CN112094829B - 一种酶活性改变的氨基脱氧分支酸合成酶突变体t426i及其应用 - Google Patents
一种酶活性改变的氨基脱氧分支酸合成酶突变体t426i及其应用 Download PDFInfo
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Abstract
本发明公开了一种酶活性改变的氨基脱氧分支酸合成酶突变体T426I及其应用,属于遗传工程和发酵工程领域。本发明将亲本谷氨酸棒杆菌△SSAAI(菌株保藏名称:△SSAAI,保藏号CGMCC No 15170)中氨基脱氧分支酸合成酶的T426位点突变成了异亮氨酸,得到的突变体酶活下降了15.2%。将该突变体构建的基因工程菌进行发酵,发现L‑丝氨酸产量提高了18.75%。该发明通过点突变有效改变氨基脱氧分支酸合成酶活力,为构建高效生产丝氨酸基因工程菌创造了条件,具有很好的工业应用价值和前景。
Description
技术领域
本发明属于遗传工程和发酵工程技术领域,具体涉及一种酶活性改变的氨基脱氧分支酸合成酶突变体T426I及其应用。
背景技术
谷氨酸棒杆菌(Corynebacterium glutamicum)是一种典型的原核模式菌株,具有遗传背景清晰,代谢改造操作方便等优势;也被FDA认证为GRAS(General Regarded AsSafe)菌株,是发酵生产氨基酸最为重要的微生物。在谷氨酸棒杆菌中,通常情况下磷酸甘油酸脱氢酶(PGDH)、磷酸丝氨酸转氨酶(PSAT)及磷酸丝氨酸磷酸化酶(PSP)参与L-丝氨酸的合成,而丝氨酸羟甲基转移酶(SHMT)及丝氨酸脱氢酶(SerDH)则参与L-丝氨酸的降解反应,四氢叶酸(THFA)是L-丝氨酸分解代谢途径中SHMT的辅酶,Michael Stolz等(MichaelStolz,Petra Peters-Wendisch,Lothar Eggeling,et al.Reduced Folate Supply as aKey to Enhanced L-Serine Production by Corynebacterium glutamicum.Applied andEnvironmental Microbiology,2007,73(3):750-755)研究表明,通过阻断叶酸途径可减少四氢叶酸的水平,然后外源添加叶酸可以提高模式菌株C.glutamicum ATCC 13032产L-丝氨酸的能力。而由于pabAB编码的氨基脱氧分支酸合成酶(ADC synthase)是叶酸组成部分对氨基苯甲酸(PABA)合成途径的关键酶(Matthews RG.One-carbon metabolism InEscherichia coli and Salmonella Cellular and Molecular Biology Volume1.Second edition.Edited by:Neidhardt FC,Curtiss R 3rd,Ingraham JL,Lin ECC,LowKB,Magasanik B,Reznikoff WS,Riley M,Schaechter M,Umbarger HE.Washington DC,ASM Press;1996:600-611),推测氨基脱氧分支酸合成酶活力的差异会直接引起胞内叶酸含量的变化,从而影响SHMT的辅酶四氢叶酸的水平,也就是说氨基脱氧分支酸合成酶(编码基因pabAB)与L-丝氨酸降解相关,氨基脱氧分支酸合成酶可促进L-丝氨酸降解,相对应的,降低其活力则可能提高丝氨酸产量。
然而如何通过对氨基脱氧分支酸合成酶进行定向改造来提高丝氨酸产量,目前未见报道,因此,本发明针对此问题提供了一种可操作性方案,对于利用谷氨酸棒杆菌生产丝氨酸具有重要意义。
发明内容
发明目的:本发明要解决的技术问题是提供一种改变氨基脱氧分支酸合成酶的方法及重组菌,为代谢工程改造高产丝氨酸菌株奠定基础。本发明在产丝氨酸菌株中对该酶的T426位点进行突变成为T426I突变株,改变了氨基脱氧分支酸合成酶的活性,明显促进了丝氨酸的积累。
技术方案:本发明的第一个目的是提供一种氨基脱氧分支酸合成酶突变体,所述突变体是在氨基酸序列如SEQ ID NO.1所示的亲本谷氨酸棒杆菌ΔSSAAI中氨基脱氧分支酸合成酶的基础上,对第426位的氨基酸进行了突变。
所述突变,在本发明的一种实施方式中,是进行了饱和突变。
所述突变体,在本发明的一种实施方式中,是将第426位的酪氨酸突变成为异亮氨酸。优选的,所述氨基脱氧分支酸合成酶突变体的氨基酸序列如SEQ ID NO.5所示。
本发明的另一个目的是提供一种编码所述亲本谷氨酸棒杆菌ΔSSAAI中氨基脱氧分支酸合成酶的编码基因,在本发明的一种实施方式中,其核苷酸序列是SEQ ID NO.2所示的序列。
本发明的另一个目的是提供一种所述突变体的基因工程菌。
所述基因工程菌,在本发明的一种实施方式中,是谷氨酸棒杆菌突变株。
本发明的另一个目的是提供所述基因工程菌的应用,在本发明的一种实施方式中,是用于发酵培养促进丝氨酸的积累。
所述基因工程菌发酵培养的方法,在本发明的一种实施方式中,是:将基因工程菌种子液即T426I突变株种子液,接种至发酵培养基,于30℃、220rpm条件下进行培养。
作为优选方案,在本发明的一种实施方式中,具体是将30℃、220rpm下培养24h的基因工程菌种子液以5%的接种量接种到发酵培养基中,于30℃、220rpm条件下培养96h。
本发明还有一个目的是本发明还要求保护所述突变体以及所述突变体发酵获得的丝氨酸在食品、饲料、化工、药物制备等方面的应用。
有益效果:本发明提供的,与现有技术相比,具有以下优势:对亲本谷氨酸棒杆菌ΔSSAAI中氨基脱氧分支酸合成酶的T426位点进行饱和突变,获得了氨基脱氧分支酸合成酶比酶活下降的突变体T426I,其比酶活较出发菌株ΔSSAAI下降了15.2%;而含有突变体的基因工程菌丝氨酸的产量提高18.75%。该发明通过点突变有效改变氨基脱氧分支酸合成酶活力,为构建高效生产丝氨酸基因工程菌创造了条件,具有很好的工业应用价值和前景。
附图说明
图1为不同氨基脱氧分支酸合成酶突变菌株丝氨酸产量对比图。
具体实施方式
下面结合附图和实施例对本发明作更进一步的说明。
实施例中涉及的各测定方法和培养基方法等参照如下:
丝氨酸浓度测定:采用高效液相色谱仪(HPLC)检测。发酵液经处理且上清液经0.22μm微孔滤膜过滤后,利用VWD(紫外检测器)检测丝氨酸含量,液相色谱方法如下:高效液相色谱仪为美国Waters公司产品,型号为1515,色谱柱为Venusil AA(4.6×250nm)液相色谱柱。柱温:30℃;流动相:75%的乙腈,流速:1mL/min;进样量:20μL。
氨基脱氧分支酸合成酶的比活测定:采用碧云天的Bradford蛋白浓度测定试剂测定;氨基脱氧分支酸合成酶酶活采用科晶生物科技有限公司ELISA检测试剂盒测定。菌液4℃,12000rpm离心10min,用0.9%的生理清洗3-4次,除去培养基,称取菌体约0.25g,用5mL的0.9%的生理盐水充分悬浮菌体,使用超声破碎约30min,4℃,12000rpm离心10min,弃去沉淀取上清,将上清稀释一定倍数,采用synergy H4多功能酶标仪测定OD450的吸光度,利用标准曲线换算出对应的氨基脱氧分支酸合成酶的酶活力。
生物量的测定:取适量的发酵液采用1mol/L的盐酸稀释至适当倍数,然后利用紫外分光光度计于562nm处测定OD562作为菌体量。
种子液体培养基(g/L):脑心浸液(BHI):37,葡萄糖:20,(NH4)2SO4:10,K2HPO4:0.2,NaH2PO4:0.3,MgSO4·7H2O:0.5;装液量:20mL/250mL;
发酵培养基(g/L):蔗糖:100,(NH4)2SO4:30,KH2PO4:3,MgSO4·7H2O:0.5,FeSO4·7H2O:0.02,MnSO4·H2O:0.02,原儿茶酸(PCA):0.03,生物素:5×10-5,VB1:4.5×10-4;装液量:25mL/250mL,CaCO3:1.5g/25mL;
谷氨酸棒杆菌感受态培养基(g/L):蛋白胨10;酵母粉5;NaCl 10;Tween-80 1;甘氨酸25;异烟肼0.04;
蔗糖筛选培养基(g/L):脑心浸液(BHI):37,蔗糖:100,(NH4)2SO4:10,K2HPO4:0.2,NaH2PO4:0.3,MgSO4·7H2O:0.5,琼脂粉:20。
电转化方法:以突变T426I为例,在超净工作台中,取100μL亲本谷氨酸棒杆菌ΔSSAAI感受态细胞于1.5mL离心管,加入5μL质粒pK18mobsacB-pabAB,混匀,在2.2KV,5ms条件下电击细胞1~2次,加入1mL BHIS,混匀,46℃温浴6min。随后30℃,120rpm往复式摇床上培养2h;室温12000rpm离心2min,弃去上清,重悬菌体,涂布于含50μg/mL Kan的电转化谷氨酸棒杆菌固体培养基上。
实施例
根据下述实施例,可以更好的理解本发明。然而,本领域的技术人员容易理解,实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
本发明的突变体命名:是以SEQ ID NO.1所示氨基酸序列为基准,采用“原始氨基酸位置替换的氨基酸”来表示突变体。比如T426I代表将位置426的氨基酸由亲本的酪氨酸(Try,T)替换为异亮氨酸(Ile,I),其他突变株T426A,T426V,T426L,T426G,T426S分别为:
T426A代表将位置426的氨基酸由亲本的酪氨酸(Try,T)替换为丙氨酸(Ala,A),T426V代表将位置426的氨基酸由亲本的酪氨酸(Try,T)替换为缬氨酸酸(Vla,V),
T426L代表将位置426的氨基酸由亲本的酪氨酸(Try,T)替换为亮氨酸(Leu,L),
T426G代表将位置426的氨基酸由亲本的酪氨酸(Try,T)替换为甘氨酸(Gly,G),
T426I代表将位置426的氨基酸由亲本的酪氨酸(Try,T)替换为异亮氨酸(Ile,S)。
如何通过定点突变技术来改变氨基脱氧分支酸合成酶的活性,难点在于突变位点的选择。
我们通过对丝氨酸高产菌株A36(菌株的保藏名称:A36,保藏号:CGMCC No15171)与出发菌株亲本谷氨酸棒杆菌ΔSSAAI(保藏名称:ΔSSAAI,保藏号:CGMCC No.15170)进行全基因组测序及比较基因组学分析,发现高产菌株A36氨基脱氧分支酸合成酶的426位氨基酸发生突变,由酪氨酸突变成为异亮氨酸。因此,本发明通过对氨基脱氧分支酸合成酶的426位进行随机饱和突变,并将突变菌株进行发酵考察该点突变对酶活力及丝氨酸积累的影响。
实施例1:氨基脱氧分支酸合成酶点突变为T426I
采用PCR方法进行定点突变,对ΔSSAAI氨基脱氧分支酸合成酶的P426位点进行饱和突变,将第426位点的酪氨酸突变成异亮氨酸。以菌株基因组为模板,选择点突变前500bp和后500bp为扩增目标,利用引物pabAB-F,pabAB-R扩增含点突变的同源臂基因片段。与经EcoR I和Xba I双酶切的质粒pK18mobsacB连接,构建回复突变质粒,电转化谷氨酸棒杆菌;于30℃培养3d,长出的单菌落即为一次重组菌落。挑取单菌落接至含50μg/mL Kan的种子液体培养基中,于30℃,120rpm的往复式摇床上进行培养,稀释涂布至10%的蔗糖筛选培养基,于30℃培养3d,长出的单菌落即为二次重组菌落。挑取单菌落,分别于无抗性种子平板和含50μg/mL Kan的种子抗性平板上划线,于30℃培养3~4d,将抗性板长不出的菌落进行菌落PCR,送至上海生工生物工程有限公司进行测序;结果正确的为突变成功的菌株,T426I突变株氨基脱氧分支酸合成酶突变体的氨基酸序列如SEQ ID NO.5所示。
其中,用于T426I突变的引物(序列分别如SEQ ID NO.3、SEQ ID NO.4所示),如表1所示。
表1定点突变引物
实施例2:氨基脱氧分支酸合成酶点突变为其他氨基酸
采用PCR方法进行定点突变,对氨基脱氧分支酸合成酶的P426位点进行饱和突变,将第426位点的酪氨酸突变成其他19种氨基酸。后续其他步骤同实施例1。用于T426进行其他非T426I突变的引物同SEQ ID NO.3、SEQ ID NO.4。
实施例1-2结果分析
(1)点突变对氨基脱氧分支酸合成酶活力的影响
将获得的系列氨基脱氧分支酸合成酶突变株进行酶活力测定,分析点突变对氨基脱氧分支酸合成酶活力的影响,采用试剂盒测定出发菌株ΔSSAAI与突变株T426I培养60h的氨基脱氧分支酸合成酶比酶活。
结果如表2所示,以未突变的氨基脱氧分支酸合成酶出发菌株为对照组,将实施例1得到的氨基脱氧分支酸合成酶点突变为T426I和实施例2得到的氨基脱氧分支酸合成酶点突变为其他氨基酸的突变株作为实验组,对各菌株培养后的氨基脱氧分支酸合成酶比酶活进行比较,发现当426位由酪氨酸突变为异亮氨酸时氨基脱氧分支酸合成酶活力下降,T426I点突变导致氨基脱氧分支酸合成酶比活力比对照下降了15.2%。而其他19个点突变导致酶活力与对照相组相比无显著变化(表中仅列出对照以及发生变化的数据)。
表2氨基脱氧分支酸合成酶的比酶活力
(2)氨基脱氧分支酸合成酶突变对丝氨酸产量的影响
选择T426I以及包括T426A,T426V,T426L,T426G,T426S在内的其他突变株在发酵培养基中进行发酵,具体方法为:将30℃、220rpm下培养24h的基因工程菌种子以5%的接种量接种到发酵培养基中,于30℃、220rpm条件下培养96h,获得丝氨酸。丝氨酸发生变化的突变株结果如图1所示,原始对照组菌株即出发菌株ΔSSAAI的L-丝氨酸产量为25.6g/L,最终突变菌株T426I的L-丝氨酸产量为30.4g/L,相较对照组出发菌株ΔSSAAI提高18.75%,而T426A,T426V,T426L,T426G,T426S突变导致丝氨酸产量反而降低,其他点突变导致丝氨酸产量没有发生变化。结合上述的“(1)点突变对氨基脱氧分支酸合成酶活力的影响”部分的结果,可以看出,相较于对照组出发菌株ΔSSAAI,突变菌株T426I的氨基脱氧分支酸合成酶比活力比对照下降了15.2%,相应的L-丝氨酸产量相较对照组出发菌株ΔSSAAI则提高了18.75%,这样的结果验证了氨基脱氧分支酸合成酶对L-丝氨酸产量的抑制影响作用。
通过对照结果,可以看出,点突变并非都是有增益促进效果的,有些点突变反而会产生抑制作用;本实施例选取T426I作为突变株,是在所有突变株中唯一获得增益促进效果的,这也说明通过对产量不同的产L-丝氨酸菌株进行基因组测序,比较高产菌株的基因突变对其产酸的影响,再进一步指导理性设计构建高产菌株具有重要意义。同时也说明氨基脱氧分支酸合成酶(编码基因pabAB)确实与L-丝氨酸降解相关,氨基脱氧分支酸合成酶可促进L-丝氨酸降解,相对应的,降低其活力则可能提高丝氨酸产量。
另外,从图1中OD562的变化可知426位氨基酸突变对菌株生长也有一定抑制作用。
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (6)
1.一种氨基脱氧分支酸合成酶突变体,其特征在于:所述酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的亲本谷氨酸棒杆菌ΔSSAAI中氨基脱氧分支酸合成酶的第426位苏氨酸进行饱和突变成为异亮氨酸,所述氨基脱氧分支酸合成酶突变体的氨基酸序列如SEQ ID NO.5所示。
2.编码根据权利要求1所述的氨基脱氧分支酸合成酶突变体的编码基因。
3.根据权利要求2所述的氨基脱氧分支酸合成酶突变体的编码基因,其特征在于:其核苷酸序列如SEQ ID NO.2所示。
4.包含权利要求2的氨基脱氧分支酸合成酶突变体编码基因的基因工程菌。
5.根据权利要求4所述的基因工程菌,其特征在于:所述基因工程菌为谷氨酸棒杆菌突变株。
6.根据权利要求4所述的一种基因工程菌应用于发酵培养生产丝氨酸。
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