CN114107157A - 一种生产n-乙酰葡萄糖胺基因工程菌的构建及应用 - Google Patents
一种生产n-乙酰葡萄糖胺基因工程菌的构建及应用 Download PDFInfo
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- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 title claims abstract description 50
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Abstract
本发明公开了一种生产N‑乙酰葡萄糖胺基因工程菌的构建方法及应用,该工程菌是通过将编码氨基葡萄糖合成酶、氨基葡萄糖乙酰转移酶和磷酸甘油酸脱氢酶基因导入大肠杆菌表达,并敲除大肠杆菌中N‑乙酰葡萄糖胺分解利用代谢途径酶的基因构建而成的重组大肠杆菌;构建的工程菌株利用葡萄糖为底物进行发酵培养合成N‑乙酰葡萄糖胺。本发明的工程菌合成N‑乙酰葡萄糖胺发酵水平高,同时副产物累积较少,具有工业化生产潜力。
Description
技术领域
本发明属于生物技术领域,具体涉及一种将基因工程改造运用于大肠杆菌构建基因工程菌生产N-乙酰葡萄糖胺。
背景技术
N-乙酰葡萄糖胺(N-acetylglucosamine,GlcNAc,CAS 134451-94-8)是葡萄糖的衍生物,具有还原性,广泛存在于各种生物体内。包括细菌、酵母、丝状真菌、植物以及动物,其中在海洋生物类动物的外骨骼中含量最高。医药方面,N-乙酰葡萄糖胺可以作为抗消炎药物,用于治疗风湿性关节炎症,其还可以提高内质网蛋白内稳态,延长细胞寿命。食品方面,N-乙酰葡萄糖胺作为膳食补充剂,具有保护软骨组织及骨关节炎的作用。此外,还可以应用于化妆品和饲料添加剂中,用途十分广泛。
N-乙酰葡萄糖胺传统的生产方法主要有甲壳素水解法及酶转化法,其中最主要的生产方法为甲壳素水解法,但该方法存在许多问题。水解法的原料为虾壳或蟹壳等,这些原料可能存在过敏原,容易引发海鲜过敏人群发生过敏反应;其次,随着市场对N-乙酰葡萄糖胺的需求量日益增长,甲壳素作为一种有限资源,可能会出现供不应求的现象;而且,在水解过程中,需要使用大量的酸和碱,会造成严重的环境污染。而使用酶解法,也存在原料有限、甲壳素酶造价昂贵、转化时间长、效率低等问题,不适合工业化生产。
大肠杆菌(Escherichia coli,E.coli)是一种革兰氏阴性、兼性厌氧细菌,由于其具有生长周期短、易于培养、代谢可塑性强、具有丰富的生物化学和生理学背景等特点,成为代谢工程和合成生物学的最佳宿主之一,也是工业中最重要的生物体之一。因此,国内外开展了利用大肠杆菌发酵生产N-乙酰葡萄糖胺的工作,利用代谢工程构建一系列代谢工程菌,通过优化发酵参数,获得生产N-乙酰葡萄糖胺的生产工艺。
发明内容
本发明的目的在于提供一种生产N-乙酰葡萄糖胺基因工程菌的构建方法,以及其在生产N-乙酰葡萄糖胺中的应用。
为解决上述技术问题,本发明提供了一种新型的发酵稳定、周期长、产物转化率高的生产N-乙酰葡萄糖胺的基因工程菌构建方法及应用。该基因工程菌首先通过RED同源重组技术敲除N-乙酰葡萄糖胺分解代谢利用途径,随后将表达氨基葡萄糖合成酶、氨基葡萄糖乙酰转移酶和磷酸甘油酸脱氢酶基因借助外源表达载体导入大肠杆菌表达。
所述氨基葡萄糖合成酶基因来源于大肠杆菌W3110,经全基因合成。
所述氨基葡萄糖乙酰转移酶基因来源于酵母菌S288C经全基因合成。
所述氨基葡萄糖合成酶、氨基葡萄糖乙酰转移酶基因导入大肠杆菌表达,是将这两个基因克隆到表达载体上后,以质粒的方式在大肠杆菌中表达。
上述高产N-乙酰葡萄糖胺基因工程菌的构建方法,具体步骤包括:敲除大肠杆菌中的manXYZ、nagBACDE基因簇,将glmS、GNA1基因分别克隆到表达载体上,转入敲除了manXYZ、nagBACDE基因簇的大肠杆菌中,获得高产N-乙酰葡萄糖胺基因工程菌。其中,该构建方法,更加具体的步骤包括:
(1)敲除大肠杆菌manXYZ、nagBACDE基因簇,获得基因簇manXYZ、nagBACDE失活的菌株;
(2)全基因合成编码氨基葡萄糖合成酶的glmS基因(SEQ ID NO.18),以及编码氨基葡萄糖乙酰转移酶的GNA1基因(SEQ ID NO.17),并克隆至表达载体;
(3)将步骤(2)获得的双基因表达载体转化入步骤(1)所得的菌株中,获得生产N-乙酰葡萄糖胺基因工程菌。
所述步骤(1)中,敲除是通过RED同源重组***,利用pKD46质粒表达RED重组酶来实现。
所述步骤(2)中,表达载体包括:质粒pBV220。
本发明还公开了一种生产N-乙酰葡萄糖胺基因工程菌的应用,即利用上述生产N-乙酰葡萄糖胺基因工程菌进行N-乙酰葡萄糖胺的生产,该生产方法包括步骤:
(1)挑取产N-乙酰葡萄糖胺基因工程菌的单菌落于LB液体培养基中,在30-35℃、220rpm好气培养8-10小时;其中,优选在31-33℃培养;
(2)将活化后的菌液接种于种子培养基,在30-35℃、220rpm好气培养10-15小时;其中,优选在31-33℃培养12-14小时;
(3)将培养好的种子液接种于发酵培养基,在30-35℃、220rpm发酵培养;其中,优选在31-33℃培养;
(4)培养至菌体浓度OD600为0.5时,升温至35-40℃继续培养,至发酵结束。其中,优选在36-38℃培养22-25小时;
所述步骤(2)中的种子培养基为LB液体培养基,配方如下:
酵母粉5g/L,胰蛋白胨10g/L,氯化钠10g/L。
所述步骤(3)中的发酵培养基配方如下:
硫酸钾1-5g/L,硫酸镁1-5g/L,磷酸氢二钠5-10g/L,磷酸氢二钾1-5g/L,磷酸二氢钾1-5g/L,氯化铵4-8g/L,氯化钠0.5-3g/L,柠檬酸4-8g/L,微量元素0.01-50mg/L,葡萄糖1-100g/L。微量元素包括:钙、锰、锌、铁、铝、钼。
本发明通过在大肠杆菌中构建一条新的生产N-乙酰葡萄糖胺的代谢通路(如图1所示),增强N-乙酰葡萄糖胺合成途径中限速酶基因表达,同时,阻断导致N-乙酰葡萄糖胺消耗与回流的基因,阻止N-乙酰葡萄糖胺的消耗与回流,使得工程菌株能积累N-乙酰葡萄糖胺。通过本发明的方法,得到一株基因工程大肠杆菌,该菌株可以利用葡萄糖合成N-乙酰葡萄糖胺,同时,副产物的累积较少,具有工业化生产的潜力。
附图说明
图1为大肠杆菌中N-乙酰葡萄糖胺合成分解代谢途径构建示意图。
具体实施方式
下面结合附图与具体实施方式对本发明作进一步详细的说明,但本发明的实施不仅限于此。
下面实施例中所使用的实验方法如无特殊说明,均为常规方法。下面实施例中所用的试剂、材料等,如无特殊说明,均可从商业途径得到。
man-P1:CCGAATTCCGATATCAAAGGAGGTAGCAAGT(SEQ ID NO.1);
man-P2:CCGAGCTCCTATTAACAATAATACGGGAGA(SEQ ID NO.2);
man-P3:CCCCTGCAGGGGCGTCTATCCTC(SEQ ID NO.3);
man-P4:CAAGCTTCGATATCCTTCCGACAAGTTCAT(SEQ ID NO.4);
man-P5:CCGAGCTCGTGTAGGCTGGAGCTGCTTC(SEQ ID NO.5);
man-P6:CCCCTGCAGATGGGAATTAGCCATGGTCC(SEQ ID NO.6);
nag-P1:CGGAATTCTGTTGGTTGTAGAGAGGTTGC(SEQ ID NO.7);
nag-P2:AGCAGCTCCAGCCTACACCCCTGATTTCGTGATTGTTG(SEQ ID NO.8);
nag-P3:GTGTAGGCTGGAGCTGCTTC(SEQ ID NO.9);
nag-P4:ATGGGAATTAGCCATGCTCC(SEQ ID NO.10);
nag-P5:ACCATGGCTAATTCCCATTGCTGGTTATGGGTGTTGTCT(SEQ ID NO.11);
nag-P6:CCCAAGCTTGGAAACGGGTGTGTACTGTGGTGTGGCT(SEQ ID NO.12);
PCR反应体系:Taq酶25μL,引物(10μmol/L)各1.5μL,模版1μL。
PCR反应过程:98℃ 10min,55℃ 15min,72℃ 5sec,30个循环。
将获得的PCR产物进行琼脂糖凝胶电泳,在2.0kb及3.0kb左右存在明显条带。
2、将RED重组酶表达质粒载体pKD46利用电击转化法转入大肠杆菌BL21(DE3),得到菌株BL21(DE3)/pKD46。
3、LB培养基中加入1%的L-***糖,30℃震荡培养菌株BL21(DE3)/pKD46至OD600达到0.5,制备感受态细胞。将步骤1制备好的DNA片段电转化入该感受态细胞内,涂布氯霉素抗性平板,得到转化子。
4、挑取转化子,用菌落PCR鉴定,菌落PCR鉴定引物如下:
manF:GCTGTTAGGCGAGCAGGAAA(SEQ ID NO.13);
manR:CCAGACATTGGCGAAGAAAA(SEQ ID NO.14);
nagF:TTCGTGGGCGAGAATGGC(SEQ ID NO.15);
nagR:CGATGATCTGACGGATAAAGT(SEQ ID NO.16);
挑取的转化子菌落经PCR扩增,能扩增出大小为1.0kb左右条带的菌落,即为manXYZ、nagBACDE双基因簇失活的菌株BL21(DE3)/Δman,Δnag。
实施例2glmS、GNA1基因双表达载体pBV220-glmS-GNA1的构建
1、根据基因序列(NCBI Gene ID:850529)全基因合成GNA1基因,两端加EcoR I、BamH I位点,具体序列如下所示(SEQ ID NO.17)。
2、根据大肠杆菌W3110基因组序列全基因合成glmS基因,两端加BamH I位点,具体序列如下所示(SEQ ID NO.18)。
3、利用常规分子生物学技术分别将GNA1、glmS基因连接至pBV220(淼灵质粒平台购买)的EcoR I、BamH I位点以及BgI II位点上,得到载体pBV220-glmS-GNA1。
实例3glmS、GNA1基因以质粒方式在BL21(DE3)/Δman,Δnag中表达
1、用液体LB培养基过夜培养含有载体pBV220-glmS-GNA1的大肠杆菌DH5α菌株,抽提质粒pBV220-glmS-GNA1。
2、培养大肠杆菌菌株BL21(DE3)/Δman,Δnag,制备感受态细胞,电击转化质粒载体pBV220-glmS-GNA1进入该菌株,获得基因工程菌株BL21(DE3)/Δman,Δnag/pBV220-glmS-GNA1,即为能生产N-乙酰葡萄糖胺的基因工程菌。
实例4工程菌株发酵实验
1、种子和发酵培养基(1L):
种子培养基:酵母粉5g,胰蛋白胨10g,氯化钠10g;
发酵培养基:硫酸钾2g,硫酸镁3g,磷酸氢二钠7g,磷酸氢二钾4g,磷酸二氢钾4g,氯化铵5g,氯化钠1g,柠檬酸5g,微量元素1mL,葡萄糖25mL。
其中,微量元素为:氯化钙60mg/L;硫酸锰10mg/L;硫酸锌50mg/L;硫酸亚铁5mg/L;三氯化铝5mg/L;钼酸钠3mg/L;硼酸0.5mg/L。
2、发酵过程
(1)挑取BL21(DE3)/Δman,Δnag/pBV220-glmS-GNA1-serA单菌落于4mL LB试管中,32℃、220rpm培养8-10小时;
(2)将活化后的菌液按照1%接种于50mL种子培养基,在32℃、220rpm培养13小时;
(3)将培养好的种子液按照1%接种于50mL发酵培养基,在32℃、220rpm发酵培养;
(4)培养至菌体浓度OD600为0.5时,升温至37℃继续培养,至发酵结束。
实例5发酵液中N-乙酰葡萄糖胺浓度的测定
将发酵液稀释至合适倍数后,采用HPLC检测,检测条件如下:
检测柱:Hedera NH2;
柱温:30℃;
流动相:乙腈与磷酸盐缓冲液的混合溶液(65∶35),流速为1mL/min;
检测波长:195nm;
经检测,24小时发酵后,发酵液中的N-乙酰葡萄糖胺浓度可达10g/L。
以上结果表明,经代谢工程技术构建的基因工程菌具有生产N-乙酰葡萄糖胺的能力,具备工业化生产的潜力。
以上已对本发明创造的较佳实例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可作出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (9)
1.一种生产N-乙酰葡萄糖胺的基因工程菌,其特征在于:所述的工程菌是通过将编码氨基葡萄糖合成酶、氨基葡萄糖乙酰转移酶和磷酸甘油酸脱氢酶基因导入大肠杆菌表达,并敲除大肠杆菌中的N-乙酰葡萄糖胺分解利用代谢途径酶的基因构建而成的重组大肠杆菌。
2.根据权利要求1所述的一种生产N-乙酰葡萄糖胺的基因工程菌,其特征在于:所述氨基葡萄糖合成酶基因来源于大肠杆菌W3110。
3.根据权利要求1所述的一种生产N-乙酰葡萄糖胺的基因工程菌,其特征在于:所述氨基葡萄糖乙酰转移酶基因来源于酵母菌S288C。
4.根据权利要求1所述的一种生产N-乙酰葡萄糖胺的基因工程菌,其特征在于:是将如SEQ ID NO.18所示的氨基葡萄糖合成酶的glmS基因、如SEQ ID NO.17所示的氨基葡萄糖乙酰转移酶的GNA1基因通过表达载体转化入敲除N-乙酰葡萄糖胺分解代谢利用途径酶的基因manXYZ、nagBACDE的大肠杆菌中表达。
5.根据权利要求4所述的一种生产N-乙酰葡萄糖胺的基因工程菌,其特征在于:表达载体为质粒pBV220。
6.根据权利要求4所述的一种生产N-乙酰葡萄糖胺的基因工程菌,其特征在于:敲除是通过RED同源重组***,利用pKD46质粒表达RED重组酶来实现。
7.根据权利要求4所述的一种生产N-乙酰葡萄糖胺的基因工程菌,其特征在于:宿主细胞为大肠杆菌BL21(DE3)。
8.一种如权利要求1至7任一所述的产N-乙酰葡萄糖胺基因工程菌在生产N-乙酰葡萄糖胺中的应用,其特征在于,生产N-乙酰葡萄糖胺的方法包括:
(1)挑取产N-乙酰葡萄糖胺基因工程菌的单菌落于LB液体培养基中,在30-35℃、220rpm好气培养8-10小时;
(2)将活化后的菌液接种于种子培养基,在30-35℃、220rpm好气培养10-15小时;
(3)将培养好的种子液接种于发酵培养基,在30-35℃、220rpm发酵培养;
(4)培养至菌体浓度OD600为0.5时,升温至35-40℃继续培养,至发酵结束。
9.根据权利要求12所述的一种生产N-乙酰葡萄糖胺的基因工程菌,其特征在于:所述基因工程菌种子培养基和发酵培养基为LB液体培养基和盐培养基,其中盐培养基配方为硫酸钾1-5g/L,硫酸镁1-5g/L,磷酸氢二钠5-10g/L,磷酸氢二钾1-5g/L,磷酸二氢钾1-5g/L,氯化铵4-8g/L,氯化钠0.5-3g/L,柠檬酸4-8g/L,微量元素0.01-50mg/L,葡萄糖1-100g/L。
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CN104293724A (zh) * | 2014-09-22 | 2015-01-21 | 上海工业生物技术研发中心 | 一种高效生产n-乙酰氨基葡萄糖的基因工程菌 |
CN104498517A (zh) * | 2014-11-29 | 2015-04-08 | 滨州市金朗生物科技有限公司 | 一种高产n-乙酰氨基葡萄糖大肠杆菌的构建及应用方法 |
CN109929791A (zh) * | 2019-04-15 | 2019-06-25 | 扬州日兴生物科技股份有限公司 | 一种积累氨基葡萄糖的重组大肠杆菌及其应用 |
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