CN109554324A - 一株产l-异亮氨酸的黄色短杆菌重组菌及其构建方法 - Google Patents

一株产l-异亮氨酸的黄色短杆菌重组菌及其构建方法 Download PDF

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CN109554324A
CN109554324A CN201811535445.4A CN201811535445A CN109554324A CN 109554324 A CN109554324 A CN 109554324A CN 201811535445 A CN201811535445 A CN 201811535445A CN 109554324 A CN109554324 A CN 109554324A
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张伟国
徐建中
王壮壮
魏佳
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Abstract

本发明公开了一株产L‑异亮氨酸的黄色短杆菌重组菌及其构建方法，属于基因工程技术领域。本发明利用基因工程手段强化了黄色短杆菌中乙酰羟基酸合酶、二羟酸还原异构酶和支链氨基酸转氨酶，并异源表达tdcB编码的分解代谢苏氨酸脱水酶，从而构建了一株L‑异亮氨酸高产菌株B.flavum I12/pEC‑XK99E‑ilvBNCE‑tdcB。该重组菌株积累异亮氨酸的产量提高到22.65g/L，比出发菌株提高了25.48％，最大生物量为达到20.95g DCW/L。此发明可将赖氨酸途经碳源转向L‑异亮氨酸，为构建L‑异亮氨酸高产菌株提供崭新的思路。

Description

一株产L-异亮氨酸的黄色短杆菌重组菌及其构建方法
技术领域
本发明涉及一株产L-异亮氨酸的黄色短杆菌重组菌及其构建方法，属于基因工程技术领域。
背景技术
L-异亮氨酸，别名L-异白氨酸，是人体八种必需氨基酸之一。广泛应用于食品、饲料、化妆品与医药行业。因此，选育一株L-异亮氨酸产量高，代谢稳定的菌株，对生产具有重要作用。代谢工程为L-异亮氨酸生产提供了有效的改造途径，但是现有的通过代谢途径改造的L-异亮氨酸生产菌依然存在产量低的问题。
L-异亮氨酸合成途径中第一关键限速酶是苏氨酸脱水酶，苏氨酸脱水酶又分为ilvA编码的合成代谢苏氨酸脱水酶和tdcB编码的分解代谢苏氨酸脱水酶，基因ilvA被广泛应用L-异亮氨酸产生菌的构建，但ilvA编码的合成代谢苏氨酸脱水酶收到L-异亮氨酸的反馈抑制，提高水平有限。由ilvBN编码乙酰羟基酸合成酶为异亮氨酸合成途径中第二限制酶，ilvBN基因催化2-酮丁酸和丙酮酸生成三种支链氨基酸。但是关键酶对生长代谢的影响较大，如何构建一株既能不影响生长，又能提高L-异亮氨酸产量的菌株，成为亟待研究的课题。
发明内容
为解决上述问题，本发明首次将来源于E.coli W3110中的tdcB异源表于产L-异亮氨酸的黄色短杆菌中。并通过提高ilvBNCE转录水平，提高L-异亮氨酸合成途径碳通量，以期提高L-异亮氨酸产量。本发明的目的是：解除L-异亮氨酸对合成途径关键酶的反馈抑制，并提高合成途径碳通量。构建一株L-异亮氨酸高产菌株，命名为：Brevibacterium flavumI12/pEC-XK99E-ilvBNCE-tdcB。
本发明的第一个目的是提供一株产L-异亮氨酸的黄色短杆菌重组菌，所述重组菌异源表达了大肠杆菌来源的分解代谢苏氨酸脱水酶，且过表达了乙酰羟基酸合酶、二羟酸还原异构酶和支链氨基酸转氨酶。
进一步地，所述的分解代谢苏氨酸脱水酶的氨基酸序列如SEQ ID NO.1所示。来源于大肠杆菌Escherichia coli W3110。
进一步地，所述的乙酰羟基酸合酶和二羟酸还原异构酶氨基酸序列如SEQ IDNO.2所示。
进一步地，所述的支链氨基酸转氨酶的氨基酸序列如SEQ ID NO.3所示。
进一步地，所述的重组菌是以黄色短杆菌B.flavumI12为宿主。黄色短杆菌B.flavumI12根据《L-异亮氨酸高产菌选育及其培养基优化》王壮壮等，生物技术通报，2019，35(1)中记载。
进一步地，所述的重组菌是以pEC-XK99E作为表达载体。
本发明的第二个目的是提供所述的黄色短杆菌重组菌的构建方法，包括如下步骤：
(1)将编码乙酰羟基酸合酶的基因ilvBN和编码二羟酸还原异构酶的基因ilvC与pEC-XK99E载体进行连接，构建质粒pEC-XK99E-ilvBNC；
(2)将编码支链氨基酸转氨酶的基因ilvE与重组质粒pEC-XK99E-ilvBNC进行连接，构建质粒pEC-XK99E-ilvBNCE；
(3)将编码分解代谢苏氨酸脱水酶的基因tdcB与重组质粒pEC-XK99E-ilvBNCE进行连接，构建质粒pEC-XK99E-ilvBNCE-tdcB；
(4)将重组质粒pEC-XK99E-ilvBNCE-tdcB转化到黄色短杆菌B.flavumI12中，构建得到黄色短杆菌重组菌。
本发明的第三个目的是提供所述的黄色短杆菌重组菌在发酵生产L-异亮氨酸中的应用。
进一步地，所述的发酵是在28～32℃，转速80～100r/min，pH 6.5～7.5条件下进行发酵。
本发明的第四个目的是提供所述的黄色短杆菌重组菌在饲料工业、医药工业或食品工业中的应用。
本发明的有益效果是：
利用基因工程手段强化了黄色短杆菌中乙酰羟基酸合酶、二羟酸还原异构酶和支链氨基酸转氨酶，并异源表达tdcB编码的分解代谢苏氨酸脱水酶，从而构建了一株L-异亮氨酸高产菌株B.flavum I12/pEC-XK99E-ilvBNCE-tdcB。该重组菌株积累异亮氨酸的产量提高到22.65g/L，比出发菌株提高了25.48％，最大生物量为达到20.95g DCW/L。此发明可将赖氨酸途经碳源转向L-异亮氨酸，为构建L-异亮氨酸高产菌株提供崭新的思路。
附图说明
图1为重组质粒的酶切验证电泳图；泳道说明：M:DNA marker DL10 000；1:pEC-XK99E-ilvBNC单酶切；2:pEC-XK99E-ilvBNC双酶切产物ilvBNCr；3:pEC-XK99E-ilvBNCE单酶切产物ilvE；4:pEC-XK99E-ilvBNCE-tdcB单酶切；5:pEC-XK99E-ilvBNCE-tdcB双酶切产物tdcB；
图2为重组菌蛋白表达图，永道说明：M:Protein Ladder；1:B.flavum I12/pEC-XK99E；2:B.flavum I12/pEC-XK99E-ilvBNC；3:B.flavum I12/pEC-XK99E-ilvBNCE；4:B.flavum I12/pEC-XK99E-ilvBNCE-tdcB；
图3为基因改造对L-异亮氨酸发酵的影响；A:B.flavum I12/pEC-XK99E；B：B.flavum I12/pEC-XK99E-ilvBNC；C：B.flavum I12/pEC-XK99E-ilvBNCE；D：B.flavumI12/pEC-XK99E-ilvBNCE-tdcB。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
L-异亮氨酸产量测定采用高效液相色谱法：取800μL发酵液，8 000r/min离心3min去除菌体，经孔径0.22μm的滤膜过滤所得滤液，用高效液相色谱Agilent 1200分析测定其中L-异亮氨酸含量。色谱柱：Agilent ZORBAX Eclipse AAA；检测器：DAD检测器；流动相：流动相A，5mL/L四氢呋喃，5g/L无水乙酸钠，200μg/L三乙胺，冰醋酸调pH至7.2；流动相B，25g/L无水乙酸钠：甲醇：乙腈＝1：2：2；色谱条件：柱温40℃，流速1.0mL/min，进样体积10.0μL，波长338nm。
表1.PCR扩增所需引物序列(下划线为酶切位点)
实施例1：重组表达载体pEC-XK99E-ilvBNC的构建
根据NCBI中C.glutamicum ATCC 13032全基因组核酸序列中的ilvBNC基因序列，其中，ilvBNC基因序列是将ilvBN基因与ilvC基因连接得到，上游引物的5’端加入限制性内切酶Sma I，在基因下游加入Xba I酶切位点，合成上下游引物ilvBNC-F和ilvBNC-R，以C.glutamicum ATCC 13032全基因组为模板PCR获得基因ilvBNC基因片段，经胶回收试剂盒纯化后，将片段与质粒pEC-XK99E用相同的限制性内切酶酶切后，过夜酶连并转化入JM109感受态细胞，经过菌落PCR验证选取可能正确的菌落，提取质粒酶切电泳验证，得到正确目的条带后(7 018和2 322bp 2个片段)，提交给通用生物系统(安徽)有限公司测序。pEC-XK99E-ilvBNC构建完成。
实施例2：重组表达载体pEC-XK99E-ilvBNCE的构建
根据NCBI中C.glutamicum ATCC 13032全基因组核酸序列中的ilvE基因序列，基因上游引物的5’端加入限制性内切酶Xba I和谷氨酸棒杆菌SD识别序列GAAAGGAGATATACC，在基因下游加入Xba I酶切位点，PCR获得基因ilvE基因片段，经去磷酸化、胶回收试剂盒纯化后，将片段与质粒pEC-XK99E-ilvBNC用相同的限制性内切酶酶切后，过夜酶连并转化入JM109感受态细胞，经过菌落PCR验证选取可能正确的菌落，提取质粒酶切电泳验证，得到正确目的条带后(9 340和681bp 2个片段)，提交给通用生物系统(安徽)有限公司测序。pEC-XK99E-ilvBNCE构建完成。
实施例3：重组表达载体pEC-XK99E-ilvBNCE-tdcB的构建
根据NCBI中E.coli W3110全基因组核酸序列中的tdcB基因序列，基因上下游引物的5’端加入限制性内切酶EcoR I和Kpn I酶切位点，PCR获得基因tdcB基因片段，经胶回收试剂盒纯化后，将片段与质粒pEC-XK99E-ilvBNCE用相同的限制性内切酶酶切后，过夜酶连并转化入JM109感受态细胞，经过菌落PCR验证选取可能正确的菌落，提取质粒酶切电泳验证，得到正确目的条带后(10021和990bp)，提交给通用生物系统(安徽)有限公司测序。pEC-XK99E-ilvBNCE-tdcB构建完成。对实施例1～3的重组质粒进行酶切验证，结果如图1所示，结果显示3个质粒单、双酶切后，经琼脂糖凝胶电泳检测，条带大小均与预测结果一致。
实施例4：重组菌株B.flavum I12/pEC-XK99E-ilvBNCE-tdcB的构建
将上述步骤中验证正确的质粒pEC-XK99E-ilvBNCE-tdcB电击转入B.flavumI12感受态，并筛选目的重组菌株B.flavum I12/pEC-XK99E-ilvBNCE-tdcB。筛选目的重组菌株的步骤如下：经含有50μg·mL-1卡那霉素的LBG固体培养基于30℃培养筛选获得第一次重组转化子。再挑取平板上长出的菌落进行菌落PCR验证单菌落。若菌落PCR有正确目的条带，则提取重组菌质粒，进一步酶切验证是否有正确目的条带。最终鉴定正确的转化子命名为：B.flavum I12/pEC-XK99E-ilvBNCE-tdcB(命名为D)。
参照上述方法，将实施例1和实施例2的重组质粒分别电击转入B.flavumI12感受态，筛选得到重组菌B.flavum I12/pEC-XK99E-ilvBNC(命名为B)和重组菌B.flavum I12/pEC-XK99E-ilvBNCE(命名为C)。
实施例5：重组菌株B.flavum I12/pEC-XK99E-ilvBNCE-tdcB中蛋白表达重组菌B.flavum I12/pEC-XK99E-ilvBNCE-tdcB、重组菌B.flavum I12/pEC-XK99E-ilvBNC和重组菌B.flavum I12/pEC-XK99E-ilvBNCE分别于30℃培养，至OD560值0.6-0.8时加入终浓度为1.0mmol/L的IPTG诱导目的蛋白表达，8h后收集菌体，对菌体进行超声破碎，并通过SDS-PAGE电泳分析。以出发菌株B.flavum I12/pEC-XK99E(命名为A)为空白对照，检验目的基因在受体菌中的表达情况。重组菌的蛋白表达图如图2所示，与对照组相比，B.flavum I12/pEC-XK99E-ilvBNC分别在66、37和19kD处出现特异性蛋白条带，这与ilvB、ilvN和ilvC基因编码的蛋白理论分子质量相一致。重组菌B.flavum I12/pEC-XK99E-ilvBNCE出现4条特异性条带，分别在66、37、19和25kD附近处，这与ilvB、ilvN、ilvC和ilvE基因编码的蛋白理论分子质量相一致。而重组菌B.flavum I12/pEC-XK99E-ilvBNCE-tdcB表达时，分别在66、37、19、25和36kD处有明显的加粗变深的条带，说明靶基因在重组细菌中成功表达。
实施例6：重组菌B.flavumI12/pEC-XK99E-ilvBNCE-tdcB摇瓶发酵
操作实验流程：菌种超低温冰箱-80℃保存→小摇瓶活化→平板活化→摇瓶培养。
菌种活化平板：每个平板用0.2mL移液器吸取0.12mL菌液涂布平板，于30℃恒温培养48h；摇瓶培养(培养体积50mL/500mL三角瓶)：温度30℃，转速90r/min，pH 6.8-7.2，培养时间72h。
发酵培养基组成(g/L)：葡萄糖110-150，(NH4)2SO430-50，玉米浆10，KH2PO41，MgSO4·7H2O 0.0.5，生物素50μg/L，CaCO3适量。
L-异亮氨酸产量和生物量结果如图3所示，重组菌株B.flavum I12/pEC-XK99E-ilvBNCE-tdcB积累异亮氨酸的产量提高到22.65g/L，比出发菌株提高了25.48％，最大生物量为达到20.95g DCW/L。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
<110> 江南大学
<120> 一株产L-异亮氨酸的黄色短杆菌重组菌及其构建方法
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Claims (10)

1.一株产L-异亮氨酸的黄色短杆菌重组菌，其特征在于，异源表达了大肠杆菌来源的分解代谢苏氨酸脱水酶，且过表达了乙酰羟基酸合酶、二羟酸还原异构酶和支链氨基酸转氨酶。
2.根据权利要求1所述的黄色短杆菌重组菌，其特征在于，所述的分解代谢苏氨酸脱水酶的氨基酸序列如SEQ ID NO.1所示。
3.根据权利要求1所述的黄色短杆菌重组菌，其特征在于，所述的乙酰羟基酸合酶和二羟酸还原异构酶的氨基酸序列如SEQ ID NO.2所示。
4.根据权利要求1所述的黄色短杆菌重组菌，其特征在于，所述的支链氨基酸转氨酶的氨基酸序列如SEQ ID NO.3所示。
5.根据权利要求1所述的黄色短杆菌重组菌，其特征在于，所述的重组菌是以黄色短杆菌B.flavumI12为宿主。
6.根据权利要求1所述的黄色短杆菌重组菌，其特征在于，所述的重组菌是以pEC-XK99E作为表达载体。
7.一种权利要求1～6任一项所述的黄色短杆菌重组菌的构建方法，其特征在于，包括如下步骤：
(1)将编码乙酰羟基酸合酶的基因ilvBN和编码二羟酸还原异构酶的基因ilvC与pEC-XK99E载体进行连接，构建质粒pEC-XK99E-ilvBNC；
(2)将编码支链氨基酸转氨酶的基因ilvE与重组质粒pEC-XK99E-ilvBNC进行连接，构建质粒pEC-XK99E-ilvBNCE；
(3)将编码分解代谢苏氨酸脱水酶的基因tdcB与重组质粒pEC-XK99E-ilvBNCE进行连接，构建质粒pEC-XK99E-ilvBNCE-tdcB；
(4)将重组质粒pEC-XK99E-ilvBNCE-tdcB转化到黄色短杆菌B.flavumI12中，构建得到黄色短杆菌重组菌。
8.权利要求1～6任一项所述的黄色短杆菌重组菌在发酵生产L-异亮氨酸中的应用。
9.根据权利要求8所述的应用，其特征在于，所述的发酵是在28～32℃，转速80～100r/min，pH 6.5～7.5条件下进行发酵。
10.权利要求1～6任一项所述的黄色短杆菌重组菌在饲料工业、医药工业或食品工业中的应用。
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CN111849953A (zh) * 2020-06-19 2020-10-30 滨州医学院 一种苏氨酸脱水酶突变体的构建及应用
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