代谢途径的ζ-胡萝卜素高产工程菌及其构建方法与应用 | |
CN110511874B (zh) | 基于杜氏藻代谢途径的链孢红素高产工程菌及其构建方法与应用 | |
CN112746062A (zh) | 与紫苏萜类物质生物合成有关的蛋白及其编码基因与应用 | |
CN110511919B (zh) | 基于杜氏藻代谢途径的番茄红素高产工程菌及其构建方法与应用 | |
CN114395571B (zh) | 三角褐指藻zep1基因、蛋白及应用 | |
CN114989274B (zh) | 一种三角褐指藻Myb类转录因子PtMYB3基因及其编码蛋白和应用 | |
CN107880134B (zh) | 一种酶促合成山奈酚的方法 | |
CN113801871B (zh) | SiLCYE调控玉米黄质等谷子类胡萝卜素合成代谢的功能及应用 | |
KR20240032944A (ko) | 람노스가 고도로 특이적인 글리코실트랜스퍼라제 및 이의 응용 | |
CN113817778B (zh) | 一种利用核仁素增强mRNA稳定表达的方法 | |
CN112029782B (zh) | 一种β-胡萝卜素羟化酶及其基因与应用 | |
CN113717912A (zh) | 一种利用膜工程构建的产番茄红素的大肠杆菌工程菌及其构建方法和应用 | |
CN108753819B (zh) | 真核表达载体、真核表达系统、二者的制备方法和应用及gdf11蛋白 | |
CN113336839A (zh) | 一种通过麦胚无细胞蛋白合成系统表达人成纤维细胞生长因子21的方法 | |
CN113046333B (zh) | 黄酮-8-羟化酶及其在8-羟基黄酮化合物合成中的应用 | |
CN114774442B (zh) | 一种产灯盏乙素的重组解脂耶氏酵母及其构建方法和用途 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |