CN113278601B - 一种腺苷酰化蛋白a6突变体及其编码基因与应用 - Google Patents

一种腺苷酰化蛋白a6突变体及其编码基因与应用 Download PDF

Info

Publication number
CN113278601B
CN113278601B CN202110531780.2A CN202110531780A CN113278601B CN 113278601 B CN113278601 B CN 113278601B CN 202110531780 A CN202110531780 A CN 202110531780A CN 113278601 B CN113278601 B CN 113278601B
Authority
CN
China
Prior art keywords
leu
ala
glu
val
gly
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
Application number
CN202110531780.2A
Other languages
English (en)
Other versions
CN113278601A (zh
Inventor
吴杰群
朱梅芳
章旭红
储消和
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University of Technology ZJUT
Original Assignee
Zhejiang University of Technology ZJUT
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Zhejiang University of Technology ZJUT filed Critical Zhejiang University of Technology ZJUT
Priority to CN202110531780.2A priority Critical patent/CN113278601B/zh
Publication of CN113278601A publication Critical patent/CN113278601A/zh
Application granted granted Critical
Publication of CN113278601B publication Critical patent/CN113278601B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
    • C07K7/62—Polymyxins; Related peptides
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

本发明公开了一种腺苷酰化蛋白A6突变体以及其编码基因，以及其作为腺苷酰化结构域关键酶在催化合成多粘菌素中的应用。所述腺苷酰化蛋白A6突变体由氨基酸序列如SEQ ID NO.1所示的腺苷酰化蛋白A6第516位氨基酸经点突变而得。本发明利用基因工程和酶工程手段，对A域的活性位点进行研究，通过构建突变体确定了与A域底物特异性相关的关键氨基酸残基(A6第516位残基)，本发明获得的突变体G516A与腺苷酰化蛋白A6相比，底物特异性发生改变，对L‑Ala的活性大幅提高，出现了对L‑Arg的活性，证明对该位点的氨基酸残基进行改造能够改变腺苷酰化蛋白的底物识别，为多粘菌素家族化合物结构的扩展提供了新的途径。

Description

一种腺苷酰化蛋白A6突变体及其编码基因与应用
(一)技术领域
本发明涉及一种腺苷酰化蛋白A6突变体以及其编码基因，以及其作为腺苷酰化结构域关键酶在催化合成多粘菌素中的应用。
(二)背景技术
多粘类芽孢杆菌是一种革兰氏阳性菌，其代谢产物多粘菌素作为多重耐药革兰阴性菌的最后一道防线，有巨大工业价值。为产生具有重要生物活性的新型多粘菌素，需要对合成多粘菌素的相关关键酶进行工程改造，如催化合成多粘菌素的非核糖体多肽合酶(NRPS)中的关键酶——A域(腺苷酰化结构域)。
NRPS是以模块化的方式将氨基酸线性组装起来，根据NRPS不同模块在装配线上的位置，可以将其分类为起始模块、延伸模块或终止模块。其中最小的延伸模块包含三个核心结构域，C域(缩合结构域)，A域(腺苷酰化结构域)和T域(巯基化结构域)。A域主要负责特异性识别底物氨基酸，将不同的氨基酸底物掺入主链。因此，A域不仅决定主链的合成效率，而且决定主链的结构多样性。对A域的研究表明其识别底物氨基酸的结合口袋主要与10个关键残基有关，不同A域的关键残基不同，因此具有不同的底物特异性，并赋予了非核糖体多肽结构的多样性。为获得更多具有重要生物活性的新型多粘菌素这一目标，设法寻找能够改变A域底物识别关键氨基酸残基，为系统改造多粘菌素的生产菌种提供理论指导。
多粘菌素中主要具有抗菌活性的成分是多粘菌素B和多粘菌素E，两者在化学结构上的区别是主链上掺入的第六个氨基酸不同，该位置的氨基酸是由多粘菌素NRPS模块6中的腺苷酰化结构域负责识别和装载。目前多粘菌素NRPS模块6中A域与底物识别相关的关键残基尚未得到鉴定，如何确定A域的活性位点并对其进行改造，从而获得更多具有重要生物活性的新型多粘菌素，具有重大工业前景。
(三)发明内容
本发明目的是提供能够改变腺苷酰化蛋白A6底物识别的腺苷酰化蛋白A6突变体以及其编码基因，以及其作为腺苷酰化结构域关键酶在催化合成多粘菌素中的应用。
本发明采用的技术方案是：
一种腺苷酰化蛋白A6突变体，由氨基酸序列如SEQ ID NO.1所示的腺苷酰化蛋白A6第516位氨基酸经点突变而得。本发明利用基因工程和酶工程手段，对A域的活性位点进行研究，通过构建突变体确定了与A域底物特异性相关的关键氨基酸残基(A6第516位残基)，为多粘菌素家族化合物结构的扩展提供了新的途径。
该腺苷酰化蛋白A6基因来源于多粘类芽孢杆菌，其氨基酸序列如SEQ ID NO.1所示，相应核苷酸序列如SEQ ID NO.2所示。
SEQ ID NO.1序列如下：
MAFEKETLFWNEKFGSDDYTLTRLPYSKAPSSLTPIMTTVGGTLSEEVAQRVLQMSKGAPLATFMILLAGVQSLLYKYTGASDILVGMPVVRKPTETRRSVNHTIILKNSLSAGATFKTLLSELRTSLPEAIQHQHIPFLKMTEKLDLQYADGITVVHTLVSLKELHLDEIGQNVVTDCSFEFSLTGGTIQLALSYNEHLYDSEFMTRVVGHLNRLLAVGLHELELEIVRADMLSEDEKFQLLQSFNDTEKDYPRDRTIHQLVEEQAKRVPEATAVVFEGRRLSYAELNERANRLARTLRSVGVLPNQLVGLMARRSLETVVGILAVLKAGGAYVPIDPEYPEERIRYILENSNAQLLLTQRELLQQVPFEGTVLALDDEQAYSDDGSNLEPASGPNDLAYVIYTSGTTGKPKGVMLEHRGLVSLKLMFADRLGITEHDRIVQFASLSFDASCWEMFKALYFGAALYIPTAETILDTRLFESYMNEHAITAAILPPTYSAYLNPDRLPSLTKLVTGGSAVSAEFVQQWKPKVHYFNAYGPTEASIVTTLWDADEEQSERRVIPIGRPLANHRIFILDTHLQLVPPGVDGELCVAGVGLARGYLNHPELTAEKFVEHPFAPGERLYRTGDLARWLPDGNIEYLGRIDHQVKIRGFRIEIGEIEEQLLKIDSVQETIVIAREGKSGQELCAYLVAGHPLTLGELRSALAQKLPNYMIPAHFVQLPRMPLTPNDKIDRKALPVPEGNALTGGAYVAPRNEAER
优选的，所述腺苷酰化蛋白A6突变体氨基酸序列如SEQ ID NO.5所示。
SEQ ID NO.5序列如下(下划线为突变位点)：
MAFEKETLFWNEKFGSDDYTLTRLPYSKAPSSLTPIMTTVGGTLSEEVAQRVLQMSKGAPLATFMILLAGVQSLLYKYTGASDILVGMPVVRKPTETRRSVNHTIILKNSLSAGATFKTLLSELRTSLPEAIQHQHIPFLKMTEKLDLQYADGITVVHTLVSLKELHLDEIGQNVVTDCSFEFSLTGGTIQLALSYNEHLYDSEFMTRVVGHLNRLLAVGLHELELEIVRADMLSEDEKFQLLQSFNDTEKDYPRDRTIHQLVEEQAKRVPEATAVVFEGRRLSYAELNERANRLARTLRSVGVLPNQLVGLMARRSLETVVGILAVLKAGGAYVPIDPEYPEERIRYILENSNAQLLLTQRELLQQVPFEGTVLALDDEQAYSDDGSNLEPASGPNDLAYVIYTSGTTGKPKGVMLEHRGLVSLKLMFADRLGITEHDRIVQFASLSFDASCWEMFKALYFGAALYIPTAETILDTRLFESYMNEHAITAAILPPTYSAYLNPDRLPSLTKLVTAGSAVSAEFVQQWKPKVHYFNAYGPTEASIVTTLWDADEEQSERRVIPIGRPLANHRIFILDTHLQLVPPGVDGELCVAGVGLARGYLNHPELTAEKFVEHPFAPGERLYRTGDLARWLPDGNIEYLGRIDHQVKIRGFRIEIGEIEEQLLKIDSVQETIVIAREGKSGQELCAYLVAGHPLTLGELRSALAQKLPNYMIPAHFVQLPRMPLTPNDKIDRKALPVPEGNALTGGAYVAPRNEAER
由于氨基酸序列的特殊性，任何SEQ ID NO.5所示氨基酸序列的多肽的片段或其变体，如其保守性变体、生物活性片段或衍生物，只要该多肽的片段或多肽变体与前述氨基酸序列同源性在95％以上，均属于本发明保护范围之列。所述改变可包括氨基酸序列中氨基酸的缺失、插入或替换；对于变体的保守性改变，所替换的氨基酸具有与原氨基酸相似的结构或化学性质，如用亮氨酸替换异亮氨酸，变体也可具有非保守性改变，如用色氨酸替换甘氨酸。
本发明还涉及编码所述腺苷酰化蛋白A6突变体的基因。
具体的，所述编码基因核苷酸序列可如SEQ ID NO.6所示(该基因编码SEQ IDNO.5所示氨基酸)。
SEQ ID NO.6序列如下(下划线为突变位点)：
atggcttttgaaaaagaaacgttgttttggaacgaaaaattcggtagcgacgattataccttgacgcggctgccttacagcaaagctccaagctccctgacgcccattatgacaaccgtcggcgggacgctttcggaggaagtggcgcagcgcgtccttcaaatgagcaagggcgctccgctggccacttttatgattttgctcgcaggtgttcagtccctcttgtataaatatacaggcgcttctgacattctggttggcatgccggttgtacggaaaccgacggagacgcgccgatccgttaatcatacgatcattttgaaaaactcgctttcggcgggtgcgacttttaaaacgcttctgagcgagttgaggacttccttgccggaagcgattcagcatcaacatattccgttcttgaaaatgacggagaagctggatctgcaatatgcagacgggataaccgtcgtccatacgctagtatcccttaaggagctgcacctggacgaaatcgggcaaaacgtggtcacggattgttcctttgaattcagcttaacgggcgggacgatacagctagcgttgtcatataacgagcacttatatgactccgagttcatgactcgggtcgttggccatctgaatcgcctgttggccgtggggcttcacgagttggaactggagatcgtgcgggcggacatgctatcggaggacgagaaatttcaattgttgcaaagctttaacgataccgagaaggactatccccgtgatcggacgattcatcaactcgtggaggagcaggcgaagcgggtgcccgaagcgacggcggttgtctttgagggtcggcggctttcgtacgcggagctgaacgaacgggcgaaccggctggcacggacgctgcgatcggtcggcgtgctgcccaatcagctggtaggcttgatggccaggagatcactggagacggtcgttggcattctggcggttctgaaagctggcggtgcctacgtgccgatcgacccggaatacccggaagaacgcatccgctacattctggagaactcgaacgcgcagctgctgctgactcaaagggagctgctgcagcaggtgccgttcgaaggaaccgtgttggcgctggatgacgagcaggcctacagcgacgatggctcgaacttggagccggccagcggtccgaatgatcttgcttatgtcatctatacgtcaggtacgacaggcaaacccaaaggggtcatgctggagcatcgcggtttggtcagcttgaagctgatgttcgcggataggctgggcatcacggagcatgaccggatcgttcaattcgccagcctgtcgttcgacgcgtcctgctgggaaatgttcaaagcgctctattttggcgcggctttgtacatcccgacggccgagacgattctcgacacccgcttgttcgagagttatatgaacgagcatgcgattacggcggcgattttgcctccaacgtacagcgcttatttgaacccggaccgccttcccagcttaacgaagctcgtaacggcaggctctgcggtatcggctgaattcgtgcagcagtggaaaccgaaggtccactatttcaatgcttacggccctaccgaagcttcgattgttacgacgctttgggatgcagatgaggagcagtcggagcgcagagtcattccgatcgggcgcccgctggccaatcaccggatttttattttggatacccacctgcagcttgtgccaccgggagtggacggcgagctgtgtgtggcaggcgtggggcttgcgagaggttacctgaaccatccggagctgacggcagagaagttcgtggaacatccgtttgcgcctggagaacgcctttaccggacgggagatctcgcccgctggctgccggacggaaatattgagtacttgggccggatcgatcatcaggtgaaaatccgtggattccggatcgagatcggcgagattgaagagcaacttctgaagatcgactccgtgcaggagacgatcgtaatcgcgcgggaaggcaaaagtggacaagaactgtgcgcttacctggtcgcgggccacccgcttacgctcggcgagttgagaagcgcgctggcgcaaaaattgccgaattacatgattccggcgcattttgttcagcttccgcggatgccgctcacgccgaacgacaaaatcgaccgcaaggctttgcccgtcccggaaggaaacgcactgaccggcggcgcgtacgtagctccccgcaatgaagccgagcgg
由于核苷酸序列的特殊性，任何SEQ ID NO.6所示多核苷酸的变体，只要其与该多核苷酸具有90％以上同源性，均属于本发明保护范围之列。所述多核苷酸的变体是指一种具有一个或多个核苷酸改变的多核苷酸序列。此多核苷酸的变体可以使生的等位变异体或非生的变异体，包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变编码氨基酸的功能。
本发明还涉及含有所述编码基因的重组菌。
本发明还涉及所述腺苷酰化蛋白A6突变体作为腺苷酰化结构域关键酶在催化合成多粘菌素中的应用。多黏菌素的生物合成基因簇参见图1，本发明体外改造的突变体来源于module 6中的A域。
本发明关键在于突变位点的选择，在已知所述腺苷酰化蛋白A6序列及其突变体位点的前提下，本领域普通技术人员可根据SEQ ID NO.1的腺苷酰化蛋白A6基因，设计定点突变的突变引物，以携带腺苷酰化蛋白A6的克隆载体为模板进行定点突变构建突变体，以质粒pET-28a或能表达该酶的载体为表达载体，将重组质粒转化E.coli BL21(DE3)细胞或能表达该酶的宿主细胞，高通量筛选验证后的阳性单克隆进行培养，获得含有本发明突变体的重组菌湿菌体。
具体的，建突变体的步骤如下：
(1)将从多粘类芽孢杆菌多粘菌素生物合成基因簇中获得的编码腺苷酰化蛋白A6的基因，克隆到表达载体中，构建A6重组表达质粒；
(2)利用定点突变技术，设计含有突变氨基酸的密码子碱基的互补引物，以重组质粒为模板进行反向PCR，得到含有定点突变目标载体片段；
(3)将含有定点突变目标载体片段的PCR反应液经过Dpn I内切酶消化，除去未突变的原质粒，通过1％琼脂糖凝胶电泳回收片段；
(4)将回收得到的含有定点突变目标载体片段转化到感受态大肠杆菌DH5α中，平板培养挑取正确复制子，并提取正确突变的质粒；
(5)将正确的重组载体转化大肠杆菌BL21(DE3)，诱导表达。
所述方法还进一步包括使用Ni-NTA1mL镍柱、脱盐柱、超滤管对腺苷酰化蛋白进行纯化。
将所述方法中步骤(5)获得的重组菌在含有50μg/L的卡那霉素的LB培养基中37℃液体培养过夜，后接入含有50μg/L的卡那霉素的TB培养基37℃培养至OD600＝0.8，迅速降温至16℃静置20min，加入最终浓度0.3mM的诱导剂IPTG诱导蛋白表达，16℃继续培养22h后离心收集菌体，用缓冲液重悬，并使用低温超高压细胞破碎仪裂解细胞，离心获得上清液即为粗酶液。
本发明的有益效果主要体现在：本发明获得的突变体G516A与腺苷酰化蛋白A6相比，底物特异性发生改变，对L-Ala的活性大幅提高，出现了对L-Arg的活性，证明对该位点的氨基酸残基进行改造能够改变腺苷酰化蛋白的底物识别，为多粘菌素家族化合物结构的扩展提供了新的途径。
(四)附图说明
图1为多黏菌素的生物合成基因簇。
图2为腺苷酰化蛋白A6及突变体I493T、G516A、V546A的蛋白表达。
图3为腺苷酰化蛋白A6及突变体I493T、G516A、V546A的蛋白纯化。
图4为突变体V514I的蛋白表达及纯化。
图5为FeCl3检测法的反应原理。
图6为腺苷酰化蛋白A6与突变体I493T底物选择性比较。
图7为腺苷酰化蛋白A6与突变体V514I底物选择性比较。
图8为腺苷酰化蛋白A6与突变体G516A底物选择性比较。
图9为腺苷酰化蛋白A6与突变体V546A底物选择性比较。
(五)具体实施方式
为了加深对本发明的理解，下面将结合具体实施例对本发明做进一步详细描述，该实施例仅用于解释本发明，并不对本发明的保护范围构成限定。
实施例1：未突变的腺苷酰化蛋白A6表达质粒的构建
(1)以实验室保存的多粘类芽孢杆菌(CICC 21777)总基因组为模板，设计带有与pET-28a(+)重组同源片段的上下游引物F/R进行PCR扩增，PCR产物用1％
琼脂糖凝胶电泳检测。检测到有目的产物后用小量DNA纯化试剂盒回收目的片段备用。
F：CTTTAAGAAGGAGATATAATGGCTTTTGAAAAAGAAACG
R：CTCGAGTGCGGCCGCAAGCCGCTCGGCTTCATTGCGGGGAGC
(2)抽提表达质粒pET-28a(+)，用Nco I和EcoR I双酶切，回收备用。
(3)将上述回收备用的载体和片段用SoSoo试剂盒进行重组，并转入大肠杆菌DH5α感受态细胞中，得到重组表达质粒pET-28a+A6克隆菌株。
实施例2：通过序列比对预测关键氨基酸残基
为预测腺苷酰化蛋白与底物结合口袋相关的关键残基，通过NCBI下载了GrsA、SlgN1、CmiS6、IdnL1、DltA这5种已通过X射线衍射确定蛋白结构及活性位点的氨基酸序列，它们的PBD编号分别为1AMU、4GR5、5JJP、5JJQ、3DHV。将这些氨基酸序列和实施例1中A6的氨基酸序列以及编码多粘菌素B生物合成基因簇模块6中腺苷酰化结构域B-A6的氨基酸序列(GenBank:JN660148.1)进行多序列比对，初步筛选得到可能与该模块A域底物特异性相关的关键残基如表1所示。
表1：通过多序列比对预测活性位点的结果
Figure BDA0003068132840000061
结论：实验室来源的腺苷酰化蛋白A6(SEQ ID NO.1)和网上报道的编码多粘菌素B生物合成基因簇模块6中腺苷酰化结构域B-A6在493、514、516、546位点存在差异，推测更可能是能改变腺苷酰化蛋白A6底物识别的关键氨基酸残基。
实施例3：腺苷酰化蛋白突变体表达质粒的构建
(1)以实施例1中构建的pET-28a+A6表达质粒为模板，设计定点突变引物I493T-F/R、V514I-F/R、G516A-F/R、V546A-F/R分别进行反向PCR。具体定点突变引物如下(下划线为突变位点)：
I493T-F：AGTCGCCGCCGTAATCGCAT
I493T-R：ATGCGATTACGGCGGCGACTTTGCCTCCAACGTACAG
V514I-F：TATGAGCTTCGTTAAGCTGG
V514I-R：
CCAGCTTAACGAAGCTCATAACGGGAGGCTCTGCGGT
G516A-F：TGCCGTTACGAGCTTCGTTA
G516A-R：
TAACGAAGCTCGTAACGGCAGGCTCTGCGGTATCGGC
V546A-F：AGCAATCGAAGCTTCGGTAG
V546A-R：
CTACCGAAGCTTCGATTGCTACGACGCTTTGGGATGC
(2)PCR产物用1％琼脂糖凝胶电泳检测。检测到有目的产物后加入Dpn I酶消化模板，用小量DNA纯化试剂盒回收目的片段备用。
(3)回收得到的突变体PCR片段化转大肠杆菌DH5α感受态细胞，得到突变体I493T(SEQ ID NO.3)、V514I(SEQ ID NO.4)、G516A(SEQ ID NO.4)、V546A(SEQ ID NO.7)的表达质粒pET-28a+I493T、pET-28a+V514I、pET-28a+G516A、pET-28a+V546A。
实施例4：腺苷酰化蛋白A6及突变体蛋白的表达与纯化
(1)将构建的质粒pET-28a+A6、pET-28a+I493T、pET-28a+V514I、pET-28a+G516A、pET-28a+V546A以及pET-28a空质粒转化到BL21(DE3)感受态细胞中，构建表达菌株。从平板上挑取单菌落接种到含有50μg/L的卡那霉素的LB培养基试管中，37℃、220rpm下恒温震荡培养过夜，按照1％接种量接种到含50μg/mL卡那霉素的TB液体培养基中。紫外分光光度法检测培养液的OD600达到0.8时，迅速降温至16℃静置20min，加入最终浓度0.3mM的诱导剂IPTG诱导蛋白表达。
(2)在16℃、220rpm条件下诱导表达22h后，将发酵液于4℃、10000rpm离心10min去除上清，收集菌体。将收集的菌体用磷酸盐缓冲液重悬，重悬后用低温超高压细胞破碎仪将菌体破碎释放胞内蛋白，破碎完成后，将破碎的液体于4℃、10000rpm离心10min收集上清。经SDS-PAGE验证蛋白表达，如图2、图3、图4所示，结果表明目的蛋白有可溶性表达。收集的上清用于后续的蛋白纯化。
(3)由于重组表达的腺苷酰化蛋白A6及突变体蛋白带有多聚组氨酸标签(Histag)，因此使用镍离子亲和层析方法分离目标蛋白。使用Ni-NTA 1mL预装重力柱纯化各个蛋白，并用脱盐柱进行脱盐处理，经超滤管浓缩后得到纯化的腺苷酰化蛋白A6蛋白及各个突变体蛋白。经SDS-PAGE验证蛋白纯化效果，如图3、图4所示。
实施例4：腺苷酰化蛋白A6及各个突变体蛋白底物特异性的测定
腺苷酰化蛋白的底物特异性测定使用FeCl3检测法，该方法基于异羟肟酸铁反应。其检测原理是腺苷酰化蛋白识别激活底物氨基酸会形成氨基酰-AMP，当有羟胺存在时，氨基酰-AMP会与羟胺反应形成异羟肟酸，异羟肟酸会与Fe3+形成红色或紫红色的络合物，可以在480nm～540nm之间的吸收波长检测到，如图5所示。
FeCl3检测法使用到的试剂包括2×腺苷化检测缓冲液(100mM Tris-HCl，pH8.0)，100mM MgSO4溶液，4M羟胺溶液，7M NaOH溶液，100mM ATP，100mM氨基酸溶液，8％三氯乙酸溶液，3.4％FeCl3溶液。具体反应体系如表2所示，其中2M的羟胺溶液需要现用现配，用移液枪取400μL 4M溶液，225μL 7M NaOH，175μL去离子水，混匀放置于冰上。
表2：FeCl3法检测腺苷化反应体系
Figure BDA0003068132840000081
FeCl3检测法具体操作方法：以一个蛋白对20种天然氨基酸进行选择性测定为例，可在100μL体系基础上扩大21倍配制成体系母液，母液中加入除氨基酸外的所有所需试剂。取21个0.2mL PCR管标记为管1，管2，管3……管21，管1～管20作为实验组加入5μL不同的100mM氨基酸溶液，管21作为对照组加入5μL去离子水作为对照，每个PCR管中分别加入95μL体系母液，置于37℃恒温培养箱进行反应，反应24h后向每个反应PCR管中加入50μL 8％三氯乙酸溶液猝灭反应，再加入50μL 3.4％FeCl3溶液，吸打混匀，显色5min，混合物以12000rpm的转速常温离心10min，小心吸取100μL上清到透明96孔板中，使用酶标仪在490nm的波长下进行检测。将实验组的吸光值减去对照组的吸光值作为该蛋白对每个氨基酸的选择性。
将腺苷酰化蛋白A6及突变体I493T、V514I、G516A、V546A的蛋白分别使用FeCl3检测法对20种天然氨基酸进行底物特异性检测，结果如图6、图7、图8、图9所示。结果表明，与腺苷酰化蛋白A6相比，突变体I493T的酶活显著降低，对所有氨基酸的活性都下降了50％以上，除此以外其他突变体的酶活都与野生型差距不大，说明493位点与腺苷酰化蛋白A6的酶活相关，在改造时应尽量避开该位点；G516A突变体与腺苷酰化蛋白A6相比，底物特异性发生改变，对L-Ala的活性大幅提高，出现了对L-Arg的活性，证明516位点是腺苷酰化蛋白A6底物识别的关键氨基酸残基，对该位点的氨基酸残基进行改造能够改变腺苷酰化蛋白的底物识别，为多粘菌素家族化合物结构的扩展提供了新的途径。
以上所述实施例仅是为充分说明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所做的等同替代或变换，均在本发明的保护范围内，本发明的保护范围以权利要求书为准。
序列表
<110> 浙江工业大学
<120> 一种腺苷酰化蛋白A6突变体及其编码基因与应用
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 760
<212> PRT
<213> 多粘类芽孢杆菌(Paenibacillus polymyxa)
<400> 1
Met Ala Phe Glu Lys Glu Thr Leu Phe Trp Asn Glu Lys Phe Gly Ser
1 5 10 15
Asp Asp Tyr Thr Leu Thr Arg Leu Pro Tyr Ser Lys Ala Pro Ser Ser
20 25 30
Leu Thr Pro Ile Met Thr Thr Val Gly Gly Thr Leu Ser Glu Glu Val
35 40 45
Ala Gln Arg Val Leu Gln Met Ser Lys Gly Ala Pro Leu Ala Thr Phe
50 55 60
Met Ile Leu Leu Ala Gly Val Gln Ser Leu Leu Tyr Lys Tyr Thr Gly
65 70 75 80
Ala Ser Asp Ile Leu Val Gly Met Pro Val Val Arg Lys Pro Thr Glu
85 90 95
Thr Arg Arg Ser Val Asn His Thr Ile Ile Leu Lys Asn Ser Leu Ser
100 105 110
Ala Gly Ala Thr Phe Lys Thr Leu Leu Ser Glu Leu Arg Thr Ser Leu
115 120 125
Pro Glu Ala Ile Gln His Gln His Ile Pro Phe Leu Lys Met Thr Glu
130 135 140
Lys Leu Asp Leu Gln Tyr Ala Asp Gly Ile Thr Val Val His Thr Leu
145 150 155 160
Val Ser Leu Lys Glu Leu His Leu Asp Glu Ile Gly Gln Asn Val Val
165 170 175
Thr Asp Cys Ser Phe Glu Phe Ser Leu Thr Gly Gly Thr Ile Gln Leu
180 185 190
Ala Leu Ser Tyr Asn Glu His Leu Tyr Asp Ser Glu Phe Met Thr Arg
195 200 205
Val Val Gly His Leu Asn Arg Leu Leu Ala Val Gly Leu His Glu Leu
210 215 220
Glu Leu Glu Ile Val Arg Ala Asp Met Leu Ser Glu Asp Glu Lys Phe
225 230 235 240
Gln Leu Leu Gln Ser Phe Asn Asp Thr Glu Lys Asp Tyr Pro Arg Asp
245 250 255
Arg Thr Ile His Gln Leu Val Glu Glu Gln Ala Lys Arg Val Pro Glu
260 265 270
Ala Thr Ala Val Val Phe Glu Gly Arg Arg Leu Ser Tyr Ala Glu Leu
275 280 285
Asn Glu Arg Ala Asn Arg Leu Ala Arg Thr Leu Arg Ser Val Gly Val
290 295 300
Leu Pro Asn Gln Leu Val Gly Leu Met Ala Arg Arg Ser Leu Glu Thr
305 310 315 320
Val Val Gly Ile Leu Ala Val Leu Lys Ala Gly Gly Ala Tyr Val Pro
325 330 335
Ile Asp Pro Glu Tyr Pro Glu Glu Arg Ile Arg Tyr Ile Leu Glu Asn
340 345 350
Ser Asn Ala Gln Leu Leu Leu Thr Gln Arg Glu Leu Leu Gln Gln Val
355 360 365
Pro Phe Glu Gly Thr Val Leu Ala Leu Asp Asp Glu Gln Ala Tyr Ser
370 375 380
Asp Asp Gly Ser Asn Leu Glu Pro Ala Ser Gly Pro Asn Asp Leu Ala
385 390 395 400
Tyr Val Ile Tyr Thr Ser Gly Thr Thr Gly Lys Pro Lys Gly Val Met
405 410 415
Leu Glu His Arg Gly Leu Val Ser Leu Lys Leu Met Phe Ala Asp Arg
420 425 430
Leu Gly Ile Thr Glu His Asp Arg Ile Val Gln Phe Ala Ser Leu Ser
435 440 445
Phe Asp Ala Ser Cys Trp Glu Met Phe Lys Ala Leu Tyr Phe Gly Ala
450 455 460
Ala Leu Tyr Ile Pro Thr Ala Glu Thr Ile Leu Asp Thr Arg Leu Phe
465 470 475 480
Glu Ser Tyr Met Asn Glu His Ala Ile Thr Ala Ala Ile Leu Pro Pro
485 490 495
Thr Tyr Ser Ala Tyr Leu Asn Pro Asp Arg Leu Pro Ser Leu Thr Lys
500 505 510
Leu Val Thr Gly Gly Ser Ala Val Ser Ala Glu Phe Val Gln Gln Trp
515 520 525
Lys Pro Lys Val His Tyr Phe Asn Ala Tyr Gly Pro Thr Glu Ala Ser
530 535 540
Ile Val Thr Thr Leu Trp Asp Ala Asp Glu Glu Gln Ser Glu Arg Arg
545 550 555 560
Val Ile Pro Ile Gly Arg Pro Leu Ala Asn His Arg Ile Phe Ile Leu
565 570 575
Asp Thr His Leu Gln Leu Val Pro Pro Gly Val Asp Gly Glu Leu Cys
580 585 590
Val Ala Gly Val Gly Leu Ala Arg Gly Tyr Leu Asn His Pro Glu Leu
595 600 605
Thr Ala Glu Lys Phe Val Glu His Pro Phe Ala Pro Gly Glu Arg Leu
610 615 620
Tyr Arg Thr Gly Asp Leu Ala Arg Trp Leu Pro Asp Gly Asn Ile Glu
625 630 635 640
Tyr Leu Gly Arg Ile Asp His Gln Val Lys Ile Arg Gly Phe Arg Ile
645 650 655
Glu Ile Gly Glu Ile Glu Glu Gln Leu Leu Lys Ile Asp Ser Val Gln
660 665 670
Glu Thr Ile Val Ile Ala Arg Glu Gly Lys Ser Gly Gln Glu Leu Cys
675 680 685
Ala Tyr Leu Val Ala Gly His Pro Leu Thr Leu Gly Glu Leu Arg Ser
690 695 700
Ala Leu Ala Gln Lys Leu Pro Asn Tyr Met Ile Pro Ala His Phe Val
705 710 715 720
Gln Leu Pro Arg Met Pro Leu Thr Pro Asn Asp Lys Ile Asp Arg Lys
725 730 735
Ala Leu Pro Val Pro Glu Gly Asn Ala Leu Thr Gly Gly Ala Tyr Val
740 745 750
Ala Pro Arg Asn Glu Ala Glu Arg
755 760
<210> 2
<211> 2280
<212> DNA
<213> 多粘类芽孢杆菌(Paenibacillus polymyxa)
<400> 2
atggcttttg aaaaagaaac gttgttttgg aacgaaaaat tcggtagcga cgattatacc 60
ttgacgcggc tgccttacag caaagctcca agctccctga cgcccattat gacaaccgtc 120
ggcgggacgc tttcggagga agtggcgcag cgcgtccttc aaatgagcaa gggcgctccg 180
ctggccactt ttatgatttt gctcgcaggt gttcagtccc tcttgtataa atatacaggc 240
gcttctgaca ttctggttgg catgccggtt gtacggaaac cgacggagac gcgccgatcc 300
gttaatcata cgatcatttt gaaaaactcg ctttcggcgg gtgcgacttt taaaacgctt 360
ctgagcgagt tgaggacttc cttgccggaa gcgattcagc atcaacatat tccgttcttg 420
aaaatgacgg agaagctgga tctgcaatat gcagacggga taaccgtcgt ccatacgcta 480
gtatccctta aggagctgca cctggacgaa atcgggcaaa acgtggtcac ggattgttcc 540
tttgaattca gcttaacggg cgggacgata cagctagcgt tgtcatataa cgagcactta 600
tatgactccg agttcatgac tcgggtcgtt ggccatctga atcgcctgtt ggccgtgggg 660
cttcacgagt tggaactgga gatcgtgcgg gcggacatgc tatcggagga cgagaaattt 720
caattgttgc aaagctttaa cgataccgag aaggactatc cccgtgatcg gacgattcat 780
caactcgtgg aggagcaggc gaagcgggtg cccgaagcga cggcggttgt ctttgagggt 840
cggcggcttt cgtacgcgga gctgaacgaa cgggcgaacc ggctggcacg gacgctgcga 900
tcggtcggcg tgctgcccaa tcagctggta ggcttgatgg ccaggagatc actggagacg 960
gtcgttggca ttctggcggt tctgaaagct ggcggtgcct acgtgccgat cgacccggaa 1020
tacccggaag aacgcatccg ctacattctg gagaactcga acgcgcagct gctgctgact 1080
caaagggagc tgctgcagca ggtgccgttc gaaggaaccg tgttggcgct ggatgacgag 1140
caggcctaca gcgacgatgg ctcgaacttg gagccggcca gcggtccgaa tgatcttgct 1200
tatgtcatct atacgtcagg tacgacaggc aaacccaaag gggtcatgct ggagcatcgc 1260
ggtttggtca gcttgaagct gatgttcgcg gataggctgg gcatcacgga gcatgaccgg 1320
atcgttcaat tcgccagcct gtcgttcgac gcgtcctgct gggaaatgtt caaagcgctc 1380
tattttggcg cggctttgta catcccgacg gccgagacga ttctcgacac ccgcttgttc 1440
gagagttata tgaacgagca tgcgattacg gcggcgattt tgcctccaac gtacagcgct 1500
tatttgaacc cggaccgcct tcccagctta acgaagctcg taacgggagg ctctgcggta 1560
tcggctgaat tcgtgcagca gtggaaaccg aaggtccact atttcaatgc ttacggccct 1620
accgaagctt cgattgttac gacgctttgg gatgcagatg aggagcagtc ggagcgcaga 1680
gtcattccga tcgggcgccc gctggccaat caccggattt ttattttgga tacccacctg 1740
cagcttgtgc caccgggagt ggacggcgag ctgtgtgtgg caggcgtggg gcttgcgaga 1800
ggttacctga accatccgga gctgacggca gagaagttcg tggaacatcc gtttgcgcct 1860
ggagaacgcc tttaccggac gggagatctc gcccgctggc tgccggacgg aaatattgag 1920
tacttgggcc ggatcgatca tcaggtgaaa atccgtggat tccggatcga gatcggcgag 1980
attgaagagc aacttctgaa gatcgactcc gtgcaggaga cgatcgtaat cgcgcgggaa 2040
ggcaaaagtg gacaagaact gtgcgcttac ctggtcgcgg gccacccgct tacgctcggc 2100
gagttgagaa gcgcgctggc gcaaaaattg ccgaattaca tgattccggc gcattttgtt 2160
cagcttccgc ggatgccgct cacgccgaac gacaaaatcg accgcaaggc tttgcccgtc 2220
ccggaaggaa acgcactgac cggcggcgcg tacgtagctc cccgcaatga agccgagcgg 2280
<210> 3
<211> 760
<212> PRT
<213> 未知(Unknown)
<400> 3
Met Ala Phe Glu Lys Glu Thr Leu Phe Trp Asn Glu Lys Phe Gly Ser
1 5 10 15
Asp Asp Tyr Thr Leu Thr Arg Leu Pro Tyr Ser Lys Ala Pro Ser Ser
20 25 30
Leu Thr Pro Ile Met Thr Thr Val Gly Gly Thr Leu Ser Glu Glu Val
35 40 45
Ala Gln Arg Val Leu Gln Met Ser Lys Gly Ala Pro Leu Ala Thr Phe
50 55 60
Met Ile Leu Leu Ala Gly Val Gln Ser Leu Leu Tyr Lys Tyr Thr Gly
65 70 75 80
Ala Ser Asp Ile Leu Val Gly Met Pro Val Val Arg Lys Pro Thr Glu
85 90 95
Thr Arg Arg Ser Val Asn His Thr Ile Ile Leu Lys Asn Ser Leu Ser
100 105 110
Ala Gly Ala Thr Phe Lys Thr Leu Leu Ser Glu Leu Arg Thr Ser Leu
115 120 125
Pro Glu Ala Ile Gln His Gln His Ile Pro Phe Leu Lys Met Thr Glu
130 135 140
Lys Leu Asp Leu Gln Tyr Ala Asp Gly Ile Thr Val Val His Thr Leu
145 150 155 160
Val Ser Leu Lys Glu Leu His Leu Asp Glu Ile Gly Gln Asn Val Val
165 170 175
Thr Asp Cys Ser Phe Glu Phe Ser Leu Thr Gly Gly Thr Ile Gln Leu
180 185 190
Ala Leu Ser Tyr Asn Glu His Leu Tyr Asp Ser Glu Phe Met Thr Arg
195 200 205
Val Val Gly His Leu Asn Arg Leu Leu Ala Val Gly Leu His Glu Leu
210 215 220
Glu Leu Glu Ile Val Arg Ala Asp Met Leu Ser Glu Asp Glu Lys Phe
225 230 235 240
Gln Leu Leu Gln Ser Phe Asn Asp Thr Glu Lys Asp Tyr Pro Arg Asp
245 250 255
Arg Thr Ile His Gln Leu Val Glu Glu Gln Ala Lys Arg Val Pro Glu
260 265 270
Ala Thr Ala Val Val Phe Glu Gly Arg Arg Leu Ser Tyr Ala Glu Leu
275 280 285
Asn Glu Arg Ala Asn Arg Leu Ala Arg Thr Leu Arg Ser Val Gly Val
290 295 300
Leu Pro Asn Gln Leu Val Gly Leu Met Ala Arg Arg Ser Leu Glu Thr
305 310 315 320
Val Val Gly Ile Leu Ala Val Leu Lys Ala Gly Gly Ala Tyr Val Pro
325 330 335
Ile Asp Pro Glu Tyr Pro Glu Glu Arg Ile Arg Tyr Ile Leu Glu Asn
340 345 350
Ser Asn Ala Gln Leu Leu Leu Thr Gln Arg Glu Leu Leu Gln Gln Val
355 360 365
Pro Phe Glu Gly Thr Val Leu Ala Leu Asp Asp Glu Gln Ala Tyr Ser
370 375 380
Asp Asp Gly Ser Asn Leu Glu Pro Ala Ser Gly Pro Asn Asp Leu Ala
385 390 395 400
Tyr Val Ile Tyr Thr Ser Gly Thr Thr Gly Lys Pro Lys Gly Val Met
405 410 415
Leu Glu His Arg Gly Leu Val Ser Leu Lys Leu Met Phe Ala Asp Arg
420 425 430
Leu Gly Ile Thr Glu His Asp Arg Ile Val Gln Phe Ala Ser Leu Ser
435 440 445
Phe Asp Ala Ser Cys Trp Glu Met Phe Lys Ala Leu Tyr Phe Gly Ala
450 455 460
Ala Leu Tyr Ile Pro Thr Ala Glu Thr Ile Leu Asp Thr Arg Leu Phe
465 470 475 480
Glu Ser Tyr Met Asn Glu His Ala Ile Thr Ala Ala Thr Leu Pro Pro
485 490 495
Thr Tyr Ser Ala Tyr Leu Asn Pro Asp Arg Leu Pro Ser Leu Thr Lys
500 505 510
Leu Val Thr Gly Gly Ser Ala Val Ser Ala Glu Phe Val Gln Gln Trp
515 520 525
Lys Pro Lys Val His Tyr Phe Asn Ala Tyr Gly Pro Thr Glu Ala Ser
530 535 540
Ile Val Thr Thr Leu Trp Asp Ala Asp Glu Glu Gln Ser Glu Arg Arg
545 550 555 560
Val Ile Pro Ile Gly Arg Pro Leu Ala Asn His Arg Ile Phe Ile Leu
565 570 575
Asp Thr His Leu Gln Leu Val Pro Pro Gly Val Asp Gly Glu Leu Cys
580 585 590
Val Ala Gly Val Gly Leu Ala Arg Gly Tyr Leu Asn His Pro Glu Leu
595 600 605
Thr Ala Glu Lys Phe Val Glu His Pro Phe Ala Pro Gly Glu Arg Leu
610 615 620
Tyr Arg Thr Gly Asp Leu Ala Arg Trp Leu Pro Asp Gly Asn Ile Glu
625 630 635 640
Tyr Leu Gly Arg Ile Asp His Gln Val Lys Ile Arg Gly Phe Arg Ile
645 650 655
Glu Ile Gly Glu Ile Glu Glu Gln Leu Leu Lys Ile Asp Ser Val Gln
660 665 670
Glu Thr Ile Val Ile Ala Arg Glu Gly Lys Ser Gly Gln Glu Leu Cys
675 680 685
Ala Tyr Leu Val Ala Gly His Pro Leu Thr Leu Gly Glu Leu Arg Ser
690 695 700
Ala Leu Ala Gln Lys Leu Pro Asn Tyr Met Ile Pro Ala His Phe Val
705 710 715 720
Gln Leu Pro Arg Met Pro Leu Thr Pro Asn Asp Lys Ile Asp Arg Lys
725 730 735
Ala Leu Pro Val Pro Glu Gly Asn Ala Leu Thr Gly Gly Ala Tyr Val
740 745 750
Ala Pro Arg Asn Glu Ala Glu Arg
755 760
<210> 5
<211> 760
<212> PRT
<213> 未知(Unknown)
<400> 5
Met Ala Phe Glu Lys Glu Thr Leu Phe Trp Asn Glu Lys Phe Gly Ser
1 5 10 15
Asp Asp Tyr Thr Leu Thr Arg Leu Pro Tyr Ser Lys Ala Pro Ser Ser
20 25 30
Leu Thr Pro Ile Met Thr Thr Val Gly Gly Thr Leu Ser Glu Glu Val
35 40 45
Ala Gln Arg Val Leu Gln Met Ser Lys Gly Ala Pro Leu Ala Thr Phe
50 55 60
Met Ile Leu Leu Ala Gly Val Gln Ser Leu Leu Tyr Lys Tyr Thr Gly
65 70 75 80
Ala Ser Asp Ile Leu Val Gly Met Pro Val Val Arg Lys Pro Thr Glu
85 90 95
Thr Arg Arg Ser Val Asn His Thr Ile Ile Leu Lys Asn Ser Leu Ser
100 105 110
Ala Gly Ala Thr Phe Lys Thr Leu Leu Ser Glu Leu Arg Thr Ser Leu
115 120 125
Pro Glu Ala Ile Gln His Gln His Ile Pro Phe Leu Lys Met Thr Glu
130 135 140
Lys Leu Asp Leu Gln Tyr Ala Asp Gly Ile Thr Val Val His Thr Leu
145 150 155 160
Val Ser Leu Lys Glu Leu His Leu Asp Glu Ile Gly Gln Asn Val Val
165 170 175
Thr Asp Cys Ser Phe Glu Phe Ser Leu Thr Gly Gly Thr Ile Gln Leu
180 185 190
Ala Leu Ser Tyr Asn Glu His Leu Tyr Asp Ser Glu Phe Met Thr Arg
195 200 205
Val Val Gly His Leu Asn Arg Leu Leu Ala Val Gly Leu His Glu Leu
210 215 220
Glu Leu Glu Ile Val Arg Ala Asp Met Leu Ser Glu Asp Glu Lys Phe
225 230 235 240
Gln Leu Leu Gln Ser Phe Asn Asp Thr Glu Lys Asp Tyr Pro Arg Asp
245 250 255
Arg Thr Ile His Gln Leu Val Glu Glu Gln Ala Lys Arg Val Pro Glu
260 265 270
Ala Thr Ala Val Val Phe Glu Gly Arg Arg Leu Ser Tyr Ala Glu Leu
275 280 285
Asn Glu Arg Ala Asn Arg Leu Ala Arg Thr Leu Arg Ser Val Gly Val
290 295 300
Leu Pro Asn Gln Leu Val Gly Leu Met Ala Arg Arg Ser Leu Glu Thr
305 310 315 320
Val Val Gly Ile Leu Ala Val Leu Lys Ala Gly Gly Ala Tyr Val Pro
325 330 335
Ile Asp Pro Glu Tyr Pro Glu Glu Arg Ile Arg Tyr Ile Leu Glu Asn
340 345 350
Ser Asn Ala Gln Leu Leu Leu Thr Gln Arg Glu Leu Leu Gln Gln Val
355 360 365
Pro Phe Glu Gly Thr Val Leu Ala Leu Asp Asp Glu Gln Ala Tyr Ser
370 375 380
Asp Asp Gly Ser Asn Leu Glu Pro Ala Ser Gly Pro Asn Asp Leu Ala
385 390 395 400
Tyr Val Ile Tyr Thr Ser Gly Thr Thr Gly Lys Pro Lys Gly Val Met
405 410 415
Leu Glu His Arg Gly Leu Val Ser Leu Lys Leu Met Phe Ala Asp Arg
420 425 430
Leu Gly Ile Thr Glu His Asp Arg Ile Val Gln Phe Ala Ser Leu Ser
435 440 445
Phe Asp Ala Ser Cys Trp Glu Met Phe Lys Ala Leu Tyr Phe Gly Ala
450 455 460
Ala Leu Tyr Ile Pro Thr Ala Glu Thr Ile Leu Asp Thr Arg Leu Phe
465 470 475 480
Glu Ser Tyr Met Asn Glu His Ala Ile Thr Ala Ala Ile Leu Pro Pro
485 490 495
Thr Tyr Ser Ala Tyr Leu Asn Pro Asp Arg Leu Pro Ser Leu Thr Lys
500 505 510
Leu Ile Thr Gly Gly Ser Ala Val Ser Ala Glu Phe Val Gln Gln Trp
515 520 525
Lys Pro Lys Val His Tyr Phe Asn Ala Tyr Gly Pro Thr Glu Ala Ser
530 535 540
Ile Val Thr Thr Leu Trp Asp Ala Asp Glu Glu Gln Ser Glu Arg Arg
545 550 555 560
Val Ile Pro Ile Gly Arg Pro Leu Ala Asn His Arg Ile Phe Ile Leu
565 570 575
Asp Thr His Leu Gln Leu Val Pro Pro Gly Val Asp Gly Glu Leu Cys
580 585 590
Val Ala Gly Val Gly Leu Ala Arg Gly Tyr Leu Asn His Pro Glu Leu
595 600 605
Thr Ala Glu Lys Phe Val Glu His Pro Phe Ala Pro Gly Glu Arg Leu
610 615 620
Tyr Arg Thr Gly Asp Leu Ala Arg Trp Leu Pro Asp Gly Asn Ile Glu
625 630 635 640
Tyr Leu Gly Arg Ile Asp His Gln Val Lys Ile Arg Gly Phe Arg Ile
645 650 655
Glu Ile Gly Glu Ile Glu Glu Gln Leu Leu Lys Ile Asp Ser Val Gln
660 665 670
Glu Thr Ile Val Ile Ala Arg Glu Gly Lys Ser Gly Gln Glu Leu Cys
675 680 685
Ala Tyr Leu Val Ala Gly His Pro Leu Thr Leu Gly Glu Leu Arg Ser
690 695 700
Ala Leu Ala Gln Lys Leu Pro Asn Tyr Met Ile Pro Ala His Phe Val
705 710 715 720
Gln Leu Pro Arg Met Pro Leu Thr Pro Asn Asp Lys Ile Asp Arg Lys
725 730 735
Ala Leu Pro Val Pro Glu Gly Asn Ala Leu Thr Gly Gly Ala Tyr Val
740 745 750
Ala Pro Arg Asn Glu Ala Glu Arg
755 760
<210> 7
<211> 760
<212> PRT
<213> 未知(Unknown)
<400> 7
Met Ala Phe Glu Lys Glu Thr Leu Phe Trp Asn Glu Lys Phe Gly Ser
1 5 10 15
Asp Asp Tyr Thr Leu Thr Arg Leu Pro Tyr Ser Lys Ala Pro Ser Ser
20 25 30
Leu Thr Pro Ile Met Thr Thr Val Gly Gly Thr Leu Ser Glu Glu Val
35 40 45
Ala Gln Arg Val Leu Gln Met Ser Lys Gly Ala Pro Leu Ala Thr Phe
50 55 60
Met Ile Leu Leu Ala Gly Val Gln Ser Leu Leu Tyr Lys Tyr Thr Gly
65 70 75 80
Ala Ser Asp Ile Leu Val Gly Met Pro Val Val Arg Lys Pro Thr Glu
85 90 95
Thr Arg Arg Ser Val Asn His Thr Ile Ile Leu Lys Asn Ser Leu Ser
100 105 110
Ala Gly Ala Thr Phe Lys Thr Leu Leu Ser Glu Leu Arg Thr Ser Leu
115 120 125
Pro Glu Ala Ile Gln His Gln His Ile Pro Phe Leu Lys Met Thr Glu
130 135 140
Lys Leu Asp Leu Gln Tyr Ala Asp Gly Ile Thr Val Val His Thr Leu
145 150 155 160
Val Ser Leu Lys Glu Leu His Leu Asp Glu Ile Gly Gln Asn Val Val
165 170 175
Thr Asp Cys Ser Phe Glu Phe Ser Leu Thr Gly Gly Thr Ile Gln Leu
180 185 190
Ala Leu Ser Tyr Asn Glu His Leu Tyr Asp Ser Glu Phe Met Thr Arg
195 200 205
Val Val Gly His Leu Asn Arg Leu Leu Ala Val Gly Leu His Glu Leu
210 215 220
Glu Leu Glu Ile Val Arg Ala Asp Met Leu Ser Glu Asp Glu Lys Phe
225 230 235 240
Gln Leu Leu Gln Ser Phe Asn Asp Thr Glu Lys Asp Tyr Pro Arg Asp
245 250 255
Arg Thr Ile His Gln Leu Val Glu Glu Gln Ala Lys Arg Val Pro Glu
260 265 270
Ala Thr Ala Val Val Phe Glu Gly Arg Arg Leu Ser Tyr Ala Glu Leu
275 280 285
Asn Glu Arg Ala Asn Arg Leu Ala Arg Thr Leu Arg Ser Val Gly Val
290 295 300
Leu Pro Asn Gln Leu Val Gly Leu Met Ala Arg Arg Ser Leu Glu Thr
305 310 315 320
Val Val Gly Ile Leu Ala Val Leu Lys Ala Gly Gly Ala Tyr Val Pro
325 330 335
Ile Asp Pro Glu Tyr Pro Glu Glu Arg Ile Arg Tyr Ile Leu Glu Asn
340 345 350
Ser Asn Ala Gln Leu Leu Leu Thr Gln Arg Glu Leu Leu Gln Gln Val
355 360 365
Pro Phe Glu Gly Thr Val Leu Ala Leu Asp Asp Glu Gln Ala Tyr Ser
370 375 380
Asp Asp Gly Ser Asn Leu Glu Pro Ala Ser Gly Pro Asn Asp Leu Ala
385 390 395 400
Tyr Val Ile Tyr Thr Ser Gly Thr Thr Gly Lys Pro Lys Gly Val Met
405 410 415
Leu Glu His Arg Gly Leu Val Ser Leu Lys Leu Met Phe Ala Asp Arg
420 425 430
Leu Gly Ile Thr Glu His Asp Arg Ile Val Gln Phe Ala Ser Leu Ser
435 440 445
Phe Asp Ala Ser Cys Trp Glu Met Phe Lys Ala Leu Tyr Phe Gly Ala
450 455 460
Ala Leu Tyr Ile Pro Thr Ala Glu Thr Ile Leu Asp Thr Arg Leu Phe
465 470 475 480
Glu Ser Tyr Met Asn Glu His Ala Ile Thr Ala Ala Ile Leu Pro Pro
485 490 495
Thr Tyr Ser Ala Tyr Leu Asn Pro Asp Arg Leu Pro Ser Leu Thr Lys
500 505 510
Leu Val Thr Ala Gly Ser Ala Val Ser Ala Glu Phe Val Gln Gln Trp
515 520 525
Lys Pro Lys Val His Tyr Phe Asn Ala Tyr Gly Pro Thr Glu Ala Ser
530 535 540
Ile Val Thr Thr Leu Trp Asp Ala Asp Glu Glu Gln Ser Glu Arg Arg
545 550 555 560
Val Ile Pro Ile Gly Arg Pro Leu Ala Asn His Arg Ile Phe Ile Leu
565 570 575
Asp Thr His Leu Gln Leu Val Pro Pro Gly Val Asp Gly Glu Leu Cys
580 585 590
Val Ala Gly Val Gly Leu Ala Arg Gly Tyr Leu Asn His Pro Glu Leu
595 600 605
Thr Ala Glu Lys Phe Val Glu His Pro Phe Ala Pro Gly Glu Arg Leu
610 615 620
Tyr Arg Thr Gly Asp Leu Ala Arg Trp Leu Pro Asp Gly Asn Ile Glu
625 630 635 640
Tyr Leu Gly Arg Ile Asp His Gln Val Lys Ile Arg Gly Phe Arg Ile
645 650 655
Glu Ile Gly Glu Ile Glu Glu Gln Leu Leu Lys Ile Asp Ser Val Gln
660 665 670
Glu Thr Ile Val Ile Ala Arg Glu Gly Lys Ser Gly Gln Glu Leu Cys
675 680 685
Ala Tyr Leu Val Ala Gly His Pro Leu Thr Leu Gly Glu Leu Arg Ser
690 695 700
Ala Leu Ala Gln Lys Leu Pro Asn Tyr Met Ile Pro Ala His Phe Val
705 710 715 720
Gln Leu Pro Arg Met Pro Leu Thr Pro Asn Asp Lys Ile Asp Arg Lys
725 730 735
Ala Leu Pro Val Pro Glu Gly Asn Ala Leu Thr Gly Gly Ala Tyr Val
740 745 750
Ala Pro Arg Asn Glu Ala Glu Arg
755 760
<210> 8
<211> 2280
<212> DNA
<213> 未知(Unknown)
<400> 8
atggcttttg aaaaagaaac gttgttttgg aacgaaaaat tcggtagcga cgattatacc 60
ttgacgcggc tgccttacag caaagctcca agctccctga cgcccattat gacaaccgtc 120
ggcgggacgc tttcggagga agtggcgcag cgcgtccttc aaatgagcaa gggcgctccg 180
ctggccactt ttatgatttt gctcgcaggt gttcagtccc tcttgtataa atatacaggc 240
gcttctgaca ttctggttgg catgccggtt gtacggaaac cgacggagac gcgccgatcc 300
gttaatcata cgatcatttt gaaaaactcg ctttcggcgg gtgcgacttt taaaacgctt 360
ctgagcgagt tgaggacttc cttgccggaa gcgattcagc atcaacatat tccgttcttg 420
aaaatgacgg agaagctgga tctgcaatat gcagacggga taaccgtcgt ccatacgcta 480
gtatccctta aggagctgca cctggacgaa atcgggcaaa acgtggtcac ggattgttcc 540
tttgaattca gcttaacggg cgggacgata cagctagcgt tgtcatataa cgagcactta 600
tatgactccg agttcatgac tcgggtcgtt ggccatctga atcgcctgtt ggccgtgggg 660
cttcacgagt tggaactgga gatcgtgcgg gcggacatgc tatcggagga cgagaaattt 720
caattgttgc aaagctttaa cgataccgag aaggactatc cccgtgatcg gacgattcat 780
caactcgtgg aggagcaggc gaagcgggtg cccgaagcga cggcggttgt ctttgagggt 840
cggcggcttt cgtacgcgga gctgaacgaa cgggcgaacc ggctggcacg gacgctgcga 900
tcggtcggcg tgctgcccaa tcagctggta ggcttgatgg ccaggagatc actggagacg 960
gtcgttggca ttctggcggt tctgaaagct ggcggtgcct acgtgccgat cgacccggaa 1020
tacccggaag aacgcatccg ctacattctg gagaactcga acgcgcagct gctgctgact 1080
caaagggagc tgctgcagca ggtgccgttc gaaggaaccg tgttggcgct ggatgacgag 1140
caggcctaca gcgacgatgg ctcgaacttg gagccggcca gcggtccgaa tgatcttgct 1200
tatgtcatct atacgtcagg tacgacaggc aaacccaaag gggtcatgct ggagcatcgc 1260
ggtttggtca gcttgaagct gatgttcgcg gataggctgg gcatcacgga gcatgaccgg 1320
atcgttcaat tcgccagcct gtcgttcgac gcgtcctgct gggaaatgtt caaagcgctc 1380
tattttggcg cggctttgta catcccgacg gccgagacga ttctcgacac ccgcttgttc 1440
gagagttata tgaacgagca tgcgattacg gcggcgattt tgcctccaac gtacagcgct 1500
tatttgaacc cggaccgcct tcccagctta acgaagctcg taacggcagg ctctgcggta 1560
tcggctgaat tcgtgcagca gtggaaaccg aaggtccact atttcaatgc ttacggccct 1620
accgaagctt cgattgttac gacgctttgg gatgcagatg aggagcagtc ggagcgcaga 1680
gtcattccga tcgggcgccc gctggccaat caccggattt ttattttgga tacccacctg 1740
cagcttgtgc caccgggagt ggacggcgag ctgtgtgtgg caggcgtggg gcttgcgaga 1800
ggttacctga accatccgga gctgacggca gagaagttcg tggaacatcc gtttgcgcct 1860
ggagaacgcc tttaccggac gggagatctc gcccgctggc tgccggacgg aaatattgag 1920
tacttgggcc ggatcgatca tcaggtgaaa atccgtggat tccggatcga gatcggcgag 1980
attgaagagc aacttctgaa gatcgactcc gtgcaggaga cgatcgtaat cgcgcgggaa 2040
ggcaaaagtg gacaagaact gtgcgcttac ctggtcgcgg gccacccgct tacgctcggc 2100
gagttgagaa gcgcgctggc gcaaaaattg ccgaattaca tgattccggc gcattttgtt 2160
cagcttccgc ggatgccgct cacgccgaac gacaaaatcg accgcaaggc tttgcccgtc 2220
ccggaaggaa acgcactgac cggcggcgcg tacgtagctc cccgcaatga agccgagcgg 2280
<210> 9
<211> 760
<212> PRT
<213> 未知(Unknown)
<400> 9
Met Ala Phe Glu Lys Glu Thr Leu Phe Trp Asn Glu Lys Phe Gly Ser
1 5 10 15
Asp Asp Tyr Thr Leu Thr Arg Leu Pro Tyr Ser Lys Ala Pro Ser Ser
20 25 30
Leu Thr Pro Ile Met Thr Thr Val Gly Gly Thr Leu Ser Glu Glu Val
35 40 45
Ala Gln Arg Val Leu Gln Met Ser Lys Gly Ala Pro Leu Ala Thr Phe
50 55 60
Met Ile Leu Leu Ala Gly Val Gln Ser Leu Leu Tyr Lys Tyr Thr Gly
65 70 75 80
Ala Ser Asp Ile Leu Val Gly Met Pro Val Val Arg Lys Pro Thr Glu
85 90 95
Thr Arg Arg Ser Val Asn His Thr Ile Ile Leu Lys Asn Ser Leu Ser
100 105 110
Ala Gly Ala Thr Phe Lys Thr Leu Leu Ser Glu Leu Arg Thr Ser Leu
115 120 125
Pro Glu Ala Ile Gln His Gln His Ile Pro Phe Leu Lys Met Thr Glu
130 135 140
Lys Leu Asp Leu Gln Tyr Ala Asp Gly Ile Thr Val Val His Thr Leu
145 150 155 160
Val Ser Leu Lys Glu Leu His Leu Asp Glu Ile Gly Gln Asn Val Val
165 170 175
Thr Asp Cys Ser Phe Glu Phe Ser Leu Thr Gly Gly Thr Ile Gln Leu
180 185 190
Ala Leu Ser Tyr Asn Glu His Leu Tyr Asp Ser Glu Phe Met Thr Arg
195 200 205
Val Val Gly His Leu Asn Arg Leu Leu Ala Val Gly Leu His Glu Leu
210 215 220
Glu Leu Glu Ile Val Arg Ala Asp Met Leu Ser Glu Asp Glu Lys Phe
225 230 235 240
Gln Leu Leu Gln Ser Phe Asn Asp Thr Glu Lys Asp Tyr Pro Arg Asp
245 250 255
Arg Thr Ile His Gln Leu Val Glu Glu Gln Ala Lys Arg Val Pro Glu
260 265 270
Ala Thr Ala Val Val Phe Glu Gly Arg Arg Leu Ser Tyr Ala Glu Leu
275 280 285
Asn Glu Arg Ala Asn Arg Leu Ala Arg Thr Leu Arg Ser Val Gly Val
290 295 300
Leu Pro Asn Gln Leu Val Gly Leu Met Ala Arg Arg Ser Leu Glu Thr
305 310 315 320
Val Val Gly Ile Leu Ala Val Leu Lys Ala Gly Gly Ala Tyr Val Pro
325 330 335
Ile Asp Pro Glu Tyr Pro Glu Glu Arg Ile Arg Tyr Ile Leu Glu Asn
340 345 350
Ser Asn Ala Gln Leu Leu Leu Thr Gln Arg Glu Leu Leu Gln Gln Val
355 360 365
Pro Phe Glu Gly Thr Val Leu Ala Leu Asp Asp Glu Gln Ala Tyr Ser
370 375 380
Asp Asp Gly Ser Asn Leu Glu Pro Ala Ser Gly Pro Asn Asp Leu Ala
385 390 395 400
Tyr Val Ile Tyr Thr Ser Gly Thr Thr Gly Lys Pro Lys Gly Val Met
405 410 415
Leu Glu His Arg Gly Leu Val Ser Leu Lys Leu Met Phe Ala Asp Arg
420 425 430
Leu Gly Ile Thr Glu His Asp Arg Ile Val Gln Phe Ala Ser Leu Ser
435 440 445
Phe Asp Ala Ser Cys Trp Glu Met Phe Lys Ala Leu Tyr Phe Gly Ala
450 455 460
Ala Leu Tyr Ile Pro Thr Ala Glu Thr Ile Leu Asp Thr Arg Leu Phe
465 470 475 480
Glu Ser Tyr Met Asn Glu His Ala Ile Thr Ala Ala Ile Leu Pro Pro
485 490 495
Thr Tyr Ser Ala Tyr Leu Asn Pro Asp Arg Leu Pro Ser Leu Thr Lys
500 505 510
Leu Val Thr Gly Gly Ser Ala Val Ser Ala Glu Phe Val Gln Gln Trp
515 520 525
Lys Pro Lys Val His Tyr Phe Asn Ala Tyr Gly Pro Thr Glu Ala Ser
530 535 540
Ile Ala Thr Thr Leu Trp Asp Ala Asp Glu Glu Gln Ser Glu Arg Arg
545 550 555 560
Val Ile Pro Ile Gly Arg Pro Leu Ala Asn His Arg Ile Phe Ile Leu
565 570 575
Asp Thr His Leu Gln Leu Val Pro Pro Gly Val Asp Gly Glu Leu Cys
580 585 590
Val Ala Gly Val Gly Leu Ala Arg Gly Tyr Leu Asn His Pro Glu Leu
595 600 605
Thr Ala Glu Lys Phe Val Glu His Pro Phe Ala Pro Gly Glu Arg Leu
610 615 620
Tyr Arg Thr Gly Asp Leu Ala Arg Trp Leu Pro Asp Gly Asn Ile Glu
625 630 635 640
Tyr Leu Gly Arg Ile Asp His Gln Val Lys Ile Arg Gly Phe Arg Ile
645 650 655
Glu Ile Gly Glu Ile Glu Glu Gln Leu Leu Lys Ile Asp Ser Val Gln
660 665 670
Glu Thr Ile Val Ile Ala Arg Glu Gly Lys Ser Gly Gln Glu Leu Cys
675 680 685
Ala Tyr Leu Val Ala Gly His Pro Leu Thr Leu Gly Glu Leu Arg Ser
690 695 700
Ala Leu Ala Gln Lys Leu Pro Asn Tyr Met Ile Pro Ala His Phe Val
705 710 715 720
Gln Leu Pro Arg Met Pro Leu Thr Pro Asn Asp Lys Ile Asp Arg Lys
725 730 735
Ala Leu Pro Val Pro Glu Gly Asn Ala Leu Thr Gly Gly Ala Tyr Val
740 745 750
Ala Pro Arg Asn Glu Ala Glu Arg
755 760

Claims (4)

1.一种腺苷酰化蛋白A6突变体，其特征在于所述腺苷酰化蛋白A6突变体氨基酸序列如SEQ ID NO.7所示。
2.编码权利要求1所述腺苷酰化蛋白A6突变体的基因。
3.含有权利要求2所述编码基因的重组菌。
4.权利要求1所述腺苷酰化蛋白A6突变体作为腺苷酰化结构域关键酶在催化合成多粘菌素中的应用。
CN202110531780.2A 2021-05-17 2021-05-17 一种腺苷酰化蛋白a6突变体及其编码基因与应用 Active CN113278601B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110531780.2A CN113278601B (zh) 2021-05-17 2021-05-17 一种腺苷酰化蛋白a6突变体及其编码基因与应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110531780.2A CN113278601B (zh) 2021-05-17 2021-05-17 一种腺苷酰化蛋白a6突变体及其编码基因与应用

Publications (2)

Publication Number Publication Date
CN113278601A CN113278601A (zh) 2021-08-20
CN113278601B true CN113278601B (zh) 2022-07-08

Family

ID=77279390

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110531780.2A Active CN113278601B (zh) 2021-05-17 2021-05-17 一种腺苷酰化蛋白a6突变体及其编码基因与应用

Country Status (1)

Country Link
CN (1) CN113278601B (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113930405B (zh) * 2021-10-21 2022-07-19 温州医科大学 一种新型热稳定磷酸化和腺苷酰化一步法催化酶及其制备方法与应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1342201A (zh) * 1999-03-03 2002-03-27 穆罕默德·A·马拉黑尔 非核糖体肽合成酶及其制备方法和用途
US20110117606A1 (en) * 2008-03-20 2011-05-19 Joergensen Hanne NRPS-PKS Gene Cluster and its Manipulation and Utility
CN102369211A (zh) * 2009-02-13 2012-03-07 诺瓦提斯公司 编码非核糖体肽合酶的生物合成簇的核酸分子及其用途
CN107995925A (zh) * 2015-03-26 2018-05-04 拜耳作物科学有限合伙公司 新型类芽孢杆菌属菌株、抗真菌化合物以及它们的使用方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1342201A (zh) * 1999-03-03 2002-03-27 穆罕默德·A·马拉黑尔 非核糖体肽合成酶及其制备方法和用途
US20110117606A1 (en) * 2008-03-20 2011-05-19 Joergensen Hanne NRPS-PKS Gene Cluster and its Manipulation and Utility
CN102369211A (zh) * 2009-02-13 2012-03-07 诺瓦提斯公司 编码非核糖体肽合酶的生物合成簇的核酸分子及其用途
CN107995925A (zh) * 2015-03-26 2018-05-04 拜耳作物科学有限合伙公司 新型类芽孢杆菌属菌株、抗真菌化合物以及它们的使用方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Crystal structure of DhbE, an archetype for aryl acid activating domains of modular nonribosomal peptide synthetases;Jurgen J. May等;《PNAS》;20020917;第99卷(第19期);第12120-12125页 *
Exploitation of the Selectivity-Conferring Code of Nonribosomal Peptide Synthetases for the Rational Design of Novel Peptide Antibiotics;Katrin Eppelmann等;《Biochemistry》;20021231;第41卷(第30期);第9718-9726页 *
Plipastatin非核糖体肽合成酶系的组合生物合成及新型脂肽的合成规律研究;高玲;《中国优秀博硕士学位论文全文数据库（博士）工程科技I辑》;20200715(第07期);第B024-9页 *
辣椒根际拮抗菌筛选、鉴定及多粘类芽孢杆菌SC2拮抗相关基因克隆;朱辉;《中国优秀博硕士学位论文全文数据库（硕士）农业科技辑》;20090315(第03期);第D046-57页 *
非核糖体肽合成酶基因腺苷酰化结构域序列克隆及分析;薛永常等;《微生物学杂志》;20190228;第39卷(第1期);第20-25页 *

Also Published As

Publication number Publication date
CN113278601A (zh) 2021-08-20

Similar Documents

Publication Publication Date Title
CN107058248B (zh) 一种重组醛酮还原酶突变体、基因、载体、工程菌及其应用
KR101455624B1 (ko) 기능성 희귀당 사이코스의 생산능을 지닌 신규 클로스트리디움 볼티에 유래의 사이코스-3-에피머화 효소 및 이를 이용한 사이코스 생산방법
KR20190027843A (ko) Rna 데그라도솜 단백질 복합체의 유전자 교란
CN113278601B (zh) 一种腺苷酰化蛋白a6突变体及其编码基因与应用
JP2007043963A (ja) Ｄｎａリガーゼ変異体
AU2002327268B8 (en) Novel glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase and the gene encoding the same
CN108048494B (zh) 一种利用生物酶合成1,3-丙二醇的方法
CN110714020A (zh) 一种高效简便纯化蛋白的方法
CN111019928B (zh) D-阿洛酮糖3-差向异构酶的编码基因、载体、重组细胞以及它们的应用
EP3371204A1 (en) Genetically modified microorganisms
CN108359652B (zh) 糖基转移酶及其应用
CN113122563B (zh) 构建r-3-氨基丁酸生产菌的方法
CN113302299B (zh) 阿洛酮糖差向异构酶变体、其生产方法以及使用其生产阿洛酮糖的方法
KR102152142B1 (ko) Cas10/Csm4를 이용한 사이클릭 올리고아데닐레이트 제조방법
CN110257312B (zh) 一种重组基因工程菌及其发酵产香兰素的应用
CN112980753B (zh) 用于外源蛋白分泌的糖苷水解酶融合表达系统
KR100844358B1 (ko) 돌연변이 ｄｎａ 중합효소들 및 그의 유전자들
KR100707758B1 (ko) 신규 글루콘산 탈수효소
CA3179180A1 (en) Methods and compositions for the production of xylitol from xylose utilizing dynamic metabolic control
CN112921024A (zh) 一种α-愈创木烯合成酶、基因及应用
KR102336516B1 (ko) 변이형 2-데옥시-실로-이노소스 합성효소
Sallam et al. A multipurpose transposon-based vector system mediates protein expression in Rhodococcus erythropolis
CN115925830B (zh) 一种内含肽变体及其在生物法制备类蛇毒肽前体中的应用
CN111518779B (zh) 一种恶唑环化酶基因及其编码蛋白和应用
CN115820585A (zh) 一种腺苷酰化蛋白a8突变体及其编码基因与应用

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