CN116590210A - 一种生产5-羟色胺的基因工程菌及其构建方法与应用 - Google Patents
一种生产5-羟色胺的基因工程菌及其构建方法与应用 Download PDFInfo
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- CN116590210A CN116590210A CN202310722800.3A CN202310722800A CN116590210A CN 116590210 A CN116590210 A CN 116590210A CN 202310722800 A CN202310722800 A CN 202310722800A CN 116590210 A CN116590210 A CN 116590210A
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- hydroxytryptamine
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- tryptophan
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Abstract
本发明提供了一种生产5‑羟色胺的基因工程菌及其构建方法与应用,利用代谢工程手段引入外源相关基因表达,并对关键酶进行密码子优化。具体为:敲除色氨酸酶tnaA;过表达芳香族氨基酸转运蛋白yddG;构建外源5‑羟色胺合成途径包括色氨酸羟化途径(人源色氨酸羟化酶变体TM2)、色氨酸脱羧途径(黑曲霉来源色氨酸脱羧酶TDC)和辅酶四氢喋呤的合成再生途径(枯草芽孢杆菌来源GTP环化水解酶ⅠmtrA、人源6‑丙酮酰四氢生物蝶呤合成酶PTPS、人源鸟蝶呤还原酶SPR、人源蝶呤‑4α‑甲醇氨脱水酶PCD、人源双氢喋呤还原酶DHPR),该菌株可高效催化色氨酸生成5‑羟色胺,具有良好的应用价值。
Description
技术领域
本发明涉及生物技术生产领域,尤其是一种生产5-羟色胺的基因工程菌及其构建方法与应用。
背景技术
5-羟色胺(5-hydroxytryptamine,5-HT)又称血清素(serotonin),是一种由L-色氨酸代谢而来的吲哚衍生物,可作为神经递质用以调控情绪、治疗失眠、清除有害自由基等多种生理功能,这也使其在医药、保健品等行业应用不断扩展,市场前景得以持续挖掘。
目前,商业上主流的5-HT生产方法仍然是植物组织提取法。然而该法原材料需求大、提取率低、步骤繁琐导致产能较低,无法满足5-HT的市场需求。此外,化学合成法由于其复杂的催化过程、昂贵的底物成本等缺陷,导致其无法被应用于大规模的工业生产中,因此急需一种更加方便快捷的方法用于合成5-HT。近年来不断发展的微生物发酵法作为一种低成本、高效可持续的生产方法,已被用于多种高价值天然产物的大规模工业化生产中,这给予了5-HT合成更加高效的方法。而大肠杆菌由于遗传稳定、背景清晰和具有成熟的基因编辑方法等优点,无疑是生产5-HT最优的底盘菌株。
发明内容
本发明所要解决的技术问题在于提供一种生产5-羟色胺的基因工程菌。
本发明所要解决的技术问题在于提供上述生产5-羟色胺的基因工程菌的构建方法。
本发明所要解决的技术问题在于提供上述生产5-羟色胺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5-羟色胺的基因工程菌,是利用代谢工程改造方法改造底盘菌株大肠杆菌E.coli W3110获得的,具体为:在基因组上敲除色氨酸酶基因tnaA;在基因组lacIZ位点整合木糖启动子诱导启动的T7RNA聚合酶(T7RNAP)基因PxylF-T7RNAP(来源于E.coliBL21(DE3));在基因组mbhA位点整合芳香族氨基酸转运蛋白基因yddG;导入色氨酸羟化-脱羧-四氢喋呤合成再生质粒pSTV28-HT11。
在E.coli W3110基因组上的lacIZ位点整合上由木糖启动子PxylF启动的T7RNA聚合酶基因,可同时失活lacI蛋白并以木糖诱导产生T7RNA聚合酶;敲除tnaA基因防止色氨酸和5-羟色胺的分解;在mbhA假基因位点使用Ptrc启动子过表达yddG基因;引入5-羟色胺合成途径质粒pSTV28-HT11,质粒中带有人源2型羟化酶短截突变体TM2基因、黑曲霉来源的TDC基因、枯草芽孢杆菌来源的mtrA和人源PTPS、SPR、PCD和DHPR基因的质粒pSTV28-HT11,以构建色氨酸羟化、脱羧途径和辅酶四氢喋呤的合成与再生途径,其中TM2、TDC由同一个PT7启动子引导表达,mtrA、PTPS、SPR由同一个Ptrc启动子引导表达,PCD、DHPR由同一个PT7启动子引导表达。
优选的,上述生产5-羟色胺的基因工程菌,所述底盘菌株大肠杆菌E.coli W3110的保藏号为ATCC 273250。
上述生产5-羟色胺的基因工程菌的构建方法,在底盘菌株E.coliW3110基础上进行进一步定向改造,具体步骤如下:
(1)在底盘菌株E.coli W3110基因组lacIZ位点上整合使用木糖启动子PxylF启动子控制T7RNA聚合酶启动得到菌株HT01;
(2)以菌株HT01为底盘菌株,敲除tnaA基因得到菌株HT02;
(3)以菌株HT02为底盘菌株,在mbhA假基因位点使用Ptrc启动子过表达yddG基因得到菌株HT03;
(4)以菌株HT03为底盘菌株,导入5-羟色胺合成途径质粒pSTV28-HT11得到菌株HT11,菌株HT11即为改造成功后的目的菌株。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述木糖启动子PxylF具有序列表SEQ ID NO.1所示核苷酸序列。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述T7RNA聚合酶具有序列表SEQ ID NO.2所示核苷酸序列。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述tnaA基因(色氨酸酶基因)来源于E.coliW3110,具有序列表SEQ ID NO.13所示核苷酸序列。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述Ptrc启动子具有序列表SEQ ID NO.3所示核苷酸序列。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述yddG基因(芳香族氨基酸转运蛋白基因)来源于E.coliW3110,具有序列表SEQ ID NO.14所示核苷酸序列。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述质粒pSTV28-HT11,是利用ClonExpress MultiS One Step Cloning Kit试剂盒,以质粒pSTV28为线性载体构建的,所述质粒pSTV28具有序列表SEQ ID NO.12所示核苷酸序列,质粒pSTV28为中拷贝质粒,具有p150A复制起始位点(ori),可降低外源基因表达对菌体造成的毒性,分别克隆由同一个PT7启动子引导表达的人源2型色氨酸羟基化酶短截突变体TM2基因、黑曲霉来源的色氨酸脱羧酶TDC基因,由同一个Ptrc启动子引导表达的枯草芽孢杆菌来源的GTP环化水解酶mtrA基因、人源6-丙酮酰四氢生物蝶呤合成酶PTPS基因和鸟蝶呤还原酶SPR基因以及由同一个PT7启动子引导表达的人源蝶呤-4α-甲醇氨脱水酶PCD基因和双氢喋呤还原酶DHPR基因,构建得到质粒pSTV28-HT11。
综上,所述TM2、TDC、mtrA、PTPS、SPR、PCD、DHPR基因克隆在质粒载体pSTV28上进行复制和表达,最终构建获得的重组质粒携带由色氨酸羟化酶突变体TM2、色氨酸脱羧酶TDC、GTP环化水解酶mtrA、6-丙酮酰四氢生物蝶呤合成酶PTPS、鸟蝶呤还原酶SPR、蝶呤-4α-甲醇氨脱水酶PCD和双氢喋呤还原酶DHPR,其中基因TM2、TDC由同一个PT7启动子驱动表达,mtrA、PTPS、SPR由同一个Ptrc启动子驱动表达,PCD、DHPR由另一个PT7启动子驱动表达。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述质粒pSTV28-HT11具有序列表SEQ ID NO.15所示的核苷酸序列。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述强启动子PT7启动子具有序列表SEQ ID NO.4所示核苷酸序列。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述编码人源2型色氨酸羟基化酶短截突变体的TM2基因具有序列表SEQ ID NO.5所示核苷酸序列。色氨酸羟化酶突变体基因TM2来源于智人,衍生于人源2型色氨酸羟化酶TPH2(humanTPH2,EC:1.14.16.4),是由人源2型色氨酸羟化酶TPH2删除了N端145个氨基酸残基和C端30个氨基酸残基,再将第2位氨基酸残基由谷氨酸突变为赖氨酸,将第97位氨基酸残基由天冬酰胺突变为异亮氨酸,将第99位氨基酸残基由脯氨酸突变为半胱氨酸后得到的。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述TDC基因(色氨酸脱羧酶基因)来源于黑曲霉,负责编码色氨酸脱羧酶(TDC),具有序列表SEQ ID NO.6所示的核苷酸序列。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述mtrA基因(GTP环化水解酶Ⅰ基因)来源于枯草芽孢杆菌,负责编码GTP环化水解酶Ⅰ(GCHⅠ),具有序列表SEQ IDNO.7所示核苷酸序列。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述PTPS基因(6-丙酮酰四氢生物蝶呤合成酶基因),来源于智人,负责编码6-丙酮酰四氢生物蝶呤合成酶(PTPS),具有序列表SEQ ID NO.8所示核苷酸序列。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述人源SPR基因(鸟蝶呤还原酶基因),来源于智人,负责编码鸟蝶呤还原酶(SPR),具有序列表SEQ ID NO.9所示核苷酸序列。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述人源PCD基因(蝶呤-4α-甲醇氨脱水酶基因),来源于智人,负责编码蝶呤-4α-甲醇氨脱水酶(PCD),具有序列表SEQ ID NO.10所示核苷酸序列。
优选的,上述生产5-羟色胺的基因工程菌的构建方法,所述人源DHPR基因(双氢喋呤还原酶基因),来源于智人,负责编码双氢喋呤还原酶(DHPR),具有序列表SEQ ID NO.11所示核苷酸序列。
上述生产5-羟色胺的基因工程菌的构建方法,所述编码人源2型色氨酸羟化酶短截突变体的TM2基因、黑曲霉TDC基因、枯草芽孢杆菌mtrA基因、人源PTPS、SPR、PCD和DHPR基因,均为经密码子优化后人工合成的。
上述生产5-羟色胺的基因工程菌在发酵生产5-羟色胺方面的应用。
优选的,上述生产5-羟色胺的基因工程菌的应用,以色氨酸为底物,使用机械搅拌式发酵罐合成5-羟色胺,具体发酵罐发酵步骤如下:
(1)种子培养:培养温度为34℃,通过自动流加25%氨水溶液维持培养pH在6.7±0.2,通过调整搅拌转速或通风量维持培养基溶氧值为30%,视OD600nm达到15为种子培养液成熟标志;
(2)发酵培养:接种量为30%,培养温度为34℃,通过自动流加25%氨水溶液维持培养pH在6.7±0.2,通过调整搅拌转速或通风量维持培养基发酵溶氧值为30%,流加80%葡萄糖溶液将罐内葡萄糖浓度控制在≤1g/L,发酵周期12h。
上述生产5-羟色胺的基因工程菌的应用,生产5-羟色胺的基因工程菌利用葡萄糖作为碳源,高效催化色氨酸合成5-羟色胺,在5L机械搅拌式发酵罐中生产5-羟色胺,发酵12h时最高可产5-羟色胺4.11g/L。
优选的,上述生产5-羟色胺的基因工程菌的应用,所述种子培养中采用的种子培养基为:葡萄糖30g/L,氯霉素15mg/L,酵母提取物6g/L,蛋白胨2g/L,柠檬酸2g/L,硫酸铵2g/L,磷酸二氢钾3g/L,七水合硫酸镁2g/L,谷氨酸2g/L,蛋氨酸0.5g/L,其余为水。
优选的,上述生产5-羟色胺的基因工程菌的应用,所述发酵培养中采用的发酵培养基为:葡萄糖20g/L,木糖10g/L,氯霉素15mg/L,酵母提取物3g/L,蛋白胨2g/L,柠檬酸2g/L,硫酸铵1g/L,磷酸二氢钾6g/L,七水合硫酸镁2g/L,七水合硫酸亚铁40mg/L,一水合硫酸锰10mg/L,色氨酸5g/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5-羟色胺的基因工程菌,为通过5-羟色胺的代谢合成途径改造得到的基因工程菌,可以葡萄糖为碳源,高效催化色氨酸生成5-羟色胺,是目前报道的利用色氨酸生产5-羟色胺的最高产量;该菌株生产速率高、发酵周期短,具有较高的工业应用价值;所述构建方法是一种定向的理性的菌种构建方法,相比传统诱变方法更为高效便捷,可操作性强,具有良好的应用前景。
附图说明
图1为质粒pSTV28-HT11图谱。
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E.coliW3110,保藏号为ATCC 273250。相应启动子、基因、质粒等见序列表。
实施例1
1.基因编辑的方法
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 genomeediting.Metabolic Engineering,2015,31:13-21.)。该方法涉及的工程质粒pREDCas9、pGRB,其中pREDCas9携带gRNA表达质粒pGRB的消除系统、λ噬菌体的Red重组系统、Cas9蛋白表达系统以及奇霉素抗性(工作浓度:100mg/L);pGRB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:100mg/L)。下列实施例2-4中涉及到的专业名词均可在该文章中解释。
2.菌株构建过程所用引物见表1
表1菌株构建过程中所用引物
实施例2
本实施例旨在说明敲除基因组tnaA基因的步骤
具体步骤如下:
①以大肠杆菌W3110基因组为模板,分别以引物lacIZ-1和lacIZ-2,lacIZ-3和lacIZ-4进行PCR扩增,得到上下游同源臂;用引物PxylF-1和PxylF-2进行PCR扩增,获得PxylF片段;以E.coli BL21(DE3)基因组为模板,用引物T7RNAP-1和T7RNAP-2进行PCR扩增,得到PT7RNAP片段。用引物PxylF-1和T7RNAP-2,以PxylF片段和PT7RNAP基因为模板进行重叠PCR,得到PxylF-T7RNAP基因片段;再以回收的上下游同源臂和PxylF-T7RNAP基因片段为模板,用引物lacIZ-1和lacIZ-4进行重叠PCR,得到需要整合的目的片段(上游同源臂-PxylF-T7RNAP基因-下游同源臂)。
②将引物PGRB-lacIZ-1和PGRB-lacIZ-2退火制得的DNA片段与质粒pGRB线载连接,构建pGRB-lacIZ,并将其化转至DH5α化转感受态中,筛选获得阳性转化子,提取质粒pGRB-lacIZ。
③将步骤①、②中得到的质粒pGRB-lacIZ和待整合的PxylF-T7RNAP基因片段一起电转至E.coli W3110/pRed-Cas9的电转感受态中,筛选阳性转化子,得到菌株E.coliHT01。
实施例3
本实施例旨在说明敲除基因组tnaA基因的步骤
①以大肠杆菌W3110基因组为模板,分别以引物tnaA-1和tnaA-2,tnaA-3和tnaA-4进行PCR扩增,得到上游同源臂和下游同源臂;以回收的上下游同源臂为模板,用引物tnaA-1和tnaA-4进行重叠PCR,得到敲除基因tnaA所需的回补片段;
②以引物PGRB-tnaA-1和PGRB-tnaA-2退火制得的DNA片段与质粒pGRB线载连接,构建pGRB-tnaA。将其化转至DH5α化转感受态细胞中,筛选获得阳性转化子,提取质粒pGRB-tnaA;
③将步骤①、②中得到的ΔtnaA整合片段和质粒pGRB-tnaA同时经电转化方式转入菌株E.coli HT01/Cas9的感受态细胞中,最终获得菌株E.coli HT02。
实施例4
本实施例旨在说明在mbhA假基因位点使用Ptrc启动子控制yddG基因过表达的步骤。
具体步骤如下:
①以大肠杆菌W3110基因组为模板,分别以mbhA-1、mbhA-2、mbhA-5、mbhA-6与yddg-3、yddG-4为引物PCR扩增得到上游同源臂、下游同源臂与目的基因片段;再以其为模板,通过重叠PCR获得ΔmbhA-Ptrc-yddG基因片段,所述基因片段由mbhA上游同源臂、Ptrc-yddG目的基因和mbhA下游同源臂组成。
②以pGRB-mbhA-S和pGRB-mbhA-A为引物,通过PCR退火程序构建pGRB-mbhA使用的含靶序列的DNA片段,并将其化转至DH5α化转感受态细胞中,筛选获得阳性转化子,提取质粒pGRB-mbhA。
③将步骤①、②中得到的ΔmbhA-Ptrc-yddG基因片段与pGRB-mbhA质粒同时经电转化方式转入菌株E.coli HT2/Cas9的感受态细胞中,最终获得菌株E.coli HT03。
实施例5
本实施例旨在说明5-羟色胺合成质粒pSTV28-HT11的构建与表达步骤。
具体步骤如下:
①以人工合成的TM2-TDC基因片段(dsDNA)、mtrA-PTPS-SPR基因片段(dsDNA)、PCD-DHPR基因片段(dsDNA)为模板,分别以引物TM2-TDC-S和TM2-TDC-A、line-pSTV28-S和line-pSTV28-A、trc-BH4-A和trc-BH4-S、T7-BH4-re-A和T7-BH4-re-S通过PCR扩增得到PT7-TM2-TDC、Ptrc-mtrA-PTPS-SPR、PT7-PCD-DHPR基因重组片段,利用引物对line-pSTV28-S/line-pSTV28-A,以质粒pSTV28为模板PCR扩增得到线性载体。
②使用ClonExpress MultiS One Step Cloning Kit试剂盒(Vazyme Biotech,Nanjing,China),通过同源重组将线性载体与PT7-TM2-TDC、Ptrc-mtrA-PTPS-SPR、PT7-PCD-DHPR基因片段连接,构建质粒pSTV28-HT11,将其化转至DH5α化转感受态细胞中,筛选获得阳性转化子,提取质粒pSTV28-HT11。
③将步骤②中得到的pSTV28-HT11质粒电转入E.coliHT03电转感受态中,经过筛选获得阳性转化子,命名为E.coliHT11。
实施例6
以E.coli HT11为5-羟色胺生产菌株,本实施例旨在说明应用该菌株的5-羟色胺的生产方法,具体培养步骤如下:
取-80℃中保藏菌种划线接种于活化斜面,34℃培养12h,并传代2次;以灭菌过后的蒸馏水将斜面上活化后的菌体洗脱并转移到5L机械搅拌式发酵罐中开始种子培养,培养温度为34℃,初始搅拌转速为200rpm,通过自动流加25%氨水溶液维持培养pH在6.7±0.2,通过调整搅拌转速或通风量维持培养基溶氧值为30%,视OD600nm达到15为种子培养液成熟标志。待种子液成熟后,保留600mL种子培养物并立即添加新鲜发酵培养基,使发酵终体积为2L开始发酵培养,培养温度为34℃,通过自动流加25%氨水溶液维持培养pH在6.7±0.2,通过调整搅拌转速或通风量维持培养基发酵溶氧值为30%。,通过流加80%葡萄糖溶液将罐内葡萄糖浓度控制在≤1g/L,发酵周期12h。
采用的斜面培养基成分:葡萄糖1g/L,蛋白胨5g/L,磷酸二氢钾0.5g/L,酵母提取物5g/L,氯化钠5g/L,七水硫酸镁0.2g/L,琼脂粉25g/L,pH7.0,121℃灭菌20mi n后分装至试管中。
采用的种子培养基成分:葡萄糖30g/L,氯霉素15mg/L,酵母提取物6g/L,蛋白胨2g/L,柠檬酸2g/L,硫酸铵2g/L,磷酸二氢钾3g/L,七水合硫酸镁2g/L,谷氨酸2g/L,蛋氨酸0.5g/L。
采用的发酵培养基成分:葡萄糖20g/L,木糖10g/L,氯霉素15mg/L,酵母提取物3g/L,蛋白胨2g/L,柠檬酸2g/L,硫酸铵1g/L,磷酸二氢钾6g/L,七水合硫酸镁2g/L,七水合硫酸亚铁40mg/L,一水合硫酸锰10mg/L,色氨酸5g/L。
实施例7
本实施例旨在验证菌株E.coli HT11的5-羟色胺生产能力,使用实施例6中的培养方法及培养基,结果如下表2所示:
表2
可见,菌株E.coliHT11在5L发酵罐12h后催化5g/L的色氨酸生成5-羟色胺的产量最高可达4.11g/L。
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,本发明菌株的构建步骤不分先后顺序,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5-羟色胺的基因工程菌,其特征在于:是利用代谢工程改造方法改造底盘菌株大肠杆菌E.coli W3110获得的,具体为:在基因组上敲除色氨酸酶基因tnaA;在基因组lacIZ位点整合木糖启动子诱导启动的T7RNA聚合酶基因PxylF-T7RNAP;在基因组mbhA位点整合芳香族氨基酸转运蛋白基因yddG;导入色氨酸羟化-脱羧-四氢喋呤合成再生质粒pSTV28-HT11。
2.根据权利要求1所述的生产5-羟色胺的基因工程菌,其特征在于:所述底盘菌株大肠杆菌E.coli W3110的保藏号为ATCC 273250。
3.权利要求1所述生产5-羟色胺的基因工程菌的构建方法,其特征在于:在底盘菌株E.coliW3110基础上进行进一步定向改造,具体步骤如下:
(1)在底盘菌株E.coli W3110基因组lacIZ位点上整合使用木糖启动子PxylF启动子控制T7RNA聚合酶启动得到菌株HT01;
(2)以菌株HT01为底盘菌株,敲除tnaA基因得到菌株HT02;
(3)以菌株HT02为底盘菌株,在mbhA假基因位点使用Ptrc启动子过表达yddG基因得到菌株HT03;
(4)以菌株HT03为底盘菌株,导入5-羟色胺合成途径质粒pSTV28-HT11得到菌株HT11,菌株HT11即为改造成功后的目的菌株。
4.根据权利要求3所述的生产5-羟色胺的基因工程菌的构建方法,其特征在于:所述木糖启动子PxylF具有序列表SEQ ID NO.1所示核苷酸序列;所述T7RNA聚合酶具有序列表SEQID NO.2所示核苷酸序列;所述tnaA基因具有序列表SEQ ID NO.13所示核苷酸序列;所述Ptrc启动子具有序列表SEQ ID NO.3所示核苷酸序列;所述yddG基因具有序列表SEQ IDNO.14所示核苷酸序列。
5.根据权利要求3所述的生产5-羟色胺的基因工程菌的构建方法,其特征在于:所述质粒pSTV28-HT11,是利用ClonExpress MultiS One Step Cloning Kit试剂盒,以质粒pSTV28为线性载体构建的,所述质粒pSTV28具有序列表SEQ ID NO.12所示核苷酸序列,质粒pSTV28为中拷贝质粒,具有p150A复制起始位点,分别克隆由同一个PT7启动子引导表达的人源2型色氨酸羟基化酶短截突变体TM2基因、黑曲霉来源的色氨酸脱羧酶TDC基因,由同一个Ptrc启动子引导表达的枯草芽孢杆菌来源的GTP环化水解酶mtrA基因、人源6-丙酮酰四氢生物蝶呤合成酶PTPS基因和鸟蝶呤还原酶SPR基因以及由同一个PT7启动子引导表达的人源蝶呤-4α-甲醇氨脱水酶PCD基因和双氢喋呤还原酶DHPR基因,构建得到质粒pSTV28-HT11。
6.根据权利要求5所述的生产5-羟色胺的基因工程菌的构建方法,其特征在于:所述质粒pSTV28-HT11具有序列表SEQ ID NO.15所示的核苷酸序列;所述PT7启动子具有序列表SEQID NO.4所示核苷酸序列;所述TM2基因具有序列表SEQ ID NO.5所示核苷酸序列;所述TDC基因具有序列表SEQ ID NO.6所示的核苷酸序列;所述mtrA基因具有序列表SEQ ID NO.7所示核苷酸序列;所述PTPS基因具有序列表SEQ ID NO.8所示核苷酸序列;所述SPR基因具有序列表SEQ ID NO.9所示核苷酸序列;所述PCD基因具有序列表SEQ ID NO.10所示核苷酸序列;所述DHPR基因具有序列表SEQ ID NO.11所示核苷酸序列。
7.权利要求1所述生产5-羟色胺的基因工程菌在发酵生产5-羟色胺方面的应用。
8.根据权利要求7所述的生产5-羟色胺的基因工程菌的应用,其特征在于:以色氨酸为底物,使用机械搅拌式发酵罐合成5-羟色胺,具体发酵罐发酵步骤如下:
(1)种子培养:培养温度为34℃,通过自动流加25%氨水溶液维持培养pH在6.7±0.2,通过调整搅拌转速或通风量维持培养基溶氧值为30%,视OD600nm达到15为种子培养液成熟标志;
(2)发酵培养:接种量为30%,培养温度为34℃,通过自动流加25%氨水溶液维持培养pH在6.7±0.2,通过调整搅拌转速或通风量维持培养基发酵溶氧值为30%,流加80%葡萄糖溶液将罐内葡萄糖浓度控制在≤1g/L,发酵周期12h。
9.根据权利要求8所述的生产5-羟色胺的基因工程菌的应用,其特征在于:所述种子培养中采用的种子培养基为:葡萄糖30g/L,氯霉素15mg/L,酵母提取物6g/L,蛋白胨2g/L,柠檬酸2g/L,硫酸铵2g/L,磷酸二氢钾3g/L,七水合硫酸镁2g/L,谷氨酸2g/L,蛋氨酸0.5g/L,其余为水。
10.根据权利要求8所述的生产5-羟色胺的基因工程菌的应用,其特征在于:所述发酵培养中采用的发酵培养基为:葡萄糖20g/L,木糖10g/L,氯霉素15mg/L,酵母提取物3g/L,蛋白胨2g/L,柠檬酸2g/L,硫酸铵1g/L,磷酸二氢钾6g/L,七水合硫酸镁2g/L,七水合硫酸亚铁40mg/L,一水合硫酸锰10mg/L,色氨酸5g/L,其余为水。
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