CN110004102A - 一种利用马来酸全细胞催化合成l-天冬氨酸的菌株与方法 - Google Patents
一种利用马来酸全细胞催化合成l-天冬氨酸的菌株与方法 Download PDFInfo
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Abstract
本发明公开了一种利用马来酸全细胞催化合成L‑天冬氨酸的菌株与方法,所述菌株分类命名为Escherichia coliΔfumABCΔgltAΔargG/pMA,其保藏号为CCTCC NO:M 2019171,构建过程包括敲除出发菌中富马酸酶编码基因、柠檬酸合酶编码基因和精氨基琥珀酸合成酶编码基因,同时将天冬氨酸酶编码基因突变N217K‑T233R‑V367G和马来酸顺反异构酶编码基因突变G8A‑G179A,将两者克隆到表达质粒上构成重组质粒,并转化至之前敲除了基因的菌株中,得到目的菌株。利用该基因工程菌全细胞催化合成L‑天冬氨酸的方法,实现了以马来酸为原料催化合成L‑天冬氨酸的路线,降低了生产成本、高效催化,具有经济性。
Description
技术领域
本发明属于基因工程及发酵工程技术领域,具体涉及一株利用马来酸全细胞催化合成L-天冬氨酸的菌株与方法。
背景技术
L-天冬氨酸在医药、食品和化工等方面有着广泛的用途。在医药方面,是氨基酸制剂的主要成分;在化工方面,可以作为制造合成树脂的原料,大量用于合成环保材料聚天门冬氨酸;尤其在食品工业方面,L-天门冬氨酸是一种良好的营养增补剂,也是糖代用品阿斯巴甜的主要生产原料。具有良好的市场前景。
目前L-天冬氨酸主要以富马酸为原料,采用生物酶法合成,以马来酸为原料的研究比较少。而马来酸相对于富马酸,价格更便宜,资源更丰富,也更容易获得。生物酶法具有反应温和、转化率高、三废易处理等优点,是L-天冬氨酸最佳的生产方式。因此,以马来酸为底物生物催化生产L-天冬氨酸具有很大意义。本发明基于此对天冬氨酸酶aspA和马来酸顺反异构酶maiA进行分子水平优化,构建了一株利用马来酸催化合成L-天冬氨酸的基因工程菌,还公布了该基因工程菌全细胞催化合成L-天冬氨酸的方法,实现了以马来酸为原料催化合成L-天冬氨酸的路线,降低了生产成本、高效催化,具有经济性。
发明内容
本发明要求解决的技术问题是,提供一株利用马来酸全细胞催化合成L-天冬氨酸的菌株,其分类命名为Escherichia coli△fumABC△gltA△argG/pMA,其保藏号为CCTCCNO:M 2019171。
本发明还要解决的技术问题是,提供上述基因工程菌的构建方法,敲除保藏号为CCTCC NO:M 2018521出发菌株富马酸酶编码基因fumABC、柠檬酸合酶编码基因gltA和精氨基琥珀酸合成酶编码基因argG,同时将来源大肠杆菌的天冬氨酸酶编码基因aspA-N217K-T233R-V367G和来源Variibacter gotjawalensis的马来酸顺反异构酶A编码基因maiA-G8A-G179A克隆到表达质粒上构成重组质粒,并将其转化至敲除了fumABC、gltA和argG基因的大肠杆菌出发菌株,得到所述基因工程菌,所述出发菌株的保藏号为CCTCC NO:M2018521,所述fumA基因序列如SEQ ID NO:1所示,所述fumB基因序列如SEQ ID NO:2所示,所述fumC基因序列如SEQ ID NO:3所示,所述aspA基因突变N217K-T233R-V367G后序列如SEQ ID NO:4所示,所述maiA基因序列来源Variibacter gotjawalensis突变G8A-G179A后如SEQ ID NO:5所示,所述gltA基因序列如SEQ ID NO:6所示,所述argG基因序列如SEQ IDNO:7所示,基因的敲除采用通用的CRISPR/Cas9技术。具体构建及选育方法如下:
(1)以SEQ ID NO:8和SEQ ID NO:9所示的核苷酸序列为引物,质粒pTarget F为模板,PCR扩增得到线性片段1;
(2)以SEQ ID NO:8和SEQ ID NO:10所示的核苷酸序列为引物,重复步骤(1)得到线性片段2;以SEQ ID NO:8和SEQ ID NO:11所示的核苷酸序列为引物,重复步骤(1)得到线性片段3;以SEQ ID NO:8和SEQ ID NO:12所示的核苷酸序列为引物,重复步骤(1)得到线性片段4
(3)将线性片段1用SpeI酶切后进行连接,并将连接后产物转化到感受态中,涂布大观霉素的LB平板筛选出阳性重组子;
(4)将阳性重组子接种于添加大观霉素的LB液培扩繁后进行质粒抽提,获得pTarget T1-1质粒;
(5)将线性片段2用SpeI酶切后进行连接转化筛选出阳性重组子进行质粒抽提,获得pTarget T1-2质粒;将线性片段3用SpeI酶切后进行连接转化筛选出阳性重组子进行质粒抽提,获得pTarget T1-3质粒;将线性片段4用SpeI酶切后进行连接转化筛选出阳性重组子进行质粒抽提,获得pTarget T1-4质粒;
(6)以SEQ ID NO:13和SEQ ID NO:14所示的核苷酸序列为引物,大肠杆菌基因组为模板,PCR扩增得到线性片段5;以SEQ ID NO:15和SEQ ID NO:16所示的核苷酸序列为引物,大肠杆菌基因组为模板,PCR扩增得到线性片段6;
(7)以SEQ ID NO:13和SEQ ID NO:16所示的核苷酸序列为引物,片段5和片段6的混合物为模板,重叠PCR扩增得到线性片段7;
(8)将pTarget T1-1质粒用EcoRI线性化后去磷酸化作为载体,与片段7进行吉布森一步克隆,用大观霉素的LB平板筛选出阳性重组子;
(9)将步骤(8)中的阳性重组子接种于添加大观霉素的LB液培扩繁后进行质粒抽提,获得pTarget T2-1质粒;
(10)以SEQ ID NO:17-SEQ ID NO:20所示的核苷酸序列为引物重复步骤(6)至步骤(9)获得pTarget T2-2质粒;以SEQ ID NO:21-SEQ ID NO:24所示的核苷酸序列为引物重复步骤(6)至步骤(9)获得pTarget T2-3质粒;以SEQ ID NO:25-SEQ ID NO:28所示的核苷酸序列为引物重复步骤(6)至步骤(9)获得pTarget T2-4质粒;
(11)将pCas质粒导入保藏号为CCTCC NO:M 2018521的出发菌株中,用卡那霉素的LB平板筛选出阳性重组子;
(12)将步骤(11)中的阳性重组子用***糖诱导,制备为感受态;
(13)将pTarget T2-1质粒导入步骤(12)的感受态中,用添加大观霉素和卡那霉素的LB平板筛选出阳性重组子;
(14)将步骤(13)中的阳性重组子用PCR的方法进行鉴定,筛选出目标菌株,并使用IPTG诱导消除pTarget T2-1质粒,获得菌株1。
(15)将步骤(14)中的菌株1用***糖诱导,制备为感受态;
(16)将pTarget T2-2质粒导入步骤(15)的感受态中,用添加大观霉素和卡那霉素的LB平板筛选出阳性重组子;
(17)将步骤(16)中的阳性重组子用PCR的方法进行鉴定,筛选出目标菌株,并使用IPTG诱导消除pTarget T2-2质粒,获得菌株2。
(18)用类似步骤(15)至步骤(17)的方法分批导入pTarget T2-3质粒、pTargetT2-4质粒进行基因敲除,并使用IPTG诱导消除导入质粒,获得基因敲除菌株Escherichiacoli△fumABC△gltA△argG;
(19)以SEQ ID NO:29~SEQ ID NO:32所示的核苷酸序列为引物,pGEMT-maiA质粒为模板,PCR扩增得到线性片段8;以SEQ ID NO:33~SEQ ID NO:38所示的核苷酸序列为引物,pGEMT-aspA质粒为模板,PCR扩增得到线性片段9;用DpnI消化后转化大肠杆菌,获得pGEMT-maiA-G8A-G179A和pGEMT-aspA-N217K-T233R-V367G;
(20)以SEQ ID NO:39和SEQ ID NO:40所示的核苷酸序列为引物,pGEMT-maiA-G8A-G179A为模板,PCR扩增得到线性片段10;以SEQ ID NO:41和SEQ ID NO:42所示的核苷酸序列为引物,pGEMT-aspA-N217K-T233R-V367G模板,PCR扩增得到线性片段11;
(21)以SEQ ID NO:39和SEQ ID NO:42所示的核苷酸序列为引物,片段10和片段11的混合物为模板,重叠PCR扩增得到线性片段12;
(22)将pTrc99a质粒用EcoRI和HindIII线性化后去磷酸化作为载体,与片段12进行吉布森一步克隆,用含氨苄抗性的LB平板筛选出阳性重组子;
(23)将步骤(22)中的阳性重组子接种于添加大观霉素的LB液培扩繁后进行质粒抽提,获得pTrc99A-maiAmut-aspAmut质粒;
(24)将步骤(18)中的Escherichia coli△fumABC△gltA△argG制备为感受态;将步骤(23)中的pTrc99A-maiAmut-aspAmut质粒转化至感受态中,用含氨苄抗性的LB平板筛选出阳性重组子,获得目的基因工程菌株。
本发明最后要解决的技术问题是,提供上述基因工程菌利用马来酸全细胞催化合成L-天冬氨酸的方法,具体过程如下:
(S1)将所述基因工程菌转接到LB培养基中,有氧培养10~12h,得到一级种子液;
(S2)将一级种子液转接到2YT培养基中培养,得到二级种子液;
(S3)待二级种子液OD600至8.5时,再接种到诱导培养基,所述诱导培养基的配方如下:
LA培养基:(NH4)2SO4 6g/L,玉米浆20g/L,KH2PO4 1g/L,MgSO4 1g/L,CaCl2 0.1mM,CaCO3 4g/L,0.8mM IPTG。
发酵过程中,分次加入灭菌葡萄糖,保证培养基中葡萄糖的浓度在1~50g/L。
步骤(S1)和(S2)中,培养温度为35~37℃,步骤(S3)中采用有氧发酵模式,溶解氧为5~40%且发酵过程中温度为28~30℃,培养过程pH用氨水调节为7.0。
将发酵获得的微生物细胞收集后用pH8.0的50mM Na2HPO4-KH2PO4缓冲液重悬后,对3.2mol/L马来酸进行转化,催化温度为35~37℃,pH值为8.0~8.2,转速为200r/min。
本发明的有益效果在于:本发明创新性地通过基因敲除的方式获得催化制备L-天冬氨酸的菌种,同时摆脱了依赖于石油基富马酸为原料的问题,选用价格更便宜,资源更丰富,也更容易获得马来酸作为原料,同时对天冬氨酸酶aspA和马来酸顺反异构酶maiA进行分子水平优化,提高稳定性及酶活,设计催化方案,最终获得利用马来酸高效催化合成L-天冬氨酸的菌株及催化方法,降低了生产成本,具有显著的经济性和社会效益。
本发明所述的生物材料,其分类命名为Escherichia coli△fumABC△gltA△argG/pMA,已保藏于中国典型培养物保藏中心(简称CCTCC),保藏编号为:CCTCC NO:M2019171,保藏日期为:2019年3月18日,保藏地址为:中国.武汉.武汉大学。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1
本实施例说明利用重叠PCR技术构建包含基因打靶序列和同源重组片段的目标质粒pTarget T2-1、pTarget T2-2、pTarget T2-3、pTarget T2-4的方法。
1、以SEQ ID NO:8和SEQ ID NO:9所示的核苷酸序列为引物,质粒pTarget F为模板,PCR扩增得到线性片段1;
2、以SEQ ID NO:8和SEQ ID NO:10所示的核苷酸序列为引物,重复步骤1得到线性片段2;以SEQ ID NO:8和SEQ ID NO:11所示的核苷酸序列为引物,重复步骤1得到线性片段3;以SEQ ID NO:8和SEQ ID NO:12所示的核苷酸序列为引物,重复步骤1得到线性片段4
3、将线性片段1用SpeI酶切后进行连接,并将连接后产物转化到感受态中,涂布大观霉素的LB平板筛选出阳性重组子;
4、将阳性重组子接种于添加大观霉素的LB液培扩繁后进行质粒抽提,获得pTarget T1-1质粒;
5、将线性片段2用SpeI酶切后进行连接转化筛选出阳性重组子进行质粒抽提,获得pTarget T1-2质粒;将线性片段3用SpeI酶切后进行连接转化筛选出阳性重组子进行质粒抽提,获得pTarget T1-3质粒;将线性片段4用SpeI酶切后进行连接转化筛选出阳性重组子进行质粒抽提,获得pTarget T1-4质粒;
6、以SEQ ID NO:13和SEQ ID NO:14所示的核苷酸序列为引物,大肠杆菌基因组为模板,PCR扩增得到线性片段5;以SEQ ID NO:15和SEQ ID NO:16所示的核苷酸序列为引物,大肠杆菌基因组为模板,PCR扩增得到线性片段6;
7、以SEQ ID NO:13和SEQ ID NO:16所示的核苷酸序列为引物,片段5和片段6的混合物为模板,重叠PCR扩增得到线性片段7;
8、将pTarget T1-1质粒用EcoRI线性化后去磷酸化作为载体,与片段7进行吉布森一步克隆,用大观霉素的LB平板筛选出阳性重组子;
9、将步骤8中的阳性重组子接种于添加大观霉素的LB液培扩繁后进行质粒抽提,获得pTarget T2-1质粒;
10、以SEQ ID NO:17-SEQ ID NO:20所示的核苷酸序列为引物重复步骤6至步骤9获得pTarget T2-2质粒;以SEQ ID NO:21-SEQ ID NO:24所示的核苷酸序列为引物重复步骤6至步骤9获得pTarget T2-3质粒;以SEQ ID NO:25-SEQ ID NO:28所示的核苷酸序列为引物重复步骤6至步骤9获得pTarget T2-4质粒。
实施例2
本实施例说明利用***糖诱导制备含cas9蛋白感受态的方法,具体步骤包括:
1、将pCas质粒导入待基因编辑的大肠杆菌重组菌,该生物材料在中国专利(申请号201811346790.3,申请日2018.11.13)的专利文献中公开,其保藏号为CCTCC NO:M2018521,用卡那霉素的LB平板筛选出阳性重组子;
2、将上述步骤中的阳性重组子用30mM的***糖诱导,于30℃环境中摇菌培养3-4小时,OD值约0.4~0.6,制备为感受态。
实施例3
本实施例说明利用CRISPR/Cas9技术敲除亲本大肠杆菌富马酸酶编码基因fumABC、柠檬酸合酶编码基因gltA和精氨基琥珀酸合成酶编码基因argG的过程。
1、将pTarget T2-1质粒导入含cas9蛋白感受态中,用添加大观霉素和卡那霉素的LB平板筛选出阳性重组子;
2、将上述中的阳性重组子用PCR的方法进行鉴定,筛选出目标菌株;
3、将步骤2中筛选出的菌株1转入添加了IPTG诱导的含卡那霉素的LB培养基中培养后,分别转点至含卡那霉素的LB培养基平板和同时含卡那霉素和大观霉素的LB培养基平板,筛选出已降解了pTarget T2-1质粒的重组菌株1。
4、将步骤3筛选出的重组菌株用***糖诱导,制备为感受态;
5、将pTarget T2-2质粒导入步骤4的感受态中,用添加大观霉素和卡那霉素的LB平板筛选出阳性重组子;
6、将上述中的阳性重组子用PCR的方法进行鉴定,筛选出目标菌株。
7、将步骤6中筛选出的菌株2转入添加了IPTG诱导的含卡那霉素的LB培养基中培养后,分别转点至含卡那霉素的LB培养基平板和同时含卡那霉素和大观霉素的LB培养基平板,筛选出已降解了pTarget T2-2质粒的重组菌株2。
8、用类似步骤5至步骤7的方法分批导入pTarget T2-3质粒、pTarget T2-4质粒进行基因敲除,并使用IPTG诱导消除导入质粒,获得基因敲除菌株Escherichia coli△fumABC△gltA△argG;
实施例4
本实施例说明pTrc99A-maiAmut-aspAmut质粒的构建及Escherichia coli△fumABC△gltA△argG/pMA基因工程菌筛选过程。
1、以SEQ ID NO:29--SEQ ID NO:32所示的核苷酸序列为引物,pGEMT-maiA质粒为模板,PCR扩增得到线性片段8;以SEQ ID NO:33--SEQ ID NO:38所示的核苷酸序列为引物,pGEMT-aspA质粒为模板,PCR扩增得到线性片段9,;用DpnI消化后转化大肠杆菌,获得pGEMT-maiA-G8A-G179A和pGEMT-aspA-N217K-T233R-V367G;
2、以SEQ ID NO:39和SEQ ID NO:40所示的核苷酸序列为引物,pGEMT-maiA-G8A-G179A为模板,PCR扩增得到线性片段10;以SEQ ID NO:41和SEQ ID NO:42所示的核苷酸序列为引物,pGEMT-aspA-N217K-T233R-V367G模板,PCR扩增得到线性片段11;
3、以SEQ ID NO:39和SEQ ID NO:42所示的核苷酸序列为引物,片段10和片段11的混合物为模板,重叠PCR扩增得到线性片段12;
4、将pTrc99a质粒用EcoRI和HindIII线性化后去磷酸化作为载体,与片段12进行吉布森一步克隆,用含氨苄抗性的LB平板筛选出阳性重组子;
5、将步骤4中的阳性重组子接种于添加大观霉素的LB液培扩繁后进行质粒抽提,获得pTrc99A-maiAmut-aspAmut质粒;
6、将菌株Escherichia coli△fumABC△gltA△argG制备为感受态;将步骤5中的pTrc99A-maiAmut-aspAmut质粒转化至感受态中,用含氨苄抗性的LB平板筛选出阳性重组子,获得目的基因工程菌株。
实施例5
本实施例说明采用有氧获得的微生物细胞利用马来酸催化合成L-天冬氨酸的能力。具体步骤如下:
1、采用LB培养基按1~2%(v/v)接种量从冻存管接入三角瓶中,有氧培养10~12h,进一步按1~2%(v/v)接种量接种至种子培养基2YT,培养4~6h后待菌体OD600至2.5~4之间,按5~10%接种诱导培养基(LA培养基,IPTG诱导),发酵过程中,分次加入灭菌葡萄糖,保证培养基中葡萄糖的浓度在1~50g/L。
2、种子培养过程温度控制在35~37℃,培养中不需调节pH。发酵过程采用有氧发酵模式,溶解氧为5~40%,发酵过程温度控制在28~30℃,培养过程pH用氨水控制在7.0。
3、将发酵获得的微生物细胞收集后用pH8.0的50mM Na2HPO4-KH2PO4缓冲液重悬后,对3.2mol/L马来酸进行转化,催化温度为35~37℃,pH值为8.0~8.2,转速为200r/min。转化过程中每隔60min取样检测反应液中的马来酸、富马酸和L-天冬氨酸的含量。结果见表1。
表1转化结果
从表上可见,3.2mol/L马来酸在420min后就全部转化完成,转化率达到98%以上。
序列表
<110> 南京工业大学
<120> 一种利用马来酸全细胞催化合成L-天冬氨酸的菌株与方法
<130> xb19042301
<160> 42
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1647
<212> DNA
<213> 敲除的fumA基因序列()
<400> 1
atgtcaaaca aaccctttca ttatcaggct ccttttccac tcaaaaaaga tgatactgag 60
tattacctgc taaccagcga acacgttagc gtatctgaat ttgaagggca ggagattttg 120
aaagtcgcac ccgaagcgtt aactctgttg gcgcgccagg cgtttcatga tgcgtcgttc 180
atgctgcgtc cggcgcacca acaacaggtg gccgacattc tgcgtgaccc ggaggccagc 240
gaaaatgata aatatgtggc gctgcaattc ctgcgtaact ccgacatcgc ggcgaaaggc 300
gttctgccaa cctgtcagga taccggcacc gcgattattg ttggtaaaaa agggcagcgt 360
gtatggaccg gtggtggtga tgaagcggcg ctggcgcgcg gtgtctataa cacttatatc 420
gaagataatc tgcgctactc gcaaaacgcg ccgctggata tgtataaaga agtgaatacc 480
ggcaccaatc tgccagcgca gatcgatctt tatgccgttg atggcgacga atacaaattc 540
ctctgtatcg ccaaaggtgg tggttcggca aacaagacgt atctctatca ggaaaccaaa 600
gcgttactga cgccggggaa actgaaaaat tacctggttg agaagatgcg cacgctgggt 660
acggcggcct gtcctccgta tcatattgcg ttcgttattg gtggaacttc tgcagaaacg 720
aaccttaaaa cggtgaaact ggcttccgcg aaatactatg atgaactgcc aacggaaggg 780
aatgagcacg gtcaggcgtt ccgcgatgtg gaactggaaa aagaattgct gatcgaagcg 840
caaaatcttg gtctgggtgc gcagtttggt ggtaaatact tcgctcacga catccgcgtg 900
attcgcctgc cacgtcacgg cgcatcctgc ccggtcggta tgggcgtctc ctgctctgct 960
gaccgtaata tcaaagcgaa gatcaaccgt caggggatct ggatcgaaaa actggaacat 1020
aatccaggca aatatatccc ggaagagctg cgcaaagcgg gagaaggcga agcggtgcgc 1080
gttgacctta accgtccgat gaaagagatc ctcgcacagt tgtcgcagta tcccgtttct 1140
acacgcttat cgcttaacgg cacgattatc gtcggtcgtg atattgctca cgccaaactg 1200
aaagagcgga tggataacgg tgaagggctg ccgcagtaca tcaaagatca tccgatttac 1260
tacgcgggtc cggccaaaac gccggaaggt tatgcctccg gttctcttgg cccaacgacc 1320
gccggacgga tggattctta tgtcgatcaa ctgcaagcgc agggcggaag tatgatcatg 1380
ctggcgaaag gcaaccgcag ccagcaggtg acggatgcct gtaaaaaaca cggcggcttc 1440
taccttggca gtatcggtgg tccggccgct gtattggcgc agggaagtat taagagcctg 1500
gaatgtgttg aatatccgga actgggaatg gaagccatct ggaaaattga agtggaagat 1560
ttcccggcgt ttatccttgt ggatgataaa ggaaatgact tcttccagca gatacaactc 1620
acacaatgca cccgctgtgt gaaataa 1647
<210> 2
<211> 1647
<212> DNA
<213> 敲除的fumB基因序列()
<400> 2
atgtcaaaca aaccctttat ctaccaggca cctttcccga tggggaaaga caataccgaa 60
tactatctac tcacttccga ttacgttagc gttgccgact tcgacggcga aaccatcctg 120
aaagtggaac cagaagccct gaccctgctg gcgcagcaag cctttcacga cgcttctttt 180
atgctccgcc cggcacacca gaaacaggtt gcggctattc ttcacgatcc agaagccagc 240
gaaaacgaca agtacgtggc gctgcaattc ttaagaaact ccgaaatcgc cgccaaaggc 300
gtgctgccga cctgccagga taccggcacc gcgatcatcg tcggtaaaaa aggccagcgc 360
gtgtggaccg gcggcggtga tgaagaaacg ctgtcgaaag gcgtctataa cacctatatc 420
gaagataacc tgcgctattc acagaatgcg gcgctggaca tgtacaaaga ggtcaacacc 480
ggcactaacc tgcctgcgca aatcgacctg tacgcggtag atggcgatga gtacaaattc 540
ctttgcgttg cgaaaggcgg cggctctgcc aacaaaacgt atctctacca ggaaaccaaa 600
gccctgctga ctcccggcaa actgaaaaac ttcctcgtcg agaaaatgcg taccctcggt 660
actgcagcct gcccgccgta ccatatcgcg tttgtgattg gcggtacgtc tgcggaaacc 720
aacctgaaaa ccgtcaagtt agcaagcgct cactattacg atgaactgcc gacggaaggg 780
aacgaacatg gtcaggcgtt ccgcgatgtc cagctggaac aggaactgct ggaagaggcc 840
cagaaactcg gtcttggcgc gcagtttggc ggtaaatact tcgcgcacga cattcgcgtt 900
atccgtctgc cacgtcacgg cgcatcctgc ccggtcggca tgggcgtctc ctgctccgct 960
gaccgtaaca ttaaagcgaa aatcaaccgc gaaggtatct ggatcgaaaa actggaacac 1020
aacccaggcc agtacattcc acaagaactg cgccaggccg gtgaaggcga agcggtgaaa 1080
gttgacctta accgcccgat gaaagagatc ctcgcccagc tttcgcaata cccggtatcc 1140
actcgtttgt cgctcaccgg caccattatc gtgggccgag atattgcaca cgccaagctg 1200
aaagagctga ttgacgccgg taaagaactt ccgcagtaca tcaaagatca cccgatctac 1260
tacgcgggtc cggcgaaaac ccctgccggt tatccatcag gttcacttgg cccaaccacc 1320
gcaggccgta tggactccta cgtggatctg ctgcaatccc acggcggcag catgatcatg 1380
ctggcgaaag gtaaccgcag tcagcaggtt accgacgcgt gtcataaaca cggcggcttc 1440
tacctcggta gcatcggcgg tccggcggcg gtactggcgc agcagagcat caagcatctg 1500
gagtgcgtcg cttatccgga gctgggtatg gaagctatct ggaaaatcga agtagaagat 1560
ttcccggcgt ttatcctggt cgatgacaaa ggtaacgact tcttccagca aatcgtcaac 1620
aaacagtgcg cgaactgcac taagtaa 1647
<210> 3
<211> 1404
<212> DNA
<213> 敲除的fumC基因序列()
<400> 3
atgaatacag tacgcagcga aaaagattcg atgggggcga ttgatgtccc ggcagataag 60
ctgtggggcg cacaaactca acgctcgctg gagcatttcc gcatttcgac ggagaaaatg 120
cccacctcac tgattcatgc gctggcgcta accaagcgtg cagcggcaaa agttaatgaa 180
gatttaggct tgttgtctga agagaaagcg agcgccattc gtcaggcggc ggatgaagta 240
ctggcaggac agcatgacga cgaattcccg ctggctatct ggcagaccgg ctccggcacg 300
caaagtaaca tgaacatgaa cgaagtgctg gctaaccggg ccagtgaatt actcggcggt 360
gtgcgcggga tggaacgtaa agttcaccct aacgacgacg tgaacaaaag ccaaagttcc 420
aacgatgtct ttccgacggc gatgcacgtt gcggcgctgc tggcgctgcg caagcaactc 480
attcctcagc ttaaaaccct gacacagaca ctgaatgaga aatcccgtgc ttttgccgat 540
atcgtcaaaa ttggtcgtac tcacttgcag gatgccacgc cgttaacgct ggggcaggag 600
atttccggct gggtagcgat gctcgagcat aatctcaaac atatcgaata cagcctgcct 660
cacgtagcgg aactggctct tggcggtaca gcggtgggta ctggactaaa tacccatccg 720
gagtatgcgc gtcgcgtagc agatgaactg gcagtcatta cctgtgcacc gtttgttacc 780
gcgccgaaca aatttgaagc gctggcgacc tgtgatgccc tggttcaggc gcacggcgcg 840
ttgaaagggt tggctgcgtc actgatgaaa atcgccaatg atgtccgctg gctggcctct 900
ggcccgcgct gcggaattgg tgaaatctca atcccggaaa atgagccggg cagctcaatc 960
atgccgggga aagtgaaccc aacacagtgt gaggcattaa ccatgctctg ctgtcaggtg 1020
atggggaacg acgtggcgat caacatgggg ggcgcttccg gtaactttga actgaacgtc 1080
ttccgtccaa tggtgatcca caatttcctg caatcggtgc gcttgctggc agatggcatg 1140
gaaagtttta acaaacactg cgcagtgggt attgaaccga atcgtgagcg aatcaatcaa 1200
ttactcaatg aatcgctgat gctggtgact gcgcttaaca cccacattgg ttatgacaaa 1260
gccgccgaga tcgccaaaaa agcgcataaa gaagggctga ccttaaaagc tgcggccctt 1320
gcgctggggt atcttagcga agccgagttt gacagctggg tacggccaga acagatggtc 1380
ggcagtatga aagccgggcg ttaa 1404
<210> 4
<211> 1437
<212> DNA
<213> aspA突变基因序列()
<400> 4
atgtcaaaca acattcgtat cgaagaagat ctgttgggta ccagggaagt tccagctgat 60
gcctactatg gtgttcacac tctgagagcg attgaaaact tctatatcag caacaacaaa 120
atcagtgata ttcctgaatt tgttcgcggt atggtaatgg ttaaaaaagc cgcagctatg 180
gcaaacaaag agctgcaaac cattcctaaa agtgtagcga atgccatcat tgccgcatgt 240
gatgaagtcc tgaacaacgg aaaatgcatg gatcagttcc cggtagacgt ctaccagggc 300
ggcgcaggta cttccgtaaa catgaacacc aacgaagtgc tggccaatat cggtctggaa 360
ctgatgggtc accaaaaagg tgaatatcag tacctgaacc cgaacgacca tgttaacaaa 420
tgtcagtcca ctaacgacgc ctacccgacc ggtttccgta tcgcagttta ctcttccctg 480
attaagctgg tagatgcgat taaccaactg cgtgaaggct ttgaacgtaa agctgtcgaa 540
ttccaggaca tcctgaaaat gggtcgtacc cagctgcagg acgcagtacc gatgaccctc 600
ggtcaggaat tccgcgcttt cagcatcctg ctgaaagaag aagtgaaaaa aatccaacgt 660
accgctgaac tgctgctgga agttaacctt ggtgcaagag caatcggtac tggtctgaac 720
acgccgaaag agtactctcc gctggcagtg aaaaaactgg ctgaagttac tggcttccca 780
tgcgtaccgg ctgaagacct gatcgaagcg acctctgact gcggcgctta tgttatggtt 840
cacggcgcgc tgaaacgcct ggctgtgaag atgtccaaaa tctgtaacga cctgcgcttg 900
ctctcttcag gcccacgtgc cggcctgaac gagatcaacc tgccggaact gcaggcgggc 960
tcttccatca tgccagctaa agtaaacccg gttgttccgg aagtggttaa ccaggtatgc 1020
ttcaaagtca tcggtaacga caccactgtt accatggcag cagaagcagg tcagctgcag 1080
ttgaacgtta tggagccggg cattggccag gccatgttcg aatccgttca cattctgacc 1140
aacgcttgct acaacctgct ggaaaaatgc attaacggca tcactgctaa caaagaagtg 1200
tgcgaaggtt acgtttacaa ctctatcggt atcgttactt acctgaaccc gttcatcggt 1260
caccacaacg gtgacatcgt gggtaaaatc tgtgccgaaa ccggtaagag tgtacgtgaa 1320
gtcgttctgg aacgcggtct gttgactgaa gcggaacttg acgatatttt ctccgtacag 1380
aatctgatgc acccggctta caaagcaaaa cgctatactg atgaaagcga acagtaa 1437
<210> 5
<211> 726
<212> DNA
<213> 来源Variibacter gotjawalensis maiA突变基因序列()
<400> 5
atgccgcaac gcatccgcct cgccgtcctc acgccatcat cgaatacagc actcgaaccg 60
ctgaccagcg cactcgcatc agcgctcccg aattgcagtg cacacttctc gcgcttcacc 120
gtcactgaga tcgcgctcac ggacagtgcg ctcggccaat tcgacgacag caagattctc 180
gcagccgccg aattgctcgc cgacgcgaag gtcgatgtca tcggctggag cggaacggcg 240
gcggggtggc taggcttcga ctcggatgtg cgtctgaccg agcgcatccg cgaacgcacc 300
ggcatcgtcg caacgacggc gatccttgcg ctcaacgaac tcattgcgct gaacaacatc 360
aagcggctcg cgctggtgac gccttacacc gaagacgtcc agcaacgcat catcgcgaac 420
taccgcgcca tcggtgtcga gatcgtcgcc gagcggcatt gcggcattcg cgtcaatcac 480
gactttgcgt tggtcgagcc gtcgcggctc aaagatatga tgcgcgaggt cgcggccgaa 540
aaaccgcaag cgatcgtcac gtactgcacc aacctgcgcg ccgcacagct cgccgaagaa 600
atcgaaagcg aactcggcat tccccttctc gacaccgtga gcacgacagt ctggggacag 660
ttgcgcgccg tcggcgccga cccttcgcaa attaaagggt ggggtcaaat tttcggctgg 720
aaatga 726
<210> 6
<211> 1284
<212> DNA
<213> 敲除的gltA基因序列()
<400> 6
atggctgata caaaagcaaa actcaccctc aacggggata cagctgttga actggatgtg 60
ctgaaaggca cgctgggtca agatgttatt gatatccgta ctctcggttc aaaaggtgtg 120
ttcacctttg acccaggctt cacttcaacc gcatcctgcg aatctaaaat tacttttatt 180
gatggtgatg aaggtatttt gctgcaccgc ggtttcccga tcgatcagct ggcgaccgat 240
tctaactacc tggaagtttg ttacatcctg ctgaatggtg aaaaaccgac tcaggaacag 300
tatgacgaat ttaaaactac ggtgacccgt cataccatga tccacgagca gattacccgt 360
ctgttccatg ctttccgtcg cgactcgcat ccaatggcag tcatgtgtgg tattaccggc 420
gcgctggcgg cgttctatca cgactcgctg gatgttaaca atcctcgtca ccgtgaaatt 480
gccgcgttcc gcctgctgtc gaaaatgccg accatggccg cgatgtgtta caagtattcc 540
attggtcagc catttgttta cccgcgcaac gatctctcct acgccggtaa cttcctgaat 600
atgatgttct ccacgccgtg cgaaccgtat gaagttaatc cgattctgga acgtgctatg 660
gaccgtattc tgatcctgca cgctgaccat gaacagaacg cctctacctc caccgtgcgt 720
accgctggct cttcgggtgc gaacccgttt gcctgtatcg cagcaggtat tgcttcactg 780
tggggacctg cgcacggcgg tgctaacgaa gcggcgctga aaatgctgga agaaatcagc 840
tccgttaaac acattccgga atttgttcgt cgtgcgaaag acaaaaatga ttctttccgc 900
ctgatgggct tcggtcaccg cgtgtacaaa aattacgacc cgcgcgccac cgtaatgcgt 960
gaaacctgcc atgaagtgct gaaagagctg ggcacgaagg atgacctgct ggaagtggct 1020
atggagctgg aaaacatcgc gctgaacgac ccgtacttta tcgagaagaa actgtacccg 1080
aacgtcgatt tctactctgg tatcatcctg aaagcgatgg gtattccgtc ttccatgttc 1140
accgtcattt tcgcaatggc acgtaccgtt ggctggatcg cccactggag cgaaatgcac 1200
agtgacggta tgaagattgc ccgtccgcgt cagctgtata caggatatga aaaacgcgac 1260
tttaaaagcg atatcaagcg ttaa 1284
<210> 7
<211> 1344
<212> DNA
<213> 敲除的argG基因序列()
<400> 7
atgacgacga ttctcaagca tctcccggta ggtcaacgta ttggtatcgc tttttctggc 60
ggtctggaca ccagtgccgc actgctgtgg atgcgacaaa agggagcggt tccttatgca 120
tatactgcaa acctgggcca gccagacgaa gaggattatg atgcgatccc tcgtcgtgcc 180
atggaatacg gcgcggagaa cgcacgtctg atcgactgcc gcaaacaact ggtggccgaa 240
ggtattgccg ctattcagtg tggcgcattt cataacacca ccggcggcct gacctatttc 300
aacacgacgc cgctgggccg cgccgtgact ggtaccatgc tggttgctgc gatgaaagaa 360
gatggcgtga atatctgggg tgacggtagc acctacaaag gaaacgatat cgaacgtttc 420
tatcgttatg gtctgctgac caatgctgaa ctgcagattt acaaaccgtg gcttgatact 480
gactttattg atgaactggg cggccgtcat gagatgtctg aatttatgat tgcctgcggt 540
ttcgactaca aaatgtctgt cgaaaaagcc tactccacag actccaacat gcttggtgca 600
acgcatgaag cgaaggatct ggaatacctc aactccagcg tcaaaatcgt caacccgatt 660
atgggcgtga aattctggga tgagagcgtg aagatcccgg cagaagaagt cacagtacgc 720
tttgaacaag gtcatccggt ggcgctgaac ggtaaaacct ttagcgacga cgtagaaatg 780
atgctggaag ctaaccgcat cggcggtcgt cacggcctgg gcatgagcga ccagattgaa 840
aaccgtatca tcgaagcgaa aagccgtggt atttacgaag ctccggggat ggcactgctg 900
cacattgcgt atgaacgcct gttgaccggt attcacaacg aagacaccat tgagcagtat 960
cacgcgcatg gtcgtcagtt gggccgtctg ctgtaccagg ggcgttggtt tgactcccag 1020
gcgctgatgc tgcgtgactc tctgcaacgc tgggttgcca gccagatcac tggtgaagtt 1080
accctggagc tgcgccgtgg gaacgattat tcaatcctga ataccgtctc agagaacctg 1140
acctacaagc cagagcgtct gacgatggaa aaaggcgact cggtgttctc gccagatgat 1200
cgtattggtc aattgaccat gcgtaacctg gatatcactg atacccgcga gaaacttttc 1260
ggttatgcca aaactggcct gctttcctcc tctgccgctt caggcgtgcc gcaggtggag 1320
aatctggaaa acaaaggcca gtaa 1344
<210> 8
<211> 28
<212> DNA
<213> 扩增引物序列F()
<400> 8
gatgactagt attataccta ggactgag 28
<210> 9
<211> 53
<212> DNA
<213> 扩增引物序列R()
<400> 9
ctagactagt atactatgat gaactgccaa gttttagagc tagaaatagc aag 53
<210> 10
<211> 53
<212> DNA
<213> 扩增引物序列R()
<400> 10
ctagactagt gtgagcgctt gctaacttga gttttagagc tagaaatagc aag 53
<210> 11
<211> 53
<212> DNA
<213> 扩增引物序列R()
<400> 11
ctagactagt gcaggtattg cttcactgtg gttttagagc tagaaatagc aag 53
<210> 12
<211> 53
<212> DNA
<213> 扩增引物序列R()
<400> 12
ctagactagt gtggtattta cgaagctccg gttttagagc tagaaatagc aag 53
<210> 13
<211> 35
<212> DNA
<213> 扩增引物序列F()
<400> 13
gctttttttg aattctggat gaacctgaat ggaga 35
<210> 14
<211> 41
<212> DNA
<213> 扩增引物序列R()
<400> 14
ctgcacctgt atgttgcaga tgttctctca cttactgcct g 41
<210> 15
<211> 41
<212> DNA
<213> 扩增引物序列F()
<400> 15
caggcagtaa gtgagagaac atctgcaaca tacaggtgca g 41
<210> 16
<211> 31
<212> DNA
<213> 扩增引物序列R()
<400> 16
gatctaagct tatgggtgca tcgtcatttg c 31
<210> 17
<211> 35
<212> DNA
<213> 扩增引物序列F()
<400> 17
gctttttttg aattcatcgc catcgtggcg gtgta 35
<210> 18
<211> 42
<212> DNA
<213> 扩增引物序列R()
<400> 18
ccaggcgctg ggccgaagag gagcttccag cctgtaactc tg 42
<210> 19
<211> 42
<212> DNA
<213> 扩增引物序列F()
<400> 19
cagagttaca ggctggaagc tcctcttcgg cccagcgcct gg 42
<210> 20
<211> 31
<212> DNA
<213> 扩增引物序列R()
<400> 20
gatctaagct tttacgtccg cgcacgtaca t 31
<210> 21
<211> 35
<212> DNA
<213> 扩增引物序列F()
<400> 21
gctttttttg aattcgttcc ggagacctgg cggca 35
<210> 22
<211> 42
<212> DNA
<213> 扩增引物序列R()
<400> 22
tttacaactt agcaatcaac cattaaggtc tccttagcgc ct 42
<210> 23
<211> 42
<212> DNA
<213> 扩增引物序列F()
<400> 23
aggcgctaag gagaccttaa tggttgattg ctaagttgta aa 42
<210> 24
<211> 31
<212> DNA
<213> 扩增引物序列R()
<400> 24
gatctaagct ttgaatttca caaggtgctt c 31
<210> 25
<211> 35
<212> DNA
<213> 扩增引物序列F()
<400> 25
gctttttttg aattcaatcc cactacgaag gccga 35
<210> 26
<211> 42
<212> DNA
<213> 扩增引物序列R()
<400> 26
agggcagggt tgatgtcgaa aaaataacac cctgcttaat ta 42
<210> 27
<211> 42
<212> DNA
<213> 扩增引物序列F()
<400> 27
taattaagca gggtgttatt ttttcgacat caaccctgcc ct 42
<210> 28
<211> 31
<212> DNA
<213> 扩增引物序列R()
<400> 28
gatctaagct ttccgcaatc cagcgccgtg t 31
<210> 29
<211> 33
<212> DNA
<213> 扩增引物序列F()
<400> 29
cgcaacgcat ccgcctcgcc gtcctcacgc cat 33
<210> 30
<211> 33
<212> DNA
<213> 扩增引物序列R()
<400> 30
cgatgatggc gtgaggacgg cgaggcggat gcg 33
<210> 31
<211> 33
<212> DNA
<213> 扩增引物序列F()
<400> 31
tgatgcgcga ggtcgcggcc gaaaaaccgc aag 33
<210> 32
<211> 33
<212> DNA
<213> 扩增引物序列R()
<400> 32
tcgcttgcgg tttttcggcc gcgacctcgc gca 33
<210> 33
<211> 33
<212> DNA
<213> 扩增引物序列F()
<400> 33
gaaagaagaa gtgaaaaaaa tccaacgtac cgc 33
<210> 34
<211> 33
<212> DNA
<213> 扩增引物序列R()
<400> 34
tcagcggtac gttggatttt tttcacttct tct 33
<210> 35
<211> 33
<212> DNA
<213> 扩增引物序列F()
<400> 35
aagttaacct tggtgcaaga gcaatcggta ctg 33
<210> 36
<211> 33
<212> DNA
<213> 扩增引物序列R()
<400> 36
gaccagtacc gattgctctt gcaccaaggt taa 33
<210> 37
<211> 33
<212> DNA
<213> 扩增引物序列F()
<400> 37
tgaacgttat ggagccgggc attggccagg cca 33
<210> 38
<211> 33
<212> DNA
<213> 扩增引物序列R()
<400> 38
acatggcctg gccaatgccc ggctccataa cgt 33
<210> 39
<211> 41
<212> DNA
<213> 扩增引物序列F()
<400> 39
aggaaacaga ccatggaatt catgccgcaa cgcatccgcc t 41
<210> 40
<211> 52
<212> DNA
<213> 扩增引物序列R()
<400> 40
tttctacctc cttataaata gtttcctttt catttccagc cgaaaatttg ac 52
<210> 41
<211> 54
<212> DNA
<213> 扩增引物序列F()
<400> 41
aaaggaaact atttataagg aggtagaaaa tgtcaaacaa cattcgtatc gaag 54
<210> 42
<211> 41
<212> DNA
<213> 扩增引物序列R()
<400> 42
tccgccaaaa cagccaagct tttactgttc gctttcatca g 41
Claims (9)
1.一株利用马来酸全细胞催化合成L-天冬氨酸的菌株,其分类命名为Escherichiacoli △fumABC△gltA△argG/pMA,其保藏号为CCTCC NO:M 2019171。
2.权利要求1所述菌株的构建方法,其特征在于,敲除保藏号为CCTCC NO:M 2018521的出发菌株富马酸酶编码基因fumABC、柠檬酸合酶编码基因gltA和精氨基琥珀酸合成酶编码基因argG,同时将天冬氨酸酶编码基因aspA-N217K-T233R-V367G和马来酸顺反异构酶编码基因maiA-G8A-G179A克隆到表达质粒上构成重组质粒,并将其转化至敲除了fumABC、gltA和argG基因的大肠杆菌出发菌株,得到所述基因工程菌,所述fumA基因序列如SEQ ID NO:1所示,所述fumB基因序列如SEQ ID NO:2所示,所述fumC基因序列如SEQ ID NO:3所示,所述aspA基因突变N217K-T233R-V367G后序列如SEQ ID NO:4所示,所述maiA基因序列来源Variibacter gotjawalensis突变G8A-G179A后如SEQ ID NO:5所示,所述gltA基因序列如SEQ ID NO:6所示,所述argG基因序列如SEQ ID NO:7所示。
3.根据权利要求2所述的构建方法,其特征在于,包括如下步骤:
(1)以SEQ ID NO:8和SEQ ID NO:9所示的核苷酸序列为引物,质粒pTarget F为模板,PCR扩增得到线性片段1;
(2)以SEQ ID NO:8和SEQ ID NO:10所示的核苷酸序列为引物,重复步骤(1)得到线性片段2;以SEQ ID NO:8和SEQ ID NO:11所示的核苷酸序列为引物,重复步骤(1)得到线性片段3;以SEQ ID NO:8和SEQ ID NO:12所示的核苷酸序列为引物,重复步骤(1)得到线性片段4;
(3)将线性片段1用SpeI酶切后进行连接,并将连接后产物转化到感受态中,涂布大观霉素的LB平板筛选出阳性重组子;
(4)将阳性重组子接种于添加大观霉素的LB液培扩繁后进行质粒抽提,获得pTargetT1-1质粒;
(5)将线性片段2用SpeI酶切后进行连接转化筛选出阳性重组子进行质粒抽提,获得pTarget T1-2质粒;将线性片段3用SpeI酶切后进行连接转化筛选出阳性重组子进行质粒抽提,获得pTarget T1-3质粒;将线性片段4用SpeI酶切后进行连接转化筛选出阳性重组子进行质粒抽提,获得pTarget T1-4质粒;
(6)以SEQ ID NO:13和SEQ ID NO:14所示的核苷酸序列为引物,大肠杆菌基因组为模板,PCR扩增得到线性片段5;以SEQ ID NO:15和SEQ ID NO:16所示的核苷酸序列为引物,大肠杆菌基因组为模板,PCR扩增得到线性片段6;
(7)以SEQ ID NO:13和SEQ ID NO:16所示的核苷酸序列为引物,片段5和片段6的混合物为模板,重叠PCR扩增得到线性片段7;
(8)将pTarget T1-1质粒用EcoRI线性化后去磷酸化作为载体,与片段7进行吉布森一步克隆,用大观霉素的LB平板筛选出阳性重组子;
(9)将步骤(8)中的阳性重组子接种于添加大观霉素的LB液培扩繁后进行质粒抽提,获得pTarget T2-1质粒;
(10)以SEQ ID NO:17~SEQ ID NO:20所示的核苷酸序列为引物重复步骤(6)至步骤(9)获得pTarget T2-2质粒;以SEQ ID NO:21~SEQ ID NO:24所示的核苷酸序列为引物重复步骤(6)至步骤(9)获得pTarget T2-3质粒;以SEQ ID NO:25~SEQ ID NO:28所示的核苷酸序列为引物重复步骤(6)至步骤(9)获得pTarget T2-4质粒;
(11)将pCas质粒导入保藏号为CCTCC NO:M 2018521的出发菌株中,用卡那霉素的LB平板筛选出阳性重组子;
(12)将步骤(11)中的阳性重组子用***糖诱导,制备为感受态;
(13)将pTarget T2-1质粒导入步骤(12)的感受态中,用添加大观霉素和卡那霉素的LB平板筛选出阳性重组子;
(14)将步骤(13)中的阳性重组子用PCR的方法进行鉴定,筛选出目标菌株,并使用IPTG诱导消除pTarget T2-1质粒,获得菌株1;
(15)将步骤(14)中的菌株1用***糖诱导,制备为感受态;
(16)将pTarget T2-2质粒导入步骤(15)的感受态中,用添加大观霉素和卡那霉素的LB平板筛选出阳性重组子;
(17)将步骤(16)中的阳性重组子用PCR的方法进行鉴定,筛选出目标菌株,并使用IPTG诱导消除pTarget T2-2质粒,获得菌株2;
(18)用步骤(15)至步骤(17)的方法分批导入pTarget T2-3质粒、pTarget T2-4质粒进行基因敲除,并使用IPTG诱导消除导入质粒,获得基因敲除菌株Escherichia coli△fumABC△gltA△argG;
(19)以SEQ ID NO:29~SEQ ID NO:32所示的核苷酸序列为引物,pGEMT-maiA质粒为模板,PCR扩增得到线性片段8;以SEQ ID NO:33~SEQ ID NO:38所示的核苷酸序列为引物,pGEMT-aspA质粒为模板,PCR扩增得到线性片段9;用DpnI消化后转化大肠杆菌,获得pGEMT-maiA-G8A-G179A和pGEMT-aspA-N217K-T233R-V367G;
(20)以SEQ ID NO:39和SEQ ID NO:40所示的核苷酸序列为引物,pGEMT-maiA-G8A-G179A为模板,PCR扩增得到线性片段10;以SEQ ID NO:41和SEQ ID NO:42所示的核苷酸序列为引物,pGEMT-aspA-N217K-T233R-V367G模板,PCR扩增得到线性片段11;
(21)以SEQ ID NO:39和SEQ ID NO:42所示的核苷酸序列为引物,片段10和片段11的混合物为模板,重叠PCR扩增得到线性片段12;
(22)将pTrc99a质粒用EcoRI和HindIII线性化后去磷酸化作为载体,与片段12进行吉布森一步克隆,用含氨苄抗性的LB平板筛选出阳性重组子;
(23)将步骤(22)中的阳性重组子接种于添加大观霉素的LB液培扩繁后进行质粒抽提,获得pTrc99A-maiAmut-aspAmut质粒;
(24)将步骤(18)中的Escherichia coli△fumABC△gltA△argG制备为感受态;将步骤(23)中的pTrc99A-maiAmut-aspAmut质粒转化至感受态中,用含氨苄抗性的LB平板筛选出阳性重组子,获得目的基因工程菌株。
4.利用权利要求1所述菌株生物催化合成L-天冬氨酸的方法,其特征在于,先发酵获得菌株细胞,然后将菌株细胞收集后用pH8.0的50mM Na2HPO4-KH2PO4缓冲液重悬后,对3.2mol/L马来酸进行转化。
5.根据权利要求4所述的菌株生物催化合成L-天冬氨酸的方法,其特征在于,所述发酵获得菌株细胞如下:
(S1)将所述菌株转接到LB培养基中,有氧培养10~12h,得到一级种子液;
(S2)将一级种子液转接到2YT培养基中培养,得到二级种子液;
(S3)待二级种子液OD600至8.5时,再接种到诱导培养基,所述诱导培养基的配方如下:
LA培养基:(NH4)2SO4 6g/L,玉米浆20g/L,KH2PO4 1g/L,MgSO4 1g/L,CaCl2 0.1mM,CaCO34g/L,0.8mM IPTG;
发酵过程中,分次加入灭菌葡萄糖,保证培养基中葡萄糖的浓度在1~50g/L。
6.根据权利要求5所述的方法,其特征在于,步骤(S1)和(S2)中,培养温度为35~37℃。
7.根据权利要求5所述的方法,其特征在于,步骤(S3)中,溶解氧控制在5~40%。
8.根据权利要求5所述的方法,其特征在于,步骤(S3)发酵过程中温度为28~30℃,培养过程pH用氨水调节为7.0。
9.根据权利要求4所述的方法,其特征在于,转化温度为35~37℃,pH值为8.0~8.2,转速为200r/min。
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