CN112708589A - 一种基因工程菌及其构建方法与在发酵产5-羟基色氨酸上的应用 - Google Patents
一种基因工程菌及其构建方法与在发酵产5-羟基色氨酸上的应用 Download PDFInfo
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Abstract
本发明公开了一种基因工程菌及其构建方法与在发酵产5‑羟基色氨酸上的应用。该工程菌株是利用已有报道的曼氏吸血虫(Schistosoma mansoni)色氨酸羟化酶基因截短片段SmT5H与已报道的辅因子BH4循环系统进行共表达构建的重组菌株。本发明首次以大肠杆菌Ecoli.BL21(DE3)为宿主实现了色氨酸羟化酶SmT5H利用可循环再生的辅因子BH4产5‑羟基色氨酸。利用该菌株进行发酵工艺优化,在无需外源添加辅因子的条件下,只添加底物色氨酸即可获得最终产物5‑羟基色氨酸。该工艺简单,成本低,原料易得,转化率高。
Description
技术领域
本发明涉及一种基因工程菌及其构建方法与在发酵产5-羟基色氨酸上的应用。
背景技术
5-羟基色氨酸(5-hydroxytryptophan,5-HTP)是一种不参与蛋白合成的天然氨基酸,由色氨酸苯环上的5’位氢原子被羟基取代而生成,化学名为5-羟基-3-吲哚基-α-氨基丙酸。在哺乳动物体内,5-HTP是神经递质血清素(Serotonin)与胺类激素褪黑素(Melatonin)的前体,对睡眠、痛觉、体温、食欲与行为等生理功能具有调节作用,己被成功用于抑郁症、失眠和偏头痛等疾病的治疗。
由于5-HTP具有很高的药用保健和市场价值,近年来对5-HTP的生产研究发展迅速。目前生产5-HTP的方法主要有天然产物提取法、化学合成法和微生物发酵法等工艺技术,其中从非洲植物加纳的种子中提取5-HTP仍然是商业生产的主要方式。然而该方法产量低、易受季节影响、原料不足、地域限制成为限制大规模生产的主要瓶颈。化学合成法如胡文辉等(CN102351775B)利用L-色氨酸甲酯/乙酯化得到L-色氨酸甲酯/乙酯盐酸盐,在碱性条件下脱盐酸得到L-色氨酸甲酯/乙酯,经乙酰化成N-乙酰-L-色氨酸甲酯/乙酯,在三乙基硅烷-三氟乙酸还原体系下还原吲哚环,在钨酸钠-30%双氧水体系下氧化吲哚环1位氮,最后在酸性条件下,脱乙酰保护基得到5-羟基色氨酸。但是总体来说化学方法合成反应体系较为复杂,步骤繁琐,因此微生物法合成以其成本低,操作简单,绿色环保,反应温和,可大量生产成为潜力巨大的5-HTP生产方式。然而目前微生物法仍存在催化的酶酶活不稳定,需外源添加昂贵的辅因子,成本高,催化效率低等问题。
发明内容
针对现有技术的不足,本发明提供了一种基因工程菌及其构建方法与在发酵产5-羟基色氨酸上的应用。该基因工程菌有效地利用已有报道的曼氏吸血虫(Schistosomamansoni)色氨酸羟化酶基因SmT5H的截短序列,且整合已报道的辅因子BH4循环系统关键酶,通过培养该基因工程菌并利用其发酵来制备5-羟基色氨酸,成本低廉,绿色环保,反应温和,可实现产业化。
为解决现有技术问题,本发明采取的技术方案为:
一种基因工程菌pET-24a-SmT5H+pCDFDuet-1-BH4 BL21(DE3),包含色氨酸羟化酶SmT5H截短基因的原核表达载体和辅因子BH4合成再生循环系统表达载体,且所述色氨酸羟化酶SmT5H截短基因和辅因子BH4合成再生循环系统在菌株内表达,形成具有活性的色氨酸羟化酶SmT5H和辅因子BH4合成再生循环系统。
作为改进的是,所述色氨酸羟化酶SmT5H基因的核苷酸如SEQ ID No.1所示。
SEQ ID No.1:
ATGACCCTGGATGATAAAGTTCCGTGGTTTCCGCGTCATATTTCAGATCTGGATAAAGTTAGCAATAGCGTGCTGATGTATGGCAAAGAACTGGATGCAGATCATCCGGGTTTTAAAGATAAAGAATATCGCAAACGTCGCATGATGTTTGCAGATATTGCACTGAACTATAAATGGGGTCAGCAGATTCCGATTGTGGAATATACCGAAATTGAAAAAACCACCTGGGGTCGTATTTATCGTGAACTGACCCGTCTGTATAAAACCAGCGCATGCCATGAATTTCAGAAAAATCTGGGTCTGCTGCAGGATAAAGCAGGCTATAATGAATTTGATCTGCCGCAGCTGCAGGTTGTTAGCGATTTCCTGAAAGCACGTACCGGTTTTTGTCTGCGTCCGGTTGCAGGTTATCTGAGCGCACGTGATTTTCTGAGCGGTCTGGCATTTCGTGTGTTTTATTGTACCCAGTATATTCGTCATCAGGCCGATCCGTTTTATACTCCGGAACCGGATTGTTGTCATGAACTGCTGGGTCATGTTCCGATGCTGGCAGATCCGAAATTTGCACGTTTTAGCCAAGAAATTGGTCTGGCAAGCCTGGGCACCAGTGATGAAGAAATCAAAAAACTGGCAACCTGCTACTTTTTCACCATTGAATTTGGTCTGTGCCGTCAGGATAATCAGCTGAAAGCATATGGTGCAGGTCTGCTGAGCAGCGTTGCAGAACTGCAGCATGCACTGAGCGATAAAGCCGTTATTAAACCGTTTATTCCGATGAAGGTGATCAACGAAGAATGTCTGGTTACCACCTTTCAGAATGGTTATTTCGAAACCAGCAGCTTTGAAGATGCAACCCGTCAGATGCGTGAATTTGTTCGTACCATTAAACGTCCGTTTGATGTGCATTATAATCCGTATACACAGAGCATCGAGATTATCAAAACCCCGAAAAGCGTTGCCAAACTGTAA
作为改进的是,所述辅因子BH4循环系统关键酶基因的核苷酸序列如SEQ ID No.2所示。
SEQ ID No.2:
atgaaagaagtgaacaaagaacagatcgaacaggcagtgcgtcagattctggaagcaatcggtgaagatccgaatcgcgaaggtctgctggataccccgaaacgcgttgccaaaatgtatgcagaagtttttagcggtctgaacgaagatccgaaagaacattttcagacaatttttggtgaaaaccatgaagaactggtgctggttaaagatattgcatttcatagtatgtgcgaacatcatctggttccgttttatggtaaagcacatgtggcatatattccacgtggtggtaaagtaacaggtctgagcaaactggcccgtgcagttgaagcagttgcaaaacgtccgcagctgcaggaacgtattaccagcacaatcgcagaaagcattgtcgaaaccctggaccctcatggtgtgatggttgttgttgaagcagaacacatgtgtatgacaatgcgcggtgtccgtaaaccaggtgcaaaaaccgttaccagcgcagtgcgcggtgtttttaaagatgatgccgcagcacgtgcagaagttctggaacatattaaacgccaggattaataaggaggtgacaatatgagcaccgaaggtggtggtcgccgctgtcaggcgcaggttagccgccgtattagctttagcgcgagccatcgtctgtattccaaatttctgagcgatgaagaaaacctgaaactgtttggtaaatgtaataacccgaatggtcatggtcataattataaagttgtggtgaccgttcatggtgaaattgatcctgccaccggtatggtcatgaatctggcagatttaaaaaaatatatggaagaagcaattatgcagccgctggatcataaaaatctggatatggatgttccgtattttgcagatgttgttagcaccaccgaaaatgttgcagtttatatttgggataatctgcagaaagttctgccggttggtgttctgtataaagttaaagtttatgaaaccgataataatattgttgtttataaaggtgaataaaaggagatataccatggaaggtggtctgggtcgtgccgtttgtctgctgacgggtgcaagccgtggttttggtcgtacactggcaccgctgctggcgagcctgctgagccctggtagcgttctggttctgagcgcacgtaatgatgaagcactgcgtcagctggaagcagaactgggtgcagaacgtagtggtctgcgcgttgttcgtgttccggcagatttaggtgcagaagcaggtctgcagcagctgctgggtgcactgcgtgaactgcctcgtcctaaaggtctgcagcgtctgctgctgattaataatgcaggtagtctgggtgatgttagcaaaggttttgtagatttaagcgattctactcaggttaataattattgggccctgaatctgacgagtatgctgtgtctgacttctagcgtactgaaagcatttcctgatagtccgggtctgaatcgtaccgtggttaatatttccagcctgtgtgcactgcagccgtttaaaggctgggcactgtattgtgccggtaaagcagcacgtgatatgctgtttcaggttctggcactggaagaaccaaatgttcgtgttctgaattatgctccgggtccgctggatacggatatgcagcagctggcgcgtgaaacatcagttgatcctgatatgcgtaaaggtctgcaggaactgaaagcaaaaggtaaactggtggattgtaaagttagcgcacagaaactgctgagcctgctggaaaaagatgaatttaaaagtggtgcacatgtggatttttatgataaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgttatctctggcggtgttgacaagagataacaacgttgatataattgagcccgtattgttagcatgtacgtttaaaccaggaaacagctatgagcaccctgaaccaggcacattgtgaagcctgccgcgcggatgcgccgcaggttagtgaagcggaactgccggaactgctgaaacagatcccggattggaacattgaagtgcgtgatggtgttatgcagctggaaaaagtttttctgtttaaaaactttaaatttgcactggcatttaccaatgccgttggtgaaattgcagaagcggaaggtcatcatccgggtctgctgaccgaatggggtaaagttaccgtgacgtggtggagccatagcattaaaggtctgcatcgtaatgattttatcatggcggcacgtacagatggtgtggcaagcggtgcggaaggtcgtaaataaaggaggtataattaatggatattatcagcgttgcgctgaaacgtcatagcaccaaagcctttgatgcaagcaaaaagctgaccccggaacaggcagaacagattaaaacgctgctgcagtatagcccgagcagcaccaacagccagccgtggcattttattgtcgcaagcaccgaagaaggtaaagcacgtgttgcaaaaagcgcagcaggtaattatgtttttaatgaacgtaaaatgctggatgcaagccatgtggttgtattttgtgcaaaaaccgcaatggatgatgtgtggctgaaactggttgttgatcaggaagatgcagatggccgttttgccaccccggaagccaaagcagcaaatgataaaggtcgtaaattttttgcagatatgcatcgtaaagatttacatgatgatgcagaatggatggcaaaacaggtatatctgaatgttggtaactttctgctgggtgttgcagcactgggtctggatgccgttccgattgaaggttttgatgcagcaattctggatgcagaatttggtctgaaagaaaaaggttatacctccctggttgttgttcctgttggtcatcattcagttgaagattttaatgcaaccctgccgaaatctcgtctgccgcagaatattacactgacggaagtttaa
上述基因工程菌pET 24a-SmT5H+pCDFDuet-1-BH4 BL21(DE3)的克隆表达,包括以下步骤:
步骤1,PCR扩增SEQ ID No.1和SEQ ID No.2所示的核苷酸序列;
步骤2,分别构建质粒载体pET 24a-SmT5H,pCDFDuet-1-BH4
将PCR扩增的DNA序列和pET 24a载体用同样的限制性内切酶BamHI和HindⅢ进行酶切,用T4DNA连接酶连接已经经过酶切回收的纯化的酶切产物,得到质粒载体pET 24a–SmT5H;同样方法获得pCDFDuet-1-BH4;
步骤3,构建克隆菌株pET 24a-SmT5H Trans1T1和pCDFDuet-1-BH4 Trans1T1
将质粒载体pET 24a-SmT5H转化至E.coli Trans 1T1,得到阳性转化子,经菌落PCR筛选并测序确定为目标菌株;同样方法获得pCDFDuet-1-BH4Trans1T1目标菌株;
步骤4,构建表达菌株pET 24a-SmT5H-pCDFDuet-1-BH4 BL21(DE3)
将克隆菌株pET 24a-SmT5H Trans1T1和pCDFDuet-1-BH4 Trans1T1提取出来的质粒共转化至E.coli BL21(DE3),挑选阳性转化子直接克隆培养,得到重组表达的基因工程菌pET 24a-SmT5H-pCDFDuet-1-BH4 BL21(DE3)。
上述基因工程菌pET 24a-SmT5H+pCDFDuet-1-BH4 BL21(DE3)在发酵产5-羟基色氨酸上的应用。
作为改进的是,上述应用,具体步骤如下:
将基因工程菌pET 24a-SmT5H+pCDFDuet-1-BH4 BL21(DE3)接入5ml含卡那霉素和链霉素的LB培养基中,25-40℃过夜培养后,按1%的接种量接种到100ml含卡那霉素和链霉素的LB培养基中,当OD600=0.4-1.0,加入终浓度为0.25mM-1mM的IPTG,于18-37℃,100-300rpm低温诱导8-24h;体外诱导表达结束后于4℃,3000-4000rpm离心7-10min收集菌体,用Tris HCL7.2缓冲液洗涤菌体三次,再用Tris HCL7.2缓冲液重悬菌体,再加入100ml的M9培养基中,37℃,100-300rpm进行发酵;发酵液6000-12000rpm离心获得上清液,用高效液相色谱检测。
有益效果:
本发明提供了一种基于曼氏吸血虫(Schistosoma mansoni)色氨酸羟化酶基因截短形式SmT5H和已报道的BH4循环再生的表达载体通过转化而获得的工程菌株Ecoli.BL21(DE3)。利用胞外表达稳定的色氨酸羟化酶SmT5H与循环再生的BH4辅因子,本发明建立起以色氨酸为底物发酵生成5-羟基色氨酸的方法。利用该工程菌进行发酵,在不需纯化色氨酸羟化酶,无需外源添加辅因子的条件下,只添加底物色氨酸即可获得最终产物5-羟基色氨酸,转化率在70%以上,收率大于80%。
附图说明
图1为本发明中色氨酸羟化酶SmT5H和BH4循环再生酶的SDS-PAGE检测结果,其中,M:蛋白标准分子量,1-重组表达菌株pET 24a-SmT5H-pCDFDuet-1-BH4BL21(DE3)诱导后离心得到的上清液,2-为重组菌株离心得到的沉淀;
图2为本发明中色氨酸和5-羟基色氨酸标准品的标准曲线,其中,(a)色氨酸,(b)5-羟基色氨酸;
图3为本发明中不同发酵时间下的5-羟基色氨酸产量;
图4为本发明中不同发酵温度获得的5-羟基色氨酸产量;
图5为本发明中不同诱导时间获得的5-羟基色氨酸产量。
具体实施方式
下面通过实施例对本发明做进一步描述,但不用于限制本发明的保护范围。实施例中的实验方法,如无特殊说明,均为常规方法。
下述实例中所用到的材料、试剂等,如无特殊说明,均可从商业途径获得;对于定量试验均设置三次重复实验,结果取平均值。
实施例1色氨酸羟化酶SmT5H基因的扩增
本发明根据已报道的色氨酸羟化酶基因SmT5H为模板,采用Primer5.0软件设计引物,通过聚合酶链式反应(PCR)的方法获得了截短的SmT5H的保守序列:
上游引物Sm-F(SEQ ID No.3):5’-ATGACCCTGGATGATAAAGTTC-3’设计有NdeI酶切位点;
下游引物Sm-R(SEQ ID No.4):5’-CCCAAGCTTCTACTTTGCCGCCGGAAT-3’设计有EcoRV酶切位点。
同样的方法,设计BH4循环系统的
上游引物BH-F(SEQ ID No.5):5’-GAATTCCCATGAAAGAAGTGAACAAAGA-3’设计有EcoRI酶切位点;
下游引物BH-R(SEQ ID No.6):5’-AAGCTTTTAAACTTCCGTCAGTGTAAT-3’设计有HindIII酶切位点。
基因组模板 | 0.5μl |
上游引物(10μM) | 1μl |
下游引物(10μM)d NTP | 1μl |
5×TransStart FastPfu Buffer | 10μl |
2.5mM dNTPs | 4μl |
TransStart FastPfu DNA Polymerase | 0.5μl |
ddH<sub>2</sub>O up to | 50μl |
PCR反应条件:95℃预变性,2min;95℃变性20s,52℃退火,20s,72℃延伸,2min,共30个循环;72℃后延伸,5min,最后4℃保温。
PCR反应结束以1%琼脂糖凝胶电泳检测,在981bp处有明亮一条带,即为色氨酸羟化酶SmT5H基因;在2994bp处有明亮一条带,即为BH4循环系统关键酶。其核苷酸序列如SEQID No.1和SEQ ID No.2所示。本发明所述的色氨酸羟化酶SmT5H基因编码323个氨基酸,分子量约35kDa,具有如SEQ ID No.9所述的氨基酸序列;BH4循环系统编码994个氨基酸,由于含有多个关键酶基因,因此分子量分布在10-30kDa,其氨基酸序列如SEQ ID No.10所述。
实施例2质粒载体pCDFDuet-1-BH4-SmT5H及克隆菌株pCDFDuet-1-BH4-SmT5HTrans1T1的制备
1、使用TaKaRa公司试剂盒(TaKaRa DNA Ligation Kit<Mighty Mix>)对实施例1得到的PCR产物进行纯化回收;
2、构建质粒载体pCDFDuet-1-BH4
将PCR扩增的BH4系统的DNA序列和pCDFDuet-1载体用同样的限制性内切酶EcoRI和HindⅢ进行酶切,酶切产物经回收纯化,并用T4DNA连接酶连接,得到质粒载体pCDFDuet-1-BH4;
3、构建质粒载体pCDFDuet-1-BH4-SmT5H
将PCR扩增的色氨酸羟化酶SmT5H的DNA序列和pCDFDuet-1-BH4载体用同样的限制性内切酶NdeI和EcoRV进行酶切,酶切产物经回收纯化,并用T4 DNA连接酶连接,得到质粒载体pCDFDuet-1-BH4-SmT5H;
4、构建克隆菌株pCDFDuet-1-BH4-SmT5H-E.coli Trans1T1
将质粒载体pCDFDuet-1-BH4-SmT5H转化至E.coli Trans 1T1:(1)取冰上冻融的20μl感受态细胞Trans 1T1,于上述10μl的连接反应液中,轻轻混合均匀;(2)在冰上静置30min后,42℃热激处理45s,然后迅速放置冰上2min;(3)在超净工作台加LB培养基800μl,37℃摇床培养1h;(4)4000g离心培养液,涂布于含有链霉素的LB平板上,37℃过夜培养,筛选阳性转化子,并测序保证获得目标菌株,然后转至液体培养基培养;(5)用TaKaRa公司试剂盒提取大量重组质粒pCDFDuet-1-BH4-SmT5H。
连接反应体系:
BH4/SmT5H基因的PCR产物 | 6μl |
pCDFDuet-1/pCDFDuet-1-BH4 | 1μl |
5×buffer | 2μl |
T<sub>4</sub>DNA连接酶 | 1μl |
连接反应的条件为:16℃过夜连接,构建重组质粒pCDFDuet-1-BH4-SmT5H。
实施例3构建重组表达菌株pCDFDuet-1-BH4-SmT5H BL21(DE3)
1、菌株培养:将已构建的重组质粒pCDFDuet-1-BH4-SmT5H从克隆宿主中提取出来,并将重组质粒再转化入E.coli.BL21(DE3)感受态细胞中,转化操作如上述实施例2中的转化步骤;最终挑取单菌落接种到含有链霉素终浓度为0.2%的5ml LB培养基中,37℃过夜培养;
2、主培养:培养12h后按1%接种量转移至100ml LB/Str培养基中,37℃震荡培养至OD600=0.4-0.6;
3、诱导表达:加入终浓度为0.05mM的IPTG诱导菌体细胞,在25℃下200rpm诱导10h;
4、收集全细胞:于4℃,6000rpm,离心8-10min收集菌体,并用10ml Tris HCL混匀后于超声波破碎处理10min,至液体呈现透明;
5、色氨酸羟化酶SmT5H以及BH4系统关键酶的提取:将上述菌液于12000rpm,4℃离心5min,取上清液即为可溶蛋白,沉淀一般为破碎细胞和少量的本底表达蛋白;
6、SDS-PAGE电泳检测表达的目的蛋白,见附图1所示。
实施例4基因工程菌pCDFDuet-1-BH4-SmT5H BL21(DE3)产5-羟基色氨酸能力检测
1、流动相配制
A相10mM磷酸钾缓冲液配制:称取磷酸二氢钾0.68g,加0.1mol/L氢氧化钠溶液15.2ml,用纯水稀释至100ml,经过抽滤后超声1h待用;
B相甲醇配制:色谱级甲醇超声备用。甲醇:10mM磷酸钾缓冲液=12:88。TC C18色谱柱,25℃,流速1ml/min,上样量10μl,检测波长276nm;使用完毕后用100%甲醇保柱。
2、标准品标准曲线的建立
分别配制0.1、0.2、0.4、0.6、0.8、1.0g/L的色氨酸与5-羟基色氨酸标准品,在276nm波长下测定其吸收峰面积。建立标准品的标准曲线,见附图2。
3、工程菌株pCDFDuet-1-BH4-SmT5H BL21(DE3)产5-羟基色氨酸测定
(1)发酵培养:将工程菌株pCDFDuet-1-BH4-SmT5H BL21(DE3)接种到含有链霉素终浓度为0.2%的5ml LB培养基中,37℃过夜培养;接着按1%接种量转移至100ml LB/Str培养基中,37℃震荡培养至OD600=0.4-0.6,加入终浓度为0.05mM的IPTG诱导菌体细胞,同时加入灭菌的2g/L色氨酸,25℃发酵培养;
(2)取样检测:取25℃下诱导与发酵同时进行的0h,24h,48h发酵液,高温煮沸5min灭活后,12000×g离心2min,上清液经0.22μm有机滤膜过滤,用安捷伦1260高效液相色谱检测色氨酸与5-羟基色氨酸含量。
实施例5工程菌株pCDFDuet-1-BH4-SmT5H BL21(DE3)最佳发酵时间的研究
(1)发酵培养:将工程菌株pCDFDuet-1-BH4-SmT5H BL21(DE3)接种到含有链霉素终浓度为0.2%的5ml LB培养基中,37℃过夜培养;接着按1%接种量转移至100ml LB/Str培养基中,37℃震荡培养至OD600=0.4-0.6。加入终浓度为0.05mM的IPTG诱导菌体细胞,同时加入灭菌的2g/L色氨酸,于25℃进行发酵培养;
(2)取样检测:分别取0h,12h,24h,36h,48h,60h,72h,84h,96h发酵液,高温煮沸5min灭活后,12000×g离心2min,上清液经0.22μm有机滤膜过滤,用安捷伦1260高效液相色谱检测色氨酸与5-羟基色氨酸含量。
实施例6工程菌株pCDFDuet-1-BH4-SmT5H BL21(DE3)最佳发酵温度的研究
(1)发酵培养:将工程菌株pCDFDuet-1-BH4-SmT5H BL21(DE3)接种到含有链霉素终浓度为0.2%的5ml LB培养基中,37℃过夜培养。接着按1%接种量转移至100ml LB/Str培养基中,37℃震荡培养至OD600=0.4-0.6,加入终浓度为0.05mM的IPTG诱导菌体细胞,同时加入灭菌的2g/L色氨酸,于18℃,20℃,25℃,30℃,35℃,37℃进行发酵培养;
(2)取样检测:分别取18℃,20℃,25℃,30℃,35℃,37℃不同温度下的72h发酵液,高温煮沸5min灭活后,12000×g离心2min,上清液经0.22μm有机滤膜过滤后,用安捷伦1260高效液相色谱检测色氨酸与5-羟基色氨酸含量。
实施例7工程菌株pCDFDuet-1-BH4-SmT5H BL21(DE3)发酵时最佳诱导时间的研究
(1)发酵培养:将工程菌株pCDFDuet-1-BH4-SmT5H BL21(DE3)接种到含有链霉素终浓度为0.2%的5ml LB培养基中,37℃过夜培养,接着按1%接种量转移至100ml LB/Str培养基中,37℃震荡培养至OD600=0.4-0.6,加入终浓度为0.05mM的IPTG诱导,以加入诱导剂后加入底物色氨酸的时间为研究对象,即0h,4h,8h,12h,16h,20h于25℃进行发酵培养;
(2)取样检测:分别取不同诱导后加入底物时间的发酵液在0h,72h的样品,高温煮沸5min灭活后,12000×g离心2min,上清液经0.22μm有机滤膜过滤,用安捷伦1260高效液相色谱检测色氨酸与5-羟基色氨酸含量。
综上所述,本发明提供了一个胞外表达稳定的色氨酸羟化酶SmT5H与循环再生的BH4辅因子共转化得到的工程菌株。该工程菌在发酵产5-羟基色氨酸时,利用诱导产酶与发酵同时进行的方法,不仅大大缩短了发酵时间而且稳定的羟化酶利用胞内循环再生的辅因子BH4,只添加底物色氨酸无需外源添加昂贵的辅因子即可发酵获得产物5-羟基色氨酸,该方法绿色环保,成本低,且产物转化率大于70%,收率80%以上。
以上所述,仅为本发明较佳的具体实施方式,本发明的保护范围不限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
序列表
<110> 南京工业大学
<120> 一种基因工程菌及其构建方法与在发酵产5-羟基色氨酸上的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 969
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgaccctgg atgataaagt tccgtggttt ccgcgtcata tttcagatct ggataaagtt 60
agcaatagcg tgctgatgta tggcaaagaa ctggatgcag atcatccggg ttttaaagat 120
aaagaatatc gcaaacgtcg catgatgttt gcagatattg cactgaacta taaatggggt 180
cagcagattc cgattgtgga atataccgaa attgaaaaaa ccacctgggg tcgtatttat 240
cgtgaactga cccgtctgta taaaaccagc gcatgccatg aatttcagaa aaatctgggt 300
ctgctgcagg ataaagcagg ctataatgaa tttgatctgc cgcagctgca ggttgttagc 360
gatttcctga aagcacgtac cggtttttgt ctgcgtccgg ttgcaggtta tctgagcgca 420
cgtgattttc tgagcggtct ggcatttcgt gtgttttatt gtacccagta tattcgtcat 480
caggccgatc cgttttatac tccggaaccg gattgttgtc atgaactgct gggtcatgtt 540
ccgatgctgg cagatccgaa atttgcacgt tttagccaag aaattggtct ggcaagcctg 600
ggcaccagtg atgaagaaat caaaaaactg gcaacctgct actttttcac cattgaattt 660
ggtctgtgcc gtcaggataa tcagctgaaa gcatatggtg caggtctgct gagcagcgtt 720
gcagaactgc agcatgcact gagcgataaa gccgttatta aaccgtttat tccgatgaag 780
gtgatcaacg aagaatgtct ggttaccacc tttcagaatg gttatttcga aaccagcagc 840
tttgaagatg caacccgtca gatgcgtgaa tttgttcgta ccattaaacg tccgtttgat 900
gtgcattata atccgtatac acagagcatc gagattatca aaaccccgaa aagcgttgcc 960
aaactgtaa 969
<210> 2
<211> 2986
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgaaagaag tgaacaaaga acagatcgaa caggcagtgc gtcagattct ggaagcaatc 60
ggtgaagatc cgaatcgcga aggtctgctg gataccccga aacgcgttgc caaaatgtat 120
gcagaagttt ttagcggtct gaacgaagat ccgaaagaac attttcagac aatttttggt 180
gaaaaccatg aagaactggt gctggttaaa gatattgcat ttcatagtat gtgcgaacat 240
catctggttc cgttttatgg taaagcacat gtggcatata ttccacgtgg tggtaaagta 300
acaggtctga gcaaactggc ccgtgcagtt gaagcagttg caaaacgtcc gcagctgcag 360
gaacgtatta ccagcacaat cgcagaaagc attgtcgaaa ccctggaccc tcatggtgtg 420
atggttgttg ttgaagcaga acacatgtgt atgacaatgc gcggtgtccg taaaccaggt 480
gcaaaaaccg ttaccagcgc agtgcgcggt gtttttaaag atgatgccgc agcacgtgca 540
gaagttctgg aacatattaa acgccaggat taataaggag gtgacaatat gagcaccgaa 600
ggtggtggtc gccgctgtca ggcgcaggtt agccgccgta ttagctttag cgcgagccat 660
cgtctgtatt ccaaatttct gagcgatgaa gaaaacctga aactgtttgg taaatgtaat 720
aacccgaatg gtcatggtca taattataaa gttgtggtga ccgttcatgg tgaaattgat 780
cctgccaccg gtatggtcat gaatctggca gatttaaaaa aatatatgga agaagcaatt 840
atgcagccgc tggatcataa aaatctggat atggatgttc cgtattttgc agatgttgtt 900
agcaccaccg aaaatgttgc agtttatatt tgggataatc tgcagaaagt tctgccggtt 960
ggtgttctgt ataaagttaa agtttatgaa accgataata atattgttgt ttataaaggt 1020
gaataaaagg agatatacca tggaaggtgg tctgggtcgt gccgtttgtc tgctgacggg 1080
tgcaagccgt ggttttggtc gtacactggc accgctgctg gcgagcctgc tgagccctgg 1140
tagcgttctg gttctgagcg cacgtaatga tgaagcactg cgtcagctgg aagcagaact 1200
gggtgcagaa cgtagtggtc tgcgcgttgt tcgtgttccg gcagatttag gtgcagaagc 1260
aggtctgcag cagctgctgg gtgcactgcg tgaactgcct cgtcctaaag gtctgcagcg 1320
tctgctgctg attaataatg caggtagtct gggtgatgtt agcaaaggtt ttgtagattt 1380
aagcgattct actcaggtta ataattattg ggccctgaat ctgacgagta tgctgtgtct 1440
gacttctagc gtactgaaag catttcctga tagtccgggt ctgaatcgta ccgtggttaa 1500
tatttccagc ctgtgtgcac tgcagccgtt taaaggctgg gcactgtatt gtgccggtaa 1560
agcagcacgt gatatgctgt ttcaggttct ggcactggaa gaaccaaatg ttcgtgttct 1620
gaattatgct ccgggtccgc tggatacgga tatgcagcag ctggcgcgtg aaacatcagt 1680
tgatcctgat atgcgtaaag gtctgcagga actgaaagca aaaggtaaac tggtggattg 1740
taaagttagc gcacagaaac tgctgagcct gctggaaaaa gatgaattta aaagtggtgc 1800
acatgtggat ttttatgata aataactagc ataacccctt ggggcctcta aacgggtctt 1860
gaggggtttt ttgttatctc tggcggtgtt gacaagagat aacaacgttg atataattga 1920
gcccgtattg ttagcatgta cgtttaaacc aggaaacagc tatgagcacc ctgaaccagg 1980
cacattgtga agcctgccgc gcggatgcgc cgcaggttag tgaagcggaa ctgccggaac 2040
tgctgaaaca gatcccggat tggaacattg aagtgcgtga tggtgttatg cagctggaaa 2100
aagtttttct gtttaaaaac tttaaatttg cactggcatt taccaatgcc gttggtgaaa 2160
ttgcagaagc ggaaggtcat catccgggtc tgctgaccga atggggtaaa gttaccgtga 2220
cgtggtggag ccatagcatt aaaggtctgc atcgtaatga ttttatcatg gcggcacgta 2280
cagatggtgt ggcaagcggt gcggaaggtc gtaaataaag gaggtataat taatggatat 2340
tatcagcgtt gcgctgaaac gtcatagcac caaagccttt gatgcaagca aaaagctgac 2400
cccggaacag gcagaacaga ttaaaacgct gctgcagtat agcccgagca gcaccaacag 2460
ccagccgtgg cattttattg tcgcaagcac cgaagaaggt aaagcacgtg ttgcaaaaag 2520
cgcagcaggt aattatgttt ttaatgaacg taaaatgctg gatgcaagcc atgtggttgt 2580
attttgtgca aaaaccgcaa tggatgatgt gtggctgaaa ctggttgttg atcaggaaga 2640
tgcagatggc cgttttgcca ccccggaagc caaagcagca aatgataaag gtcgtaaatt 2700
ttttgcagat atgcatcgta aagatttaca tgatgatgca gaatggatgg caaaacaggt 2760
atatctgaat gttggtaact ttctgctggg tgttgcagca ctgggtctgg atgccgttcc 2820
gattgaaggt tttgatgcag caattctgga tgcagaattt ggtctgaaag aaaaaggtta 2880
tacctccctg gttgttgttc ctgttggtca tcattcagtt gaagatttta atgcaaccct 2940
gccgaaatct cgtctgccgc agaatattac actgacggaa gtttaa 2986
<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgaccctgg atgataaagt tc 22
<210> 4
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cccaagcttc tactttgccg ccggaat 27
<210> 5
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gaattcccat gaaagaagtg aacaaaga 28
<210> 6
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aagcttttaa acttccgtca gtgtaat 27
Claims (5)
1.一种基因工程菌pCDFDuet-1-BH4-SmT5H BL21(DE3),其特征在于,包含色氨酸羟化酶SmT5H截短基因的原核表达载体和辅因子BH4合成再生循环系统表达载体,且所述色氨酸羟化酶SmT5H截短基因和辅因子BH4合成再生循环系统在菌株内表达,形成具有活性的色氨酸羟化酶SmT5H和辅因子BH4合成再生循环系统。
2.根据权利要求1所述的一种基因工程菌pCDFDuet-1-BH4-SmT5H BL21(DE3),其特征在于:所述色氨酸羟化酶SmT5H基因的核苷酸如SEQ ID No.1所示;所述辅因子BH4循环系统关键酶基因的核苷酸序列如SEQ ID No.2所示。
3.基于权利要求1所述的一种基因工程菌pCDFDuet-1-BH4-SmT5H BL21(DE3)的构建方法,其特征在于,包括以下步骤:
步骤1,PCR扩增SEQ ID No.1和SEQ ID No.2所示的核苷酸序列;
步骤2,分别构建质粒载体pET 24a-SmT5H, pCDFDuet-1-BH4
将PCR扩增的DNA序列和pET 24a载体用同样的限制性内切酶BamHI和HindⅢ进行酶切,用T4DNA连接酶连接已经经过酶切回收的纯化的酶切产物,得到质粒载体pET 24a–SmT5H;同样方法获得pCDFDuet-1-BH4;
步骤3,构建克隆菌株pET 24a -SmT5HTrans1T1和pCDFDuet-1-BH4Trans1T1
将质粒载体pET 24a -SmT5H转化至E.coliTrans 1T1,得到阳性转化子,经菌落PCR筛选并测序确定为目标菌株;同样方法获得pCDFDuet-1-BH4Trans1T1目标菌株;
步骤4,构建表达菌株pET 24a -SmT5H- pCDFDuet-1-BH4 BL21(DE3)
将克隆菌株pET 24a -SmT5H Trans1T1和pCDFDuet-1-BH4 Trans1T1提取出来的质粒共转化至E.coli BL21(DE3),挑选阳性转化子直接克隆培养,得到重组表达的基因工程菌pET 24a -SmT5H- pCDFDuet-1-BH4 BL21(DE3)。
4.基于权利要求1所述的基因工程菌pET 24a-SmT5H+pCDFDuet-1-BH4 BL21(DE3)在发酵产5-羟基色氨酸上的应用。
5.根据权利要求4所述的应用,其特征在于,具体步骤如下:将基因工程菌pET 24a -SmT5H+pCDFDuet-1-BH4 BL21(DE3)接入5ml含卡那霉素和链霉素的LB培养基中,25-40℃过夜培养后,按1%的接种量接种到100 ml含卡那霉素和链霉素的LB培养基中,当OD600=0.4-1.0,加入终浓度为0.25 mM-1 mM的IPTG,于18-37℃,100-300rpm低温诱导8-24 h;体外诱导表达结束后于4℃,3000-4000rpm离心7-10 min收集菌体,用Tris HCL7.2缓冲液洗涤菌体三次,再用Tris HCL7.2缓冲液重悬菌体,再加入100 ml的M9培养基中,37℃,100-300rpm进行发酵;发酵液6000-12000rpm离心获得上清液,用高效液相色谱检测。
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