CN117603923A - 单核非血红素铁酶、基因及其表达载体、菌株及其用途 - Google Patents
单核非血红素铁酶、基因及其表达载体、菌株及其用途 Download PDFInfo
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Abstract
本发明公开了一种单核非血红素铁酶、基因及其表达载体、菌株及其用途,所述单核非血红素铁酶的氨基酸序列如SEQ ID NO:1所示。本发明提供了一种来源于嗜热偶氮螺旋菌(Azospirillum thermophilum)的高活性单核非血红素铁酶AtEgtB,可以将组氨酸甜菜碱高效转化为组氨酸‑半胱氨酸亚砜偶联物,能显著提高麦角硫因的生产水平。本发明还提供了一种基因工程菌株Lumy‑EgtDBE,应用于高效合成麦角硫因,可达到每24小时126 mg/L的生产速率,相对于目前已有的麦角硫因生物合成法而言,大幅提高了麦角硫因的合成速率,是具有广泛应用前景的绿色生物合成技术。
Description
技术领域
本发明涉及生物领域,特别是涉及一种单核非血红素铁酶、基因及其表达载体、菌株及其用途。
背景技术
麦角硫因(ERG)是一种含硫组氨酸衍生物,最初于1909年在麦角真菌Claviceps purpure中发现。ERG在各种生物中无处不在,例如植物、动物和微生物中,虽然ERG可以由大多数真菌和细菌合成,但人类只能通过特定的阳离子转运蛋白(OCTN1)从食物中获取ERG。
ERG是一种强抗氧化剂,具有许多生理功能,例如保护皮肤细胞免受紫外线照射,维持氧化还原稳态和促进神经元干细胞分化。因此,ERG在医药和化妆品领域有广泛的应用。然而,目前生产的ERG由于产量低,成本高,无法满足市场需求。工程菌株的构建和微生物发酵生产ERG因其高产、低成本等优点成为研究热点。
在多数真菌中,麦角硫因的合成途径从L-组氨酸开始,以S-腺苷甲硫氨酸(SAM)作为甲基供体,通过甲基转移酶EgtD,将L-组氨酸甲基化形成组氨酸甜菜碱(THM)。EgtB是一种单核非血红素铁酶,利用半胱氨酸为硫供体,把组氨酸甜菜碱转化为组氨酸-半胱氨酸亚砜偶联物。之后,吡哆醛-5-磷酸依赖性β裂解酶EgtE将该偶联物转化为麦角硫因。
随着麦角硫因多种生物合成途径的发现,已有广泛关于构建ERG生产的基因工程菌株研究。Osawa等人在大肠杆菌中异源表达来自包皮分枝杆菌的EgtBCDE基因,并在培养72小时后产生24 mg / L ERG(Osawa, R., et al. Heterologous and high productionof ergothioneine in Escherichia coli. J. Agric. Food Chem. 2018, 66, 1191−1196)。为了进一步提高ERG产量,作者共表达EgtA,改造大肠杆菌以提高L-半胱氨酸的产生,并加强SAM的补充。工程菌株在经过补料分批发酵的优化培养基中216小时后产生1.3g/L ERG(Tanaka, N., et al. Gramscale fermentative production of ergothioneinedriven by over- production of cysteine in Escherichia coli. Sci. Rep. 2019,9, 1895.)。
但是,通过这些研究发现,大量的THM会随着ERG的合成而积累,EgtB催化的反应步骤成为了ERG生物合成的核心步骤。为了解决这个问题,Kamide等人筛选了许多EgtB 直系同源物(Kamide, T., et al. High production of ergothioneine in Escherichia coli using the sulfoxide synthase from Methylobacterium strains. J. Agric.Food Chem. 2020, 68, 6390−6394.),鉴定出了一种具有更好性能的基因,即来源于沙西科兰甲基杆菌的EgtB(MpEgtB),并与包皮分枝杆菌的EgtDE一起克隆到修饰的大肠杆菌中。虽然该转化菌株在锥形瓶中192小时后产生657 mg/L ERG,但这种方式的ERG生产速率依然较慢,为每24小时82 mg/L,因此亟需开发活性更高的EgtB和基因工程菌株以提高ERG的生产水平。
发明内容
为解决上述技术问题,本发明提出了一种实现辐致暗化漂白的方法及系统。
本发明的目的通过以下技术方案实现:
一种单核非血红素铁酶,所述单核非血红素铁酶的氨基酸序列如SEQ ID NO:1所示。
一种单核非血红素铁酶基因,所述单核非血红素铁酶基因的核苷酸序列编码SEQID NO:1所示的氨基酸序列。
进一步的改进,所述单核非血红素铁酶基因的核苷酸序列如SEQ ID NO:2所示。
一种表达载体,所述表达载体含有编码SEQ ID NO:1所示氨基酸序列的核苷酸序列。
进一步的改进,所述核苷酸序列如SEQ ID NO:2所示。
一种菌株,所述菌株内含有NcEgtD基因、AtEgtB基因和MtEgtE基因,AtEgtB基因的核苷酸序列如SEQ ID NO:2所示;NcEgtD基因的核苷酸序列如SEQ ID NO:3所示,MtEgtE基因的核苷酸序列SEQ ID NO:4所示。
一种单核非血红素铁酶的用途,所述单核非血红素铁酶的氨基酸序列如SEQ IDNO:1所示,所述单核非血红素铁酶作为以组氨酸甜菜碱为底物生产麦角硫因的催化剂。
进一步的改进,所述单核非血红素铁酶催化组氨酸甜菜碱生产麦角硫因的方法如下:
构建反应体系,其中每200 μL反应体系中含有5 μM所述单核非血红素铁酶、100 μM ~ 1 mM组氨酸甜菜碱、3 mM SAM二盐酸盐、0.3 mM Fe2SO4、2 mM TCEP、2 mM 抗坏血酸盐、50 mM Tris-HCl,pH=8.0;反应温度为25℃,反应时间为20s至60s。
一种菌株的用途,所述菌株包含两个pETDuet-1载体,其中一个pETDuet-1载体的MCSⅠ的BamHI和HindⅢ位点连接SEQ ID NO:3所示的核苷酸序列,MCSⅡ的NdeⅠ和XhoⅠ位点连接SEQ ID NO:2所示的核苷酸序列;另一个pETDuet-1载体的MCSⅠ的BamHI和HindⅢ位点连接SEQ ID NO:4所示的核苷酸序列;所述菌株用于以组氨酸、甲硫氨酸和硫代硫酸钠为底物生产麦角硫因。
进一步的改进,以组氨酸、甲硫氨酸和硫代硫酸钠为底物生产麦角硫因的方法如下:
培养所述菌株至OD600达到0.6~0.8得到生产菌液;将生产菌液加入液体培养基中进行发酵,液体培养基包括如下组分:0.2 g/L组氨酸,0.2 g/L甲硫氨酸,2 g/L硫代硫酸钠,辅因子8 μg/L Fe2SO4,诱导剂0.04 mM IPTG,溶剂为水;生产菌液与液体培养基的体积比为1:10;生产菌液和液体培养基中军含有100mg/L氨苄青霉素和100mg/L壮观霉素。
本发明的有益效果在于:
本发明提供了一种来源于嗜热偶氮螺旋菌(Azospirillum thermophilum)的高活性单核非血红素铁酶AtEgtB,可以将组氨酸甜菜碱高效转化为组氨酸-半胱氨酸亚砜偶联物,能显著提高麦角硫因的生产水平。本发明还提供了一种基因工程菌株Lumy-EgtDBE,应用于高效合成麦角硫因,可达到每24小时126 mg/L的生产速率,相对于目前已有的麦角硫因生物合成法而言,大幅提高了麦角硫因的合成速率,是具有广泛应用前景的绿色生物合成技术。
附图说明
利用附图对本发明做进一步说明,但附图中的内容不构成对本发明的任何限制。
图1 为本发明内容中AtEgtB参与的从组氨酸开始制备麦角硫因的合成途径示意图。
图2 为本发明内容中AtEgtB与其它EgtB(NcEgtB ; TtEgtB ; CmEgtB ; NhEgtB; MpEgtB)在25℃条件下的酶活对比。
图3 为本发明内容中基因工程菌株Lumy-EgtDBE的麦角硫因产量。
具体实施方式
为了使发明的目的、技术方案及优点更加清楚明白,以下结合附图及实例,对本发明进行进一步的详细说明。
本发明使用的菌株和生长条件如下:
表达宿主BL21(DE3)购自Invitrogen公司,所有大肠杆菌在含有100 mg/L氨苄青霉素的LB培养基中,37 °C培养。
其中,所述LB液体培养基配方是:蛋白胨 10 g/L,酵母膏 5 g/L,NaCl 10 g/L,pH7.0;LB固体培养基在LB液体培养基中添加20 g/L琼脂;121 ℃高温高压蒸汽灭菌20 min。
质粒为pETDuet-1(购自Novagen公司)衍生质粒,用于表达目的基因)
实施例1 单核非血红素铁酶AtEgtB表达质粒的构建:
(1)利用在线软件JCat,根据大肠杆菌的密码子偏好性将单核非血红素铁酶AtEgtB的编码基因(AtEgtB)进行密码子优化,优化后的AtEgtB核苷酸序列如SEQ ID NO:2所示。
(2)将步骤(1)中优化后的基因序列送金唯智公司(GENEWIZ)进行基因合成,AtEgtB合成至pETDuet-1的BamHI和HindⅢ酶切位点中间,获得重组表达质粒pETDuet-AtEgtB。
实施例2 单核非血红素铁酶的表达与纯化:
(1)单核非血红素铁酶AtEgtB的表达
将3 μL实施例1步骤(2)中得到重组质粒pETDuet- AtEgtB通过热激的方法转化入大肠杆菌BL21(DE3)感受态细胞中。将热激后的菌液涂布到含有100 mg/L氨苄青霉素的LB固体培养基平板上,37℃恒温培养箱中培养12小时。在平板上挑取单菌落至5mL LB液体培养基中,37℃摇床中培养,摇床转速为200转/分钟;将培养后的菌液进行PCR扩增验证,获得基因工程菌株。将菌液接至1L LB液体培养基(含有100mg/L氨苄青霉素)中,37℃摇床中培养约2小时,摇床转速为180转/分钟,菌液OD600值在0.6~0.8时,将菌液置于4℃预冷10分钟,之后添加1 mM IPTG 于16℃摇床中诱导表达,转速为180转/分钟,16小时。
(2)单核非血红素铁酶AtEgtB的纯化
将菌液转移至200 mL离心瓶中,于4℃,3500rpm,离心10分钟收集菌体。磷酸盐缓冲液将菌体洗涤2次后,加入50 mL细胞裂解液,准备细胞破碎。细胞裂解液为现配现用,按顺序依次加入50 mL浓度为25 mM的咪唑缓冲液,30 μL Triton X 100,15 μL β-巯基乙醇,200 μL PMSF和50 μL核酸酶。菌体重悬后于4℃高压破碎2分钟,压力800~900 bar,收集细胞裂解液,冷冻离心机中4℃,3500 rpm,离心50分钟。将上清液即时过孔径为0.22 μm的滤膜,过滤后的样品使用镍柱亲和层析纯化。10 mL去离子水过柱清洗、25 mM咪唑缓冲液润洗镍柱后,将细胞上清液过柱,之后依次用5 mL 25 mM咪唑缓冲液冲洗小分子杂蛋白;5 mL50 mM咪唑缓冲液竞争目的蛋白,收集流出样;10 mL 200 mM咪唑缓冲液竞争目的蛋白,收集流出样;5 mL 1M咪唑缓冲液清洗镍柱。最后用去离子水冲洗镍柱。收集的流出样与3×蛋白loading buffer混合后,煮沸10分钟变性,之后通过聚丙烯酰胺凝胶电泳检验纯化结果。得到的蛋白样品混有高浓度咪唑,再于4 ℃,3000~4000 rpm超滤换液,每次用pH 8.0的Tris-HCl稀释10倍,重复3次,得到换液浓缩后的蛋白样品。最后按体积比1:1将蛋白溶液与蛋白储存液混合,完成酶的制备。分装后液氮速冻,于-80 ℃保存。其中蛋白储存液的配方是pH 8.0的5%甘油、Tris-HCl混合液。
实施例3单核非血红素铁酶AtEgtB的酶活测定:
以实施例2步骤(2)收集的纯酶AtEgtB为催化剂构建反应体系,200 μL体系中,AtEgtB的添加量为5 μM,底物为组氨酸甜菜碱(100 μM ~ 1 mM),辅因子为3 mM SAM二盐酸盐、0.3 mM Fe2SO4,还原剂为2 mM TCEP和2 mM 抗坏血酸盐,缓冲液为50 mM Tris-HCl,pH=8.0,反应温度为25℃。设置4组反应,分别在反应第20s、30s、40s、60s时加入10 μL 8 MHCl终止反应,每组反应重复操作3次。反应上清液通过ESI-MS和HPLC检测。色谱柱为C18反相柱,Bischoff,250×4.6 mm,流动相为含有2%乙腈的水溶液和0.1% TFA。同样地,选择粗糙脉孢霉(Neurospora crassa)来源的NcEgtB、喜热梭孢壳(Thermothelomyces thermophilus)来源的TtEgtB、蛹虫草(Cordyceps militaris)来源的CmEgtB、汉氏硝化杆菌(Nitrobacter hamburgensis)来源的NhEgtB,和沙西科兰甲基杆菌(Methylobacterium pseudosasicola)来源的MpEgtB,测定各EgtB的酶活并进行比较,结果见附图2:实验条件下,AtEgtB的酶活约为MpEgtB的8.7倍。
实施例4 工程菌的构建与发酵:
利用在线软件JCat,将粗糙脉孢霉(Neurospora crassa)来源的NcEgtD进行密码子优化,优化后的NcEgtD序列(SEQ ID NO:3)与pETDuet-1载体MCSⅠ的BamHI和HindⅢ位点连接。同样地,将嗜热偶氮螺菌(Azospirillum thermophilum)来源的AtEgtB进行密码子优化,优化后的AtEgtB序列(SEQ ID NO:2)与pETDuet-1载体MCSⅡ的NdeⅠ和XhoⅠ位点连接,得到表达质粒pETDuet-NcEgtD-AtEgtB;将耐热分枝杆菌(Mycobacterium thermoresistibile)来源的MtEgtE进行密码子优化,优化后的MtEgtE序列(SEQ ID NO:4)与pCDFDuet-1载体MCSⅠ的BamHI和HindⅢ位点连接,得到表达质粒pCDFDuet-MtEgtE。将上述两种质粒进行大肠杆菌BL21(DE3)感受态细胞的共转化,涂布于抗性为氨苄青霉素(100mg/L)和壮观霉素(100 mg/L)的平板,37℃培养12h,挑取单菌落进行菌落PCR验证目的基因。
挑选验证成功的5个转化子进行发酵对比,命名其中麦角硫因产量最高的菌株为基因工程菌株Lumy-EgtDBE。Lumy-EgtDBE包含NcEgtD、AtEgtB和MtEgtE三种基因,用于生产麦角硫因。
具体的发酵步骤为:将上述获得的基因工程菌株Lumy-EgtDBE,用接种环接种到5mL LB液体培养基(含有100mg/L氨苄青霉素和100mg/L壮观霉素)中进行菌株活化,37℃过夜培养。随后,按照1%体积比将活化后的菌液接种到50 mL LB液体培养基(含有100 mg/L氨苄青霉素和100 mg/L壮观霉素)中,37℃,200 rpm,培养1~2 h至OD600达到0.6~0.8,预冷后加入底物0.2 g/L组氨酸,0.2 g/L甲硫氨酸,2 g/L硫代硫酸钠,辅因子8 μg/L Fe2SO4,诱导剂0.04 mM IPTG,25℃,200rpm,诱导培养24~48 h;取样后于进行产物麦角硫因的检测。
实施例5 麦角硫因的检测:
将实施例4得到的样品于100℃金属浴中煮沸10分钟,12000 rpm 离心10分钟,将上清液用0.22 μm孔径的滤膜进行过滤。用HPLC法检测麦角硫因的生成量,使用Agilent1260 Infinity Ⅱ超高效液相色谱仪,色谱柱为Eclipse Plus-C18 柱(2.1×50 mm),柱温30℃,麦角硫因的检测波长为258 nm,流动相为95 %的0.1%磷酸水溶液和5 %的乙腈,流速为0.2 mL / min。
最后应当说明的是,以上实施例仅用于说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当了解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (10)
1.一种单核非血红素铁酶,其特征在于,所述单核非血红素铁酶的氨基酸序列如SEQID NO:1所示。
2.一种单核非血红素铁酶基因,其特征在于,所述单核非血红素铁酶基因的核苷酸序列编码SEQ ID NO:1所示的氨基酸序列。
3.如权利要求1所述的单核非血红素铁酶基因,其特征在于,所述单核非血红素铁酶基因的核苷酸序列如SEQ ID NO:2所示。
4.一种表达载体,其特征在于,所述表达载体含有编码SEQ ID NO:1所示氨基酸序列的核苷酸序列。
5.如权利要求4所述的表达载体,其特征在于,所述核苷酸序列如SEQ ID NO:2所示。
6.一种菌株,其特征在于,所述菌株内含有NcEgtD基因、AtEgtB基因和MtEgtE基因,AtEgtB基因的核苷酸序列如SEQ ID NO:2所示;NcEgtD基因的核苷酸序列如SEQ ID NO:3所示,MtEgtE基因的核苷酸序列SEQ ID NO:4所示。
7.一种单核非血红素铁酶的用途,其特征在于,所述单核非血红素铁酶的氨基酸序列如SEQ ID NO:1所示,所述单核非血红素铁酶作为以组氨酸甜菜碱为底物生产麦角硫因的催化剂。
8.如权利要求8所述的单核非血红素铁酶的用途,其特征在于,所述单核非血红素铁酶催化组氨酸甜菜碱生产麦角硫因的方法如下:
构建反应体系,其中每200 μL反应体系中含有5 μM所述单核非血红素铁酶、100 μM ~1 mM组氨酸甜菜碱、3 mM SAM二盐酸盐、0.3 mM Fe2SO4、2 mM TCEP、2 mM 抗坏血酸盐、50mM Tris-HCl,pH=8.0;反应温度为25℃,反应时间为20s至60s。
9.一种菌株的用途,其特征在于,所述菌株包含两个pETDuet-1载体,其中一个pETDuet-1载体的MCSⅠ的BamHI和HindⅢ位点连接SEQ ID NO:3所示的核苷酸序列,MCSⅡ的NdeⅠ和XhoⅠ位点连接SEQ ID NO:2所示的核苷酸序列;另一个pETDuet-1载体的MCSⅠ的BamHI和HindⅢ位点连接SEQ ID NO:4所示的核苷酸序列;所述菌株用于以组氨酸、甲硫氨酸和硫代硫酸钠为底物生产麦角硫因。
10.如权利要求9所述的菌株的用途,其特征在于,以组氨酸、甲硫氨酸和硫代硫酸钠为底物生产麦角硫因的方法如下:
培养所述菌株至OD600达到0.6~0.8得到生产菌液;将生产菌液加入液体培养基中进行发酵,液体培养基包括如下组分:0.2 g/L组氨酸,0.2 g/L甲硫氨酸,2 g/L硫代硫酸钠,辅因子8 μg/L Fe2SO4,诱导剂0.04 mM IPTG,溶剂为水;生产菌液与液体培养基的体积比为1:10;生产菌液和液体培养基中军含有100mg/L氨苄青霉素和100mg/L壮观霉素。
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