CN113201524A - 肌醇-3-磷酸合酶突变体及其在构建高产谷氨酰胺的谷氨酸棒杆菌中的应用 - Google Patents
肌醇-3-磷酸合酶突变体及其在构建高产谷氨酰胺的谷氨酸棒杆菌中的应用 Download PDFInfo
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- CN113201524A CN113201524A CN202110502341.9A CN202110502341A CN113201524A CN 113201524 A CN113201524 A CN 113201524A CN 202110502341 A CN202110502341 A CN 202110502341A CN 113201524 A CN113201524 A CN 113201524A
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- Prior art keywords
- corynebacterium glutamicum
- glutamine
- inositol
- phosphate synthase
- culture medium
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
本发明涉及生物工程领域,具体涉及一种肌醇‑3‑磷酸合酶突变体及其在构建高产谷氨酰胺的谷氨酸棒杆菌中的应用。本发明发现,肌醇‑3‑磷酸合酶第84位丝氨酸突变为其他氨基酸后,有助于在谷氨酸棒杆菌中增强其产生谷氨酰胺的能力。与未发生上述突变的野生型菌株相比,本发明中谷氨酸棒杆菌产生谷氨酰胺的能力得到增强,谷氨酰胺产量从28.5g/L提高到30.7g/L,产酸提高7.7%,有利于对谷氨酰胺高产菌株的筛选和构建。
Description
技术领域
本发明涉及生物工程领域,具体涉及一种肌醇-3-磷酸合酶突变体及其在构建高产谷氨酰胺的谷氨酸棒杆菌中的应用。
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,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。
发明内容
本发明意外发现,当肌醇-3-磷酸合酶第84位丝氨酸突变为其他氨基酸后,有助于增强谷氨酸棒杆菌产生谷氨酰胺的能力。据此,本发明提供了肌醇-3-磷酸合酶突变体及其在构建高产谷氨酰胺的谷氨酸棒杆菌中的应用。
第一方面,本发明提供了一种肌醇-3-磷酸合酶突变体,其第84 位丝氨酸突变为其他氨基酸。
本发明中所提到的“肌醇-3-磷酸合酶”实际上目前在谷氨酸棒杆菌中的命名并不统一。比如,在谷氨酸棒杆菌ATCC 13032中该氨基酸序列所对应的酶的命名有两个——肌醇-1-磷酸合酶 (myo-inositol-1-phosphate synthase)和肌醇-3-磷酸合酶 (inositol-3-phosphate synthase)。而在谷氨酸棒杆菌ATCC 14067中则只有一个名字——肌醇-3-磷酸合酶(inositol-3-phosphate synthase)。同时,有文献(如Can Chen等,Myo-inositol-1-phosphate synthase(Ino-1) functions as a protection mechanism inCorynebacterium glutamicum under oxidative stress)在应用ATCC 13032的改造菌时将 myo-inositol-1-phosphate synthase命名为Ino-1,模式菌谷氨酸棒杆菌 MB001中inositol-3-phosphate synthase也标注为ino1,而在谷氨酸棒杆菌ATCC 1406中该酶暂无基因名字简写。不过,本领域人员知晓,上述命名均指代同一功能蛋白,依据本发明的突变体也能在上述的相应酶中找到对应的氨基酸序列并进行相同效果的突变,其也在本发明的保护范围内。
作为优选,所述肌醇-3-磷酸合酶突变体的第84位丝氨酸突变为丙氨酸、酪氨酸、赖氨酸、苏氨酸、苯丙氨酸或精氨酸。
更优选的,所述肌醇-3-磷酸合酶突变体的第84位丝氨酸突变为丙氨酸。
作为优选,所述的肌醇-3-磷酸合酶突变体源于谷氨酸棒杆菌,更优选其氨基酸序列如SED ID NO:1所示。
本发明中的“突变”是指通过对编码蛋白的基因进行突变,进而引起氨基酸发生改变。所述突变方法可以选自诱变、PCR定点突变法、和/或同源重组等方法中的一种。本发明中,优选通过同源重组的突变方法,将肌醇-3-磷酸合酶第84位丝氨酸突变为丙氨酸、酪氨酸、赖氨酸、苏氨酸、苯丙氨酸或精氨酸。
在一些优选的实施方式中,将肌醇-3-磷酸合酶核酸序列中第250~252位的三个碱基TCC突变为GCC,使得肌醇-3-磷酸合酶第84位氨基酸由丝氨酸(S)突变为丙氨酸(A),其氨基酸序列如SED ID NO:1 所示。
第二方面,本发明提供一种编码所述的肌醇-3-磷酸合酶突变体的核酸。
作为优选,所述核酸的核苷酸序列如SED ID NO:2所示。
第三方面,本发明提供所述的肌醇-3-磷酸合酶突变体或核酸在以下任一方面的应用:
(1)构建高产谷氨酰胺的谷氨酸棒杆菌;
(2)鉴别高产谷氨酰胺的谷氨酸棒杆菌;
(3)筛选高产谷氨酰胺的谷氨酸棒杆菌。
进一步的,本发明提供一种谷氨酸棒杆菌,其表达上述所提到的肌醇-3-磷酸合酶突变体;和/或,其含有上述所提到的核酸。
作为优选,所述的谷氨酸棒杆菌以保藏编号为CGMCC No.13405 的谷氨酸棒杆MHZ-0513-3为出发菌株构建得到。其中,谷氨酸棒杆 MHZ-0513-3已在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。
本发明还提供了高产谷氨酰胺的谷氨酸棒杆菌的构建方法,构建含所述核酸的重组质粒,将所述重组质粒转化谷氨酸棒杆菌 MHZ-0513-3。
第四方面,本发明还提供所述的谷氨酸棒杆菌在生产谷氨酰胺中的应用。
进一步的,本发明提供一种生产谷氨酰胺的方法,其包括:培养所述的谷氨酸棒杆菌,以产生、积累和收集谷氨酰胺。
作为优选方案,所述的方法包括:将所述的谷氨酸棒杆菌接种至斜面培养基进行斜面培养,挑取斜面培养基上的菌苔接种至种子培养基进行种子培养,然后将种子培养物转入发酵培养基发酵;
其中,所述斜面培养基中含有如下组分:脑心浸液37g/L,琼脂粉1.8%;
所述种子培养基中含有如下组分:葡萄糖50g/L,尿素5g/L, KH2PO4 2.0g/L,MgSO4·7H2O 1.0g/L,玉米浆30g/L;且pH值为7.0;
所述发酵培养基中含有如下组分:葡萄糖90g/L,(NH4)2SO4 40g/L,KH2PO4 2.0g/L,MgSO4·7H2O 1.0g/L,玉米浆10g/L,CaCO3 50 g/L;且pH值为pH 7.0。
在一些优选的实施方案中,所述斜面培养具体为:将所述的谷氨酸棒杆菌在33℃下培养24h。
在一些优选的实施方案中,所述种子培养具体为:挑取斜面培养基上1/4的平板菌苔,接种于量程大小为200ml三角瓶进行种子培养,装液量50ml/瓶,在33℃,100rpm下振荡培养至对数生长中后期,培养时间约为5h。
在一些优选的实施方式中,所述发酵具体为:将种子培养物于量程大小500ml三角摇瓶中进行发酵培养,装液量为20ml/瓶,接种量为 10%,而后在33℃、150rpm下培养48h。
基于上述技术方案,本发明的有益效果如下:
本发明发现,肌醇-3-磷酸合酶第84位丝氨酸突变为其他氨基酸后,有助于在谷氨酸棒杆菌中增强其产生谷氨酰胺的能力。与未发生上述突变的野生型菌株相比,本发明中谷氨酸棒杆菌产生谷氨酰胺的能力得到增强,谷氨酰胺产量从28.5g/L提高到30.7g/L,产酸提高 7.7%,有利于对谷氨酰胺高产菌株的筛选和构建。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
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
引物 | 序列(由上至下依次为SED ID NO:3~11) |
UP-1F | ctagTCTAGAAAAGCGAAGGTGGTAGTGCCACA |
UP-1R | TCGGCGATTTTGATAGTGCAGTTTTGTGA |
DN-2F | TCACAAAACTGCACTATCAAAATCGCCGATGTCCCACA |
DN-2R | aaaaCTGCAGTTCCACCTTTCCAGACAGTGGA |
P82 | CTCGTATGTTGTGTGGAATTGTG |
P85 | CGCCCTGAGTGCTTGCGGCA |
鉴定-F | ACCGAGGCTTCACAAAACTGCACTATCAAAATAGCCGAT |
ID-F | AGAAGTGGCAATCACCTGGTCATT |
ID-R | TTTGTACTCAAGGTTGAGGGGAA |
实施例中所使用的出发菌株MHZ-0513-3已在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。
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。
实施例1突变菌株Q8的分离及生产谷氨酰胺的性能
已知谷氨酸棒杆菌MHZ-0513-3(菌株保藏编号为CGMCC No.13405)可作为谷氨酰胺生产菌来进行谷氨酰胺发酵,对其进行分离、纯化、复壮。
活化的步骤如下:
将甘油管中的MHZ-0513-3菌株在斜面培养基上活化,33℃培养 24h,接脑心浸液液体试管33℃过夜培养,稀释105,106,107涂布于斜面培养基平板,从平板上挑选不同的菌株Q1~Q12进行摇瓶发酵。意外发现一株菌Q8谷氨酰胺含量从28.7g/L提高到30.9g/L。
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;
种子培养基为: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;
发酵培养基为:葡萄糖90g/L,(NH4)2SO4 40g/L,KH2PO4 2.0g/L, MgSO4·7H2O1.0g/L,玉米浆10g/L,CaCO3 50g/L,pH 7.0。
发酵结果如表2所示。
表2菌株谷氨酰胺含量检测
菌株 | OD<sub>562</sub> | L-谷氨酰胺(g/L) |
Q1 | 43.4±0.015 | 28.7±0.12 |
Q2 | 43.1±0.021 | 28.8±0.06 |
Q3 | 42.8±0.001 | 28.5±0.20 |
Q4 | 43.2±0.017 | 28.3±0.13 |
Q5 | 42.7±0.023 | 28.9±0.04 |
Q6 | 43.5±0.013 | 28.1±0.23 |
Q7 | 45.1±0.011 | 26.8±0.07 |
Q8 | 43.3±0.014 | 30.9±0.02 |
Q9 | 43.1±0.034 | 27.8±0.30 |
Q10 | 45.7±0.026 | 27.4±0.21 |
Q11 | 43.6±0.012 | 29.0±0.18 |
Q12 | 44.1±0.034 | 27.8±0.30 |
实施例2测序发现肌醇-3-磷酸合酶核酸序列发生点突变
鉴于Q8菌株谷氨酰胺含量有明显提高,因此对谷氨酰胺合成和代谢途径的相关基因进行了测序,但这些基因均与MHZ-0513-3相一致,因此对菌株MHZ-0513-3进行了全基因组测序,测序结果表明肌醇-3-磷酸合酶第84位氨基酸由丝氨酸(TCC)突变为丙氨酸(GCC),其氨基酸序列如SED ID NO:1所示,核苷酸序列如SED ID NO:2所示(包含上游同源臂及外侧鉴定引物部分)。
已知肌醇-3-磷酸合酶与细胞壁和AcCys-GlcN-Ins(MSH)合成相关,失活会降低菌体在氧化应激条件下的存活率,推测点突变后可能导致酶活提高,增强了菌体在恶劣环境条件下的抗逆性,从而提高谷氨酰胺含量。
实施例3重组质粒pK18-ino-1S84A的构建及在MHZ-0513-3菌株引入点突变
利用Phusion超保真聚合酶(New England BioLabs),UP-1F/ UP-1R,DN-2F/-DN-2R作为引物对,以谷氨酸棒杆菌MHZ-0513-3 的基因组为模板,制备重组片段,PCR程序为,98℃变性10s,50℃复性20s,72℃延伸15s,循环30次,所得片段经琼脂糖凝胶回收试剂盒(天根)纯化,随后以UP-1F/DN-2R为引物对,以上下游同源臂为模板,制备重组片段,PCR程序为,98℃变性10s,50℃复性20s, 72℃延伸30s,循环30次,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/PstI进行消化,同时将pK18-mobsacB利用 XbaI/PstI进行消化,并用T4DNA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/PstI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-ino-1S84A。将 pK18-ino-1S84A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的重组子利用鉴定引物鉴定-F/ DN-2R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ID-F/ID-R扩增测序,验证获得的为目的突变菌株,并命名为MHZ-0513-3-ino-1S84V(肌醇-3-磷酸合酶编码基因记作ino-1,编码的氨基酸序列如SED ID NO:1所示。)。
实施例4 MHZ-0513-3-ino-1S84V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上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,接种于上述种子培养基中,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3所示(OD562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)。
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;
种子培养基为: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;
发酵培养基为:葡萄糖90g/L,(NH4)2SO4 40g/L,KH2PO4 2.0g/L, MgSO4·7H2O1.0g/L,玉米浆10g/L,CaCO3 50g/L,pH 7.0。
表3突变菌株谷氨酰胺含量检测
菌株 | OD<sub>562</sub> | Gln(g/L) | 产酸提高率% |
MHZ-0513-3 | 43.4±0.21 | 28.5±0.021 | -- |
MHZ-0513-3-ino-1<sup>S84V</sup> | 43.9±0.013 | 30.7±0.012 | 7.7% |
如表3所示,在MHZ-0513-3中ino-1的84位氨基酸由丝氨酸(S) 突变为丙氨酸(A),即TCC突变为GCC后,谷氨酰胺从28.5g/L提高到30.7g/L,产酸提高7.7%,由此可见,确实是肌醇-3-磷酸合酶发生点突变后导致谷氨酰胺产量提升。
实施例5肌醇-3-磷酸合酶84位氨基酸突变为其他氨基酸后突变菌株生产谷氨酰胺的性能
鉴于肌醇-3-磷酸合酶84位氨基酸由丝氨酸(S)突变为丙氨酸 (A)后,谷氨酰胺产量有所提升,接着研究了第84位氨基酸由丝氨酸(S)突变为突变为酪氨酸(Y)、赖氨酸(K)、苏氨酸(T)、苯丙氨酸(F)或精氨酸(R),菌株构建方法参考实施例3,突变菌株发酵结果如下表4:
表4
菌株 | OD<sub>562</sub> | Gln(g/L) | 产酸提高率% |
MHZ-0513-3 | 43.4±0.21 | 28.5±0.021 | -- |
MHZ-0513-3-ino-1<sup>S84Y</sup> | 43.9±0.013 | 30.6±0.012 | 7.4% |
MHZ-0513-3-ino-1<sup>S84K</sup> | 43.3±0.08 | 29.8±0.130 | 4.6% |
MHZ-0513-3-ino-1<sup>S84T</sup> | 43.4±0.16 | 29.4±0.031 | 3.2% |
MHZ-0513-3-ino-1<sup>S84F</sup> | 42.9±0.14 | 30.4±0.059 | 6.7% |
MHZ-0513-3-ino-1<sup>S84R</sup> | 43.8±0.19 | 29.9±0.014 | 4.9% |
发酵结果表明,肌醇-3-磷酸合酶84位氨基酸由丝氨酸(S)突变为酪氨酸(Y)、赖氨酸(K)、苏氨酸(T)、苯丙氨酸(F)或精氨酸(R)后,突变株谷氨酰胺产量均有提升,在这几种氨基酸中,突变为丙氨酸(A)后效果最好。
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> 肌醇-3-磷酸合酶突变体及其在构建高产谷氨酰胺的谷氨酸棒杆菌中的应用
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ggcatcaacg tgctgcgtgg cccgactctc gacggcctgg gcgatcatta ccgcgcgacc 780
atcgacgagt ccaccgccga gccagtcgac gttgtccagg cgcttatcga cgcaaaagcc 840
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Claims (10)
1.肌醇-3-磷酸合酶突变体,其特征在于,其第84位丝氨酸突变为其他氨基酸。
2.根据权利要求1所述的肌醇-3-磷酸合酶突变体,其特征在于,其第84位丝氨酸突变为丙氨酸、酪氨酸、赖氨酸、苏氨酸、苯丙氨酸或精氨酸。
3.根据权利要求2所述的肌醇-3-磷酸合酶突变体,其特征在于,其源于谷氨酸棒杆菌,优选其氨基酸序列如SEDID NO:1所示。
4.编码权利要求1-3中任一项所述的肌醇-3-磷酸合酶突变体的核酸;优选其核苷酸序列如SEDID NO:2所示。
5.权利要求1-3中任一项所述的肌醇-3-磷酸合酶突变体或权利要求4所述的核酸在以下任一方面的应用:
(1)构建高产谷氨酰胺的谷氨酸棒杆菌;
(2)鉴别高产谷氨酰胺的谷氨酸棒杆菌;
(3)筛选高产谷氨酰胺的谷氨酸棒杆菌。
6.一种谷氨酸棒杆菌,其特征在于,其表达权利要求1-3中任一项所述的肌醇-3-磷酸合酶突变体;和/或,其含有权利要求4所述的核酸。
7.根据权利要求6所述的谷氨酸棒杆菌,其特征在于,其以保藏编号为CGMCC No.13405的谷氨酸棒杆MHZ-0513-3为出发菌株构建得到。
8.权利要求6或7所述的谷氨酸棒杆菌在生产谷氨酰胺中的应用。
9.一种生产谷氨酰胺的方法,其特征在于,其包括:培养权利要求6或7所述的谷氨酸棒杆菌,以产生、积累和收集谷氨酰胺。
10.根据权利要求9所述的方法,其特征在于,其包括:将权利要求6或7所述的谷氨酸棒杆菌接种至斜面培养基进行斜面培养,挑取斜面培养基上的菌苔接种至种子培养基进行种子培养,然后将种子培养物转入发酵培养基发酵;
其中,所述斜面培养基中含有如下组分:脑心浸液37g/L,琼脂粉1.8%;
所述种子培养基中含有如下组分:葡萄糖50g/L,尿素5g/L,KH2PO4 2.0g/L,MgSO4·7H2O1.0g/L,玉米浆30g/L;且pH值为7.0;
所述发酵培养基中含有如下组分:葡萄糖90g/L,(NH4)2SO440g/L,KH2PO4 2.0g/L,MgSO4·7H2O 1.0g/L,玉米浆10g/L,CaCO3 50g/L;且pH值为pH 7.0。
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CN112812985A (zh) * | 2020-11-11 | 2021-05-18 | 新疆阜丰生物科技有限公司 | 一种提高谷氨酰胺发酵产酸的方法 |
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS57163489A (en) * | 1981-04-02 | 1982-10-07 | Ajinomoto Co Inc | Production of l-glutamine through fermentation process |
US20020197605A1 (en) * | 1999-12-16 | 2002-12-26 | Satoshi Nakagawa | Novel Polynucleotides |
CN110951661A (zh) * | 2019-12-26 | 2020-04-03 | 新疆梅花氨基酸有限责任公司 | 一种高产l-谷氨酰胺的谷氨酸棒杆菌及其构建方法与应用 |
-
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- 2021-05-08 CN CN202110502341.9A patent/CN113201524B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS57163489A (en) * | 1981-04-02 | 1982-10-07 | Ajinomoto Co Inc | Production of l-glutamine through fermentation process |
US20020197605A1 (en) * | 1999-12-16 | 2002-12-26 | Satoshi Nakagawa | Novel Polynucleotides |
CN110951661A (zh) * | 2019-12-26 | 2020-04-03 | 新疆梅花氨基酸有限责任公司 | 一种高产l-谷氨酰胺的谷氨酸棒杆菌及其构建方法与应用 |
Non-Patent Citations (1)
Title |
---|
佚名: "MULTISPECIES: inositol-3-phosphate synthase [Corynebacterium]", 《NCBI REFERENCE SEQUENCE》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112812985A (zh) * | 2020-11-11 | 2021-05-18 | 新疆阜丰生物科技有限公司 | 一种提高谷氨酰胺发酵产酸的方法 |
CN112812985B (zh) * | 2020-11-11 | 2023-01-10 | 新疆阜丰生物科技有限公司 | 一种提高谷氨酰胺发酵产酸的方法 |
CN114277003A (zh) * | 2021-12-14 | 2022-04-05 | 廊坊梅花生物技术开发有限公司 | 谷氨酰胺合酶突变体及其应用 |
CN114277003B (zh) * | 2021-12-14 | 2023-06-06 | 廊坊梅花生物技术开发有限公司 | 谷氨酰胺合酶突变体及其应用 |
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