CN116200316A - 一种基因工程菌及其在制备唾液酸乳糖中的应用 - Google Patents

一种基因工程菌及其在制备唾液酸乳糖中的应用 Download PDF

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CN116200316A
CN116200316A CN202111450746.9A CN202111450746A CN116200316A CN 116200316 A CN116200316 A CN 116200316A CN 202111450746 A CN202111450746 A CN 202111450746A CN 116200316 A CN116200316 A CN 116200316A
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gene
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genetically engineered
engineered bacterium
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CN116200316B (zh
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吴燕
唐静
王舒
田振华
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Hongmo Biotechnology Shanghai Co ltd
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Hongmo Biotechnology Shanghai Co ltd
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Abstract

本发明公开了一种基因工程菌及其在制备唾液酸乳糖中的应用。所述基因工程菌具有N‑乙酰神经氨酸生物合成途径,其含有多拷贝的用于编码唾液酸合酶的基因neuB,且所述基因neuB被强启动子启动表达。利用本发明基因工程菌生产唾液酸乳糖具有产量高且整体成本低的优势。

Description

一种基因工程菌及其在制备唾液酸乳糖中的应用
技术领域
本发明属于生物工程领域,具体涉及一种基因工程菌及其在制备唾液酸乳糖中的应用。
背景技术
人乳低聚糖(HMO)是人乳中营养价值较高的成分之一,根据单糖组成和结构特点,HMOs可分为中性岩藻糖基、中性非岩藻糖基、唾液酸等,其中唾液酸化的HMO(SHMO)占比高于20%,且种类较丰富,在SHMO中含量较丰富的有3’-唾液酸乳糖(3’-Sialyllactose,3’-SL)、6’-唾液酸乳糖(6’-Sialyllactose,6’-SL)、唾液酸乳-N-四糖a(LST-a)等,具有免疫调节,帮助大脑发育及调节肠道菌群等功能,因此,唾液酸在婴儿早期发育阶段的脑快速生长及其免疫系统的发育过程总发挥着重要作用,但目前市场上婴儿配方物中唾液酸类物质含量较低,因此,有必要提供足够质量和数量的唾液酸,其质量和数量足以补充婴儿配方物和其他营养组合物。
目前关于唾液酸乳糖(sialyllactose,SL)的生产有化学和酶法合成、生物合成等方式,但化学合成或酶法合成在实际生成过程中均存在许多困难,例如,立体化学的控制、特定连接的形成、原料的可用性等,相比于化学合成和酶法合成,利用合成生物学技术通过微生物代谢合成的方式更经济、高效,有研究证明可以通过生物合成的方式以葡萄糖为底物合成唾液酸(Sialic acid,SA),最丰富的唾液酸似乎是N-乙酰神经氨酸(NANA、NeuNAc、Neu5Ac),其方法简单,成本较低,且产量可观。
CN111133112A中介绍了采用失活lacZ和araA基因、缺失N-乙酰神经氨酸分解代谢基因簇nagAB、nanAEKT、缺失wzxC-wcaJ基因、缺失fucl、fucK基因,并且整合了galETKM、lacY、cscBKAR、glmS、glmM、glmU、Gna1、slr1975、neuBC、ppsA到大肠杆菌基因组中的方法得到Neu5Ac生产菌株,用于唾液酸转移酶的筛选,筛选出了一些具有唾液酸转移酶活性的唾液酸转移酶,可用于3’-SL,6’-SL的制备。但是因为这些基因都是整合到基因组上,得到的产量并不高。
发明内容
本发明所要解决的技术问题是为克服现有技术中唾液酸乳糖合成产量较低、生产成本高的缺陷,提供一种基因工程菌及其在制备唾液酸乳糖中的应用。利用本发明基因工程菌生产唾液酸乳糖具有产量高且整体成本低的优势。
本发明人创造性地通过CRISPR/Cas9技术对野生型菌株BL2l(DE3)中与N-乙酰神经氨酸(Neu5Ac)的胞内降解相关基因进行敲除构建一种用于生产Neu5Ac的非天然存在的大肠杆菌菌株,在该菌株中构建至少含有一种异源酶的Neu5Ac或SL合成途径,并选择性地将天然存在的N-乙酰神经氨酸(Neu5Ac)分解代谢途径失效,最终得到的基因工程菌能够利用发酵液中存在的单一廉价外源碳源自身代谢的方式合成Neu5Ac或SL。
本发明主要通过以下技术方案解决上述技术问题。
本发明的技术方案之一为:一种基因工程菌,其具有N-乙酰神经氨酸生物合成途径,其含有多拷贝的用于编码唾液酸合酶的基因neuB,且所述基因neuB被强启动子启动表达。
较佳地,本发明中所述基因工程菌中的N-乙酰神经氨酸分解代谢途径被失效。所述失效的途径可为本领域常规,本发明中优选通过敲除N-乙酰神经氨酸分解代谢途径中的全部或者部分基因实现所述失效的目的。
该全部或者部分基因可为编码N-乙酰甘露糖胺激酶的基因nanK、编码N-乙酰甘露糖胺-6-磷酸差向异构酶的基因nanE和编码N-乙酰神经氨酸醛缩酶的基因nanA中的一种或多种。
进一步地,所述基因工程菌中编码乳糖操纵子β-半乳糖苷酶的基因LacZ也可被敲除。
技术方案之一中所述多拷贝可以通过本领域的常规方式实现,例如通过复制子、基因组中多位点整合以及外源质粒插入中的一种或多种方式实现。在本发明一优选实施方案中,通过外源质粒插入的方式实现。
技术方案之一中所述强启动子可为本领域常规,优选Tet启动子。
本发明中所述的基因工程菌优选还包含编码唾液酸合酶的基因neuB、编码N-乙酰葡糖胺2-差向异构酶的基因slr1975、编码N-乙酰葡糖胺-6-磷酸磷酸酶的基因YqaB、编码葡糖胺-6-磷酸乙酰转移酶的基因Gna1、编码L-谷氨酰胺-D-果糖-6-磷酸转氨酶的基因glmS以及编码磷酸烯醇丙酮酸合酶的基因ppsA。
较佳地,所述基因neuB、所述基因slr1975、所述基因YqaB、所述基因Gna1、所述基因glmS以及所述基因ppsA串联连接于质粒载体1上。
其中,所述质粒载体1中的基因优选满足以下条件中的一种或多种:
所述基因neuB的GenBank登录号为AF305571;
所述基因slr1975的GenBank登录号为BAL35720;
所述基因YqaB来自BL21基因组;
所述基因Gna1的GenBank登录号为NP_116637;
所述基因glmS的核酸序列如SEQ ID NO:66所示;
所述基因ppsA来自BL21基因组。
本发明中所述质粒载体1的骨架优选pACYCDuet。
本发明中所述的基因工程菌较佳地还包含编码N-乙酰神经氨酸胞苷酰转移酶的基因和编码唾液酸转移酶的基因;该两种基因优选串联地连接于质粒载体2上。
其中,N-乙酰神经氨酸胞苷酰转移酶优选NCBI登录号为WP_003512903.1的N-乙酰神经氨酸胞苷酰转移酶,其优选含有如SEQ ID NO:61所示的核酸序列。
本发明中所述唾液酸转移酶可为本领域常规,较佳地为α-2,6-唾液酸转移酶或α-2,3-唾液酸转移酶。其中,所述α-2,6-唾液酸转移酶优选NCBI登录号为BAF91416.1的酶,其优选含有如SEQ ID NO:62所示的核酸序列。所述α-2,3-唾液酸转移酶优选为NCBI登录号为AJC62560.1的酶,其优选含有如SEQ ID NO:63所示的核酸序列。
本发明中所述质粒载体2的骨架优选pET28a。
本发明中,所述基因工程菌的出发菌优选大肠杆菌(Escherichia coli)BL21(DE3)。
本发明的技术方案之二为:一种生产N-乙酰神经氨酸的方法,其包括:培养如技术方案之一所述的基因工程菌。
本发明的技术方案之三为:一种发酵生产唾液酸乳糖的方法,其包括:采用如技术方案之一所述的基因工程菌,在发酵培养基中加入乳糖进行发酵,从发酵液中提取唾液酸乳糖。
任选地,当所述基因工程菌中含有α-2,3-唾液酸转移酶基因时,获得3’-唾液酸乳糖;当所述基因工程菌中含有α-2,6-唾液酸转移酶基因时,获得6’-唾液酸乳糖。
本发明中所述发酵培养基较佳地为TB培养基;其含有12g/L胰蛋白栋、24g/L酵母提取物、4mL/L甘油、2.31g/L KH2PO4以及12.54g/L K2HPO4
在技术方案之三所述的方法中,较佳地:在培养至OD值为0.6~0.8时使用IPTG进行诱导培养。
此外,在所述诱导培养结束后补加2g/L MgSO4·7·H2O、20g/L甘油、1mL/L微量元素母液和5g/L乳糖;所述微量元素母液优选含有54.4g/L柠檬酸铁铵、9.8g/L MnCl2·4H2O、1.6g/L CoCl2·6H2O、1g/L CuCl2·2H2O、1.9g/LH3BO3、9g/L ZnSO4·7H2O、1.1g/LNa2MoO4·2H2O、1.5g/L Na2SeO3和1.5g/L NiSO4·6H2O。
本发明中,所述发酵培养条件较佳地为30℃、250rpm振荡培养。
本发明中术语之后的数字,例如质粒载体1和质粒载体2中的“1”和“2”没有实际含义,仅为区分相同术语。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:
本发明中所得到的基因工程菌一方面能够获得较高产量的唾液酸乳糖,摇瓶发酵24小时后所得唾液酸乳糖产量可达2.5~3g/L;另一方面,发酵过程中,该基因工程菌能够利用单一廉价外源碳源通过自身代谢的方式合成唾液酸乳糖。
附图说明
图1为lacZ敲除验证图谱。
图2为pTargetF质粒图谱。
图3为表达质粒SL006图谱。
图4为pET28a-neuB质粒图谱。
图5为pTac-neuB质粒图谱。
图6为pTet-neuB质粒图谱。
图7为SL023质粒图谱。
图8为SL023-Tet质粒图谱。
图9为SL023-Tac质粒图谱。
图10为Tet启动子调控neuB表达时发酵液Neu5Ac HPLC检测图谱。
图11为6’-SL标准品检测图谱,出峰时间为17.818min。
图12为Tet启动子调控neuB表达时发酵液6’-SL HPLC检测图谱。
图13为SL037质粒图谱。
图14为3’-SL标准品检测图谱,出峰时间为15.049min。
图15为Tet启动子调控neuB表达时发酵液3’-SL HPLC检测图谱。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。
为更进一步阐述本发明所采取的技术手段及其效果,以下结合附图和本发明的优选实施例进行详细描述。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
BL21(DE3)菌株购至Novagen公司,货号69450-M;大肠杆菌Trans10感受态细胞购自北京全式金生物技术有限公司;质粒提取试剂盒、胶回收试剂盒购买自生工生物工程(上海)股份有限公司,SDS-PAGE试剂盒购买自上海雅酶生物科技有限公司。
唾液酸乳糖的HPLC检测方法:色谱柱:Sepax HP-Amide(250×4.6mm,5um)。缓冲盐:10mM甲酸铵(pH为3.0)。流动相:乙腈:缓冲盐=70:30。流速:1.0mL/min;浓度:1mg/mL;检测波长:210nm;进样量:10μl;柱温:35℃。
实施例1底盘菌株SLIS026的构建
1.1构建含有CRISPR/Cas9敲除系统所需小向导RNA(small guide RNA,sgRNA)质粒
根据表3设计的引物(擎科合成)以pTargetF质粒(图谱见图2)或BL21基因组为模板进行各片段的特异性扩增,采用Takala公司的高保真酶Primer Star Mix进行PCR反应,反应体系如下:
表1 PCR扩增反应体系
Figure BDA0003385837230000061
PCR扩增程序如下:
表2 PCR反应程序
Figure BDA0003385837230000071
扩增产物取5μl进行1%的琼脂电泳,检测扩增结果。采用凝胶回收试剂盒对目的片段进行切胶回收。采用NEB的多片段重组酶将目的片段进行连接重组,连接重组产物转化至大肠杆菌感受态细胞Trans 10。加入灭菌的LB液体培养基,37℃250rpm震荡培养1h;
(2)挑点至事先加入壮观霉素的LB固体平板上,37℃倒置过夜培养;
(3)待长出白色单菌落后,挑取白色单菌落于含有2mL LB液体培养基的离心管中(含50μg/mL壮观霉素),37℃180rpm震荡培养6h;
(4)对菌液进行PCR检测,将取验证为阳性的菌液500μl送擎科公司测序,并将剩余菌液保存于20%甘油中。
(5)扩大培养测序验证正确的菌种,采用生工的质粒提取试剂盒进行质粒提取。分别得到含有BL21基因组的sgRNA质粒,命名为pTargetF-△LacZ、pTargetF-△nanKE、pTargetF-△nanA。
表3 lacZ、nanAKE敲除sgRNA质粒构建引物信息
Figure BDA0003385837230000072
Figure BDA0003385837230000081
1.2 lacZ、nanAKE基因敲除
1.2.1 BL21菌株lacZ(GA001)基因敲除
(1)BL21感受态细胞制备:对-80℃保存的菌种BL21进行划线培养单菌落;挑取单菌落接种于5mL LB培养基中,37℃条件下200rpm震荡培养值OD为0.5左右(约3h),然后将培养物冰浴30min;将菌液转移到预冷的无菌离心管中,于4℃条件下4000rpm离心10min,弃上清,收集菌体;用预冷的无菌水重悬菌体,于4℃条件下4000rpm离心10min,弃上清;用含0.1M CaCl2溶液重悬菌体2次,4℃条件下4000rpm离心10min,弃上清;最后用适量15%甘油的0.1M CaCl2溶液重悬细胞,以每管100μl分装至1.5mL离心管中,迅速放入液氮中速冻,保存于-80℃。
(2)3μl pCas-sac质粒加入100μL E.coli BL21感受态中,于冰上放置30min,之后进行42℃热激45s,立即放到冰上2~5min;加入800μL LB后放置到30℃摇床孵育45min,涂板(Km抗性,LB培养基),倒置于30℃培养箱中,培养过夜;挑点至LB培养基(Kana抗性)中,培养数小时后进行保菌(甘油终浓度30%)。
(3)挑取pCas-sac/BL21转化子接种LB筛管(卡拉抗性)30℃培养至OD为0.2时添加终浓度2g/L的阿拉伯糖进行诱导,OD为0.4时进行感受态制备,制备方法同操作(1);
(4)将构建正确的pTargetF-ΔLacZ质粒通过热激法转化至pCas-sac/BL21感受态细胞,复苏后涂布LB平板(k+、spe+)置于30℃培养过夜;
(5)对抗性平板上单菌落进行PCR验证,验证引物见表4,测序验证图谱见图1,验证得到LacZ基因敲除的菌株;
(6)对发生LacZ基因敲除的菌株挑点摇菌、并添加终浓度为10mM的鼠李糖进行诱导用于sgRNA质粒pTargetF-ΔLacZ丢失处理;
(7)划线验证pTargetF-ΔLacZ质粒是否发生丢失(引物见表4),并将已完成sgRNA丢失的LacZ基因已敲除的菌株命名为SLSI020。
1.2.2基于SLIS020菌株对Neu5Ac降解相关基因nanKAE基因的敲除
(1)SLIS020感受态制备和敲除操作同1.2.1,使用pTargetF-△nanA质粒进行nanA基因敲除,方法同1.2.1,获得nanA基因敲除的菌株,命名为SLIS024。
(2)对SLIS024菌株进行nanKE基因双敲除,使用pTargetF-△nanE质粒进行敲除,方法同1.2.1,并将完成nanKE基因敲除的菌株命名为SLIS026。
(3)SLIS026菌株进行sgRNA质粒的丢失,方法同1.2.1。
(4)SLIS026菌株进行pCas-SAC质粒的丢失:将已完成sgRNA丢失的SLIS026菌种接种无抗LB平板,置于42℃培养,并用表4中pCas-SAC验证引物进行PCR验证,以确保获得无pCas-SAC质粒的底盘菌株SLIS026。
表4 LacZ、nanAKE等基因敲除验证引物
Figure BDA0003385837230000101
实施例2构建N-乙酰神经氨酸(Neu5Ac)合成相关表达质粒以及Neu5Ac的制备
2.1用于Neu5Ac合成的表达质粒构建
(1)基因glmS*(GA010)是大肠杆菌L-谷氨酰胺-D-果糖-6-磷酸转氨酶基因的突变形式(Metab Eng.,2005May;7(3):201-14),其核酸序列如SEQ ID NO:66所示;Gna1(GA009,GenBank:NP_116637)编码来自酿酒酵母的葡糖胺-6-磷酸乙酰转移酶;基因slr1975(GA006,GenBank:BAL35720)编码集胞藻属种PCC6803 N-乙酰葡糖胺2-差向异构酶;基因neuB(GA005,GenBank:AF305571)编码空肠弯曲杆菌的唾液酸合酶;基因ppsA编码大肠杆菌BL21(DE3)的磷酸烯醇丙酮酸合酶;基因YqaB编码N-乙酰葡糖胺-6-磷酸磷酸酶。其中slr1975、Gnal、glmS、neuB、启动子Tet、Tac基因序列均由生工生物工程(上海)股份有限公司合成并连接到puc57载体上,YqaB、ppsA均来自BL21基因组。
(2)按照表5所列引物、模板进行PCR扩增,获取目的片段,PCR反应体系、条件同实施例1中1.1。
(3)使用胶回收试剂盒对扩增的DNA片段进行切胶回收,使用NEB公司的多片段重组试剂盒进行重组、转化、平板筛选获取阳性单菌落,然后挑点、摇菌、经擎科生物科技有限公司测序验证获得正确的质粒。
表5表达质粒SL006、pET28a-neuB等质粒构建所需引物
Figure BDA0003385837230000111
Figure BDA0003385837230000121
(4)启动子序列:
Tac启动子的序列如SEQ ID NO:64所示。
Tet启动子的序列如SEQ ID NO:65所示。
2.2 N-乙酰神经氨酸(Neu5Ac)的制备
2.2.1产N-乙酰神经氨酸(Neu5Ac)E.coli菌株的构建
以完成基因敲除的菌株SLIS026为基础制备感受态细胞,具体方法同1.2.1,然后将质粒SL006单独,以及分别和pET28a-neuB、pTac-neuB、pTet-neuB其一共转入SLIS026感受态细胞中,在LB平板(卡那霉素50μg/mL,氯霉素25μg/mL)上筛选正确的克隆。经PCR验证相应地得到4种(SL006、SL006+pET28a-neuB、SL006+pTet-neuB、SL006+pTac-neuB)携带Neu5Ac合成途径的菌株E.coli SLIS026-SA。
2.2.2 SLIS026-Neu5Ac菌株生产N-乙酰神经氨酸(Neu5Ac)
(1)TB培养基:胰蛋白栋12g、酵母提取物24g,甘油4mL,2.31g KH2PO4,12.54gK2HPO4,去离子水定容至1000mL,最后按100mL/瓶分装到三角瓶中并包扎好,121℃下灭菌30min,室温储存。
(2)LB培养基:分别称取胰蛋白胨10g,酵母提取物5g,NaCl 10g,琼脂15g,加入蒸馏水溶解混匀,lmol/L NaOH调节pH至7.2后,定容至1L,最后分装到三角瓶中并包扎好,121℃下灭菌30min,4℃储存,LB液体不添加琼脂。
(3)200g/L MgSO4·7H2O母液:称取10gMgSO4·7H2O溶液去离子水中,待完全溶解后定容到50mL,然后121℃下灭菌30min,室温保存。
(4)微量元素母液:54.4g/L柠檬酸铁铵、9.8g/L MnCl2·4H2O、1.6g/L CoCl2·6H2O、1g/L CuCl2·2H2O、1.9g/LH3BO3、9g/L ZnSO4·7H2O、1.1g/L Na2MoO4·2H2O、1.5g/LNa2SeO3、1.5g/L NiSO4·6H2O,按照以上浓度配制微量元素母液,121℃下灭菌30min,4℃储存备用。
(5)1000g/L甘油:称取1000g甘油,用去离子水定容到1L,然后121℃下灭菌30min,室温保存。
(6)将菌株接种至5mL的LB培养基中(卡那霉素50μg/mL,氯霉素25μg/mL),37℃,250rpm培养4h。然后按照种子液:培养基=1:100的比例接种至新鲜的TB培养基,37℃、250rpm培养至OD600=0.6-0.8,并加入终浓度为0.1mM的IPTG在25℃、250rpm的条件下培养15h进行蛋白诱导表达。
(7)蛋白诱导表达结束后向摇瓶中添加1mL 200g/L MgSO4·7H2O(终浓度2g/L)、1mL微量元素母液(终浓度0.1%)、2mL 1000g/L的甘油(终浓度20g/L),并置于30℃、250rpm条件下培养24h后取样检测Neu5Ac含量。
(8)样品处理方式:将完成发酵培养的菌液置于121℃下灭菌20min,然后对其进行离心(4℃,4000rpm离心15min),去沉淀保留上清,并使用经灭菌处理的TB培养基定容至100mL保存备用,并取样1mL,过0.22μm滤膜,采用离子对检测,测得采用了Tet启动子的菌株获得的Neu5Ac产量最高,为2.9g/L的Neu5Ac,图10为Neu5Ac检测图谱。
实施例3构建6’-SL(6’-唾液酸乳糖)合成相关表达质粒以及6’-SL的制备
3.1 6’-SL合成质粒构建
(1)全基因合成N-乙酰神经氨酸胞苷酰转移酶CSS(登录号为WP_003512903.1)(GA031)的基因SEQ ID NO:61;登录号为BAF91416.1的α-2,6-唾液酸转移酶(6ST,GA025)基因SEQ ID NO:62,基因序列均由生工生物工程(上海)股份有限公司合成并连接到pET28a载体上。
(2)按照表6所列引物、模板进行PCR扩增,获取目的片段,PCR反应体系、条件同实施例1中1.1。
(3)使用胶回收试剂盒对扩增的DNA片段进行切胶回收,使用NEB公司的多片段重组试剂盒进行重组、转化、平板筛选获取阳性单菌落,然后挑点、摇菌、经擎科生物科技有限公司测序验证获得正确的质粒。
表6表达质粒SL023构建所需引物
Figure BDA0003385837230000141
Figure BDA0003385837230000151
3.2利用SLIS026菌株进行6’-SL的生产
3.2.1产6’-SL E.coli菌株的构建
以完成基因敲除的菌株SLIS026为基础制备感受态细胞,具体方法同1.2.1,然后按照以下质粒组合SL006+pET28a-neuB+SL023、SL006+pET28a-neuB+SL023-Tet、SL006+pET28a-neuB+SL023-Tac、SL006+pTet-neuB+SL023、SL006+pTet-neuB+SL023-Tac、SL006+pTet-neuB+SL023-Tet、SL006+pTac-neuB+SL023、SL006+pTac-neuB+SL023-Tac、SL006+pTac-neuB+SL023-Tet分别转入SLIS026感受态细胞中,在LB平板(卡那霉素50μg/mL,氯霉素25μg/mL、氨苄100μg/mL)上筛选正确的克隆,得到携带6’-SL合成途径的菌株E.coliSLIS026-6SL。
3.2.2 SLIS026-6SL菌株生产6’-SL
(1)TB培养基:胰蛋白栋12g、酵母提取物24g,甘油4mL,2.31g KH2PO4,12.54gK2HPO4,去离子水定容至1000mL,最后按100mL/瓶分装到三角瓶中并包扎好,121℃下灭菌30min,室温储存。
(2)LB培养基:分别称取胰蛋白胨10g,酵母提取物5g,NaCl 10g,琼脂15g,加入蒸馏水溶解混匀,lmol/L NaOH调节pH至7.2后,定容至1L,最后分装到三角瓶中并包扎好,121℃下灭菌30min,4℃储存,LB液体不添加琼脂。
(3)200g/L MgSO4·7H2O母液:称取10g MgSO4·7H2O溶液去离子水中,待完全溶解后定容到50mL,然后121℃下灭菌30min,室温保存。
(4)微量元素母液:54.4g/L柠檬酸铁铵、9.8g/L MnCl2·4H2O、1.6g/L CoCl2·6H2O、1g/L CuCl2·2H2O、1.9g/LH3BO3、9g/L ZnSO4·7H2O、1.1g/L Na2MoO4·2H2O、1.5g/LNa2SeO3、1.5g/L NiSO4·6H2O,按照以上浓度配制微量元素母液,121℃下灭菌30min,4℃储存备用。
(5)1000g/L甘油:称取1000g甘油,用去离子水定容到1L,然后121℃下灭菌30min,室温保存。
(6)250g/L乳糖:250g乳糖溶于去离子水中(加热溶解),并定容到1L,然后121℃下灭菌30min,室温保存。
(7)将菌株接种至5mL的LB培养基中(卡那霉素50μg/mL,氯霉素25μg/mL),37℃,250rpm培养4h。然后按照种子液:培养基=1:100的比例接种至新鲜的TB培养基,37℃、250rpm培养至OD600=0.6-0.8,并加入终浓度为0.1mM的IPTG在25℃、250rpm的条件下培养15h进行蛋白诱导表达。
(8)蛋白诱导表达结束后向摇瓶中添加1mL 200g/L MgSO4·7H2O(终浓度2g/L)、100μl微量元素母液(终浓度0.1%)、2mL 1000g/L的甘油(终浓度20g/L)和2mL 250g/L乳糖(终浓度5g/L)并置于30℃、250rpm条件下培养24h后取样检测终产物SL含量。
(9)样品处理方式:取发酵液2-3mL,采用反复冻融的方式破碎细胞,破碎后置于沸水中煮20min,然后对其进行离心(4℃,12000rpm离心5min),去沉淀保留上清,过0.22μm滤膜,对6’-SL含量进行检测,各处理在发酵24h时6’-SL含量见附表1,其中处理使用Tet启动子调控neuB表达时即组合SL006+pTet-neuB+SL023的6’-SL含量最高,可达到2.5g/L,SL检测结果见附图,图11为6’-SL标准品检测图谱,图12为6’-SL发酵液检测图谱。
表7 6’-SL含量检测结果
Figure BDA0003385837230000161
Figure BDA0003385837230000171
实施例4利用SLIS026菌株进行3’-SL的生产
4.1 3’-SL合成相关表达质粒构建
(1)全基因合成编码登录号为AJC62560.1的α-2,3-唾液酸转移酶(3ST,GA040)基因SEQ ID NO:63,由生工生物工程(上海)股份有限公司合成并连接到pET28a载体上。
(2)按照表8所列引物、模板进行PCR扩增,获取目的片段,PCR反应体系、条件同实施例1中1.1。
(3)使用胶回收试剂盒对扩增的DNA片段进行切胶回收,使用NEB公司的多片段重组试剂盒进行重组、转化、平板筛选获取阳性单菌落,然后挑点、摇菌、经擎科生物科技有限公司测序验证获得正确的质粒。
表8表达质粒SL037构建所需引物
Figure BDA0003385837230000172
4.2在发酵过程中生产3’-SL
4.2.1产3’-SL E.coli菌株的构建
以完成基因敲除的菌株SLIS026为基础制备感受态细胞,具体方法同1.2.1,然后参照6’-SL发酵结果最好的质粒组合将质粒组合SL006+pTet-neuB+SL037转入SLIS026感受态细胞中,在LB平板(卡那霉素50μg/mL,氯霉素25μg/mL、氨苄100μg/mL)上筛选正确的克隆,得到携带3’-SL合成途径的菌株E.coli SLIS026-3SL。
4.2.2 SLIS026-3SL菌株生产3’-SL
(1)实验过程中所需培养基种类以及配制同3.2。
(2)将菌株接种至5mL的LB培养基中(卡那霉素50μg/mL,氯霉素25μg/mL),37℃,250rpm培养4h。然后按照种子液:培养基=1:100的比例接种至新鲜的TB培养基,37℃、250rpm培养至OD600=0.6-0.8,并加入终浓度为0.1mM的IPTG在25℃、250rpm的条件下培养15h进行蛋白诱导表达。
(3)蛋白诱导表达结束后向摇瓶中添加1mL 200g/L MgSO4·7H2O(终浓度2g/L)、100μl微量元素母液(终浓度0.1%)、2mL 1000g/L的甘油(终浓度20g/L)和2mL 250g/L乳糖(终浓度5g/L)并置于30℃、250rpm条件下培养24h后取样检测终产物SL含量。
(4)样品处理方式:样品处理方式同3.2,对3’-SL进行定量检测,结果显示在发酵24h时3’-SL含量可达到3g/L,其中图14为3’-SL标准品检测图谱,图15为3’-SL发酵液检测图谱。
SEQUENCE LISTING
<110> 弈柯莱生物科技(上海)股份有限公司
<120> 一种基因工程菌及其在制备唾液酸乳糖中的应用
<130> P21018933C
<160> 84
<170> PatentIn version 3.5
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<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> GA001-P1-F4
<400> 1
tgccgaccgt ctagagtcga cctgcagaag cttag 35
<210> 2
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> GA001-P1-R4
<400> 2
aactggcgtt acccaactta atcactagta ttatacctag gactg 45
<210> 3
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> GA001-P1-F1
<400> 3
tagtgattaa gttgggtaac gccagtttta gagctagaaa tagcaag 47
<210> 4
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> GA001-P1-R1
<400> 4
gttccggaat tcaaaaaaag caccgactcg gtgcc 35
<210> 5
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> GA001-LF
<400> 5
gctttttttg aattccggaa cgggaaggcg actggagtg 39
<210> 6
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> GA001-P1-LR
<400> 6
ggtgcgggcc tcgacggcca gtgaatccgt aatcatg 37
<210> 7
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> GA001-P1-RR
<400> 7
tcgactctag acggtcggca aagaccagac cgttc 35
<210> 8
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> GA001-P1-RF
<400> 8
ctggccgtcg aggcccgcac cgatcgccct tc 32
<210> 9
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> pT14-F2
<400> 9
gctttttttg aattcgggaa ttccgcagac accatctg 38
<210> 10
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> pT-G08-R1
<400> 10
ccaaaagtta atgatgggtg aagtacagtc attac 35
<210> 11
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> pT-G08-F2
<400> 11
gtacttcacc catcattaac ttttggtttt gac 33
<210> 12
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> pT-G08-R2
<400> 12
gtcgactcta gagcgtgcgc tgttctttat cgg 33
<210> 13
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> pT-G08-F3
<400> 13
cagcgcacgc tctagagtcg acctgcagaa g 31
<210> 14
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> pT-G08-R3
<400> 14
accacgcgtg gcttgcagat ttactagtat tatacctag 39
<210> 15
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> pT-G08-F1
<400> 15
agtaaatctg caagccacgc gtggttttag agctagaaat a 41
<210> 16
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> pT4-1R1
<400> 16
ctaaaaccga tcgtcgtgaa cttcctaact agtattatac ctagg 45
<210> 17
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> pT15-F2
<400> 17
gctttttttg aattcgcagc gcctgaatcg gccatgag 38
<210> 18
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> pT15-1R2
<400> 18
gaggtatttg ttgtttcccc tcgctcgccc ctacc 35
<210> 19
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> pT15-1F3
<400> 19
gcgaggggaa acaacaaata cctctgaagt gatgcttg 38
<210> 20
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> pT15-R3
<400> 20
caggtcgact ctagaccgcc cgctggcgcg taaaaaac 38
<210> 21
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> pT15-P1-F1
<400> 21
actagtatcg acggtttata cgtggggttt tagagctaga aatag 45
<210> 22
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> pT15-R1
<400> 22
gcgaattcaa aaaaagcacc gactcggtgc cac 33
<210> 23
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> pT15-F4
<400> 23
gcgggcggtc tagagtcgac ctgcagaagc ttag 34
<210> 24
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> pT15-1R4
<400> 24
aaccccacgt ataaaccgtc gatactagta ttatacctag g 41
<210> 25
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Lacz-YZ1-F
<400> 25
cgcgctgtta gcgggcccat taagttctg 29
<210> 26
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> LAC-YZ2-R
<400> 26
ggtcttcatc cacgcgcgcg tacatcgg 28
<210> 27
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> nanA-YZ-F1
<400> 27
catggtgatg tagcctggcg caaagcc 27
<210> 28
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> nanA-YZ-R
<400> 28
atgggcctta tgaacgcatt tgattcgc 28
<210> 29
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> nanE-YZ-F
<400> 29
gctcgtgcaa ttccgctttt ttctcgac 28
<210> 30
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> nanE-YZ-R
<400> 30
cggttatttc gataccgacc agcgtgcag 29
<210> 31
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> CX-targetF-F
<400> 31
cagcgagtca gtgagcgag 19
<210> 32
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> CX-targetF-R
<400> 32
gacattgcac tccaccgct 19
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Kan-F
<400> 33
gaaggagaaa actcaccgag 20
<210> 34
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Pcr4-R1
<400> 34
cagctgcata aaattgcgat tggcaaaacc atc 33
<210> 35
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> ACY-GA5-F1
<400> 35
aataaggaga tataatgaaa gaaatcaaaa tcc 33
<210> 36
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> ACY-GA5-R
<400> 36
ggtgagcgat catggtatat ctccttttag gcgaaatctt cataag 46
<210> 37
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> ACY-GA6-F
<400> 37
accatgatcg ctcaccgtcg tcaggaactg g 31
<210> 38
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> ACY-GA6-R
<400> 38
gctcgtacat ggtatatctc cttttaagag accggcagtt g 41
<210> 39
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> ACY-GA16-F
<400> 39
gatataccat gtacgagcgt tatgcagg 28
<210> 40
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> AVY-GA16-R
<400> 40
cgggtaagct catggtatat ctcctttcac agcaagcgaa catcc 45
<210> 41
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> ACY-GA9-F
<400> 41
taccatgagc ttacccgatg gattttatat aag 33
<210> 42
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> ACY-GA9-R
<400> 42
cgataccgca catggtatat ctccttctat tttctaattt gc 42
<210> 43
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> ACY-GA10-F
<400> 43
taccatgtgc ggtatcgttg gtgctatcg 29
<210> 44
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> ACY-GA10-R
<400> 44
gccattgttg gacatggtat atctcctttt attccacggt cacggatt 48
<210> 45
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> ACY-GA28-F
<400> 45
ccatgtccaa caatggctcg tcaccgctgg tg 32
<210> 46
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> ACY-GA28-R
<400> 46
ccgcaagctt ttatttcttc agttcagcca gg 32
<210> 47
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> ACY-F
<400> 47
gaaataaaag cttgcggccg cataatgc 28
<210> 48
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> PACY-R
<400> 48
gatttctttc attatatctc cttattaaag ttaaac 36
<210> 49
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> 28a-G5F2
<400> 49
gatttcgcct aagcttgcgg ccgcactcga gcac 34
<210> 50
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> 28a-G5R2
<400> 50
gatttctttc atatggctgc cgcgcggcac cagg 34
<210> 51
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 28a-G5F1
<400> 51
ctggattttg atttctttca tatggctgcc gc 32
<210> 52
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> 28a-G5R1
<400> 52
ccgcaagctt aggcgaaatc ttcataagac agctg 35
<210> 53
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Tac-F1
<400> 53
gaaatttgac aattaatcat cggctcgtat aatgtg 36
<210> 54
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Tac-R1
<400> 54
ggctgctgcc catgtatatc tccttcttaa agttaaac 38
<210> 55
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Tac-F2
<400> 55
gaaggagata tacatgggca gcagccatca tcatc 35
<210> 56
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Tac-R2
<400> 56
gatgattaat tgtcaaattt cgcgggatcg agatctc 37
<210> 57
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Tet-F1
<400> 57
gatcccgcga aatgttgaca ctctatcatt gatag 35
<210> 58
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Tet-R1
<400> 58
tgatttcttt catttgtata tctccttctt aaag 34
<210> 59
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Tet-F2
<400> 59
gatatacaaa tgaaagaaat caaaatccag aac 33
<210> 60
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Tet-R2
<400> 60
gatagagtgt caacatttcg cgggatcgag atctcg 36
<210> 61
<211> 697
<212> DNA
<213> Artificial Sequence
<220>
<223> N-乙酰神经氨酸胞苷酰转移酶(CSS)的优化核苷酸序列
<400> 61
atgaaaaaca tggcgatcat cccggcgcgt gcgggtagca aaggcctgaa agataaaaac 60
atcaaactgc tgaacggcaa accgatgctg gcgtacacca tcgaagcggc gaaagaaagc 120
ggcctgttcg aagaaatcat ggttagcacc gatagcgaaa aatacgcgga aatcgcgaaa 180
cagtggggtg cgagcgttcc gttcctgcgt ccggttgaac tgagcaacga taccgcgagc 240
agctgggatg ttgttaaata cgttatcgaa aaatacaaag aactgggccg tgaatttgat 300
accgtggcgc tgctgcaacc gaccagcccg ctgcgtaaca gcgcggacat catcaaaggt 360
tacgaaatca tgaaagaaaa acacgcgaac agcgttatcg cggtttgcga agcggaacac 420
agcccgctgt ggatgaacat cctgccggaa aaccacagca tggcgggctt catccgtccg 480
gatgcggcga acgtgccgcg tcagagcatc ccgacctact accgtatcaa cggcgcgctg 540
tacatcgtta aagtggaata cctgatgaac agcaacgaca tctacagcga taaaagctac 600
gcgctgatca tggataaaga aaacagcatc gacatcgatg atatgttcga tttcaaaatc 660
gcgagcctga tcctgcgtga tcgtaaacag taagctt 697
<210> 62
<211> 1449
<212> DNA
<213> Artificial Sequence
<220>
<223> α-2,6-唾液酸转移酶(6ST)的核苷酸序列
<400> 62
atgaacgata atcaaaatac ggtggacgtg gtggtctcaa ccgtcaacga taacgtgatc 60
gaaaacaaca cgtaccaagt caaaccgatc gataccccga ccacgttcga ctcatactcg 120
tggattcaga cgtgcggcac cccgatcctg aaagatgacg aaaaatatag cctgtctttt 180
gatttcgttg ccccggaact ggatcaagac gaaaaattct gtttcgaatt taccggcgat 240
gtggatggta aacgttatgt gacgcagacc aacctgacgg tggttgcacc gaccctggaa 300
gtttacgtcg atcatgcttc actgccgtcg ctgcagcaac tgatgaaaat catccagcag 360
aaaaacgaat acagccagaa tgaacgcttt atttcttggg gccgtatccg cctgacggaa 420
gataacgcgg aaaaactgaa tgcccatatt tatccgctgg caggcaacaa taccagccag 480
gaactggtgg acgcagttat cgattacgct gactctaaaa accgtctgaa tctggaactg 540
aacacgaata ccggccacag tttccgtaac attgcgccga tcctgcgcgc caccagctct 600
aaaaacaaca tcctgatctc caacatcaac ctgtacgatg acggtagtgc tgaatatgtg 660
tccctgtaca actggaaaga taccgacaat aaatcacaga aactgagtga ttcctttctg 720
gttctgaaag actacctgaa tggcatcagt tccgaaaaac cgaacggtat ttatagcatc 780
tacaattggc atcagctgta tcactcatcg tattacttcc tgcgtaaaga ttacctgacg 840
gtggaaacca aactgcacga cctgcgcgaa tatctgggcg gttcactgaa acaaatgtcg 900
tgggatacct ttagccagct gtctaaaggc gacaaagaac tgttcctgaa cattgttggt 960
tttgatcagg aaaaactgca gcaagaatac cagcaaagcg aactgccgaa tttcgtcttt 1020
acgggcacca cgacctgggc aggcggtgaa accaaagaat attacgctca gcaacaggtg 1080
aacgtcgtga acaatgcgat taatgaaacc tctccgtatt acctgggccg tgaacatgac 1140
ctgtttttca aaggtcaccc gcgcggcggt attatcaatg atattatcct gggctcattc 1200
aacaatatga ttgacatccc ggccaaagtt tcgtttgaag tcctgatgat gacgggtatg 1260
ctgccggata ccgttggcgg tattgcgagc agcctgtatt ttagtatccc ggccgaaaaa 1320
gtgtccttca ttgtttttac cagttccgat acgatcaccg atcgcgaaga cgcgctgaaa 1380
agtccgctgg tccaagtgat gatgaccctg ggcattgtga aagaaaaaga tgtgctgttc 1440
tggtgctaa 1449
<210> 63
<211> 1116
<212> DNA
<213> Artificial Sequence
<220>
<223> α-2,3-唾液酸转移酶的核苷酸序列
<400> 63
atgggcctga aaaaagcgtg cctgaccgtt ctgtgcctga tcgttttctg cttcggcatc 60
ttctacacct tcgatcgcgt taaccagggc gaacgcaacg cggtttctct gctgaaagaa 120
aaactgttca acgaagaagg tgaaccggtt aacctgatct tctgctacac catcctccag 180
atgaaagttg cggaacgtat catggcgcag catccgggcg aacgcttcta cgttgtgctg 240
atgagcgaaa accgcaacga aaaatacgac tactacttca accagatcaa agataaagcg 300
gaacgtgcgt acttcttcca cctgccgtac ggtctgaaca aatccttcaa cttcatcccg 360
acgatggcgg aactgaaagt taaatctatg ctgctgccga aagtgaaacg catctacctg 420
gcgtccctgg aaaaagtgag catcgcggca ttcctgtcta cctacccgga cgcagaaatc 480
aaaaccttcg atgatggcac cggcaacctg atccagtcca gctcttacct gggcgacgag 540
ttctctgtta acggcaccat caaacgtaac ttcgcacgta tgatgatcgg cgattggtcc 600
atcgcgaaaa cccgtaacgc gtctgatgaa cactacacca tcttcaaagg cctgaaaaac 660
atcatggatg atggtcgtcg taaaatgacc tacctgccgc tgttcgatgc gtctgaactg 720
aaaaccggtg acgaaaccgg cggcaccgtt cgtatcctgc tgggcagccc ggataaagaa 780
atgaaagaaa tcagcgaaaa agcggcgaaa aacttcaaaa tccagtacgt tgcgccgcac 840
ccgcgccaga cctacggcct gtctggcgtt accactctga acagcccgta cgtgatcgaa 900
gattacatcc tgcgtgaaat caagaaaaac ccgcacaccc gctacgaaat ctacaccttc 960
ttcagcggtg cggcgctgac catgaaagac ttcccgaacg ttcacgttta cgcgctgaaa 1020
ccggcgagcc tgccggaaga ttactggctg aaaccggtgt acgcgctgtt cacccagagc 1080
ggcatcccga tcctgacctt cgacgataaa aactaa 1116
<210> 64
<211> 119
<212> DNA
<213> Artificial Sequence
<220>
<223> Tac启动子
<400> 64
ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca 60
caggaaacag aattgcccct ctagaaataa ttttgtttaa ctttaagaag gagatatac 119
<210> 65
<211> 124
<212> DNA
<213> Artificial Sequence
<220>
<223> Tet启动子
<400> 65
gttgacactc tatcattgat agagttattt taccactccc tatcagtgat agagaaaagt 60
gaaatgaata gttcgacaaa aatctagaaa taattttgtt taactttaag aaggagatat 120
acaa 124
<210> 66
<211> 1830
<212> DNA
<213> Artificial Sequence
<220>
<223> Glms
<400> 66
atgtgcggta tcgttggtgc tatcgcacag cgtgatgtag cgaaaatcct cctggaaggt 60
ctgcgtcgtc tcgaataccg tggttacgac tctgccggtc tggcagtagt ggatgcagaa 120
ggtcacatga ctcgtctgcg tcgtctgggt aaagtgcaga tgctcgcgca ggcggcggaa 180
gaacacccac tccacggtgg tacgggtatc gcacacactc gttgggcaac ccacggtgaa 240
ccgtctgagg tcaacgcaca cccgcatgtt agcgagcaca tcgtagtcgt tcacaacggt 300
atcatcgaga accacgaacc actccgtgag gaactcaaag cccgtggtta caccttcgta 360
agcgaaaccg acacggaagt tatcgcccac ctcgttaact gggaactcaa acagggtggt 420
actctgcgtg aagcagttct gcgtgccatt ccacagctgc gtggtgcata cggtaccgtg 480
atcatggact ctcgtcatcc ggataccctg ctcgccgcac gttctggttc tccactcgtt 540
atcggtctgg gtatgggtga gaacttcatc gcctctgatc agctggccct gctcccagtt 600
acccgtcgct tcatcttcct ggaagagggt gacatcgccg aaatcacccg tcgttccgtt 660
aacatcttcg acaaaacggg tgcggaagtt aaacgtcagg acatcgagtc taacctgcag 720
tatgacgctg gtgacaaagg catctaccgt cactacatgc agaaagagat ctacgaacag 780
ccgaacgcga tcaaaaacac cctgaccggt cgtatctctc acggtcaggt tgacctgtct 840
gagctgggtc caaacgcgga cgaactcctg tccaaagtcg agcacatcca gatcctggct 900
tgtggtacct cttacaactc cggtatggtt tctcgttact ggttcgaatc tctggcaggt 960
atcccatgcg acgttgaaat cgcctccgaa ttccgttatc gtaaatctgc ggtacgtcgt 1020
aactccctca tgatcaccct gtctcagtct ggtgaaaccg ctgatactct ggcaggtctg 1080
cgtctcagca aagaactggg ttacctgggt tctctggcca tctgcaacgt tccgggttct 1140
agcctggttc gtgagtctgt gctggctctg atgaccaacg cgggtacgga gatcggtgtt 1200
gcctctacca aagcgttcac tacccagctc actgtcctgc tgatgctggt tgccaaactg 1260
tctcgtctca aaggcctcga cgctagcatc gaacacgaca tcgtacacgg tctgcaggcc 1320
ctcccatctc gtatcgagca gatgctgccg caggacaaac gtatcgaagc actggcagaa 1380
gacttcagcg acaaacacca cgcgctgttt ctgggtcgtg gtgaccagta cccaattgcg 1440
ctggaaggtg ccctgaaact gaaagagatc agctacatcc atgcagaggc atacgcagcg 1500
ggtgagctga aacatggtcc actggccctg atcgacgcag atatgccggt tattgtggtt 1560
gctccgaaca acggcctgct ggagaaactg aaatccaaca tcgaggaagt acgtgcgcgt 1620
ggtggtcagc tgtacgtgtt tgctgaccag gacgcgggtt tcgtttccag cgacaacatg 1680
cacatcatcg aaatgccgca tgttgaagag gtaatcgcgc caatcttcta caccgtaccg 1740
ctgcagctgc tggcgtacca tgtagccctg atcaaaggta cggacgttga ccagccgcgt 1800
aacctggcga aatccgtgac cgtggaataa 1830
<210> 67
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> SL23-F1
<400> 67
gatatacata tgaacgataa tcaaaatacg gtgg 34
<210> 68
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> SL23-R1
<400> 68
ccatggtata tctcctttta gcaccagaac agcacatctt tttc 44
<210> 69
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> SL23-F2
<400> 69
gtgctaaaag gagatatacc atggaaaaac agaacatcgc gg 42
<210> 70
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> SL023-R2
<400> 70
gtgcggccgc aagcttaaga ttctttgtgg ttcaggatg 39
<210> 71
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> SL023-F3
<400> 71
caaagaatct taagcttgcg gccgcactcg agcacc 36
<210> 72
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> SL23-R3
<400> 72
gattatcgtt catatgtata tctccttctt aaag 34
<210> 73
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Tet-F1
<400> 73
gatcccgcga aatgttgaca ctctatcatt gatag 35
<210> 74
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> SL023-Tet-R1
<400> 74
tgattatcgt tcatttgtat atctccttct taaag 35
<210> 75
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> SL23-Tet-F1
<400> 75
gatatacaaa tgaacgataa tcaaaatacg gtgg 34
<210> 76
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Tet-R2
<400> 76
gatagagtgt caacatttcg cgggatcgag atctcg 36
<210> 77
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> Tac-F1
<400> 77
cgcgaaattt gacaattaat catcggctcg tataatgtg 39
<210> 78
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> SL023-Tac-R1
<400> 78
tgattatcgt tcatgtatat ctccttctta aag 33
<210> 79
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> SL23-Tac-F1
<400> 79
gagatataca tgaacgataa tcaaaatacg gtgg 34
<210> 80
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Tac-R2
<400> 80
gatgattaat tgtcaaattt cgcgggatcg agatctcg 38
<210> 81
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> SL37-F1
<400> 81
tcgcctaaaa ggagatatac catgggcctg aaaaaagcgt gcct 44
<210> 82
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> SL37-R1
<400> 82
tttccatggt atatctcctt ttagttttta tcgtcgaagg tcagg 45
<210> 83
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> SL37-F2
<400> 83
aaggagatat accatggaaa aacagaacat cgcggttatc c 41
<210> 84
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> SL37-R2
<400> 84
ggtatatctc cttttaggcg aaatcttcat aagacagctg c 41

Claims (11)

1.一种基因工程菌,其具有N-乙酰神经氨酸生物合成途径,其特征在于,其含有多拷贝的用于编码唾液酸合酶的基因neuB,且所述基因neuB被强启动子启动表达。
2.如权利要求1所述的基因工程菌,其特征在于,所述基因工程菌中的N-乙酰神经氨酸分解代谢途径被失效;较佳地:
所述基因工程菌中的N-乙酰神经氨酸分解代谢途径中的全部或者部分基因被敲除;优选敲除编码N-乙酰甘露糖胺激酶的基因nanK、编码N-乙酰甘露糖胺-6-磷酸差向异构酶的基因nanE和编码N-乙酰神经氨酸醛缩酶的基因nanA中的一种或多种;
和/或,所述基因工程菌中编码乳糖操纵子β-半乳糖苷酶的基因LacZ被敲除。
3.如权利要求1所述的基因工程菌,其特征在于,所述多拷贝通过复制子、基因组中多位点整合以及外源质粒插入中的一种或多种方式实现;优选通过外源质粒插入的方式实现;
和/或,所述强启动子为Tet启动子。
4.如权利要求1~3任一项所述的基因工程菌,其特征在于,进一步包含编码唾液酸合酶的基因neuB、编码N-乙酰葡糖胺2-差向异构酶的基因slr1975、编码N-乙酰葡糖胺-6-磷酸磷酸酶的基因YqaB、编码葡糖胺-6-磷酸乙酰转移酶的基因Gna1、编码L-谷氨酰胺-D-果糖-6-磷酸转氨酶的基因glmS以及编码磷酸烯醇丙酮酸合酶的基因ppsA;优选地所述基因neuB、所述基因slr1975、所述基因YqaB、所述基因Gna1、所述基因glmS以及所述基因ppsA串联连接于质粒载体1上;
其中,质粒载体1中的基因优选满足以下条件中的一种或多种:
所述基因neuB的GenBank登录号为AF305571;
所述基因slr1975的GenBank登录号为BAL35720;
所述基因YqaB来自BL21基因组;
所述基因Gna1的GenBank登录号为NP_116637;
所述基因glmS的核酸序列如SEQ ID NO:66所示;
所述基因ppsA来自BL21基因组;
优选地所述质粒载体1为pACYCDuet。
5.如权利要求1~4任一项所述的基因工程菌,其特征在于,其还包含编码N-乙酰神经氨酸胞苷酰转移酶的基因和编码唾液酸转移酶的基因;
较佳地,编码N-乙酰神经氨酸胞苷酰转移酶的基因和编码唾液酸转移酶的基因串联连接于质粒载体2上;优选地N-乙酰神经氨酸胞苷酰转移酶的NCBI登录号为WP_003512903.1,优选含有如SEQ ID NO:61所示的核酸序列;
所述唾液酸转移酶为α-2,6-唾液酸转移酶或α-2,3-唾液酸转移酶,优选地所述α-2,6-唾液酸转移酶为NCBI登录号为BAF91416.1的酶,优选含有如SEQ ID NO:62所示的核酸序列;所述α-2,3-唾液酸转移酶为NCBI登录号为AJC62560.1的酶,优选含有如SEQ ID NO:63所示的核酸序列;优选地所述质粒载体2为pET28a。
6.如权利要求1~5任一项所述的基因工程菌,其特征在于,出发菌为大肠杆菌(Escherichia coli)BL21(DE3)。
7.一种生产N-乙酰神经氨酸的方法,其特征在于,培养如权利要求1~4、6任一项所述的基因工程菌获得。
8.一种发酵生产唾液酸乳糖的方法,其特征在于,采用如权利要求1~6任一项所述的基因工程菌,在发酵培养基中加入乳糖进行发酵,从发酵液中提取唾液酸乳糖;
任选地,当所述基因工程菌中含有α-2,3-唾液酸转移酶基因时,获得3’-唾液酸乳糖;当所述基因工程菌中含有α-2,6-唾液酸转移酶基因时,获得6’-唾液酸乳糖。
9.如权利要求8所述的方法,其特征在于,所述发酵培养基为TB培养基;所述TB培养基含有12g/L胰蛋白栋、24g/L酵母提取物、4mL/L甘油、2.31g/L KH2PO4以及12.54g/L K2HPO4
10.如权利要求8所述的方法,其特征在于,在培养至OD值为0.6~0.8时使用IPTG进行诱导培养;
和/或,在所述诱导培养结束后补加2g/L MgSO4·7H2O、20g/L甘油、1mL/L微量元素母液和5g/L乳糖;所述微量元素母液优选含有54.4g/L柠檬酸铁铵、9.8g/L MnCl2·4H2O、1.6g/LCoCl2·6H2O、1g/L CuCl2·2H2O、1.9g/LH3BO3、9g/L ZnSO4·7H2O、1.1g/L Na2MoO4·2H2O、1.5g/L Na2SeO3和1.5g/L NiSO4·6H2O。
11.如权利要求8~10任一项所述的方法,其特征在于,所述发酵培养条件为30℃、250rpm振荡培养。
CN202111450746.9A 2021-11-30 2021-11-30 一种基因工程菌及其在制备唾液酸乳糖中的应用 Active CN116200316B (zh)

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CN202111450746.9A CN116200316B (zh) 2021-11-30 2021-11-30 一种基因工程菌及其在制备唾液酸乳糖中的应用
PCT/CN2022/124823 WO2023098308A1 (en) 2021-11-30 2022-10-12 A genetically engineered bacterium and its application in the preparation of sialyllactose
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