CN114196606B - 胞内nad+含量提高的基因工程菌及其构建方法 - Google Patents
胞内nad+含量提高的基因工程菌及其构建方法 Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种胞内NAD+含量提高的基因工程菌及其构建方法,以及提高基因工程菌胞内氧化型辅酶Ⅱ含量的发酵方法。本发明通过采用共表达增加内源NAD的基因与NAD激酶的组合,并且在发酵培养基中添加不同的前体物质,将多基因与多种类的前体物质结合,扩大代谢网络通量,大肠杆菌胞内的辅酶含量在经过代谢工程和生化工程两个手段的调控后可达到30μmol/g DCW。相比出发菌株BL21/pCDFDuet提高了近4倍,NADP+生成的效率明显提高,大肠杆菌胞内NAD+的含量也进一步增加。
Description
技术领域
本发明涉及一种胞内NAD+含量提高的基因工程菌及其构建方法。
背景技术
大多数产生工业上重要化合物的代谢反应都依赖于电子携带的辅助因子,如NADH和NADPH。特别地,NADP+和NADPH在药物的生物合成中起着至关重要的作用,同时也是脂质生物合成所必需的,因此,NADPH的再生速率通常是过量生产所需化学物质的限速步骤,同时保持细胞的强劲生长。因此,提高NADPH再生率可以增加途径生产率和产物产量。
细胞内 NADPH 的主要供应渠道是 HMP途径和 NAD 激酶,其中HMP 途径利用氧化态辅酶Ⅱ(NADP+)作为电子受体生成还原态辅酶Ⅱ(NADPH),NAD激酶则催化辅酶Ⅰ发生磷酸化生成辅酶Ⅱ。HMP途径在产生NADPH的,会消耗其他底物,如葡萄糖;而 NAD激酶则只调节辅酶的转变,不消耗其他碳源底物。NAD+激酶利用ATP或多聚磷酸盐[poly(P)]作为磷酸供体,催化NAD+发生磷酸化,生成NADP+,在胞内增加NAD+的供应可以大幅度增加NADP+的积累。
NADPH主要是在磷酸戊糖(PP)途径中作为两种酶的辅助因子产生的,葡萄糖-6-磷酸脱氢酶(G6PDH)催化6-磷酸葡萄糖氧化为6-磷酸葡萄糖内酯,6-磷酸葡萄糖酸脱氢酶(6PGDH)催化6-磷酸葡萄糖酸氧化脱羧为5-磷酸核酮糖,由zwf,gnd,prs基因表达。
NAD+生物合成主要有两条途径:从头合成途径和补救合成途径(图1)。从头合成途径以天冬氨酸或色氨酸为前体,通过一系列步骤生成喹啉酸(QA),接着经基因nadC编码的喹啉酸磷酸核糖转移酶催化生成烟酸单核苷酸(NaMN),再由基因nadD编码的腺苷转移酶催化腺苷化生成烟酸腺嘌呤二核苷酸(NaAD),最后在基因nadE编码的NAD + 合成酶催化下氨基化成NAD+。补救途径是基因pncA编码的烟酰胺酶水解烟酰胺生成烟酸,再经基因pncB编码的烟酸磷酸核糖转移酶催化烟酸生成烟酸单核苷酸(NaMN),最后生成NAD+。当细胞内大量存在NAD+前体烟酰胺(NAM)、烟酸(NA)时,补救途径对胞内NAD+的含量起到重要的作用。
大肠杆菌由于遗传背景清楚、易操作、易调控、培养基要求简单且生长周期短等优点,被广泛应用于科研工作中。然而,其细胞内的辅酶含量较低,无法起到增强相关催化反应效率的作用。因此,通过基因工程的手段增强细胞内NAD+含量对于提高胞内氧化型辅酶Ⅱ的含量具有重要作用。
发明内容
本发明的目的是提供一种胞内NAD+含量提高的基因工程菌及其构建方法,以及提高基因工程菌胞内氧化型辅酶Ⅱ含量的发酵方法。
本发明采用的技术方案是:
一种胞内NAD+含量提高的基因工程菌,由如下方法构建获得:将谷氨酸棒杆菌(Corynebacterium)来源的ppnk基因,与大肠杆菌(Escherichia coli)来源的zwf、gnd、prs、pncB、nadD、nadE、CglpncB、CglnadD、CglnadE中一种共表达至大肠杆菌,获得所述胞内NAD+含量提高的基因工程菌。
具体的,所述ppnk基因核苷酸序列如SEQ ID NO.1所示。
具体的,所述zwf基因核苷酸序列如SEQ ID NO.2所示,所述gnd基因核苷酸序列如SEQ ID NO.3所示,所述prs基因核苷酸序列如SEQ ID NO.4所示,所述pncB基因核苷酸序列如SEQ ID NO.5所示,所述nadD基因核苷酸序列如SEQ ID NO.6所示,所述nadE基因核苷酸序列如SEQ ID NO.7所示,所述CglpncB基因核苷酸序列如SEQ ID NO.8所示,所述CglnadD基因核苷酸序列如SEQ ID NO.9所示,所述CglnadE基因核苷酸序列如SEQ ID NO.10所示。
本发明还涉及构建所述基因工程菌的方法,所述方法包括:
(1)以谷氨酸棒杆菌为模版,PCR克隆扩增得到目的基因ppnk片段,将目的基因产物纯化后,连接到pCDFDuet载体的第一个多克隆位点,转化、筛选阳性克隆;
(2)以大肠杆菌或谷氨酸棒杆菌为模版,克隆得到zwf、gnd、prs、pncB、nadD、nadE、CglpncB、CglnadD或CglnadE基因片段,连接到pCDFDuet载体的第二个多克隆位点,转化、筛选阳性克隆,得到重组质粒;
(3)将重组质粒导入大肠杆菌感受态,得到所述胞内NAD +含量提高的基因工程菌。
本发明还涉及一种提高基因工程菌胞内氧化型辅酶Ⅱ含量的发酵方法,所述方法包括:将所述高辅酶含量的基因工程菌接种至发酵培养基,添加50~100mg/L的功能促进因子,进行有氧发酵;所述功能促进因子为下列之一:色氨酸、天冬氨酸、喹啉酸、烟酸、烟酰胺。
优选的,所述功能促进因子为烟酸或烟酰胺。
本发明的有益效果主要体现在:本发明通过采用共表达增加内源NAD的基因与NAD激酶的组合,并且在发酵培养基中添加不同的前体物质,将多基因与多种类的前体物质结合,扩大代谢网络通量,大肠杆菌胞内的辅酶含量在经过代谢工程和生化工程两个手段的调控后可达到30μmol/g DCW。相比出发菌株BL21/pCDFDuet提高了近4倍,NADP +生成的效率明显提高,大肠杆菌胞内NAD+的含量也进一步增加。
附图说明
图1为辅酶NADP + 合成途径示意图;L-Trp:L-色氨酸;Asp:天冬氨酸;QA:喹啉酸;NA:烟酸;NaMN:烟酸单核苷酸;NaAD:烟酸腺嘌呤二核苷酸;NAD+:烟酰胺腺嘌呤二核苷酸;NAM:烟酰胺;NR:烟酰胺核苷;NMN:烟酰胺单核苷酸:NAD+:烟酰胺腺嘌呤二核苷酸磷酸。
图2为实施例1与激酶基因共表达重组菌株胞内NADP+和NADPH总含量。
图3为实施例2与激酶基因共表达重组菌株胞内NADP+和NADPH总含量。
图4为实施例3与激酶基因共表达重组菌株胞内NADP+和NADPH总含量。
图5为实施例4添加不同前体物质后E.coli BL21/pCDFDuet-ppnk-CglpncB胞内NADP+ 和NADPH的总含量。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
发酵培养基:NaCl 10g/L蛋白胨10g/L,酵母提取物5g/L,pH7.2,以去离子水配制,使用前加入氨苄青霉素100μg/mL。
发酵条件:初始温度为37℃,摇床转速为200r/min,菌体OD 600 达到0.6~1.0时,加入终浓度为0.1~10mM的IPTG,16~37℃诱导10~15h,添加前体物质的浓度为1~100mg/L。
实施例1:
(1)构建过量表达ppnk基因分别与大肠杆菌来源的zwf、gnd、prs基因过表达的表达质粒:根据NCBI上报道的谷氨酸棒杆菌来源的ppnk的基因序列,大肠杆菌基因zwf的序列(Gene ID: 946370),大肠杆菌基因gnd的序列(Gene ID: 946554),大肠杆菌基因prs的序列(Gene ID: 945772)设计上下游引物,在pcDFduet的第一个多克隆位点克隆ppnk基因,在pcDFduet的第二个多克隆位点克隆基因。
ppnk-F: accatcatcaccacagccagATGACAGCACCTACCAACGCA
ppnk-R: gccgagctcgaattcggatcCCCTGCGCTGCGCGGGTC
pCDFDuet-1-F: GATCCGAATTCGAGCTCGG
pCDFDuet-1-R: CTGGCTGTGGTGATGATGGTG
zwf-F: agatctcaattggatATGGCGGTAACGCAAACAGC
zwf-R: gcgtggccggccgatTTACTCAAACTCATTCCAGGAACGACC
gnd-F: agatctcaattggatATGTCCAAGCAACAGATCGGC
gnd-R: gcgtggccggccgatTTAATCCAGCCATTCGGTATGGAACAC
prs-F: agatctcaattggatGTGCCTGATATGAAGCTTTTTGCTG
prs-R: gcgtggccggccgatTTAGTGTTCGAACATGGCAGAGATCG
pCDFDuet-2-F: atcggccggccacg
pCDFDuet-1-R: atccaattgagatctgccatatgtatatctcc
以野生型谷氨酸棒杆菌ATCC13032为模版,PCR克隆扩增得到目的基因ppnk片段,反应条件为:98℃预变性30s;98℃变性10s;55℃退火30s;72℃分别延伸30s,30个循环;72℃延伸10min。将目的基因产物纯化后,连接到pCDFDuet载体的第一个多克隆位点,转化筛选阳性克隆并测序。
以E.coli BL21(DE3)为模板,分别进行PCR克隆扩增得到目的基因zwf、gnd、prs片段,反应条件为:98℃预变性30s;98℃变性10s;55℃退火30s;72℃分别延伸30s,30个循环;72℃延伸10min。将目的基因产物纯化后,连接到pCDFDuet载体的第二个多克隆位点,转化筛选阳性克隆并测序,得到重组质粒pCDFduet-ppnk-zwf、pCDFduet-ppnk-gnd、pCDFduet -ppnk-prs。
(2)将质粒pCDFduet-ppnk、pCDFduet-ppnk-zwf、pCDFduet-ppnk-gnd、pCDFduet -ppnk-prs分别导入E.coli BL21(DE3)感受态,得到重组菌株E.coli BL21/ pCDFduet-ppnk、E.coli BL21/pCDFduet - ppnk-zwf、E.coli BL21/ pCDFduet- ppnk-gnd和E.coliBL21/pCDFduet -ppnk-prs。
(3)对重组菌株进行发酵培养,接种量为按体积的1%,IPTG诱导浓度为0.1~10mM,测定其胞内的NAD+含量。三种重组菌的胞内辅酶Ⅱ都有大幅度提升,其中效果比较明显的有:E.coli BL21/pCDFduet - ppnk-zwf和E.coli BL21/ pCDFduet- ppnk-gnd。其胞内的辅酶含量分别达到16.582μmol/g DCW和15.312μmol/g DCW,与对照(WT和E.coli BL21/pCDFduet-ppnk,WT为E.coli BL21)相比提高了2.7倍和2.5倍(图2)。
实施例2:
(1)构建过量表达ppnk基因分别与大肠杆菌来源的pncB、nadD、nadE过表达的表达质粒:根据NCBI上报道的中基因谷氨酸棒杆菌来源的ppnk的基因序列,大肠杆菌基因pncB的序列(Gene ID:8182321),大肠杆菌基因nadD的序列(Gene ID:8180157),大肠杆菌基因nadE的序列(Gene ID:8179982)设计上下游引物,在pcDFduet的第一个多克隆位点克隆ppnk基因,在pcDFduet的第二个多克隆位点克隆基因。
ppnk-F: accatcatcaccacagccagATGACAGCACCTACCAACGCA
ppnk-R: gccgagctcgaattcggatcCCCTGCGCTGCGCGGGTC
pCDFDuet-1-F: GATCCGAATTCGAGCTCGG
pCDFDuet-1-R: CTGGCTGTGGTGATGATGGTG
pncB-F: agatctcaattggatATGACACAATTCGCTTCTCCTGT
pncB-R: gcgtggccggccgatTTAACTGGCTTTT
nadD-F: agatctcaattggatATGAAATCTTTACAGGCTCTGTTTGGC
nadD-R: gcgtggccggccgatTCAGCGATACAA
nadE-F: agatctcaattggatATGACATTGCAACAACAAATAATAAAGGCG
nadE-R: gcgtggccggccgatTTACTTTTTCCAGAAATCATCGAAAACGGTAATTGG
pCDFDuet-2-F: atcggccggccacg
pCDFDuet-1-R: atccaattgagatctgccatatgtatatctcc
以野生型谷氨酸棒杆菌ATCC 13032为模版,PCR克隆扩增得到目的基因ppnk片段,反应条件为:98℃预变性30s;98℃变性10s;55℃退火30s;72℃分别延伸30s,30个循环;72℃延伸10min。将目的基因产物纯化后,连接到pCDFDuet载体的第一个多克隆位点,转化筛选阳性克隆并测序。
以E.coli BL21(DE3)其为模板,分别进行PCR克隆扩增得到目的基因pncB、nadD、nadE片段,反应条件为:98℃预变性30s;98℃变性10s;55℃退火30s;72℃分别延伸30s,30个循环;72℃延伸10min。将目的基因产物纯化后,连接到pCDFDuet载体的第二个多克隆位点,转化筛选阳性克隆并测序,得到重组质粒pCDFduet-ppnk-pncB、pCDFduet-ppnk-nadD、pCDFduet-ppnk-nadE。
(2)将质粒pCDFduet-ppnk-pncB、pCDFduet-ppnk-nadD、pCDFduet-ppnk-nadE导入E.coli BL21(DE3)感受态,得到重组菌株E.coli BL21/pCDFduet-ppnk-pncB、E.coliBL21/pCDFduet-ppnk-nadD、和E.coli BL21/ pCDFduet-ppnk-nadE。
(3)对重组菌株进行发酵培养,接种量为按体积的1%,IPTG诱导浓度为0.1-10mM,测定其胞内的NAD+ 含量。其中效果比较明显的有:E.coli BL21/pCDFduet -pncB和E.coliBL21/ pCDFduet -nadE。其胞内的辅酶含量分别达到13.286μmol/g DCW和14.847μmol/gDCW,与对照(WT和E.coli BL21/ pCDFduet-ppnk)相比提高了2.2倍和2.4倍(图3)。
实施例3:
(1)构建过量表达ppnk基因分别与谷氨酸棒杆菌来源的Cg-CglpncB、Cg-CglnadD、Cg-nadE基因过表达的表达质粒:根据NCBI上报道的中基因谷氨酸棒杆菌来源的ppnk的基因序列,谷氨酸棒杆菌基因CglpncB的序列(GeneID:58310407),大肠杆菌基因CglnadD的序列(GeneID:58310976),大肠杆菌基因CglnadE的序列(GeneID:58310391)设计上下游引物,在pcDFduet的第一个多克隆位点克隆ppnk基因,在pcDFduet的第二个多克隆位点克隆基因。
ppnk-F: accatcatcaccacagccagATGACAGCACCTACCAACGCA
ppnk-R: gccgagctcgaattcggatcCCCTGCGCTGCGCGGGTC
pCDFDuet-1-F: GATCCGAATTCGAGCTCGG
pCDFDuet-1-R: CTGGCTGTGGTGATGATGGTG
CglpncB -F: agatctcaattggatgtgaataccaatccgtctgaattctcct
CglpncB -R: gcgtggccggccgatctaagcggccggcgg
CglnadD-F: agatctcaattggatatgactacaacggtgaaacgcc
CglnadD -R: gcgtggccggccgatttatgcttggttttggcccttgg
CglnadE -F: agatctcaattggatatgacaaacactcaaaccgagatcattaatga
CglnadE -R: gcgtggccggccgatttaacgccaccaattttcctgcg
pCDFDuet-2-F: atcggccggccacg
pCDFDuet-1-R: atccaattgagatctgccatatgtatatctcc
以野生型谷氨酸棒杆菌为模版,PCR克隆扩增得到目的基因ppnk片段,反应条件为:98℃预变性30s;98℃变性10s;55℃退火30s;72℃分别延伸30s,30个循环;72℃延伸10min。将目的基因产物纯化后,连接到pCDFDuet载体的第一个多克隆位点,转化筛选阳性克隆并测序。
以E.coli BL21(DE3)其为模板,分别进行PCR克隆扩增得到目的基因CglpncB、CglnadD、CglnadE片段,反应条件为:98℃预变性30s;98℃变性10s;55℃退火30s;72℃分别延伸30s,30个循环;72℃延伸10min。将目的基因产物纯化后,连接到pCDFDuet载体的第二个多克隆位点,转化筛选阳性克隆并测序,得到重组质粒pCDFduet-ppnk-CglpncB、pCDFduet-ppnk-CglnadD、pCDFduet-ppnk-CglnadE。
(2)将质粒pCDFduet-ppnk-CglpncB、pCDFduet-ppnk-CglnadD、pCDFduet-ppnk-CglnadE导入E.coli BL21(DE3)感受态,得到重组菌株E.coli BL21/pCDFduet-ppnk-CglpncB、E.coli BL21/ pCDFduet-ppnk-CglnadD、和E.coli BL21/ pCDFduet-ppnk-CglnadE。
(3)对重组菌株进行发酵培养,接种量为按体积的1%,IPTG诱导浓度为0.1-10mM,测定其胞内的NAD+ 含量。其中效果比较明显的有:E.coli BL21/pCDFduet-cglpncB和E.coli BL21/ pCDFduet -cgnadE。其胞内的辅酶含量分别达到21.587μmol/g DCW和20.231μmol/g DCW,与对照(WT和E.coli BL21/ pCDFduet-ppnk)相比提高了3.59倍和3.3倍(图4)。
实施例4:
(1)配制功能因子促进剂:将色氨酸、天冬氨酸、喹啉酸、烟酸、烟酰胺分别用灭菌后的超纯水溶解,配制为终浓度5g/L的溶液。
(2)对初始菌株E.coli BL21/pCDFDuet-ppnk-CglpncB进行发酵培养,采用50ml摇瓶有氧发酵。按1%接种量从冻存管里接入5mL的LB试管中,同时接入终浓度为100μg/mL的氨苄青霉素,培养12h后再按1%的接种量转接于50mL的LB发酵培养基中,分别添加终浓度为50mg/L的功能因子促进剂,37℃、200r/min培养至OD 600 为0.6~2.0时添加终浓度为0.1-10mmol/L的IPTG,16~37℃,200r/min诱导。
(3)测定添加不同前体物质培养后的初始菌株E.coli BL21/pCDFDuet- ppnkcgpncB胞内NADP+和NADPH总含量。结果如图5所示,添加的前体物质对于辅酶Ⅱ的积累都有促进作用,其中添加了烟酸或烟酰胺后对胞内辅酶的生成量促进作用较大,相比空白对照组,分别提高了近23%、19%。
序列表
<110> 浙江工业大学
<120> 胞内NAD+含量提高的基因工程菌及其构建方法
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 960
<212> DNA
<213> Corynebacterium
<400> 1
atgacagcac ctaccaacgc aggggagctg cgtcgcgtcc ttctggtgcc acatactggt 60
cgttcttcga atatcgaatc cgcgatcctg gcagcgaaat tattagacga tgcggggatt 120
gatgtccgtg tccttattaa tgatgccgac gaccctattg ctgaacactc tgtattgggc 180
cgctttaccc acgttcgtca cgccgcagac gcggcagatg gagctgaact tgtccttgtt 240
ttggggggag acggcacctt cttgcgtgct gctgatatgg ctcacgcggt tgatctgccg 300
gttctgggaa tcaatttagg tcatgtgggc ttcctggcgg aatgggaaag tgatagtctg 360
gaggaagcat tgaagcgtgt gattgaccgt gactaccgca tcgaagatcg catgacactt 420
acagttgttg tcctggacgg gggtggtgaa gagattggac gtggctgggc acttaacgaa 480
gtgagcatcg aaaacctgaa tcgccgcggc gttttggatg caacgttgga ggtagacgct 540
cgtccagtag catccttcgg gtgcgacggg gtcttaatct ctactcccac aggctcgaca 600
gcttacgcct ttagcgctgg gggcccagtc ctgtggcctg aactggacgc gattttggtc 660
gtgcctaata acgcccatgc gttgtttacg aagcccttgg tcgtgtcgcc gaagagtaca 720
gtcgctgtcg aaagtaactc cgatactagc gcagctatgg cagtaatgga cggtttccgc 780
cccattccga tgccccctgg gagtcgcgta gaagtcactc gtggggaacg tccagtccgc 840
tgggtacgtc ttgactcctc acccttcact gatcgtttgg tcagcaaact tcgcttacct 900
gtgacgggct ggcgtggccc gcaaaagcag gcagaaaata aagacccgcg cagcgcaggg 960
<210> 2
<211> 1476
<212> DNA
<213> Escherichia coli
<400> 2
atggcggtaa cgcaaacagc ccaggcctgt gacctggtca ttttcggcgc gaaaggcgac 60
cttgcgcgtc gtaaattgct gccttccctg tatcaactgg aaaaagccgg tcagctcaac 120
ccggacaccc ggattatcgg cgtagggcgt gctgactggg ataaagcggc atataccaaa 180
gttgtccgcg aggcgctcga aactttcatg aaagaaacca ttgatgaagg tttatgggac 240
accctgagtg cacgtctgga tttttgtaat ctcgatgtca atgacactgc tgcattcagc 300
cgtctcggcg cgatgctgga tcaaaaaaat cgtatcacca ttaactactt tgccatgccg 360
cccagcactt ttggcgcaat ttgcaaaggg cttggcgagg caaaactgaa tgctaaaccg 420
gcacgcgtag tcatggagaa accgctgggg acgtcgctgg cgacctcgca ggaaatcaat 480
gatcaggttg gcgaatactt cgaggagtgc caggtttacc gtatcgacca ctatcttggt 540
aaagaaacgg tgctgaacct gttggcgctg cgttttgcta actccctgtt tgtgaataac 600
tgggacaatc gcaccattga tcatgttgag attaccgtgg cagaagaagt ggggatcgaa 660
gggcgctggg gctattttga taaagccggt cagatgcgcg acatgatcca gaaccacctg 720
ctgcaaattc tttgcatgat tgcgatgtct ccgccgtctg acctgagcgc agacagcatc 780
cgcgatgaaa aagtgaaagt actgaagtct ctgcgccgca tcgaccgctc caacgtacgc 840
gaaaaaaccg tacgcgggca atatactgcg ggcttcgccc agggcaaaaa agtgccggga 900
tatctggaag aagagggcgc gaacaagagc agcaatacag aaactttcgt ggcgatccgc 960
gtcgacattg ataactggcg ctgggccggt gtgccattct acctgcgtac tggtaaacgt 1020
ctgccgacca aatgttctga agtcgtggtc tatttcaaaa cacctgaact gaatctgttt 1080
aaagaatcgt ggcaggatct gccgcagaat aaactgacta tccgtctgca acctgatgaa 1140
ggcgtggata tccaggtact gaataaagtt cctggccttg accacaaaca taacctgcaa 1200
atcaccaagc tggatctgag ctattcagaa acctttaatc agacgcatct ggcggatgcc 1260
tatgaacgtt tgctgctgga aaccatgcgt ggtattcagg cactgtttgt acgtcgcgac 1320
gaagtggaag aagcctggaa atgggtagac tccattactg aggcgtgggc gatggacaat 1380
gatgcgccga aaccgtatca ggccggaacc tggggacccg ttgcctcggt ggcgatgatt 1440
acccgtgatg gtcgttcctg gaatgagttt gagtaa 1476
<210> 3
<211> 1407
<212> DNA
<213> Escherichia coli
<400> 3
atgtccaagc aacagatcgg cgtagtcggt atggcagtga tgggacgcaa ccttgcgctc 60
aacatcgaaa gccgtggtta taccgtctct attttcaacc gttcccgtga gaagacggaa 120
gaagtgattg ccgaaaatcc aggcaagaaa ctggttcctt actatacggt gaaagagttt 180
gtcgaatctc tggaaacgcc tcgtcgcatc ctgttaatgg tgaaagcagg tgcaggcacg 240
gatgctgcta ttgattccct caaaccatat ctcgataaag gagacatcat cattgatggt 300
ggtaacacct tcttccagga cactattcgt cgtaatcgtg agctttcagc agagggcttt 360
aacttcatcg gtaccggtgt ttctggcggt gaagaggggg cgctgaaagg tccttctatt 420
atgcctggtg gccagaaaga agcctatgaa ttggtagcac cgatcctgac caaaatcgcc 480
gccgtagctg aagacggtga accatgcgtt acctatattg gtgccgatgg cgcaggtcac 540
tatgtgaaga tggttcacaa cggtattgaa tacggcgata tgcagctgat tgctgaagcc 600
tattctctgc ttaaaggtgg cctgaacctc accaacgaag aactggcgca gacctttacc 660
gagtggaata acggtgaact gagcagttac ctgatcgaca tcaccaaaga tatcttcacc 720
aaaaaagatg aagacggtaa ctacctggtt gatgtgatcc tggatgaagc ggctaacaaa 780
ggtaccggta aatggaccag ccagagcgcg ctggatctcg gcgaaccgct gtcgctgatt 840
accgagtctg tgtttgcacg ttatatctct tctctgaaag atcagcgtgt tgccgcatct 900
aaagttctct ctggtccgca agcacagcca gcaggcgaca aggctgagtt catcgaaaaa 960
gttcgtcgtg cgctgtatct gggcaaaatc gtttcttacg cccagggctt ctctcagctg 1020
cgtgctgcgt ctgaagagta caactgggat ctgaactacg gcgaaatcgc gaagattttc 1080
cgtgctggct gcatcatccg tgcgcagttc ctgcagaaaa tcaccgatgc ttatgccgaa 1140
aatccacaga tcgctaacct gttgctggct ccgtacttca agcaaattgc cgatgactac 1200
cagcaggcgc tgcgtgatgt cgttgcttat gcagtacaga acggtattcc ggttccgacc 1260
ttctccgcag cggttgccta ttacgacagc taccgtgctg ctgttctgcc tgcgaacctg 1320
atccaggcac agcgtgacta ttttggtgcg catacttata agcgtattga taaagaaggt 1380
gtgttccata ccgaatggct ggattaa 1407
<210> 4
<211> 948
<212> DNA
<213> Escherichia coli
<400> 4
gtgcctgata tgaagctttt tgctggtaac gccaccccgg aactagcaca acgtattgcc 60
aaccgcctgt acacttcact cggcgacgcc gctgtaggtc gctttagcga tggcgaagtc 120
agcgtacaaa ttaatgaaaa tgtacgcggt ggtgatattt tcatcatcca gtccacttgt 180
gcccctacta acgacaacct gatggaatta gtcgttatgg ttgatgccct gcgtcgtgct 240
tccgcaggtc gtatcaccgc tgttatcccc tactttggct atgcgcgcca ggaccgtcgc 300
gtccgttccg ctcgtgtacc aatcactgcg aaagtggttg cagacttcct ctccagcgtc 360
ggtgttgacc gtgtgctgac agtggatctg cacgctgaac agattcaggg tttcttcgac 420
gttccggttg ataacgtatt tggtagcccg atcctgctgg aagacatgct gcagctgaat 480
ctggataacc caattgtggt ttctccggac atcggcggcg ttgtgcgtgc ccgcgctatc 540
gctaagctgc tgaacgatac cgatatggca atcatcgaca aacgtcgtcc gcgtgcgaac 600
gtttcacagg tgatgcatat catcggtgac gttgcaggtc gtgactgcgt actggtcgat 660
gatatgatcg acactggcgg tacgctgtgt aaagctgctg aagctctgaa agaacgtggt 720
gctaaacgtg tatttgcgta cgcgactcac ccgatcttct ctggcaacgc ggcgaacaac 780
ctgcgtaact ctgtaattga tgaagtcgtt gtctgcgata ccattccgct gagcgatgaa 840
atcaaatcac tgccgaacgt gcgtactctg accctgtcag gtatgctggc cgaagcgatt 900
cgtcgtatca gcaacgaaga atcgatctct gccatgttcg aacactaa 948
<210> 5
<211> 1203
<212> DNA
<213> Escherichia coli
<400> 5
atgacacaat tcgcttctcc tgttctgcac tcgttgctgg atacagatgc ttataagttg 60
catatgcagc aagccgtgtt tcatcactat tacgatgtgc atgtcgcggc ggagtttcgt 120
tgccgaggtg acgatctgct gggtatttat gccgatgcta ttcgtgaaca ggttcaggcg 180
atgcagcacc tgcgcctgca ggatgatgaa tatcagtggc tttctgccct gcctttcttt 240
aaggccgact atcttaactg gttacgcgag ttccgcttta acccggaaca agtcaccgtg 300
tccaacgata atggcaagct ggatattcgt ttaagcggcc cgtggcgtga agtcatcctc 360
tgggaagttc ctttgctggc ggttatcagt gaaatggtac atcgctatcg ctcaccgcag 420
gccgacgttg cgcaagccct cgacacgctg gaaagcaaat tagtcgactt ctcggcgtta 480
accgccggtc ttgatatgtc gcgcttccat ctgatggatt ttggcacccg tcgccgtttt 540
tctcgcgaag tacaagaaac catcgttaag cgtctgcaac aggaatcctg gtttgtgggc 600
accagcaact acgatctggc gcgtcggctt tccctcacgc cgatgggaac acaggcacac 660
gaatggttcc aggcacatca gcaaatcagc ccggatctag ccaacagcca gcgagctgca 720
cttgctgcct ggctggaaga gtatcccgac caacttggca ttgcattaac cgactgcatc 780
actatggatg ctttcctgcg tgatttcggt gtcgagttcg ctagtcggta tcagggcctg 840
cgtcatgact ctggcgaccc ggttgaatgg ggtgaaaaag ccattgcaca ttatgaaaag 900
ctgggaattg atccacagag taaaacgctg gttttctctg acaatctgga tttacgcaaa 960
gcggttgagc tataccgcca cttctcttcc cgcgtgcaat taagttttgg tattgggact 1020
cgcctgacct gcgatatccc ccaggtaaaa cccctgaata ttgtcattaa gttggtagag 1080
tgtaacggta aaccggtggc gaaactttct gacagccctg gcaaaactat ctgccatgat 1140
aaagcgtttg ttcgggcgct gcgcaaagcg ttcgaccttc cgcatattaa aaaagccagt 1200
taa 1203
<210> 6
<211> 642
<212> DNA
<213> Escherichia coli
<400> 6
atgaaatctt tacaggctct gtttggcggc acctttgatc cggtgcacta tggtcatcta 60
aaacccgtgg aaacgctggc gaatttgatt ggtctgacgc gggtcacaat catccctaat 120
aatgttcctc cgcatcgtcc ccagccggaa gcgaacagcg tgcagcgtaa acacatgctt 180
gaactggcga ttgccgacaa gccattattt actcttgatg aacgcgagct aaagcgcaat 240
gccccctctt acactgcgca aacactgaaa gagtggcggc aggaacaagg accggacgtg 300
ccgctggcgt ttattattgg tcaggattca ctgctgacct ttccgacctg gtacgaatac 360
gaaacgatac tcgacaatgc acatttgatc gtctgtcggc gtccaggtta cccacttgaa 420
atggcgcaac cgcaatacca gcaatggctg gaagatcatt tgacacataa cccggaagat 480
cttcaccttc agcctgccgg taaaatttat ctggctgaaa cgccgtggtt taacatctcg 540
gcgaccatca tccgcgaacg tttgcaaaac ggtgaatcat gtgaggattt attgccggaa 600
ccggtactga cttacattaa ccaacaaggc ttgtatcgct ga 642
<210> 7
<211> 828
<212> DNA
<213> Escherichia coli
<400> 7
atgacattgc aacaacaaat aataaaggcg ctgggcgcaa aaccgcagat taatgctgaa 60
gaggaaattc gtcgtagtgt cgattttctg aaaagctacc tgcaaactta tccgttcatt 120
aaatcactgg tgctcgggat cagcggcggt caggactcca cgcttgccgg aaagctgtgc 180
cagatggcga ttaatgagct gcgcctggaa accggcaacg aatcactgca atttattgcc 240
gtacgcctgc cctatggtgt tcaggccgac gaacaagatt gccaggatgc cattgccttt 300
attcaaccgg atcgcgtatt aaccgttaat atcaagggcg cggtattggc cagcgaacag 360
gcattgcggg aagcaggcat tgaactgagc gattttgtcc gtggcaatga aaaagcgcgt 420
gagcggatga aagcacaata tagcattgcg ggtatgacca gcggtgtcgt ggtgggcacc 480
gatcatgcag cagaagccat taccggattc ttcactaaat atggtgacgg cggtacggac 540
attaatccgc tgtatcgtct caacaaacgt cagggtaaac agttactggc ggcattagct 600
tgcccggaac acctttataa gaaagcgcca acggccgatc tggaagatga tcgcccttct 660
ctgccagatg aagtggcact cggcgtgacc tatgacaata tcgacgacta tctggaaggg 720
aaaaacgtac ctcaacaggt cgccagaaca atagagaact ggtatctgaa aaccgaacat 780
aaacgccgtc cgccaattac cgttttcgat gatttctgga aaaagtaa 828
<210> 8
<211> 1341
<212> DNA
<213> Escherichia coli
<400> 8
gtgaatacca atccgtctga attctcctca aaccgttcaa cagctctcct tactgataaa 60
tatgagctga ccatgcttca agcagcgctc gctgatggtt ctgcagaacg cccctcaacg 120
tttgaggtct ttagccgccg cctccccaac gagcgccgat acggtgtcgt cgcaggaaca 180
gcacgagtgc tgaaggcgat tcgtgacttt gtattcacag aggaacaact cgccgatctt 240
gactttttag acgaccgtac cctggaatac ctccgcaact accgattcac cggccaagtt 300
gatggctacc gcgaaggcga aatctacttc ccgcagtccc ctcttctgac tgtgcgtggc 360
acgtttgcag aatgcgtcat cctagaaact gtcattttgt ccatcatgaa tgcagattct 420
gccgtcgctt ccgccgctgc gcgcatggtc accgcagctg atggtcgccc catcatcgaa 480
atgggatcca ggcgcaccca cgaatactcg gcagtcaccg catcccgcgc agcatacctc 540
gctggattct ccaccacctc caacctcgag gcggcctacc gctacggaat tccagcatcc 600
ggaacctccg cccacgcatg gactttgctg cacatcaacg atgacggcac ccccaacgaa 660
gcagcagctt tcaaagcaca ggttgaatcc ctcggcgtgg acaccacctt gctggtagat 720
acttatgaca tcacccaagg tgtggccacc gccattgaag ttgcaggtcc agaccttggt 780
ggcgtacgta tcgactccgg cgacctaggt gtgcttgccc gaaaggtccg caagcagctc 840
gacgatctca acgcccacaa caccaagatt gtggtctcct ccgacctgga tgaattcgcc 900
atcgcgggtc ttcgcggcga accagttgac gtctttggcg ttggcacctc cgttgtcaca 960
ggttctggcg caccaaccgc tggcctcgtg tacaagatcg tggaagttgc cggtcaccct 1020
gtggccaagc gttcccgaaa caaggaaagc tacggtggtg gcaagaaggc tgtgcgcacc 1080
caccgcaagt ccggtaccgc aatcgaagaa atcgtctacc cattcaatgc cgaagcacca 1140
gatactggaa agctcgacac tttgagcctg accatcccat tgatgcgcga cggtgaaatc 1200
gttccaggtt tgcctacttt ggaagattcc cgagcgtatt tggccaagca attggtctct 1260
ttaccatggg aaggccttgc actgtctcgc gatgagcctg ttttgcacac tcgtttcgtg 1320
ggtttcccgc cggccgctta g 1341
<210> 9
<211> 657
<212> DNA
<213> Escherichia coli
<400> 9
atgactacaa cggtgaaacg ccgcgctcgc attggcatca tgggtggcac atttgacccc 60
attcataatg gtcaccttgt ggcgggctca gaggtagcgg atcgattcga tcttgatctg 120
gtggtgtacg ttcccaccgg acagccatgg caaaaggcga acaagaaagt cagcccagcg 180
gaagatcgtt acctgatgac ggtgatcgcc actgcctcta atccacggtt tatggtatcg 240
cgggttgata ttgatcgggg aggggatact tacacgatcg ataccctgca agatttgagc 300
aagcaatacc cggacgccca gctgtacttc atcaccggtg ccgatgcact ggcacagatc 360
gtgacgtggc gcgattggga gaaaaccttc gaacttgccc acttcgttgg agtgactcga 420
cccggttatg aattggatgg aaacatcatt ccggaaatgc accaagatcg agtctcattg 480
gtggatatcc ccgccatggc tatttcctcc acggactgca gagaacgctc cagcgaagaa 540
cgccctgttt ggtatcttgt ccctgatggc gtggtgcaat acattgccaa acgccaactc 600
tatcgacctg aaggatccga taaggatatg gatcccaagg gccaaaacca agcataa 657
<210> 10
<211> 834
<212> DNA
<213> Escherichia coli
<400> 10
atgacaaaca ctcaaaccga gatcattaat gaactaaagg tgagcccagc aatcgacgtg 60
gccaaggaag ttgaattccg tgtgcagttc ctcgtcgatt acctgcgggc ttcccataca 120
aaaggctttg ttcttggtat ttcaggtggc caggattcca ctcttgcggg acgactcacg 180
cagctggcag tagagcgcat tcgtgcggaa gaaaacagca cggattatgt cttctacgca 240
gttcgcctcc cctacgcgat ccaggcagat gaggacgatg cgcaagttgc attggaattc 300
atcgcacctg acaagagcgt gaccgtcaac gttaaagacg caacggacgc caccgaagca 360
actgttgcag ctgctttgga acttcctgag ctgaccgact tcaatcgggg caatattaaa 420
gctcgccaac gcatggttgc ccagtacgca atcgcaggcc agttgggctt gctggttatt 480
ggcactgatc acgcggctga aaacgtcacg gggttcttca ccaaattcgg tgatggcgca 540
gctgacctgc ttcctttggc aggtttgagc aagcgtcaag gagctgccat tctggagcac 600
ctgggtgcac cttcaagcac gtggaccaag gttcctaccg ctgatttgga agaggatcgc 660
ccagcgttgc cagatgagga agcacttggt gtgtcgtatg cggacatcga taattacctg 720
gaaaacaagc ccgatgtcag tgaaaaagcc cagcagcgca ttgagcacct gtggaaggtg 780
ggccagcaca agcgccacct ccctgctacc ccgcaggaaa attggtggcg ttaa 834
Claims (4)
1.一种胞内NAD+含量提高的基因工程菌,由如下方法构建获得:将谷氨酸棒杆菌来源的ppnk基因,与谷氨酸棒杆菌来源的CglpncB、CglnadD、CglnadE中一种共表达至大肠杆菌,获得所述胞内NAD+含量提高的基因工程菌;其中,所述ppnk基因核苷酸序列如SEQ ID NO.1所示,所述CglpncB基因核苷酸序列如SEQ ID NO.8所示,所述CglnadD基因核苷酸序列如SEQ ID NO.9所示,所述CglnadE基因核苷酸序列如SEQ ID NO.10所示。
2.构建权利要求1所述基因工程菌的方法,所述方法包括:
(1)以谷氨酸棒杆菌为模版,PCR克隆扩增得到目的基因ppnk片段,将目的基因产物纯化后,连接到pCDFDuet载体的第一个多克隆位点,转化、筛选阳性克隆;
(2)以谷氨酸棒杆菌为模版,克隆得到CglpncB、CglnadD或CglnadE基因片段,连接到pCDFDuet载体的第二个多克隆位点,转化、筛选阳性克隆,得到重组质粒;
(3)将重组质粒导入大肠杆菌感受态,得到所述胞内NAD +含量提高的基因工程菌。
3.一种提高基因工程菌胞内氧化型辅酶Ⅱ含量的发酵方法,所述方法包括:将权利要求1所述基因工程菌接种至发酵培养基,添加50~100mg/L的功能促进因子,进行有氧发酵;所述功能促进因子为下列之一:色氨酸、天冬氨酸、喹啉酸、烟酸、烟酰胺。
4.如权利要求3所述的方法,其特征在于所述功能促进因子为烟酸或烟酰胺。
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