CN117363553A - 一种生产2’-脱氧腺苷的基因工程菌及其构建方法与应用 - Google Patents
一种生产2’-脱氧腺苷的基因工程菌及其构建方法与应用 Download PDFInfo
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- CN117363553A CN117363553A CN202311666347.5A CN202311666347A CN117363553A CN 117363553 A CN117363553 A CN 117363553A CN 202311666347 A CN202311666347 A CN 202311666347A CN 117363553 A CN117363553 A CN 117363553A
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- C12Y403/02002—Adenylosuccinate lyase (4.3.2.2)
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- C12Y—ENZYMES
- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
- C12Y603/04—Other carbon-nitrogen ligases (6.3.4)
- C12Y603/04004—Adenylosuccinate synthase (6.3.4.4)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
本发明提供了一种生产2’‑脱氧腺苷的基因工程菌及其构建方法与应用,所述基因工程菌是通过对E.coli和Bacillus subtilis的嘌呤代谢相关途径进行分析,以野生型E.coli.W3110为底盘微生物并在底盘微生物E.coliW3110中异源引入嘌呤操纵子,通过基因组整合和质粒过表达2’‑脱氧腺苷生物合成途径关键酶基因,敲除2’‑脱氧腺苷分解途径等相关分子改造获得的,具有遗传背景清晰、可持续改造等优点,该菌株发酵生产2’‑脱氧腺苷操作简单、能以廉价碳源直接合成2’‑脱氧腺苷,采用分批补料发酵法在发酵罐中48h发酵2’‑脱氧腺苷产量可达到3.2g/L,具有很好的工业应用前景。
Description
技术领域
本发明涉及化合物生物技术及发酵工程技术生产领域,尤其是一种生产2’-脱氧腺苷的基因工程菌及其构建方法与应用。
背景技术
2’-脱氧腺苷作为脱氧核糖核苷的一种,参与了几乎参与所有生物细胞的遗传信息的传递,影响着蛋白质的合成以及多糖的代谢,并对生物体内细胞的生长、增殖、分化和抑制等具有十分重要的调控作用。它不但是基因药物与基因工程研究的重要原材料,而且它还自身具有很好的生理活性,2’-脱氧腺苷可抑制由糖诱导的胰岛素释放,并且能够降低特异性磷酸二脂酶抑制剂或腺苷环化酶激活剂对于胰岛素分泌的促进作用。此外,2’-脱氧腺苷还可作为抗病毒、抗癌、抗艾滋病药物的重要中间体。
目前合成2’-脱氧腺苷的方法有水解法、化学合成法和生物催化法。水解法通过水解含有DNA的原料来制备2’-脱氧腺苷,该方法具有原料供应不稳定和分离过程复杂的缺点。化学合成法以腺苷为底物经酯化,酰化剂和缚酸剂的酰化作用,再通过还原和纯化处理得到2’-脱氧腺苷,其缺点为对设备的要求高,产率较低、反应步骤繁琐,反应条件苛刻,且反应时间较长。目前报导的生物催化法以腺嘌呤为前体物,过表达嘌呤核苷磷酸化酶(deoD)、尿苷磷酸化酶(udp)和硫脒磷酸化酶(deoA)基因,实现了2’-脱氧腺苷的合成,该方法的缺点为前体物价格昂贵,产量普遍很低,存在耗时长、产率低等问题,很难实现工业化生产。
微生物发酵法已经被作为大多数天然小分子产物大规模生产的主流方式,因此,构建一株遗传背景清晰、产量较为可观的2’-脱氧腺苷生产菌株是目前亟待解决的技术问题。
发明内容
本发明所要解决的技术问题在于提供一种生产2’-脱氧腺苷的基因工程菌。
本发明所要解决的另一技术问题在于提供上述生产2’-脱氧腺苷的基因工程菌的构建方法。
本发明所要解决的另一技术问题在于提供上述生产2’-脱氧腺苷的基因工程菌的应用。
为解决上述技术问题,本发明的技术方案是:
一种生产2’-脱氧腺苷的基因工程菌,为菌株dAR-09,是通过下述基因改造方法获得的:首先通过在出发菌株E.coliW3110的基因组yjiV位点引入异源的B.subtilis168菌株嘌呤操纵子基因pur(EKBCSQLFMNHD),由Ptrc启动子启动,得到菌株dAR-01;然后在基因组yghX、ygaY位点引入异源的B. subtilis168菌株腺苷酸琥珀酸合酶基因purA和腺苷酸琥珀酸裂解酶基因purB,分别由PT7启动子、Ptrc启动子启动,并分别得到菌株dAR-02、dAR-03;然后敲除腺苷脱氨酶基因add、嘌呤核苷磷酸化酶deoD、黄嘌呤碱磷酸化酶xapA、核苷二磷酸激酶基因ndk,阻断分解途径,分别得到菌株dAR-04、dAR-05、dAR-06、dAR-07;然后在基因组ycgH位点双拷贝5'-脱氧核苷酸酶基因yfbR和腺苷酸激酶基因adk,由PT7启动子启动得到菌株dAR-08;最后导入pET-28a(+)-T4-nrdBCA质粒,得到改造成功后的目的菌株dAR-09。
优选的,上述生产2’-脱氧腺苷的基因工程菌,所述出发菌株是野生型E.coliW3110(ATCC 273250)。
优选的,上述生产2’-脱氧腺苷的基因工程菌,所述嘌呤操纵子基因pur(EKBCSQLFMNHD)、腺苷酸琥珀酸合酶基因purA和腺苷酸琥珀酸裂解酶基因purB均来自于腺苷工程菌株B. subtilis168(ATCC 23857),其中,所述嘌呤操纵子基因pur(EKBCSQLFMNHD)的核苷酸序列如SEQ ID NO.1所示(NCBI-GeneID分别为:purE:939481;purK:936039;purB:936048;purC:939389;purS:936041;purQ:938760;purL:939233;purF:936046;purM:936044;purN:936045;purH:936053;purD:938741);所述腺苷酸琥珀酸合酶基因purA的核苷酸序列如SEQ ID NO.2所示(purA;NCBI-GeneID:935805);所述腺苷酸琥珀酸裂解酶基因purB的核苷酸序列如SEQ ID NO.3所示(purB;NCBI-GeneID:936048)。
优选的,上述生产2’-脱氧腺苷的基因工程菌,所述腺苷脱氨酶基因add的核苷酸序列如SEQ ID NO.4所示(add;NCBI-GeneID:945851);所述嘌呤核苷磷酸化酶deoD的核苷酸序列如SEQ ID NO.5所示(deoD;NCBI-GeneID:945654);所述黄嘌呤碱磷酸化酶xapA的核苷酸序列如SEQ ID NO.6所示(xapA;NCBI-GeneID:946878);所述核苷二磷酸激酶ndk的核苷酸序列如SEQ ID NO.7所示(ndk;NCBI-GeneID:945611)。
优选的,上述生产2’-脱氧腺苷的基因工程菌,所述5'-脱氧核苷酸酶基因yfbR的核苷酸序列如SEQ ID NO.8所示(yfbR;NCBI-GeneID:946771);所述腺苷酸激酶基因adk的核苷酸序列如SEQ ID NO.9所示(adk;NCBI-GeneID:945097)。
优选的,上述生产2’-脱氧腺苷的基因工程菌,所述质粒pET-28a(+)-T4-nrdBCA,以pET-28a(+)质粒为骨架,携带硫酸卡那霉素抗性基因和由PT7启动子表达的噬菌体埃希氏菌体T4 nrdBCA基因,具有更适宜在大肠杆菌中高效表达的特性,所述pET-28a(+)质粒作为线性载体的核苷酸序列如SEQ ID NO.11所示。
优选的,上述生产2’-脱氧腺苷的基因工程菌,所述硫酸卡那霉素抗性基因的核苷酸序列如SEQ ID NO.14所示,所述噬菌体埃希氏菌体T4 nrdBCA基因的核苷酸序列如SEQID NO.10所示。
上述核糖核苷酸还原酶nrdBCA基因由噬菌体埃希氏菌体核糖核苷酸还原酶Ia类β 亚基基因nrdB、NrdC硫氧还蛋白基因nrdC、类NrdA需氧 NDP 还原酶大亚基基因nrdA经密码子优化人工合成并连接在一起。
优选的,上述生产2’-脱氧腺苷的基因工程菌,所述Ptrc启动子具有序列表SEQ IDNO.12所示核苷酸序列,PT7启动子具有序列表SEQ ID NO.13所示核苷酸序列。
上述生产2’-脱氧腺苷的基因工程菌的构建方法,具体步骤如下:
(1)通过在E. coliW3110的基因组yjiV位点引入异源的嘌呤操纵子基因pur(EKBCSQLFMNHD)(核苷酸序列如SEQ ID NO.1所示),由Ptrc启动子启动,增强嘌呤代谢途径的碳代谢流;
(2)通过在基因组yghX、ygaY位点引入异源的腺苷酸琥珀酸合酶基因purA(核苷酸序列如SEQ ID NO.2所示)和腺苷酸琥珀酸裂解酶基因purB(核苷酸序列如SEQ ID NO.3所示),分别由PT7启动子、Ptrc启动子(PT7的核苷酸序列如SEQ ID NO.13、Ptrc的核苷酸序列如SEQ ID NO.12所示)启动;
(3)敲除腺苷脱氨酶基因add(核苷酸序列如SEQ ID NO.4所示),敲除嘌呤核苷磷酸化酶deoD(核苷酸序列如SEQ ID NO.5所示),敲除黄嘌呤碱磷酸化酶xapA(核苷酸序列如SEQ ID NO.6所示),敲除核苷二磷酸激酶ndk(核苷酸序列如SEQ ID NO.7所示),阻断2’-脱氧腺苷的分解途径;
(4)通过在基因组ycgH位点过表达5'-脱氧核苷酸酶基因yfbR(核苷酸序列如SEQID NO.8所示)和腺苷酸激酶基因adk(核苷酸序列如SEQ ID NO.9所示),由PT7强启动子启动(核苷酸序列如SEQ ID NO.13所示),强化2’-脱氧腺苷的生物合成途径;
(5)通过pET-28a(+)质粒线性载体(核苷酸序列如SEQ ID NO.11所示)过表达噬菌体埃希氏菌体 T4核糖核苷酸还原酶nrdBCA(核苷酸序列如SEQ ID NO.10所示)。
上述生产2’-脱氧腺苷的基因工程菌在生产2’-脱氧腺苷方面的应用。
优选的,上述生产2’-脱氧腺苷的基因工程菌的应用,通过发酵罐培养的方法获得2’-脱氧腺苷。
优选的,上述生产2’-脱氧腺苷的基因工程菌的应用,所述发酵罐培养的具体方法为:
(1)菌种活化:将菌种从甘油管接至斜面培养基活化培养11-13 h;将菌种从斜面培养基中接至茄形瓶培养基中继续活化扩大培养10-12h,培养温度:35℃;
(2)种子培养:培养过程 pH 维持在 6.7±0.1,温度维持在35±0.2℃,溶氧维持在35%-50%,培养至OD为20时转接发酵罐中进行发酵培养;
(3)发酵培养:按20%的接种量将种子液接种至发酵培养基中进行发酵培养,发酵培养过程中控制pH6.7±0.1,温度35±0.2℃,溶氧30%-50%;培养过程中通过流加 80%(m/v)葡萄糖溶液维持发酵过程菌体所需的碳源,期间通过添加25%的氨水来调节pH,使pH维持在6.7±0.1。
优选的,上述生产2’-脱氧腺苷的基因工程菌的应用,采用的种子培养基为:葡萄糖30g/L,酵母浸粉6g/L,蛋白胨4g/L,柠檬酸1g/L,(NH4)2SO43g/L,MgSO4·7H2O 0.4g/L,KH2PO41.5g/L,FeSO4·7H2O 10mg/L,MnSO45mg/L,Vb1、Vb3、Vb5、Vb12各2mg/L,VH 1mg/L,硫酸卡那霉素50mg/L,其余为水。
优选的,上述生产2’-脱氧腺苷的基因工程菌的应用,采用的发酵培养基为:葡萄糖30g/L,酵母浸粉6g/L,蛋白胨2g/L,柠檬酸2g/L,(NH4)2SO43g/L,MgSO4·7H2O 1.5g/L,KH2PO44g/L,FeSO4·7H2O 20mg/L,MnSO410mg/L,Vb1、Vb3、Vb5、Vb12各2 mg/L,VH 1 mg/L,硫酸卡那霉素50mg/L,其余为水。
上述培养基均可采用标准方法制备获得。
有益效果:
上述生产2’-脱氧腺苷的基因工程菌,通过对E.coli和Bacillus subtilis的嘌呤代谢相关途径进行分析,以野生型E.coli.W3110为底盘微生物并在底盘微生物E.coliW3110中异源引入嘌呤操纵子,通过基因组整合和质粒过表达2’-脱氧腺苷生物合成途径关键酶基因,敲除2’-脱氧腺苷分解途径等相关分子改造获得,具有遗传背景清晰、可持续改造等优点,该菌株发酵生产2’-脱氧腺苷操作简单、能以廉价碳源直接合成2’-脱氧腺苷,采用分批补料发酵法在发酵罐中48h发酵2’-脱氧腺苷产量可达到3.2g/L,具有很好的工业应用前景。
附图说明
图1为2’-脱氧腺苷工程菌株dAR-09的构建方法图解。
图2为2’-脱氧腺苷工程菌株dAR-09中pET-28a(+)-T4-nrdBCA质粒的构建方法图解。
具体实施方式
为了使本领域的技术人员更好的理解本发明的技术方案,下面结合具体实施方式对本发明所述技术方案作进一步的详细说明。
实施例中涉及到的百分号“%”,若未特别说明,指质量百分比,溶液的百分比指100mL中含有溶质的克数,液体之间的百分比,是指在25℃时溶液的体积比例。
实施例中所用的出发菌株为野生型E.coliW3110,保藏号ATCC 273250;菌株B. subtilis168,保藏号ATCC 23857;相应启动子和基因等见序列表。
实施例1
采用的基因编辑方法参照文献(Li Y,Lin Z,Huang C,et al. Metabolicengineering of Escherichia coli using CRISPR-Cas9 meditated genome editing.Metabolic Engineering,2015,31:13-21.)。该方法涉及的工程质粒pGRB是以pUC18为骨架,包括启动子J23100、gRNA-Cas9结合区域序列和终止子序列以及氨节青霉素抗性(工作浓度:100 mg/L)。下列实施例中涉及到的基因整合、构建质粒等专业名词均可在该文章中解释。菌株构建过程中用到的引物见表1。
表1菌株构建过程中所涉及的引物
引物名称 | 序列号 | 引物序列(5’-3’) |
pGRB-yjiV-s | SEQ ID NO.15 | AGTCCTAGGTATAATACTAGTATCCCGCATTTCTTAAAGTCGTTTTAGAGCTAGAA |
pGRB-yjiV-a | SEQ ID NO.16 | TTCTAGCTCTAAAACGACTTTAAGAAATGCGGGATACTAGTATTATACCTAGGACT |
yjiV-up-s | SEQ ID NO.17 | GTGCTGGAGGGATGATTGTTGGGAG |
yjiV-up-purEKB-a | SEQ ID NO.18 | AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGCAGTACTTCCTGCTGGCT |
purEKB-s | SEQ ID NO.19 | TGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCAGCCGCTAGTAGGAATCATCA |
purEKB-a | SEQ ID NO.20 | TATAGTGAACGCCGGTAATTCGCCTAGAAGAAATCAACCAGCGCACTATGCTAAACCTAAACGTTCAAAG |
yjiV-dn-purEKB-s | SEQ ID NO.21 | TGCGCTGGTTGATTTCTTCTAGGCGAATTACCGGCGTTCACTATA |
yjiV-dn-a | SEQ ID NO.22 | TCACCTCCACCAGCACATCC |
pGRB-1-s | SEQ ID NO.23 | CTGAACAACATCATATTTAAATGAACATAACTCAATTTGTAGGCTAGCATAACCCCTTGGGGC |
pGRB-1-a | SEQ ID NO.24 | GCCCCAAGGGGTTATGCTAGCCTACAAATTGAGTTATGTTCATTTAAATATGATGTTGTTCAG |
1-1-up-s | SEQ ID NO.25 | AGAAATTCGCTGTGGAAATCCG |
1-1-up-a | SEQ ID NO.26 | TAGAAGAAGCCGCTAAAAGCTTCTTCTATGCTAAACCTAAACGTTCAAAGAT |
purCSQ-s | SEQ ID NO.27 | CTTTGAACGTTTAGGTTTAGCATAGAAGAAGCTTTTAGCGGCTTCTTCTA |
purCSQ-a | SEQ ID NO.28 | CCTACAAATTGAGTTATGTTCATTCAAGCAGTAGTGACATGAGTTTCC |
yjiV-DN-purCSQ-s | SEQ ID NO.29 | ATGAACATAACTCAATTTGTAGGCGAATTACCGGCGTTCACTATA |
pGRB-4-s | SEQ ID NO.30 | ATGCACAGGAGACTTTCTGATGCGCTGGTTGATTTCTTCTAGGGTCATAGTAATCCAGCAACT |
pGRB-4-a | SEQ ID NO.31 | AGTTGCTGGATTACTATGACCCTAGAAGAAATCAACCAGCGCATCAGAAAGTCTCCTGTGCAT |
4-1-up-s | SEQ ID NO.32 | GAGGATTTTCTTACGGCGATTACT |
4-1-up-a | SEQ ID NO.33 | TCAAGCAGTAGTGACATGAACAATTTAAGACCGTCGGC |
purL-s | SEQ ID NO.34 | GCCGACGGTCTTAAATTGTTCATGTCACTACTGCTTGA |
purL-a | SEQ ID NO.35 | CCATAATTATTGCGGATATTCCTTTAAGCCTTTGATTTCAGCAAGCAT |
yjiV-dn-purL-s | SEQ ID NO.36 | AGGAATATCCGCAATAATTATGGCGAATTACCGGCGTTCACTATA |
pGRB-5-s | SEQ ID NO.37 | ATGCACAGGAGACTTTCTGAAGGAATATCCGCAATAATTATGGGTCATAGTAATCCAGCAACT |
pGRB-5-a | SEQ ID NO.38 | AGTTGCTGGATTACTATGACCCATAATTATTGCGGATATTCCTTCAGAAAGTCTCCTGTGCAT |
5-1-up-s | SEQ ID NO.39 | AGATACAGCGCACATTACAACACAG |
5-1-up-a | SEQ ID NO.40 | TTAAGCCTTTGATTTCAGCAAGCAT |
purFMN-s | SEQ ID NO.41 | ATGCTTGCTGAAATCAAAGGCTTAA |
purFMN-a | SEQ ID NO.42 | CCTAGAAGAAATCAACCAGCGCATCATGCCTTTTCACCTCTGTTATT |
yjiV-dn-purFMN-s | SEQ ID NO.43 | TGCGCTGGTTGATTTCTTCTAGGCGAATTACCGGCGTTCACTATA |
4-2-up-s | SEQ ID NO.44 | GAAGGAGGAGAACTGGGATGC |
4-2-up-a | SEQ ID NO.45 | GCACGTTTAATGGTCATTCATGCCTTTTCACCT |
purHD-s | SEQ ID NO.46 | AGGTGAAAAGGCATGAATGACCATTAAACGTGC |
purHD-a | SEQ ID NO.47 | CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTATTTTTGGGCAGCC |
yjiV-dn-purHD-s | SEQ ID NO.48 | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGAATTACCGGCGTTCACTATA |
pGRB-yghX-s | SEQ ID NO.49 | AGTCCTAGGTATAATACTAGTCCCACTTTGCCTGTCGCTTGGTTTTAGAGCTAGAA |
pGRB-yghX-a | SEQ ID NO.50 | TTCTAGCTCTAAAACCAAGCGACAGGCAAAGTGGGACTAGTATTATACCTAGGACT |
yghx-up-s | SEQ ID NO.51 | GCGCAACGTAGAACAGGAATT |
yghx-up-a | SEQ ID NO.52 | TAAAGTTAAACAAAATTATTTCTAGACCCTATAGTGAGTCGTATTAGATTGAAGCGCCTTTACTACTCC |
purA-s | SEQ ID NO.53 | TAGGGTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGTCTTCAGTAGTTGTAGTAGGTACGCA |
purA-a | SEQ ID NO.54 | AGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTAGTTCGCACGGTACACACTGC |
yghX-dn-s | SEQ ID NO.55 | TGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGGTCATAGTAATCCAGCAACTCTTGTG |
yghX-dn-a | SEQ ID NO.56 | GAGCAGGTATTTACGTGAACCG |
pGRB-ygaY-s | SEQ ID NO.57 | AGTCCTAGGTATAATACTAGTCACTGATGGCGCTGGCATTAGTTTTAGAGCTAGAA |
pGRB-ygaY-a | SEQ ID NO.58 | TTCTAGCTCTAAAACTAATGCCAGCGCCATCAGTGACTAGTATTATACCTAGGACT |
ygaY-up-s | SEQ ID NO.59 | CCTACAAACCACATCGCACATT |
ygaY-up-a | SEQ ID NO.60 | AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGAATTTGTGCCACGACTGAGAA |
purB-s | SEQ ID NO.61 | TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGATCGAACGTTATTCAAGACCTG |
purB-a | SEQ ID NO.62 | CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCTATGCTAAACCTAAACGTTCAAAGAT |
ygaY-dn-s | SEQ ID NO.63 | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCTTGCCGCTCCACC |
ygaY-dn-a | SEQ ID NO.64 | GGAGTAGGGCTTTCCATAGAGTGT |
pGRB-add-s | SEQ ID NO.65 | AGTCCTAGGTATAATACTAGTATTGAAGATGCAGCCCGTCAGTTTTAGAGCTAGAA |
pGRB-add-a | SEQ ID NO.66 | TTCTAGCTCTAAAACTGACGGGCTGCATCTTCAATACTAGTATTATACCTAGGACT |
add-up-s | SEQ ID NO.67 | CGGTGAAGAAGGTCGTGGTT |
add-up-a | SEQ ID NO.68 | TTTGCTCGCGGGATAACCTTAATGGCAGGGTGGTATCAATC |
add-dn-s | SEQ ID NO.69 | GATTGATACCACCCTGCCATTAAGGTTATCCCGCGAGCAAA |
add-dn-a | SEQ ID NO.70 | ATCCCGAGGCAGTTATGTGAA |
pGRB-deoD-s | SEQ ID NO.71 | AGTCCTAGGTATAATACTAGTACTGGGTATTGATGCTCGCGGTTTTAGAGCTAGAA |
pGRB-deoD-a | SEQ ID NO.72 | TTCTAGCTCTAAAACCGCGAGCATCAATACCCAGTACTAGTATTATACCTAGGACT |
deoD-up-s | SEQ ID NO.73 | TGACGCCACCATCAAAGAGA |
deoD-up-a | SEQ ID NO.74 | CCATTTCCACGCCGAGAAGGTCGCCTGGCATCAAAA |
deoD-dn-s | SEQ ID NO.75 | TTTTGATGCCAGGCGACCTTCTCGGCGTGGAAATGG |
deoD-dn-a | SEQ ID NO.76 | TGGCAACAAGGCGTGAGAAC |
pGRB-xapA-s | SEQ ID NO.77 | AGTCCTAGGTATAATACTAGTTTGCGACCTTAAAGTCGTTGGTTTTAGAGCTAGAA |
pGRB-xapA-a | SEQ ID NO.78 | TTCTAGCTCTAAAACCAACGACTTTAAGGTCGCAAACTAGTATTATACCTAGGACT |
xapA-up-s | SEQ ID NO.79 | GTATTGGTAAGAGCCGGGACA |
xapA-up-a | SEQ ID NO.80 | CCTTCCGCCATATTGGTAATCACTCGTGGCGTGAAATCAGG |
xapA-dn-s | SEQ ID NO.81 | CCTGATTTCACGCCACGAGTGATTACCAATATGGCGGAAGG |
xapA-dn-a | SEQ ID NO.82 | TTCCTTGTCGCATCATTTGG |
pGRB-ndk-s | SEQ ID NO.83 | AGTCCTAGGTATAATACTAGTATCGTGGTTTCCGTGCTGGAGTTTTAGAGCTAGAA |
pGRB-ndk-a | SEQ ID NO.84 | TTCTAGCTCTAAAACTCCAGCACGGAAACCACGATACTAGTATTATACCTAGGACT |
ndk-up-s | SEQ ID NO.85 | TCAATTGCTGGTGATGGATGACG |
ndk-up-a | SEQ ID NO.86 | GCAGATTCGACGGAATCAGAACCGCATAAAAGCCACGTGCCTG |
ndk-dn-s | SEQ ID NO.87 | CAGGCACGTGGCTTTTATGCGGTTCTGATTCCGTCGAATCTGC |
ndk-dn-a | SEQ ID NO.88 | GTTGAGATCCAGCAGGTTGATTTTTCC |
ycgH-up-s | SEQ ID NO.89 | TAAACTCGTCAGCGGCACAA |
ycgH-up-a | SEQ ID NO.90 | CTTAAAGTTAAACAAAATTATTTCTAGACCCTATAGTGAGTCGTATTAGGTAGGCGTTTCTGTTGATTCTG |
pGRB-ycgH-s | SEQ ID NO.91 | AGTCCTAGGTATAATACTAGTTATGCGTCTGAACGACCGTGGTTTTAGAGCTAGAA |
pGRB-ycgH-a | SEQ ID NO.92 | TTCTAGCTCTAAAACCACGGTCGTTCAGACGCATAACTAGTATTATACCTAGGACT |
ycgH-dn-s | SEQ ID NO.93 | GGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGGCGTGTCGGATTATCGTTCG |
ycgH-dn-a | SEQ ID NO.94 | GATTCAGGTTGCCATTTACGC |
ycgH-adk-s | SEQ ID NO.95 | GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGCGTATCATTCTGCTTGGCG |
ycgH-adk-a | SEQ ID NO.96 | GATTCTCTGCCATTCAATGGATCCATTAGCCGAGGATTTTTTCCAGATC |
ycgH-yfbR-s | SEQ ID NO.97 | GGATCCATTGAATGGCAGAGAATCATGAAACAGAGCCATTTTTTTGCC |
ycgH-yfbR-a | SEQ ID NO.98 | CAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTACAGCGGTGAATCCTGGCTAATC |
nrdB-s | SEQ ID NO.99 | ATGGGTCGCGGATCCGAATTCATGAGTACAGTTTTTAATACAAATCCAGTT |
nrdA-a | SEQ ID NO.100 | CTCGAGTGCGGCCGCAAGCTTTTACAATTTACACGCTGCACAATC |
如图1所示,2’-脱氧腺苷工程菌株dAR-09的构建方法为:以野生型E.coli.W3110为底盘微生物,异源表达枯草芽孢杆菌嘌呤操纵子基因pur(EKBCSQLFMNHD),过表达腺苷酸琥珀酸合成酶基因purA和腺苷琥珀酸裂解酶基因purB;敲除了腺苷脱氨酶基因add、嘌呤核苷磷酸化酶基因deoD、黄嘌呤碱磷酸化酶基因xapA,核苷二磷酸激酶基因ndk;过表达5'-脱氧核苷酸酶基因yfbR、腺苷酸激酶基因adk;最后导入pET-28a(+)-T4-nrdBCA质粒,得到菌株dAR-09。具体方法如下:
1.1嘌呤核苷操纵子基因pur(EKBCSQLFMNHD)的整合
由于B. subtilis168嘌呤核苷操纵子基因全长12804bp,通过分段整合的方法(具体方法参照CN114774341A说明书[0096]段)将其依次整合在基因组yjiV位点,这五段分别为purEKB、purCSQ、purL、purFMN、purHD。
1.1.1基因purEKB的整合
以pGRB-yjiV-s、pGRB-yjiV-a为引物,构建出pGRB-yjiV质粒。以yjiV-up-s、yjiV-up-purEKB-a、purEKB-s、purEKB-a、yjiV-DN-purEKB-s、yjiV-DN-s为引物,再分别以B.subtilis168和E.coli W3110的基因组为模板,获得上游同源臂yjiV-up,下游同源臂yjiV-DN,目的基因purEKB;然后再获得融合片段yjiV::purEKB。最后将pGRB-yjiV质粒与重叠片段yjiV::purEKB电转至出发菌株E.coliW3110的电转感受态细胞中;再以yjiV-up-s、yjiV-DN-s为鉴定引物,筛选阳性转化子,获得菌株dAR-01-purEKB。
1.1.2基因purCSQ的整合
以pGRB-1-s、pGRB-1-a为引物,构建出pGRB-1质粒。以1-1-up-s、1-1-up-a、purCSQ-s、purCSQ-s、yjiV-DN-purCSQ-s、yjiV-DN-s为引物,再分别以B. subtilis168和E.coliW3110的基因组为模板,获得上游同源臂1-1-up,下游同源臂1-1-DN,目的基因purCSQ;然后再获得融合片段1::purEKB。最后将pGRB-1质粒与重叠片段1::purEKB电转至dAR-01-purEKB的电转感受态细胞中;再以1-1-up-s、yjiV-DN-s为鉴定引物,筛选阳性转化子,获得菌株dAR-01-purEKBCSQ。
1.1.3基因purL的整合
以pGRB-4-s、pGRB-4-a为引物,构建出pGRB-4质粒。以4-1-up-s、4-1-up-a、purL-s、purL-a、yjiV-DN-purL-s、yjiV-DN-s为引物,分别以B. subtilis168和E.coliW3110的基因组为模板,获得上游同源臂4-1-up,下游同源臂4-1-DN,目的基因purL;然后再获得融合片段4::purL。最后将pGRB-yjiV质粒与重叠片段4::purL电转至dAR-01-purEKBCSQ的电转感受态细胞中;再以4-1-up-s、yjiV-DN-s为鉴定引物,筛选阳性转化子,获得菌株dAR-01-purEKBCSQL。
1.1.4基因purFMN的整合
以pGRB-5-s、pGRB-5-a为引物,构建出pGRB-5质粒。以5-1-up-s、5-1-up-a、purFMN-s、purFMN-a、yjiV-DN-purFMN-s、yjiV-DN-s为引物,分别以B. subtilis168和E.coliW3110的基因组为模板,获得上游同源臂5-1-up,下游同源臂5-1-DN,目的基因purFMN;然后再获得融合片段5::purEKB。最后将pGRB-5质粒与重叠片段5::purFMN电转至dAR-01-purEKBCSQL的电转感受态细胞中;再以5-1-up-s、yjiV-DN-s为鉴定引物,筛选阳性转化子,获得菌株dAR-01-purEKBCSQLFMN。
1.1.5基因purHD的整合
以pGRB-4-s、pGRB-4-a为引物,构建出pGRB-4质粒。然后以4-2-up-s、4-2-up-a、purHD-s、purHD-a、yjiV-DN-purL-s、yjiV-DN-s为引物,分别以B. subtilis168和E.coliW3110的基因组为模板,获得上游同源臂4-2-up,下游同源臂4-2-DN,目的基因purHD;然后再获得融合片段4::purHD。最后将pGRB-4质粒与重叠片段4::purHD电转至Hx-1-purEKBCSQLFMN的电转感受态细胞中;再以4-2-up-s、yjiV-DN-s为鉴定引物,筛选阳性转化子,获得菌株dAR-01-purEKBCSQLFMNHD(即为菌株dAR-01)。
1.2基因purA的整合
以pGRB-yghX-s、pGRB-yghX-a为引物,构建出pGRB-yghX质粒。然后以yghx-up-s、yghx-up-a、purA-s、purA-a、yghX-DN-s、yghX-DN-a为引物,分别以B. subtilis168和E.coliW3110的基因组为模板,获得上游同源臂yghX-up,下游同源臂yghX-DN,目的基因purA;然后再获得融合片段yghX::purA。最后将pGRB-yghX质粒与重叠片段yghX::purA电转至dAR-01的电转感受态细胞中;再以yghx-up-s、yghX-DN-a为鉴定引物,筛选阳性转化子,获得菌株dAR-02。
1.3基因purB的整合
以pGRB-ygaY-s、pGRB-ygaY-a为引物,构建出pGRB-ygaY质粒。然后以ygaY-up-s、ygaY-up-a、purB-s、purB-a、ygaY-DN-s、ygaY-DN-a为引物,分别以B. subtilis 168和E.coliW3110的基因组为模板,获得上游同源臂ygaY-up,下游同源臂ygaY-DN,目的基因purB;然后再获得融合片段ygaY::purB。最后将pGRB-ygaY质粒与重叠片段ygaY::purB电转至dAR-02的电转感受态细胞中;再以ygaY-up-s、ygaY-DN-a为鉴定引物,筛选阳性转化子,获得菌株dAR-03。
1.4基因add的敲除
以pGRB-add-s、pGRB-add-a为引物,构建出pGRB-add质粒。然后以add-up-s、add-up-a、add-dn-s、add-dn-a为引物,以E.coliW3110的基因组为模板,获得上游同源臂add-up,下游同源臂add-dn;然后再获得融合片段∆add。最后将pGRB-add质粒与重叠片段∆add电转至dAR-03的电转感受态细胞中;再以add-up-s、add-dn-a为鉴定引物,筛选阳性转化子,获得菌株dAR-04。
1.5基因deoD的敲除
以pGRB-deoD-s、pGRB-deoD-a为引物,构建出pGRB-deoD质粒。然后以deoD-up-s、deoD-up-a、deoD-dn-s、deoD-dn-a为引物,以E.coliW3110的基因组为模板,获得上游同源臂deoD-up,下游同源臂deoD-dn;然后再获得融合片段∆deoD。最后将pGRB-deoD质粒与重叠片段∆deoD电转至dAR-04的电转感受态细胞中;再以deoD-up-s、deoD-dn-a为鉴定引物,筛选阳性转化子,获得菌株dAR-05。
1.6基因xapA的敲除
以pGRB-xapA-s、pGRB-xapA-a为引物,构建出pGRB-xapA质粒。然后以xapA-up-s、xapA-up-a、xapA-dn-s、xapA-dn-a为引物,以E.coliW3110的基因组为模板,获得上游同源臂xapA-up,下游同源臂xapA-dn;然后再获得融合片段∆xapA。最后将pGRB-xapA质粒与重叠片段∆xapA电转至dAR-05的电转感受态细胞中;再以xapA-up-s、xapA-dn-a为鉴定引物,筛选阳性转化子,获得菌株dAR-06。
1.7基因ndk的敲除
以pGRB-ndk-s、pGRB-ndk-a为引物,构建出pGRB-ndk质粒。然后以ndk-up-s、ndk-up-a、ndk-dn-s、ndk-dn-a为引物,以E.coliW3110的基因组为模板,获得上游同源臂ndk-up,下游同源臂ndk-dn;然后再获得融合片段∆ndk。最后将pGRB-ndk质粒与重叠片段∆ndk电转至dAR-06的电转感受态细胞中;再以ndk-up-s、ndk-dn-a为鉴定引物,筛选阳性转化子,获得菌株dAR-07。
1.8基因yfbR和adk的整合
以pGRB-ycgH-s、pGRB-ycgH-a为引物,构建出pGRB-ycgH质粒。然后以ycgH-up-s、ycgH-up-a、ycgH-dn-s、ycgH-dn-a、ycgH-adk-s、ycgH-adk-a、ycgH-yfbR-s、ycgH-yfbR-a为引物,以E.coliW3110的基因组为模板,获得上游同源臂ycgH-up,下游同源臂ycgH-dn,目的片段yfbR和adk;然后再获得融合片段ycgH::yfbR+adk。最后将pGRB-ycgH质粒与重叠片段ycgH::yfbR+adk电转至dAR-07的电转感受态细胞中;再以ycgH-up-s、ycgH-dn-a为鉴定引物,筛选阳性转化子,获得菌株dAR-08。
1.9pET-28a(+)-T4-nrdBCA质粒的表达
如图2所示,以限制性内切酶EcoRⅠ和HindⅢ双酶切pET-28a(+)质粒,得到携带有硫酸卡那霉素抗性基因的pET-28a(+)线性载体。然后以nrdB-s、nrdA-a为引物,以人工合成经密码子优化后nrdBCA基因片段为模板,获得目的片段nrdBCA。使用ClonExpressMultiSOne Step Cloning Kit试剂盒(Vazyme Biotech, Nanjing, China),通过同源重组将pET-28a(+)线性载体与基因片段nrdBCA连接,得到pET-28a(+)-T4-nrdBCA质粒。将其化转至DH5α化转感受态细胞中,筛选获得阳性转化子,提取pET-28a(+)-T4-nrdBCA质粒。最后将pET-28a(+)-T4-nrdBCA质粒电转至dAR-08的电转感受态细胞中,筛选阳性转化子,获得菌株dAR-09。
实施例2
采用实施例1中所获得的菌株dAR-09在5L发酵罐中发酵生产2’-脱氧腺苷,具体步骤如下:
(1)菌种活化:将菌种从甘油管接至斜面培养基活化培养11-13 h;将菌种从斜面培养基中接至茄形瓶培养基中继续活化扩大培养10-12 h;培养温度:35℃。
(2)种子培养:采用的种子培养基为葡萄糖30g/L,酵母浸粉6g/L,蛋白胨4g/L,柠檬酸1g/L,(NH4)2SO43g/L,MgSO4·7H2O 0.4g/L,KH2PO41.5g/L,FeSO4·7H2O 10mg/L,MnSO45mg/L,Vb1、Vb3、Vb5、Vb12各2mg/L,VH 1mg/L,硫酸卡那霉素50mg/L,其余为水;培养过程pH维持在 6.7±0.2,温度维持在35℃±0.2,溶氧维持在35%-50%之间,培养至OD600nm为20时转接发酵罐中进行发酵培养。
(3)发酵培养:按20%的接种量将种子液接种至发酵培养基中进行发酵培养,采用的发酵培养基为葡萄糖30g/L,酵母浸粉6g/L,蛋白胨2g/L,柠檬酸2g/L,(NH4)2SO43g/L,MgSO4·7H2O 1.5g/L,KH2PO44g/L,FeSO4·7H2O 20mg/L,MnSO410mg/L,Vb1、Vb3、Vb5、Vb12各2mg/L,VH 1 mg/L,硫酸卡那霉素50mg/L,其余为水;发酵pH维持在6.7±0.2,温度维持在35±0.2℃,溶氧维持在30%-50%之间;培养过程中通过流加 80%(m/v)葡萄糖溶液维持发酵过程菌体所需的碳源,期间通过添加25%的氨水来调节pH。
5 L发酵罐发酵48h时,2’-脱氧腺苷的产量可达到3.2g/L,为现有报道中最高。
可见,本发明所述生产2’-脱氧腺苷的基因工程菌可以廉价葡萄糖为底物,从头合成价值更高的2’-脱氧腺苷,具有遗传性能稳定、遗传背景清晰、无缺陷型、生产效率高等优点,具有非常好的工业化前景。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,本技术领域技术人员以本发明的方法或以本方法为基础进行的菌种改造等改进和润饰均视为本发明的保护范围。
Claims (10)
1.一种生产2’-脱氧腺苷的基因工程菌,其特征在于:是通过下述基因改造方法获得的:首先通过在出发菌株E.coliW3110的基因组yjiV位点引入异源的B.subtilis 168菌株嘌呤操纵子基因pur(EKBCSQLFMNHD),由Ptrc启动子启动,得到菌株dAR-01;然后在基因组yghX、ygaY位点引入异源的B. subtilis 168菌株腺苷酸琥珀酸合酶基因purA和腺苷酸琥珀酸裂解酶基因purB,分别由PT7启动子、Ptrc启动子启动,并分别得到菌株dAR-02、dAR-03;然后敲除腺苷脱氨酶基因add、嘌呤核苷磷酸化酶deoD、黄嘌呤碱磷酸化酶xapA、核苷二磷酸激酶基因ndk,阻断分解途径,分别得到菌株dAR-04、dAR-05、dAR-06、dAR-07;然后在基因组ycgH位点双拷贝5'-脱氧核苷酸酶基因yfbR和腺苷酸激酶基因adk,由PT7启动子启动得到菌株dAR-08;最后导入pET-28a(+)-T4-nrdBCA质粒,得到改造成功后的目的菌株。
2.根据权利要求1所述的生产2’-脱氧腺苷的基因工程菌,其特征在于:所述嘌呤操纵子基因pur(EKBCSQLFMNHD)的核苷酸序列如SEQ ID NO.1所示;所述腺苷酸琥珀酸合酶基因purA的核苷酸序列如SEQ ID NO.2所示;所述腺苷酸琥珀酸裂解酶基因purB的核苷酸序列如SEQ ID NO.3所示;所述腺苷脱氨酶基因add的核苷酸序列如SEQ ID NO.4所示;所述嘌呤核苷磷酸化酶deoD的核苷酸序列如SEQ ID NO.5所示;所述黄嘌呤碱磷酸化酶xapA的核苷酸序列如SEQ ID NO.6所示;所述核苷二磷酸激酶ndk的核苷酸序列如SEQ ID NO.7所示;所述5'-脱氧核苷酸酶基因yfbR的核苷酸序列如SEQ ID NO.8所示;所述腺苷酸激酶基因adk的核苷酸序列如SEQ ID NO.9所示。
3.根据权利要求1所述的生产2’-脱氧腺苷的基因工程菌,其特征在于:所述质粒pET-28a(+)-T4-nrdBCA,以pET-28a(+)质粒为骨架,携带硫酸卡那霉素抗性基因和由PT7启动子表达的噬菌体埃希氏菌体T4 nrdBCA基因,所述pET-28a(+)质粒的核苷酸序列如SEQ IDNO.11所示。
4.根据权利要求1所述的生产2’-脱氧腺苷的基因工程菌,其特征在于:所述硫酸卡那霉素抗性基因的核苷酸序列如SEQ ID NO.14所示,所述噬菌体埃希氏菌体T4 nrdBCA基因的核苷酸序列如SEQ ID NO.10所示。
5.根据权利要求1所述的生产2’-脱氧腺苷的基因工程菌,其特征在于:所述Ptrc启动子具有序列表SEQ ID NO.12所示核苷酸序列,PT7启动子具有序列表SEQ ID NO.13所示核苷酸序列。
6.权利要求1-5之一所述生产2’-脱氧腺苷的基因工程菌的构建方法,其特征在于:具体步骤如下:
(1)通过在E. coliW3110的基因组yjiV位点引入异源的嘌呤操纵子基因pur(EKBCSQLFMNHD),由Ptrc启动子启动,增强嘌呤代谢途径的碳代谢流;
(2)通过在基因组yghX、ygaY位点引入异源的腺苷酸琥珀酸合酶基因purA和腺苷酸琥珀酸裂解酶基因purB,分别由PT7启动子、Ptrc启动子启动;
(3)敲除腺苷脱氨酶基因add,敲除嘌呤核苷磷酸化酶deoD,敲除黄嘌呤碱磷酸化酶xapA,敲除核苷二磷酸激酶ndk,阻断2’-脱氧腺苷的分解途径;
(4)通过在基因组ycgH位点过表达5'-脱氧核苷酸酶基因yfbR和腺苷酸激酶基因adk,由PT7强启动子启动,强化2’-脱氧腺苷的生物合成途径;
(5)通过pET-28a(+)质粒线性载体过表达噬菌体埃希氏菌体 T4核糖核苷酸还原酶nrdBCA。
7.权利要求1-5之一所述生产2’-脱氧腺苷的基因工程菌在生产2’-脱氧腺苷方面的应用。
8.根据权利要求7所述的生产2’-脱氧腺苷的基因工程菌的应用,其特征在于:通过发酵罐培养的方法获得2’-脱氧腺苷。
9.根据权利要求8所述的生产2’-脱氧腺苷的基因工程菌的应用,其特征在于:所述发酵罐培养的具体方法为:
(1)菌种活化:将菌种从甘油管接至斜面培养基活化培养11-13 h;将菌种从斜面培养基中接至茄形瓶培养基中继续活化扩大培养10-12h,培养温度:35℃;
(2)种子培养:培养过程 pH 维持在 6.7±0.1,温度维持在35±0.2℃,溶氧维持在35%-50%,培养至OD为20时转接发酵罐中进行发酵培养;
(3)发酵培养:按20%的接种量将种子液接种至发酵培养基中进行发酵培养,发酵培养过程中控制pH6.7±0.1,温度35±0.2℃,溶氧30%-50%;培养过程中通过流加 80%葡萄糖溶液维持发酵过程菌体所需的碳源,期间通过添加25%的氨水来调节pH,使pH维持在6.7±0.1。
10.根据权利要求9所述的生产2’-脱氧腺苷的基因工程菌的应用,其特征在于:采用的种子培养基为:葡萄糖30g/L,酵母浸粉6g/L,蛋白胨4g/L,柠檬酸1g/L,(NH4)2SO4 3g/L,MgSO4·7H2O 0.4g/L,KH2PO41.5g/L,FeSO4·7H2O 10mg/L,MnSO45mg/L,Vb1、Vb 3、Vb 5、Vb 12各2mg/L,VH 1mg/L,硫酸卡那霉素50mg/L,其余为水;采用的发酵培养基为:葡萄糖30g/L,酵母浸粉6g/L,蛋白胨2g/L,柠檬酸2g/L,(NH4)2SO4 3g/L,MgSO4·7H2O 1.5g/L,KH2PO44g/L,FeSO4·7H2O 20mg/L,MnSO4 10mg/L,Vb1、Vb 3、Vb 5、Vb 12各2 mg/L,VH 1 mg/L,硫酸卡那霉素50mg/L,其余为水。
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