CN117946227A - 一种钠/脯氨酸共转运蛋白突变体及其重组微生物与应用 - Google Patents
一种钠/脯氨酸共转运蛋白突变体及其重组微生物与应用 Download PDFInfo
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Abstract
本发明涉及微生物工程技术领域,具体公开了一种钠/脯氨酸共转运蛋白突变体及其重组微生物与应用。本发明的钠/脯氨酸共转运蛋白突变体,以野生型钠/脯氨酸共转运蛋白的氨基酸序列为参考序列,所述钠/脯氨酸共转运蛋白突变体含有第116位天冬酰胺被其他氨基酸取代的突变。具有本发明突变体的发酵菌,其生产L‑谷氨酰胺的能力得到了明显提升,产量高、副产物少。本发明为发酵生产L‑谷氨酰胺提供了一种新的高效率生产方式。
Description
技术领域
本发明涉及微生物工程技术领域,具体地说,涉及一种钠/脯氨酸共转运蛋白突变体及其重组微生物与应用。
背景技术
谷氨酰胺是一种非必需氨基酸。化学名称为2-氨基-4-氨甲酰基丁酸。谷氨酰胺是蛋白质合成中的编码氨基酸,可促进蛋白质的合成,抑制蛋白质的分解,可用于治疗胃及十二指肠溃疡,在医药行业中有重要作用。
目前,谷氨酰胺最常用的生产方法为发酵法,主要以谷氨酸棒杆菌(Corynebacterium glutamicum)为生产菌发酵生产谷氨酰胺。谷氨酸棒状杆菌是异养需氧型,为革兰氏阳性菌,具有生长速度快、非致病、对自身代谢物弱降解能力的特性。发酵法具有原料来源广泛、生产成本低、产品质量可控、产物单一等优点。但目前生产谷氨酰胺的菌种的发酵性能仍较差,含有副产物脯氨酸,谷氨酰胺转化率不理想,工业上对谷氨酰胺的需求量极高,现有的菌种根本无法满足大规模工业化生产的需求。已知putP编码脯氨酸转运蛋白,失活putP可以减少脯氨酸的合成(2017,叶邦策,Systematic pathway engineeringof Corynebacterium glutamicum S9114 for l-ornithine production)。削弱脯氨酸的合成,减少副产物积累,促进谷氨酸转化为谷氨酰胺具有重要意义。
发明内容
本发明的目的在于提供一种可改善细菌的L-谷氨酰胺生产能力的钠/脯氨酸共转运蛋白突变体,及含有其的重组微生物和应用。
本发明的具体技术方案如下:
一种钠/脯氨酸共转运蛋白突变体,其以野生型钠/脯氨酸共转运蛋白的氨基酸序列(NCBI编号为AST20430.1)为参考序列,所述钠/脯氨酸共转运蛋白突变体含有第116位天冬酰胺(N)被其他氨基酸取代的突变。
优选,所述其他氨基酸为丝氨酸(S)、赖氨酸(K)或组氨酸(H)。
本发明研究发现,当将钠/脯氨酸共转运蛋白(PutP,由putP编码)进行突变,可使包含该突变体的重组微生物生产L-谷氨酰胺的能力提升,生产副产物脯氨酸的能力下降,有利于提升谷氨酰胺工业化生产的效率。
优选,所述钠/脯氨酸共转运蛋白突变体的氨基酸序列如SEQ ID NO.2所示。
本发明还提供一种DNA分子,其以SEQ ID NO.1为参考序列,所述DNA分子含有第346-348位碱基由AAC突变为AGC、AAG或CAC的突变。
本发明另提供一种重组微生物,所述重组微生物表达上述钠/脯氨酸共转运蛋白突变体。
本发明通过对多种出发菌株中的putP基因进行多种修饰,发现重组菌株产生L-谷氨酰胺的能力与未修饰的菌株相比均得到增强,且优于文献已报道ΔputP菌株。
优选,所述重组微生物还表达肌醇3磷酸合酶突变体;所述肌醇3磷酸合酶突变体以野生型肌醇-3-磷酸合酶的氨基酸序列(NCBI编号为AST22023.1)为参考序列,含有第84位氨基酸由丝氨酸突变为丙氨酸的突变。
本发明发现同时表达上述钠/脯氨酸共转运蛋白突变体和肌醇-3-磷酸合酶突变体,可进一步提升重组微生物的谷氨酰胺生产能力。
本发明中重组微生物的出发菌株为棒杆菌、肠杆菌或枯草芽孢杆菌。优选,所述重组微生物的出发菌株为棒杆菌,更优选为谷氨酸棒杆菌。
本发明另提供一种上述重组微生物的如下任一种应用:
(1)在发酵生产L-谷氨酰胺或其衍生物中的应用;
(2)在用于生产L-谷氨酰胺或其衍生物的微生物遗传育种中的应用;
(3)在提高发酵生产L-谷氨酰胺或其衍生物产量上的应用;
(4)在发酵生产L-谷氨酰胺或其衍生物时,降低副产物脯氨酸生成中的应用。
L-谷氨酰胺的衍生物例如为精氨酸,色氨酸等。
本发明还提供一种L-谷氨酰胺或其衍生物的生产方法,其包括以重组微生物进行发酵培养的步骤,所述重组微生物如上所述。
本发明的有益效果至少在于:
本发明提供了一种钠/脯氨酸共转运蛋白突变体,含有其的重组微生物在发酵生产谷氨酰胺或其衍生物时,可获得更高的产量,且副产物脯氨酸产量降低,提高了生产性能。
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.3-26)
引物 | 序列(5’-3’) |
UP1-F | ctagTCTAGACACCACTGCTTAAAAGATTTTGCT |
UP1-R | AAGCGCGCGAGATTTATCGCGAAGTCGGCTCTCAAAGAATGAAGGCAGGGT |
DN1-F | CGACTTCGCGATAAATCTCGCGCGCTT |
DN1-R | cccAAGCTTATGCGACGACCTTGCTTTGCTTC |
ID1-F | ATTCATTTCACCTATGTTGA |
ID1-R | ATAAGCCCTTGTGTCAGTGA |
鉴定1-F | ACCCTGCCTTCATTCTTTGAGCG |
鉴定2-F | TGCCTTCATTCTTTGAGACG |
鉴定4-F | TCACCCTGCCTTCATTCTTTGATC |
UP2-F | ctagTCTAGATTAACTTTCTTTCGAAAGCT |
UP2-R | TTCGCGATAAATCTCGCGCGCTTCCGCATCTTCCCTGATTGAGGA |
DN2-F | AAGCGCGCGAGATTTATCGCGAA |
DN2-R | cccAAGCTTAGATAGGGGTGATTTTCATG |
ID2-F | ATCACCTTGGCGTTTGGTTC |
ID2-R | GGTTATTTGAAAAATCACCA |
UP3-F | ctagTCTAGATGCTCCACGCGGCAGATGATAT |
UP3-R | TGCAGCTTCCTCTGGTGGCAGTTCGAAGAGGTCCTTGTCCACTGGAGCGT |
DN3-F | TTCGAACTGCCACCAGAGGAAGCT |
DN3-R | cccAAGCTTAAATGAAGGTCTGGGAGACA |
ID3-F | AAACCGTTGACGTGCGCGAT |
ID3-R | TGTAACGCAAGAAGCAAATA |
鉴定3-F | ACGCTCCAGTGGACAAGGACCTCGT |
P82 | CTCGTATGTTGTGTGGAATTGTG |
P85 | CGCCCTGAGTGCTTGCGGCA |
本发明的出发菌分别为MHZ-0513-3,MHZ-0513-3-ino-1S84A,谷氨酸棒杆菌ATCC14067-glnAY405F。其中,MHZ-0513-3已在中国专利CN106701649A中公开,其分类命名为谷氨酸棒杆菌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,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.13405。MHZ-0513-3-ino-1S84A的构建方法见中国专利CN202110502341.9。谷氨酸棒杆菌ATCC 14067为模式菌(可市售获得),以其为出发菌株,参照中国专利CN200610089484.7的记载引入点突变glnAY405F,该突变解除了腺苷酰化修饰,有利于谷氨酰胺的积累,因此本发明构建了菌株ATCC14067-glnAY405F。
实施例1质粒pK18-putPN116S构建和重组菌株MHZ-0513-3-putPN116S、MHZ-0513-3-ΔputP构建
菌株MHZ-0513-3-putPN116S具体构建过程如下:
利用Phusion超保真聚合酶(New England BioLabs),以谷氨酸棒杆菌ATCC 14067的基因组为模板,以UP1-F/UP1-R为引物,制备重组片段UP1,以DN1-F/DN1-R为引物,制备重组片段DN1,所得片段经琼脂糖凝胶回收试剂盒(天根)纯化,随后以UP1、DN1为模板,以UP1-F/DN1-R为引物制备重组片段,所得重组片段经琼脂糖凝胶回收试剂盒(天根)纯化,然后利用XbaI/HindIII进行消化,同时将pK18-mobsacB(武汉淼灵生物科技有限公司)利用XbaI/HindIII进行消化,并用T4 DNA连接酶(TransGen Biotech)将片段与载体进行连接,转化Trans1T1感受态细胞(TransGen Biotech),挑取卡那抗性克隆,XbaI/HindIII酶切鉴定得到片段插入pK18mobsacB的阳性克隆,进一步通过用P82/P85引物测序(Invitrogen公司)鉴定插入的片段正确。将所得到的质粒命名为pK18-putPN116S。将pK18-putPN116S转入谷氨酸棒杆菌MHZ-0513-3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33℃,倒置培养。将筛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33℃,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。将所筛选出的菌株进一步进行表型验证,挑选KanS的重组子利用鉴定1-F/DN1-R验证点突变重组子,通过摸索退火温度,获得含点突变的重组子,将得到的阳性重组子用ID1-F/ID1-R扩增测序,验证获得的为目的突变菌株,并命名为MHZ-0513-3-putPN116S。
同理MHZ-0513-3-ΔputP构建方法同上,所用到的引物分别为UP2-F、UP2-R、DN2-F、DN2-R、ID2-F、ID2-R。
实施例2putPN116S突变菌株构建
菌株MHZ-0513-3、MHZ-0513-3-ino-1S84A是在模式菌谷氨酸棒杆菌ATCC 14067改造后获得,基因putP完全相同,将质粒pK18-putPN116S电转到MHZ-0513-3-ino-1S84A和ATCC14067-glnAY405F,菌株ATCC 14067-glnAY405F构建方法同上,所用引物为UP3-F、UP3-R、DN3-F、DN3-R、鉴定3-F、ID3-F、ID3-R、P82、P85。获得改造菌株命名为MHZ-0513-3-ino-1S84A-putPN116S、14067-glnAY405F-putPN116S。
实施例3MHZ-0513-3-putPN116S突变菌株以及putPN116S突变菌株生产谷氨酰胺的性能
发酵验证谷氨酰胺产量的方法:将冻存于-80℃甘油管中的菌株接种于下述斜面培养基中进行活化,于33℃下培养24h后长出菌苔,从新鲜活化的斜面上挑取菌苔,接种于下述种子培养基中,于33℃,100rpm下振荡培养至对数生长中后期,培养时间为5h,制得种子液,以10%的接种量将上述种子液接种至装有20ml发酵培养基的500ml摇瓶中,在33℃,150rpm振荡培养48h。结果如表2所示(OD562为培养液在562nm的浊度并表示细胞量,Gln(g/L)表示积累的L-谷氨酰胺的量,Pro(g/L)表示副产物脯氨酸生成量)。
培养基配方如下:
斜面培养基:脑心浸液37g/L,琼脂1.8%,121℃0.1MPa灭菌20min;
种子培养基:葡萄糖50g/L,尿素5g/L,KH2PO4 2.0g/L,MgSO4·7H2O 1.0g/L,玉米浆30g/L,pH 7.0;
发酵培养基:葡萄糖90g/L,(NH4)2SO4 40g/L,KH2PO4 2.0g/L,MgSO4·7H2O 1.0g/L,玉米浆10g/L,CaCO3 50g/L,pH 7.0。
表2突变菌株谷氨酰胺含量检测
如表2所示,在MHZ-0513-3中putPN116S第116位氨基酸由天冬酰胺(N)突变为丝氨酸(S),即AAC突变为AGC后,获得菌株MHZ-0513-3-putPN116S,摇瓶发酵谷氨酰胺产量从28.9g/L提高到33.2g/L,产酸提高14.9%,脯氨酸减少到0.5g/L,性能优于ΔputP菌株。由此可见,putPN116S突变更利于谷氨酰胺的积累,降低副产物脯氨酸。为确认该点突变在不同菌株中是否有效,将其引入菌株MHZ-0513-3-ino-1S84A和谷氨酸棒杆菌ATCC 14067-glnAY405F。MHZ-0513-3-ino-1S84A中putPN116S第116位氨基酸由天冬酰胺(N)突变为丝氨酸(S),即AAC突变为AGC后,获得菌株MHZ-0513-3-ino-1S84A-putPN116S,摇瓶发酵谷氨酰胺产量从30.7g/L提高到35.3g/L,产酸提高15.0%,脯氨酸由4.8g/L降低到0.3g/L,ATCC 14067-glnAY405F中putPN116S第116位氨基酸由天冬酰胺(N)突变为丝氨酸(S),获得菌株14067-glnAY405F-putPN116S,摇瓶发酵谷氨酰胺产量从0.6g/L提高到0.8g/L,产酸提高33.3%,脯氨酸减少到0.1g/L。由此可见,putPN116S突变在不同的菌株中均利于谷氨酰胺的积累,并且可以降低副产物脯氨酸。同理,将该基因整合到其他可以合成谷氨酰胺的菌株,如肠杆菌、枯草芽孢杆菌、棒杆菌等,均有利于谷氨酰胺或其衍生物的积累。
实施例4putP第116位氨基酸突变成其他氨基酸生产谷氨酰胺的性能
鉴于putP第116位氨基酸由天冬酰胺(N)突变为丝氨酸(S)后,谷氨酰胺产量有所提升,本发明接着研究了第116位氨基酸由天冬酰胺(N)突变为赖氨酸(K)、组氨酸(H),具体为AAC突变为AAG,CAC,菌株构建方法参考实施例1,其中摸索退火温度所用的鉴定引物分别为鉴定2-F/DN1-R、鉴定4-F/DN1-R,其他引物相同,突变菌株发酵性能测试方法参见实施例3,结果如下表3:
表3突变菌株谷氨酰胺含量检测
菌株 | OD562 | Gln(g/L) | 产酸提高率% | Pro(g/L) |
MHZ-0513-3-ino-1S84A | 43.5 | 30.7 | -- | 4.8 |
MHZ-0513-3-ino-1S84A-putPN116K | 43.1 | 34.5 | 12.4 | 0.7 |
MHZ-0513-3-ino-1S84A-putPN116H | 43.7 | 33.9 | 10.4 | 1.3 |
发酵结果表明,第116位氨基酸由天冬酰胺(N)突变为赖氨酸(K)、组氨酸(H)后,突变株谷氨酰胺产量均有提升,且效果都优于对照菌株MHZ-0513-3-ino-1S84A,其中突变为赖氨酸(K)后,谷氨酰胺产量提高到34.5g/L,产酸提高12.4%,脯氨酸从4.8g/L降低到0.7g/L。显而易见,该位点突变成其他氨基酸也有利于谷氨酰胺及其衍生物的生产。
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。
Claims (10)
1.一种钠/脯氨酸共转运蛋白突变体,其特征在于,以野生型钠/脯氨酸共转运蛋白的氨基酸序列为参考序列,所述钠/脯氨酸共转运蛋白突变体含有第116位天冬酰胺被其他氨基酸取代的突变。
2.根据权利要求1所述的钠/脯氨酸共转运蛋白突变体,其特征在于,所述其他氨基酸为丝氨酸、赖氨酸或组氨酸。
3.根据权利要求2所述的钠/脯氨酸共转运蛋白突变体,其特征在于,所述钠/脯氨酸共转运蛋白突变体的氨基酸序列如SEQ ID NO.2所示。
4.一种DNA分子,其特征在于,以SEQ ID NO.1为参考序列,所述DNA分子含有第346-348位碱基由AAC突变为AGC、AAG或CAC的突变。
5.一种重组微生物,其特征在于,所述重组微生物表达权利要求1-3任一项所述的钠/脯氨酸共转运蛋白突变体。
6.根据权利要求5所述的重组微生物,其特征在于,所述重组微生物还表达肌醇-3-磷酸合酶突变体;所述肌醇-3-磷酸合酶突变体以野生型肌醇-3-磷酸合酶的氨基酸序列为参考序列,含有第84位氨基酸由丝氨酸突变为丙氨酸的突变。
7.根据权利要求5或6所述的重组微生物,其特征在于,所述重组微生物的出发菌株为棒杆菌、肠杆菌或枯草芽孢杆菌。
8.根据权利要求7所述的重组微生物,其特征在于,所述重组微生物的出发菌株为棒杆菌。
9.权利要求5-8任一项所述的重组微生物的如下任一种应用:
(1)在发酵生产L-谷氨酰胺或其衍生物中的应用;
(2)在用于生产L-谷氨酰胺或其衍生物的微生物遗传育种中的应用;
(3)在提高发酵生产L-谷氨酰胺或其衍生物产量上的应用;
(4)在发酵生产L-谷氨酰胺或其衍生物时,降低副产物脯氨酸生成中的应用。
10.一种L-谷氨酰胺或其衍生物的生产方法,其特征在于,包括以重组微生物进行发酵培养的步骤,所述重组微生物如权利要求5-8任一项所述。
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