CN116656754A - 一种异亮氨酸生产方法、乙酰羟酸合酶突变体及重组微生物与应用 - Google Patents
一种异亮氨酸生产方法、乙酰羟酸合酶突变体及重组微生物与应用 Download PDFInfo
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Abstract
本发明涉及微生物工程技术领域，具体公开了一种异亮氨酸生产方法、乙酰羟酸合酶突变体及重组微生物与应用。本发明的异亮氨酸生产方法，包括以重组微生物进行发酵培养的步骤，所述重组微生物相比于出发菌株，表达乙酰羟酸合酶ilvIH或乙酰羟酸合酶突变体ilvHF134L、ilvHF134V或ilvHF134A；所述重组微生物的出发菌株为棒杆菌。本发明生产异亮氨酸的方法，生产效率高，副产物少，适于工业化推广。
Description
技术领域
本发明涉及微生物工程技术领域，具体地说，涉及一种异亮氨酸生产方法、乙酰羟酸合酶突变体及重组微生物与应用。
背景技术
已知支链氨基酸(例如：缬氨酸、亮氨酸、异亮氨酸)在各种工业应用中用于生产各种产品，例如人类营养增强剂，动物饲料添加剂，医疗产品成分和化妆品成分。支链氨基酸的生产方法有三种：提取法、化学合成法、微生物发酵法。提取法和化学合成法由于原料来源受限制、生产成本高、污染环境，难以实现工业化生产。微生物发酵法具有原料成本低、反应条件温和、容易实现大规模生产等优点，是目前支链氨基酸最主要的方法。但因菌种的发酵性能仍较差，且副产物较高，导致转化率仍较低，不能满足大规模工业化生产的需求。
支链氨基酸的生物合成具有共同的前体物(丙酮酸)和相同的酶(乙酰羟酸合酶)，因此难以通过微生物发酵生物合成单一种类的支链氨基酸。乙酰羟酸合酶是支链氨基酸生物合成的第一个酶，也是支链氨基酸生物合成的关键酶，催化两分子丙酮酸生成乙酰乳酸(乙酰乳酸是缬氨酸和亮氨酸的前体物)，同时也催化a-酮基丁酸和丙酮酸生成a-乙酰羟基丁酸(a-乙酰羟基丁酸是异亮氨酸的前体物)。因此，乙酰羟酸合酶是支链氨基酸生物合成非常重要的酶。
乙酰羟酸合酶由两个亚基组成，分别是大亚基和小亚基，大亚基具有催化活性，小亚基起到反馈抑制的调控作用，受缬氨酸、亮氨酸、异亮氨酸的反馈抑制。在谷氨酸棒杆菌中，大亚基由ilvB基因编码，小亚基由ilvN基因编码；而在大肠杆菌中，大亚基由ilvI基因编码，小亚基由ilvH基因编码。中国专利CN 110506112 A、CN 110724679A报道了乙酰羟酸合酶突变体、包含其的微生物和用其生产L-支链氨基酸的方法，公开了乙酰羟酸合酶大亚基IlvB蛋白的突变体。中国专利CN201610647430.1报道了一种谷氨酸棒状杆菌及应用，公开了乙酰羟酸合酶小亚基IlvN蛋白的突变体。仍有必要进一步对支链氨基酸的生物合成进行研究，以提供一种新的提升支链氨基酸中异亮氨酸产率，并降低副产物缬氨酸、亮氨酸的含量的方法。
发明内容
本发明的目的是提供一种能够高效率生产支链氨基酸的重组微生物及方法。
本发明的技术方案如下：
一种异亮氨酸生产方法，其包括以重组微生物进行发酵培养的步骤，所述重组微生物相比于出发菌株，表达乙酰羟酸合酶ilvIH或乙酰羟酸合酶突变体ilvHF134L、ilvHF134V或ilvHF134A；所述重组微生物的出发菌株为棒杆菌；
乙酰羟酸合酶ilvIH的氨基酸序列如SEQ ID NO.21所示，乙酰羟酸合酶突变体ilvHF134L的氨基酸序列如SEQ ID NO.22，乙酰羟酸合酶突变体ilvHF134V的氨基酸序列如SEQID NO.23所示，乙酰羟酸合酶突变体ilvHF134A的氨基酸序列如SEQ ID NO.24所示。
本发明将大肠杆菌(Escherichia coli str.K-12substr.MG1655)来源的乙酰羟酸合酶(由ilvIH基因编码)引入出发菌株的细胞内，并在ilvH基因引入特异的突变位点，从而成功创造出了能够高效生产异亮氨酸的新微生物，进而将其应用到异亮氨酸的生产中，使得能够高效率地生成异亮氨酸，并且降低了副产物缬氨酸、亮氨酸的含量。
本发明的异亮氨酸生产方法中，乙酰羟酸合酶ilvIH的核苷酸序列如SEQ IDNO.17所示，乙酰羟酸合酶突变体ilvHF134L的核苷酸序列如SEQ ID NO.18，乙酰羟酸合酶突变体ilvHF134V的核苷酸序列如SEQ ID NO.19所示，乙酰羟酸合酶突变体ilvHF134A的核苷酸序列如SEQ ID NO.20所示。
本发明的异亮氨酸生产方法中，所述出发菌株为谷氨酸棒杆菌(Corynebacteriumglutamicum)、北京棒杆菌(Corynebacterium pekinense)或黄色短杆菌(Breviabacteriumflavum)。
本发明还提供一种乙酰羟酸合酶突变体，其以大肠杆菌野生型乙酰羟酸合酶的氨基酸序列为参考序列，所述乙酰羟酸合酶突变体含有第134位亮氨酸、缬氨酸或丙氨酸取代的突变。
本发明将乙酰羟酸合酶的第134位氨基酸，由苯丙氨酸(F)突变为除苯丙氨酸之外的其他氨基酸，优选为亮氨酸(L)、缬氨酸(V)或丙氨酸(A)，从而使表达该突变体的棒杆菌可以高效生产异亮氨酸。
当乙酰羟酸合酶基因ilvH第134位氨基酸由苯丙氨酸(F)突变为亮氨酸(L)、缬氨酸(V)、丙氨酸(A)后，异亮氨酸产量均有提升，缬氨酸、亮氨酸产量均有下降，尤其以苯丙氨酸(F)突变为丙氨酸(A)后效果最好。
上述突变位点可应用于谷氨酸棒杆菌，但不限于谷氨酸棒杆菌，也可用于如北京棒杆菌或黄色短杆菌等，用于产缬氨酸、异亮氨酸、亮氨酸等支链氨基酸或其衍生物。
本发明的乙酰羟酸合酶突变体，具有如SEQ ID NO.22-24任一所示的氨基酸序列。
本发明还提供一种编码上述乙酰羟酸合酶突变体的核酸。
优选，所述核酸具有如SEQ ID NO.18-20任一所示的核苷酸序列。
本发明另提供一种含有上述核酸的生物材料，所述生物材料为表达盒、载体或宿主细胞。
本发明又提供一种重组微生物，所述重组微生物表达乙酰羟酸合酶ilvIH或乙酰羟酸合酶突变体，所述乙酰羟酸合酶ilvIH如上所述，所述乙酰羟酸合酶突变体如上所述；所述重组微生物的出发菌株为棒杆菌；
优选，编码所述乙酰羟酸合酶突变体的核酸具有如SEQ ID NO.18-20任一所示的核苷酸序列；
和/或，所述重组微生物的出发菌株为谷氨酸棒杆菌、北京棒杆菌或黄色短杆菌。
ilvIH基因编码的乙酰羟酸合酶是支链氨基酸生物合成的第一个酶，也是支链氨基酸生物合成的关键酶，催化两分子丙酮酸生成乙酰乳酸(乙酰乳酸是缬氨酸和亮氨酸的前体物)，同时也催化a-酮基丁酸和丙酮酸生成a-乙酰羟基丁酸(a-乙酰羟基丁酸是异亮氨酸的前体物)。
本发明通过基因工程手段在出发菌株细胞内引入大肠杆菌(E.coli str.K-12substr.MG1655)来源的乙酰羟酸合酶(由ilvIH基因编码)，获得乙酰羟酸合酶增强菌株，使得所述微生物产异亮氨酸的能力与未修饰的菌株相比增强。
进一步，本发明通过对ilvH基因修饰，将乙酰羟酸合酶的第134位氨基酸由苯丙氨酸(F)突变为亮氨酸(L)、缬氨酸(V)或丙氨酸(A)，实现了对乙酰羟酸合酶的突变，获得乙酰羟酸合酶突变菌株，使得所述微生物产副产物缬氨酸和亮氨酸的能力下降，产异亮氨酸的能力与未修饰的菌株相比增强，最终提高了异亮氨酸的产量。
本发明还提供一种上述乙酰羟酸合酶突变体，或核酸，或生物材料，或重组微生物的如下任一种应用：
(1)在发酵生产缬氨酸、亮氨酸和/或异亮氨酸及其衍生物中的应用；
(2)在用于生产缬氨酸、亮氨酸和/或异亮氨酸及其衍生物的微生物遗传育种中的应用；
(3)在提高发酵生产异亮氨酸及其衍生物产量上的应用。
本发明的有益效果至少在于：
本发明的乙酰羟酸合酶增强菌株2-ilvIH的异亮氨酸产量较出发菌株显著提高，异亮氨酸产量为4.6g/L，比出发菌株产量提高1.9g/L，提高了70％。
本发明的乙酰羟酸合酶突变菌株2-ilvIHF134L、2-ilvIHF134V、2-ilvIHF134A的异亮氨酸产量和转化率较突变之前进一步提高，且副产物缬氨酸和亮氨酸产量大幅下降，菌株的生长正常，仍能够保持较好的生长性能。其中，乙酰羟酸合酶突变菌株2-ilvIHF134A表现最为突出，异亮氨酸产量为6.5g/L，比出发菌株产量提高3.8g/L，提高了141％；副产物缬氨酸产量为2.2g/L，较出发菌株缬氨酸产量下降63％；副产物亮氨酸产量为1.5g/L，较出发菌株亮氨酸产量下降46％。
本发明提供了一种新的高效生产异亮氨酸的方法，副产物少，生产效率高，适于工业化推广。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。下述实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。
本发明实施例中涉及的引物名称及序列如表1所示。
表1引物序列(SEQ ID No.1-16)
实施例1构建乙酰羟酸合酶增强菌株2-ilvIH
以MHZ-1012-2为出发菌株，在其细胞内引入大肠杆菌(E.coli str.K-12substr.MG1655)来源的乙酰羟酸合酶基因ilvIH，构建乙酰羟酸合酶增强菌株2-ilvIH。
野生型ilvH核苷酸序列如SEQ ID NO.17所示，氨基酸序列如SEQ ID NO.21所示。
本发明的出发菌株MHZ-1012-2为谷氨酸棒杆菌，分类命名：谷氨酸棒杆菌，Corynebacterium glutamicum，于2016年11月30日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏中心地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏编号为CGMCC No.13406，公开于中国专利CN201611250330.1。
具体构建方法如下：
1、质粒pK18mobsacB-ilvIH的构建
利用Phusion超保真聚合酶(New England BioLabs)，以出发菌株MHZ-1012-2的基因组为模板，以UP-1F/UP-1R为引物，制备重组片段UP-1；以DN-4F/DN-4R为引物，制备重组片段DN-1；以大肠杆菌MG1655(在NCBI的编号为：NC_000913.3)的基因组为模板，以ilvIH-2F/ilvIH-2R为引物，制备重组片段ilvIH-2，以ilvIH-3F/ilvIH-3R为引物，制备重组片段ilvIH-3；以重组片段ilvIH-2和重组片段ilvIH-3共为模板，以ilvIH-2F/ilvIH-3R为引物，制备重组片段ilvIH；以质粒pk18-mob-sacB为模板，以pk18-5F/pk18-5R为引物获得片段pk18-1，经琼脂糖凝胶回收试剂盒(天根)纯化，随后按照吉普森组装试剂盒配置体系进行反应，反应体系表2所示。
表2吉普森组装反应体系
| 组分 | UP-1 | DN-1 | ilvIH | pk18-1 | CE Buffer | CE Exnase | 无菌水 |
| 体积/μL | 1 | 1 | 1 | 2 | 4 | 2 | 9 |
将配制的反应体系于37℃反应30min，吸取10μL转化Trans1T1感受态细胞(TransGen Biotech)，挑取单克隆，通过菌落PCR鉴定插入的片段正确，进一步酶切鉴定得到片段插入pK18mobsacB的阳性克隆，最后将质粒送至金唯智生物科技有限公司进行测序，将所得到的测序正确的质粒命名为pK18mobsacB-ilvIH。
2、乙酰羟酸合酶增强菌株2-ilvIH的构建
将按上述步骤1所述方法构建获得的重组质粒pK18mobsacB-ilvIH转入出发菌株MHZ-1012-2中，在含有15mg/L的卡那霉素的选择培养基上选择交换重组子。培养的温度为30℃，倒置培养。将筛选获得的转化子过夜培养于普通液体脑心浸液培养基中，培养温度为30℃，回转摇床220rpm振荡培养。此培养过程中，转化子发生第二次重组，通过基因交换将载体序列从基因组中除去。将培养物做连续梯度稀释(10-2连续稀释至10-4)，稀释液涂布在含有10％蔗糖的普通固体脑心浸液培养基上，33℃静置培养48h。对在此培养基上长出的转化子进行鉴定。通过PCR扩增目的序列，核苷酸测序分析，获得目的菌株，命名为2-ilvIH。
实施例2构建乙酰羟酸合酶突变菌株2-ilvIHF134L、2-ilvIHF134V、2-ilvIHF134A
以MHZ-1012-2为出发菌株，在其细胞内引入经过修饰的乙酰羟酸合酶突变体，构建乙酰羟酸合酶突变菌株2-ilvIHF134L、2-ilvIHF134V、2-ilvIHF134A。
ilvHF134L突变体核苷酸序列如SEQ ID NO.18所示，氨基酸序列如SEQ ID NO.22所示。
ilvHF134V突变体核苷酸序列如SEQ ID NO.19所示，氨基酸序列如SEQ ID NO.23所示。
ilvHF134A突变体核苷酸序列如SEQ ID NO.20所示，氨基酸序列如SEQ ID NO.24所示。
具体构建方法如下。
1、质粒pK18mobsacB-ilvIHF134L、pK18mobsacB-ilvIHF134V、pK18mobsacB-ilvIHF134A的构建
按实施例1所述质粒pK18mobsacB-ilvIH的构建方法，将引物ilvIH-2R、ilvIH-3F分别替换为ilvIHF134L-2R、ilvIHF134L-3F，构建获得的质粒命名为pK18mobsacB-ilvIHF134L。
按实施例1所述质粒pK18mobsacB-ilvIH的构建方法，将引物ilvIH-2R、ilvIH-3F分别替换为ilvIHF134V-2R、ilvIHF134V-3F，构建获得的质粒命名为pK18mobsacB-ilvIHF134V。
按实施例1所述质粒pK18mobsacB-ilvIH的构建方法，将引物ilvIH-2R、ilvIH-3F分别替换为ilvIHF134A-2R、ilvIHF134A-3F，构建获得的质粒命名为pK18mobsacB-ilvIHF134A。
2、乙酰羟酸合酶突变菌株2-ilvIHF134L、2-ilvIHF134V、2-ilvIHF134A的构建
按上述实施例1所述乙酰羟酸合酶增强菌株2-ilvIH的构建方法，将质粒pK18mobsacB-ilvIH分别替换为pK18mobsacB-ilvIHF134L、pK18mobsacB-ilvIHF134V、pK18mobsacB-ilvIHF134A，依次获得目的突变菌株，分别命名为：2-ilvIHF134L、2-ilvIHF134V、2-ilvIHF134A。
实施例3摇瓶发酵验证
将上述构建得到的突变菌株与出发菌株MHZ-1012-2一同进行摇瓶发酵，比较生产性能。
1、摇瓶发酵使用的培养基如下：
(1)固体活化平板：BHI培养基37g/L，琼脂粉20g/L。
(2)种子培养基：葡萄糖20g/L，蛋白胨10g/L，酵母膏5g/L，尿素1.5g/L，KH2PO4 4g/L，K2HPO4 8g/L，MgSO4·7H2O 0.5g/L，生物素100μg/L，盐酸硫胺素1000μg/L，泛酸钙2000μg/L，烟酰胺2000μg/L，pH调至7.0。
(3)发酵培养基：葡萄糖100g/L、豆粕提取物9.75g/L、玉米浆干粉14.4g/L、MgSO4·7H2O 2g/L、KH2PO4·12H2O 2g/L、FeSO4·7H2O 0.01g/L、MnSO4·H2O0.01g/L、VB10.01g/L、(NH4)2SO4 50g/L，蒸馏水配制，pH调至7.0。
2、摇瓶发酵的方法如下：
(1)种子培养：在平板上刮取1环菌体接种到含有50ml种子培养基的500ml锥形瓶中，然后在30℃条件下、以110rpm振荡培养15-17h至OD562值为16-18；
(2)摇瓶发酵：按10％的接种量转接至装有25ml发酵培养基的500ml锥形瓶中，然后在30℃条件下、以135rpm振荡培养持续48小时。
(3)使用分光光度计在562纳米波长下检测菌体浓度，记为OD562；使用HPLC检测发酵液中氨基酸的含量。发酵结果如表3所示。
表3摇瓶发酵结果
注：*表示与出发菌株相比具有显著差异(P＜0.05)。
以上结果表明，出发菌株MHZ-1012-2的异亮氨酸产量仅为2.7g/L，乙酰羟酸合酶增强菌株2-ilvIH的异亮氨酸产量和转化率较出发菌株显著提高，异亮氨酸产量为4.6g/L，比出发菌株产量提高1.9g/L，提高了70％；乙酰羟酸合酶突变菌株2-ilvIHF134L、2-ilvIHF134V、2-ilvIHF134A的异亮氨酸产量和转化率较突变前进一步提高，且副产物缬氨酸和亮氨酸产量大幅下降，菌株的生长正常，仍能够保持较好的生长性能。其中，乙酰羟酸合酶突变菌株2-ilvIHF134A表现最为突出，异亮氨酸产量为6.5g/L，比出发菌株产量提高3.8g/L，提高了141％；副产物缬氨酸产量为2.2g/L，较出发菌株缬氨酸产量下降63％；副产物亮氨酸产量为1.5g/L，较出发菌株亮氨酸产量下降46％。
由此可见，本发明提供的乙酰羟酸合酶突变体及乙酰羟酸合酶突变菌株对目标产物异亮氨酸的产量提升具有显著的促进作用，对副产物缬氨酸、亮氨酸产量有显著的降低作用。该乙酰羟酸合酶突变体及其重组微生物为产缬氨酸、亮氨酸、异亮氨酸及以其为前体的衍生物的生产菌株的构建提供借鉴。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 廊坊梅花生物技术开发有限公司
<120> 一种异亮氨酸生产方法、乙酰羟酸合酶突变体及重组微生物与应用
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Claims (10)
1.一种异亮氨酸生产方法，其特征在于，包括以重组微生物进行发酵培养的步骤，所述重组微生物相比于出发菌株，表达乙酰羟酸合酶ilvIH或乙酰羟酸合酶突变体ilvHF134L、ilvHF134V或ilvHF134A；所述重组微生物的出发菌株为棒杆菌；
乙酰羟酸合酶ilvIH的氨基酸序列如SEQ ID NO.21所示，乙酰羟酸合酶突变体ilvHF134L的氨基酸序列如SEQ ID NO.22，乙酰羟酸合酶突变体ilvHF134V的氨基酸序列如SEQ IDNO.23所示，乙酰羟酸合酶突变体ilvHF134A的氨基酸序列如SEQ ID NO.24所示。
2.根据权利要求1所述的异亮氨酸生产方法，其特征在于，乙酰羟酸合酶ilvIH的核苷酸序列如SEQ ID NO.17所示，乙酰羟酸合酶突变体ilvHF134L的核苷酸序列如SEQ ID NO.18，乙酰羟酸合酶突变体ilvHF134V的核苷酸序列如SEQ ID NO.19所示，乙酰羟酸合酶突变体ilvHF134A的核苷酸序列如SEQ ID NO.20所示。
3.根据权利要求1或2所述的异亮氨酸生产方法，其特征在于，所述出发菌株为谷氨酸棒杆菌、北京棒杆菌或黄色短杆菌。
4.一种乙酰羟酸合酶突变体，其特征在于，以大肠杆菌野生型乙酰羟酸合酶的氨基酸序列为参考序列，所述乙酰羟酸合酶突变体含有第134位亮氨酸、缬氨酸或丙氨酸取代的突变。
5.根据权利要求4所述的乙酰羟酸合酶突变体，其特征在于，具有如SEQ ID NO.22-24任一所示的氨基酸序列。
6.编码权利要求4或5所述的乙酰羟酸合酶突变体的核酸。
7.根据权利要求6所述的核酸，其特征在于，具有如SEQ ID NO.18-20任一所示的核苷酸序列。
8.含有权利要求6或7所述的核酸的生物材料，所述生物材料为表达盒、载体或宿主细胞。
9.一种重组微生物，其特征在于，所述重组微生物表达乙酰羟酸合酶ilvIH或乙酰羟酸合酶突变体，所述乙酰羟酸合酶ilvIH如权利要求1或2所述，所述乙酰羟酸合酶突变体如权利要求4或5所述；所述重组微生物的出发菌株为棒杆菌；
优选，编码所述乙酰羟酸合酶突变体的核酸具有如SEQ ID NO.18-20任一所示的核苷酸序列；
和/或，所述重组微生物的出发菌株为谷氨酸棒杆菌、北京棒杆菌或黄色短杆菌。
10.权利要求4或5所述的乙酰羟酸合酶突变体，或权利要求6或7所述的核酸，或权利要求8所述的生物材料，或权利要求9所述的重组微生物的如下任一种应用：
(1)在发酵生产缬氨酸、亮氨酸和/或异亮氨酸及其衍生物中的应用；
(2)在用于生产缬氨酸、亮氨酸和/或异亮氨酸及其衍生物的微生物遗传育种中的应用；
(3)在提高发酵生产异亮氨酸及其衍生物产量上的应用。
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