CN111849953B - 一种苏氨酸脱水酶突变体的构建及应用 - Google Patents
一种苏氨酸脱水酶突变体的构建及应用 Download PDFInfo
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- CN111849953B CN111849953B CN202010563346.8A CN202010563346A CN111849953B CN 111849953 B CN111849953 B CN 111849953B CN 202010563346 A CN202010563346 A CN 202010563346A CN 111849953 B CN111849953 B CN 111849953B
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- threonine dehydratase
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Abstract
本发明涉及一种苏氨酸脱水酶突变体的构建及应用，其特征在于，所述的改造是将大肠杆菌来源的苏氨酸脱水酶中的441位脯氨酸替换为亮氨酸。本发明通过结构生物学的方法，预测了该位点的突变，通过将苏氨酸脱水酶进行位点突变，解除了异亮氨酸对苏氨酸脱水酶的反馈抑制。将上述突变的基因连接到表达质粒上，并在宿主菌如大肠杆菌中过量表达，显著提高了异亮氨酸的产量。与同样过量表达野生型苏氨酸脱水酶基因的质粒重组菌株相比，含有本发明苏氨酸脱水酶突变体的大肠杆菌产异亮氨酸能力提高一倍。
Description
技术领域
本发明属于生物技术领域，具体涉及一种用于L-异亮氨酸(isoleucine)、L-亮氨酸(leucine)及L-缬氨酸(valine)生产关键酶苏氨酸脱水酶突变体的构建及其应用，即通过改造源自肠杆菌科的酶苏氨酸脱水酶，并将其转入肠杆菌科中。使其能够将苏氨酸(葡萄糖)转化为α-酮戊二酸进一步转化为L-异亮氨酸、L-亮氨酸或L-缬氨酸，从而在肠杆菌中实现L-异亮氨酸、L-亮氨酸或L-缬氨酸的大量积累。
背景技术
L-异亮氨酸是人体八种必需氨基酸之一，同时又是三种支链氨基酸之一，是合成人体激素与酶类的原料，因其特殊的结构和功能，在人类生命代谢中占有特别重要的地位。目前，L-异亮氨酸的应用涉及医药品、保健品及精细化工品行业，其中以L-异亮氨酸为原料的产品包含配制营养型复合氨基酸输液制剂和保健品等，例如以L-异亮氨酸为主要原料生产的肝安干糖浆、治肝灵口服液，对治疗各种肝脏疾病具有显著疗效，因此其在食品和医药行业具有广泛的应用及商业价值。国际上氨基酸生产厂家主要有日本味之素、协和发酵、田边制药以及德国Degussa等4家公司，均以发酵法生产。日本在L-异亮氨酸产量、品质和技术水平均居世界领先地位。L-异亮氨酸产酸率为35g/L左右、提取率在70％。日本味之素公司占有全球医药及食品用氨基酸市场60％的市场份额，味之素公司巴西分公司也将于2006年正式生产L-异亮氨酸。近年来，随着国内各生产厂家产量的增加，我国L-异亮氨酸的产量增大，主要有湖北宜药集团公司宜昌三峡药业有限公司、无锡晶石氨基酸公司(年产120吨药用L-异亮氨酸)、南宁安力泰药业有限责任公司(年产量100吨)等。
我国生产L-异亮氨酸存在的主要技术水平问题为：生产规模较小、发酵技术落后、菌株产酸水平低发酵产物的提取技术落后等问题。其中最关键的技术问题为菌株产酸水平低，而菌株产酸低的根本原因为无法解决L-异亮氨酸生产途径中关键酶的反馈抑制问题。苏氨酸脱水酶催化苏氨酸脱氨生成α-酮戊二酸其受到了产物L-异亮氨酸的反馈抑制作用，其是L-异亮氨酸生产途径中的最主要的限速步骤。改造苏氨酸脱水酶以解除L-异亮氨酸对其的反馈抑制作用是解决菌株产酸水平低的关键。
发明内容
本发明的所要解决的问题是，通过计算机辅助设计获得一种不受异亮氨酸别构调控作用的苏氨酸脱水酶的突变基因，该突变基因所编码的突变型酶能够提高L-异亮氨酸产酸菌株的生产能力。本发明还提供编码这一突变基因的核苷酸或其核苷酸序列的片段、类似物或者衍生物。
本发明的苏氨酸脱水酶突变基因，其特征在于，所述的改造是将大肠杆菌来源的苏氨酸脱水酶中的441位点由脯氨酸突变为亮氨酸。
上述的苏氨酸脱水酶突变基因的核苷酸序列苷酸序列为SEQ ID NO:1。
本发明的更进一步要解决的问题是提供编码上述苏氨酸脱水酶突变体的基因，该基因的核苷酸序列为SEQ ID NO:2。
本发明的更进一步要解决的问题是考虑到密码子的简并性，编码本发明苏氨酸脱水酶突变体的基因序列可以有多种，但优选为埃希氏属的偏好密码子。
本发明的更进一步要解决的问题是本发明还包括有表达苏氨酸脱水酶突变体的重组质粒。
本发明的更进一步要解决的问题是本发明还包括有表达苏氨酸脱水酶突变体的重组质粒转化或转导至宿主细胞及其后代。
本发明的更进一步要解决的问题是本发明还包括有表达苏氨酸脱水酶突变体的重组质粒转化或转导至宿主细胞及其后代，提高宿主细胞异亮氨酸产量的方法。
附图说明
图1是野生型(WT)和突变型(P441L)TD在不同浓度异亮氨酸存在时的相对酶活
具体实施方式
下面结合实例对本发明做进一步说明，在实施例中未注明具体条件的实验方法，通常可按常规条件，如萨姆布鲁克(J.Sambrook)等编写的《分子克隆实验指南》中所述的条件，或按照设备或试剂生产厂商所建议的条件运行。
实施例1、苏氨酸脱水酶突变体的获得
1.苏氨酸脱水酶表达质粒及定点突变的实现
苏氨酸脱水酶在大肠杆菌中由ilvA基因通过PCR由大肠杆菌基因组获得。同样采用PCR的方法扩增质粒pET28a，获得共同的重复序列，采用In Fusion酶进行连接，获得质粒pET28a-ilvA。实验中采用的引物见表1。
基因的定点突变利用Stratagene系列
XL-Ⅱ定点突变试剂盒实现，通过引物P441L-F/P441L-R(见表1)对质粒pET28a-ilvA通过PCR引入突变位点P441L，即将ilvA 441位的脯氨酸替换为亮氨酸。获得的质粒经过PCR产物回收，除去PCR体系中的酶及缓冲体系中的盐离子后，采用Dpn1酶切1h，除去甲基化的模板质粒DNA。处理后的质粒进行化学转化，转入感受态细胞E.coli TransT1。正确突变质粒命名为pET28a-ilvA-P441L，携带的TD 441位突变体核苷酸序列为SEQ ID NO:2，翻译的氨基酸序列为SEQ ID NO:1。
表1：引物表
实施例2、ilvA突变体的体外效果检测
1、蛋白的表达及纯化
首先从平板上挑BL21(DE3)(pET28a-ilvA)(过量表达ilvA野生型基因)，BL21(DE3)(pET28a-ilvA)(过量表达ilvA-P441L突变型基因)的单菌落接于5ml含50μg/ml卡纳青霉素的LB培养基中，37度200rpm培养5小时至OD600为1.0左右。取2ml OD为1.0左右的一级种子液，转接于100ml含100μg/ml卡纳青霉素的新鲜LB培养基中，采用20度培养过夜。收集菌体后，超声波破碎菌体10分钟,超声1秒停3秒，用镍柱对蛋白进行纯化。通过BCA(Bicinchoninic Acid)法进行蛋白定量，用SDS-PAGE的方法确定蛋白纯度。结果表明蛋白纯度均大于90％。
2、体外实验(TD活力测定)
采用纯化后的蛋白测定异亮氨酸对其的别构调控程度。酶活反应体系为苏氨酸10mM，PLP 20mM，磷酸钾缓冲液50mM(pH＝7.5)，在测定调控效果时异亮氨酸浓度分别为10mM、2.5mM、1mM、0.5mM、0.2mM、0.1mM、0.01mM，以及适量的TD，总反应体系为0.2ml。置于96孔板上检测信号变化。各个数据均进行了3次平行试验及2次重复试验以保证试验结果的可靠性。
酶活检测结果如图1所示，在两者的初始酶活几乎相同的情况下，野生型TD在浓度为0.1mM以上异亮氨酸存在时，其酶活仅为无异亮氨酸的10％左右。而其突变型TD在低浓度异亮氨酸存在时，异亮氨酸由抑制剂变为了激活剂，而当10mM异亮氨酸存在时，其几乎不受异亮氨酸的反馈抑制作用。
实施例3：突变体的应用
1、发酵方法
将上述构建的菌株中在摇瓶中培养，培养基成分如下：
表2：培养基的配方组分
发酵液通过加入0.5M的HCl中和CaCO3后，测定OD600值。每隔3小时会加入50-100μL氨水调节pH≥7。在37℃和200rpm的条件下振荡培养至OD600约为0.8，加入诱导剂IPTG至终浓1mmol·L-1，继续培养28小时，离心收集菌体。
2、发酵结果：
在相同的发酵条件下，过量表达TD基因的三株菌，生长及耗糖情况基本一致，但在28小时残糖及苏氨酸耗尽时，过量表达突变型的TD可以产3.81g/L的异亮氨酸，而过量表达野生型的TD只能产1.23g/L的酸，突变体比野生型菌株脂肪酸产量提高200％。
表3：发酵28h，异亮氨酸终产量
上述结果表明，本发明的突变体的重组质粒转入大肠杆菌后，都具有显著提高异亮氨酸产量的作用，提高异亮氨酸的产量是提高异亮氨酸为前体的其它化合物产量的基础，因而具有很好的应用前景。
序列表
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gccctgcaca acattcgcgg cgaacggctg gcgcatattc tttccggtgc caacgtgaac 960
ttccacggcc tgcgctacgt ctcagaacgc tgcgaactgg gcgaacagcg tgaagcgttg 1020
ttggcggtga ccattccgga agaaaaaggc agcttcctca aattctgcca actgcttggc 1080
gggcgttcgg tcaccgagtt caactaccgt tttgccgatg ccaaaaacgc ctgcatcttt 1140
gtcggtgtgc gcctgagccg cggcctcgaa gagcgcaaag aaattttgca gatgctcaac 1200
gacggcggct acagcgtggt tgatctctcc gacgacgaaa tggcgaagct acacgtgcgc 1260
tatatggtcg gcggacgtcc atcgcatccg ttgcaggaac gcctctacag cttcgaattc 1320
ctggaatcac cgggcgcgct gctgcgcttc ctcaacacgc tgggtacgta ctggaacatt 1380
tctttgttcc actatcgcag ccatggcacc gactacgggc gcgtactggc ggcgttcgaa 1440
cttggcgacc atgaaccgga tttcgaaacc cggctgaatg agctgggcta cgattgccac 1500
gacgaaacca ataacccggc gttcaggttc tttttggcgg gttag 1545
<210> 3
<211> 22
<212> DNA/RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
tgagatccgg ctgctaacaa ag 22
<210> 4
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 4
agtcatgcta gccatatggc tgc 23
<210> 5
<211> 33
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggctagca tgactatggc tgactcgcaa ccc 33
<210> 7
<211> 35
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 7
agcagccgga tctcactaac ccgccaaaaa gaacc 35
<210> 6
<211> 30
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 6
cgcgcccggt gattcctgga attcgaagct 30
<210> 8
<211> 30
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 8
caggaatcac cgggcgcgct gctgcgcttc 30
Claims (6)
1.一种苏氨酸脱水酶突变体，是对苏氨酸脱水酶进行遗传改造获得的，其特征在于，所述的改造是将大肠杆菌来源的苏氨酸脱水酶中的441位点由脯氨酸突变为亮氨酸;所述突变体的氨基酸序列为SEQ ID NO:1。
2.一种基因，所述的基因用于编码权利要求1所述的苏氨酸脱水酶突变体。
3.如权利要求2所述的基因，其核苷酸序列为SEQ ID NO:2。
4.一种重组质粒，其特征在于所述的重组质粒用于表达权利要求1所述的苏氨酸脱水酶突变体。
5.一种提高异亮氨酸、亮氨酸或缬氨酸产量的方法，是在大肠杆菌中表达权利要求1所述的苏氨酸脱水酶突变体。
6.如权利要求5所述的方法，其特征在于是将权利要求4所述的重组质粒转入大肠杆菌中。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10132947A1 (de) * | 2000-09-12 | 2002-03-21 | Degussa | Für das rodA-Gen kodierte Nukleotidsequenzen |
| KR20180134695A (ko) * | 2017-06-09 | 2018-12-19 | 대상 주식회사 | L-로이신 생산능이 개선된 변이 균주 및 이를 이용한 l-로이신의 제조 방법 |
| CN109554324A (zh) * | 2018-12-14 | 2019-04-02 | 江南大学 | 一株产l-异亮氨酸的黄色短杆菌重组菌及其构建方法 |
| CN110305829A (zh) * | 2019-06-25 | 2019-10-08 | 天津科技大学 | 一种生产l-异亮氨酸的基因工程菌及其应用 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10132947A1 (de) * | 2000-09-12 | 2002-03-21 | Degussa | Für das rodA-Gen kodierte Nukleotidsequenzen |
| KR20180134695A (ko) * | 2017-06-09 | 2018-12-19 | 대상 주식회사 | L-로이신 생산능이 개선된 변이 균주 및 이를 이용한 l-로이신의 제조 방법 |
| CN109554324A (zh) * | 2018-12-14 | 2019-04-02 | 江南大学 | 一株产l-异亮氨酸的黄色短杆菌重组菌及其构建方法 |
| CN110305829A (zh) * | 2019-06-25 | 2019-10-08 | 天津科技大学 | 一种生产l-异亮氨酸的基因工程菌及其应用 |
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