CN109337854B - 一株敲除胞外核酸酶ExeR的谷氨酸棒杆菌及其构建方法与应用 - Google Patents
一株敲除胞外核酸酶ExeR的谷氨酸棒杆菌及其构建方法与应用 Download PDFInfo
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Abstract
本发明公开了一株敲除胞外核酸酶ExeR的谷氨酸棒杆菌,该菌株中胞外核酸酶ExeR基因失活,本发明中还公开了上述敲除胞外核酸酶ExeR的谷氨酸棒杆菌的构建方法。本发明研究了敲除谷氨酸棒状杆菌中胞外核酸酶对生物膜的作用,进一步验证eDNA作为细菌生物膜中不可或缺的成分,并成功使谷氨酸棒状杆菌成膜能力加强,为后面应用连续化发酵提供实验基础。
Description
技术领域
本发明涉及敲除胞外核酸酶ExeR的谷氨酸棒杆菌及其构建方法与应用,属于生物技术领域。
背景技术
生物被膜又被称为生物膜,是细菌为在环境中更好生长和繁殖的生存形式。生物膜的形成分为四个阶段:吸附,增值,成熟和分散,分散出来的细胞再次进入第一个阶段如此以往的循环,其中吸附时期分为可逆与不可逆阶段。在生物膜形成过程中细胞分泌出胞外物质,其中主要成分为蛋白、多聚糖、eDNA,这些化学成分聚合为致密的包涵体,从而促使了生物膜的形成。以往的研究表明蛋白作为支柱支撑着生物膜空间结构,多糖作为粘合剂充实整个空间,但最新研究表明eDNA是生物膜的灵魂主宰,并且从生物膜初始阶段就参与整个形成过程,无论是从时空上还是空间上,eDNA都无处不在。
已经有研究表明eDNA可以增加菌体初始对载体以及菌体自身之间的粘附,从而促使生物膜的后期形成,并且可以稳固生物膜的结构。生物膜的形成从第一批细胞到载体上就已经开始了,细胞开始分泌出DNA,DNA作为一种载体与细胞之间的粘合剂使得细胞与载体之间无法分开,接着以相同的方法拉近细胞与细胞之间的距离。随后细胞便会分泌出多糖和蛋白用来巩固DNA形成的网状结构,使生物膜不易被破坏。这三样胞外分泌物质是生物膜形成过程中不可缺少的。
发明内容
本发明要解决的技术问题是,提供一株敲除胞外核酸酶ExeR的谷氨酸棒杆菌以解决现有技术中谷氨酸棒杆菌成膜能力弱,不能用于连续化发酵的问题。
本发明还要解决的技术问题是,提供上述敲除胞外核酸酶ExeR的谷氨酸棒杆菌的构建方法。
本发明最后要解决的技术问题是,提供上述敲除胞外核酸酶ExeR的谷氨酸棒杆菌在发酵制备脯氨酸中的应用。
一株敲除胞外核酸酶ExeR的谷氨酸棒杆菌,该菌株中胞外核酸酶ExeR基因失活,所述胞外核酸酶ExeR基因失活是指该菌株中胞外核酸酶ExeR基因不能正常表达得到具有功能的胞外核酸酶ExeR。本发明中是通过缺失胞外核酸酶ExeR基因中的部分基因来使其失活,若是通过其他基因缺失或基因替换来使胞外核酸酶ExeR基因失活都在本发明的保护范围之内。
其中,所述谷氨酸棒杆菌为谷氨酸棒状杆菌ATCC13032。
其中,所述胞外核酸酶ExeR的基因序列如SEQ ID NO:2所示。
其中,胞外核酸酶ExeR失活后的基因序列如SEQ ID NO:3所示。
上述敲除胞外核酸酶ExeR的谷氨酸棒杆菌的构建方法,包括如下步骤:
(1)构建ExeR基因打靶片段,所述打靶片段的核苷酸序列如SEQ ID NO:1所示;
(2)将步骤(1)得到的ExeR基因打靶片段构建到质粒上,得到重组质粒;
(3)将重组质粒转化谷氨酸棒状杆菌,通过基因重组得到敲除胞外核酸酶ExeR的谷氨酸棒杆菌。
步骤(2)中,所述质粒为pk18mobsacB。
步骤(3)中,所述谷氨酸棒状杆菌为谷氨酸棒状杆菌ATCC13032。
上述敲除胞外核酸酶ExeR的谷氨酸棒杆菌在微生物发酵中的应用。
上述敲除胞外核酸酶ExeR的谷氨酸棒杆菌在发酵制备脯氨酸中的应用。
有益效果:
谷氨酸棒状杆菌是产氨基酸的重要菌株之一,但是它成膜能力是非常弱的,将出发菌株用于连续化发酵的作用几乎微乎其微,没有起到连续化发酵中周期短、产量高的特点,并且没有任何实验对谷氨酸棒状杆菌进行生物膜的研究。本发明重点研究了敲除谷氨酸棒状杆菌中胞外核酸酶对生物膜的作用,进一步验证eDNA作为细菌生物膜中不可或缺的成分,并成功使谷氨酸棒状杆菌成膜能力加强,为后面应用连续化发酵提供实验基础。本发明也扩充了eDNA在细菌菌群中起作用的数量,为日后验证eDNA对所有细菌成膜起到作用提供了研究证明。
附图说明
图1谷氨酸棒状杆菌ATCC13032与重组菌成膜效果电镜图,其中A为谷氨酸棒状杆菌ATCC13032,B为重组菌。
图2谷氨酸棒状杆菌ATCC13032与重组菌脯氨酸产量对比图。
图3谷氨酸棒状杆菌ATCC13032与重组菌脯氨酸发酵周期对比图。
图4构建pk18mobsacB/△ExeR重组质粒验证图,泳道1为Marker,泳道2为pk18mobsacB/△ExeR重组质粒验,泳道3为pk18mobsacB质粒。
图5菌落PCR验证图,敲除成功的重组菌经菌落PCR是3000bp,出发菌经菌落PCR后仍为原碱基对数量4751bp,泳道1为出发菌,泳道2、3为重组菌,泳道4为Marker。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1:构建基因敲除质粒。
用于ExeR基因敲除的重组质粒pk18mobsacB/△ExeR是通过Cross-over PCR获得的。左同源臂(1526bp)引物ExeR-L-F、ExeR-L-R和右同源臂(1552bp)引物ExeR-R-F、ExeR-R-R分别以出发菌株谷氨酸棒状杆菌ATCC13032基因组为模板进行PCR分别获得左同源臂片段和右同源臂片段。分别纯化左同源臂片段和右同源臂片段,再将其等量混合作为模板,以ExeR-L-F、ExeR-R-R为引物进行Cross-over PCR,获得将左右同源臂连接到一起、且ExeR内部有缺失的打靶片段,所述打靶片段的核苷酸序列如SEQ ID NO:1所示。将打靶片段与经BamH1单酶切后的pk18mobsacB质粒一步克隆,获得用于基因敲除的质粒pk18mobsacB/△ExeR,。
其中,引物ExeR-L-F、ExeR-L-R、ExeR-R-F、ExeR-R-R的序列如下:
ExeR-L-F(SEQ ID NO:5):aattcgagctcggtacccggggatcctcacttcgcgacctcaactccc(前20bp质粒上的,粗体字为BamH1酶切位点);
ExeR-L-R(SEQ ID NO:6):acagacaatttgttgctggtctcagg;
ExeR-R-F(SEQ ID NO:7):cctgagaccagcaacaaattgtctgtgcattggcagcctcgaccacg;
ExeR-R-R(SEQ ID NO:8):gcctgcaggtcgactctagaggatcccagacctgcgagccgacgg(后20bp质粒上的,粗体字为BamH1酶切位点)。
上下同源臂PCR体系:
PCR扩增的反应体系如下,总体系为50μl:
Buffer 25ul
dNTPs 10ul
F1 1.5ul
F2 1.5ul
模板 1ul
ddH20 19ul
PCR扩增程序如下:
95℃预变性5min;94℃变性30sec,54℃退火30sec,60℃延伸1.5min,30个循环;72℃延伸10min,4℃保存。
缺失中间部分进行Cross-over PCR的体系如下:
PCR扩增的反应体系如下,总体系为50μl:
Buffer 25ul
dNTPs 10ul
F1 1.5ul
F2 1.5ul
模板1 1ul
模板2 1ul
ddH20 18ul
PCR扩增程序如下:
95℃预变性5min;94℃变性30sec,54℃退火30sec,60℃延伸3min,30个循环;72℃延伸10min,4℃保存。
实施例2:谷氨酸棒状杆菌ATCC13032中ExeR基因的敲除。
谷氨酸棒状杆菌ATCC13032中ExeR基因敲除是通过两次同源重组的原理,通过电转化先将pk18mobsacB/△ExeR重组质粒转入谷氨酸棒状杆菌ATCC13032的感受态中进行基因打靶。在含卡那霉素的LB平板上筛选经过第一次同源重组的卡那霉素抗性克隆,即pk18mobsacB/△ExeR质粒通过同源重组整合到了谷氨酸棒状杆菌ATCC13032的基因组上。将转化子转接到LB液体培养基中,30℃、200rpm/min振荡培养过夜后涂布于含10%蔗糖的LB平板上,通过蔗糖致死基因(sacB基因)负筛选出经过第2次同源重组的克隆。将转化子分别转接到含有30mg/L卡那霉素的LB平板和LB平板上生长,能在LB平板上生长,但对卡那霉素敏感的菌株即是经过第2次同源重组的克隆。经过第2次同源重组的不一定是基因缺失的突变,也可能是恢复突变,需要对基因组进一步的PCR验证。PCR验证图如图5所示。
重组菌株构造成功后进行96孔板以及6孔板实验,以及FESEM和CLSM电镜实验。如图1所示,电镜照片可以直观具体的看出生物膜多少及形态,图1中A、B分别为出发菌谷氨酸棒状杆菌ATCC13032和ExeR基因失活的谷氨酸棒状杆菌ATCC13032,均用DAPI染剂染色,用CLAM进行检测,可以明显看出重组菌成膜效果比出发菌成膜效果好。经过验证改造后的菌株确实比出发菌株生物膜形成量多,这为以后做连续化发酵提供了基础。
实施例3:基因重组菌发酵产脯氨酸。
活化培养基每升组分如下:葡萄糖10-20g、蛋白胨8-15g、酵母粉5-12g、氯化钠8-15g。
种子培养基每升组分如下:葡萄糖25-35g、玉米浆15-25g、硫酸铵5-10g、七水硫酸镁0.1-1g、磷酸二氢钾0.5-2g、尿素1-5g。
发酵培养基每升组分如下:葡萄糖80-120g、玉米浆20-25g、硫酸铵20-30g、七水硫酸镁0.1-1g、磷酸二氢钾0.5-2g、尿素1-5g。
每50ml离心管加5ml活化培养基,分别接出发菌和重组菌,在28-34℃,200~250rpm/min条件下活化18h。
活化完成后分别倒入装有50ml种子培养基的500ml摇瓶中,在28-34℃,200~250rpm/min条件下培养12h。
将海绵作为载体,放入倒有发酵液的摇瓶当中一起灭菌,115℃,15min。
每500ml的摇瓶倒入50ml的发酵培养基,接3-5ml种子液,在28-34℃,200-250rpm/min条件下发酵72h。从图1可以看出,改造后的菌株固定化产量比出发菌高出40%。从图2可以看出,改造后的菌株固定化发酵周期比出发菌缩短31%。
利用出发菌和本发明构建的重组菌进行连续发酵实验,通过7批次发酵实验,发酵结果见表1。由表中数据可以看出,在连续化发酵到第五批的时候发酵产量趋于稳定,在第五批到第七批的时候产量没有明显增高,本研究认为连续化发酵到第七批时产量达到最高,出发菌和重组菌第七批次产量分别为13g/L和15.1g/L,均比出发菌的初始产量高。
表1固定化菌株连续发酵实验
序列表
<110> 南京工业大学
<120> 一株敲除胞外核酸酶ExeR的谷氨酸棒杆菌及其构建方法与应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4751
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tcacttcgcg acctcaactc ccaccacatc gaagtcttcg atgacggtgt ttgcgaggag 60
ggtttcagca attttcttta ggtcagcttc ggtgacggaa tcatctacct caagctcgaa 120
gcgctttccc tgacggacat cggaaacgcc agaaactccg atacgtccga gggcgcggtg 180
taccgcctgc ccctggggat ccagaatctc agccttaggc atgacattga caactacacg 240
ggccacggta ttttccctta ctcaagaaat ggggaggaca atgttttacg agcacaagtg 300
taactgttgc cactggtcaa acctagccag cccttagata gggagattct cctcgattgc 360
ttccactacc tcagctgcag atggttccgt ccgaggagca aagcgcttaa tcgtattacc 420
ttctgcatct accaggaatt tctcaaaatt ccactcgatt tcgctaccat cagttgcctc 480
tttgagcacc ttgtacaggg ggtgggcacc ctccccattc acctcggttt tgctcaagag 540
cgggaaggtg acgtcgtact gattttgcgc gaaagcacac acctcagcgt cggttccagg 600
ttcctggccg ttgaattgat tgcagggcac gccaatgaca aagaagcctc gatcttggta 660
ttcctcatac agtttttgaa gcccttcata ctgtggcgtg agtccgcact tggatgccac 720
gttcacgatg agcaaaaggt ggcccgccca atccgccatg gtggtttctg tgccgtcgtt 780
gagagttacg ctgatgtcat gaatagaagt cataatcgca accctagttg agggggagga 840
tttagtgcat catctaaata aaggtcagct aataggtgaa ctttggtgag accaaaggtg 900
aactgccagg tcgaccaaat tgctcgccaa gcagactccg aaaaacacgg gtaattcata 960
tggcttgtat ctaatccata ctgaacagag gacctctcct atgtctcgca tttctgcgcg 1020
cactctggca atcgcacttg ccggtgcaac cgcggccagc ctggcagttg ttccagcagc 1080
aacagctaat cctgccggaa ccgctcctgt catcaacgaa atctacggag gcggtggaaa 1140
cagcggatcg ttgttctcca acgacttcat tgagctctac aacccaacct caggggacat 1200
ttccctcgac ggttggagcg ttacctacta cgcagccaac ggtaactccg gcggaaccac 1260
aaacctgacc ggaaacatcc ctgccaacgg ttactacctc atccagcaac gcgcaggcag 1320
caacaacacc ggcgctctgc ctaccccaga cgccaccggt aacttggcaa tgggtgcctc 1380
ccaaggatca gttgcactga ccgacaactc tggcctaacc gctgaccttg tcggattcgg 1440
tggcacgtcc atgtttgaag gaacagctgc tgcacctgag accagcaaca aattgtctgt 1500
tcaacgcaaa gaagttggcg ctgactctga taacaactcc gtagacttcg agactggagc 1560
tccaactcca acgtcctcgg gaggatccgc tcctgttgac ccaggcgagc cagaaactcc 1620
agtaaaccct ggggaaacag tctccatcgc acaaatccaa ggaaccggtc tcgctacccc 1680
actcgagggt cagaccgtca ccaccgaagg tattgtcact gccgtttacg cagaaggtgg 1740
cttcaacggt tactacatcc agacacctgg atctggtact gcaccaaagg ttgctggcga 1800
cgcatccgac ggcatcttcg tctacgtggg aagcaatggt tcctacccag agctcggcgc 1860
atctgtcacc gtcactggca aggccaccga acactacgag atgactcagc taggcaactc 1920
ctccttcacc gtttcggaca ccgcattcga gccagtaacc ccactcgaac tggacaccgt 1980
tcctactggc gatgacattc gcgaagcata cgaaggcatg ctgctgaagc caaccggcgc 2040
tcacaccgtg accaacaact acgcaaccaa caccttcggt gaaattgccc tcgccccagg 2100
taacgagcct ttgtaccagg ccactcaaat ggtggcaccg ggagccgaag cgattgcgta 2160
cgaggcggaa aacgtcgcaa agcaaattac gctggatgac ggacgctccg gcaactacac 2220
tcgcggcgac tccagcacgc ctatggcatg gcttgtgcag gacggtggcg agaccatcaa 2280
gtccatccgc accggcgacc aggtggaatt ccaggcacca gtaatcttcg attaccgcta 2340
cgacctgtgg aaattccagc caaccacccc tgtcaccggc aacaccgcaa gctccgacct 2400
tcctatcacc tgggatgaca cccgcgcggc tgagctagct tcaatcaatg acgttgctgg 2460
cgaattccac atcgcaagct tcaacgtgct caactacttc acctctctcg gcgaagatga 2520
accaggctgc agcgcataca gggatatcaa caacacccca gtcaccgcca acaactgtaa 2580
cgtccgtggc gcttacaccg aagaagcact cgaagatcag cagagcaaga tcgtcgaagc 2640
aatcaaccgc cttgacgtcg atgttcttgg acttgaagaa atcgaaaaca ccgcgaccgt 2700
caccggcgac gtctcccgtc gcgatgacgc actcaatacc ctcgtcgcag cactcaacga 2760
agcagttgga tccgatcgct gggcggccgt cgaatctcca gaacaattgg gcaccgatga 2820
agactacatc cgcgtcgcct tcatctacga ccaaaccacc gtcaagcccg tcggcgaatc 2880
ccgaatcttc gacgacgcag ccttcaccgg caccgcacgc cagccactcg cacaggaatt 2940
ccagccactc aacgacagcg agaaatcctt cgtcggcgta gtcaaccact tcaagtccaa 3000
gggctctgtc actcgtggag acgccgacac cggcgacggc caaggcaaca acgccaacgt 3060
tcgcgtcgca caggcacagg cactcatcga ccacctggaa aaccaggacg actgggcatc 3120
caagccaatc ttcatcctcg gcgacaccaa ctcctacgcc aaggaaaccg cgatgaccac 3180
cctttacggc gctggctaca ccaacatcgc caccgaattc gacgctggct acagctacca 3240
gttctccggc cgcattggca gcctcgacca cgcactcggc aacgaagcag ccatgaagca 3300
cgtcatcgac gccgaggtct gggacatcaa cgctgacgaa gcaatcgcat tcgaatactc 3360
ccgtcgactc aacaacacct ccgacgtatt cgagaacaac gtcttccgct cctccgacca 3420
cgacccgatc aaggtcggat tcaacctcag cgagaccact gagcccacca ttccggtaga 3480
gcccactgat cctgcagaac ctaccgatcc aactacccca gttaagccaa ctgatccggt 3540
agagaccacg gatccatctg agccaaccga ccctgcagaa cctactgatc cagctgaacc 3600
aactgaccct gaggaaacga agaagccaga ggagccgaag aaccctggtt cctccaacgg 3660
aagctcccaa tacgccacca ttgcagcaat catcgcagca atcctaggtg ccattgcttt 3720
ggccttccag ttcttcccat tcaagttcta attaactctt agggagtaat cccctgccct 3780
cgcattacgc gagtgcaggg gattttgcat ttcggggcct ccgcgttcct gcgtcttgaa 3840
aacagcgatt taagaggtcg tgttttcaga atgcctatca ccttggggag ggtgccttga 3900
ggcgcttaga agcgaatctg agagggcact ttttgcagaa ccggggtttg aaatccggca 3960
tttcttgaag cgaccagaca ggcgcgacaa gattggcgaa aaagtcaaag ttttggcacg 4020
tgtgtccggt ttccagtggt tcaaaccaga caggcgtgcc aaaagctcgc aagatgtcag 4080
atttttgtca cgcctgtctg gtctctccca attgggtgcc ccgaacctcc ccaactaggc 4140
cagttgtgcg gccgcctgag aataggtttg cacctgagca cccaaatcca aggcagctaa 4200
tgcagcttcc cggaagtcag ctgcgacttt agggttggtg agatttcgcg ccgatgcgga 4260
caagcgcact gtatttcctt tttccgctcc acgagtgaca gccttgatga cattcttgcg 4320
ccaccatttt gcagcaccaa aaccttcacc cacggagagg tttctacacg ccaagaattc 4380
gccggtgtcg aggttatgta atcccctggt cgaggcggcg acgttgaaat caaattctgg 4440
gagtaccgcg aaggtgcctt ctgacattct ccaacgaggt ggggcaaaga tttggaattc 4500
gaagccaatt ttctgcattt gcctaatggc accggtaagg cgaagacgtg cttcgtgccg 4560
ttcaaggttg gcgaattctg agcgacgtcc ctgaactgct tggtcgaaac cgttgaggat 4620
gagttcgtgg ccgcgttcgc gttgttgttc catccacgcg agggtccctt tgtctttggc 4680
gagacgccag tccccatcga tgcggggtgc gacaagtaat gagacctcga tgccgtcggc 4740
tcgcaggtct g 4751
<210> 2
<211> 2751
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgtctcgca tttctgcgcg cactctggca atcgcacttg ccggtgcaac cgcggccagc 60
ctggcagttg ttccagcagc aacagctaat cctgccggaa ccgctcctgt catcaacgaa 120
atctacggag gcggtggaaa cagcggatcg ttgttctcca acgacttcat tgagctctac 180
aacccaacct caggggacat ttccctcgac ggttggagcg ttacctacta cgcagccaac 240
ggtaactccg gcggaaccac aaacctgacc ggaaacatcc ctgccaacgg ttactacctc 300
atccagcaac gcgcaggcag caacaacacc ggcgctctgc ctaccccaga cgccaccggt 360
aacttggcaa tgggtgcctc ccaaggatca gttgcactga ccgacaactc tggcctaacc 420
gctgaccttg tcggattcgg tggcacgtcc atgtttgaag gaacagctgc tgcacctgag 480
accagcaaca aattgtctgt tcaacgcaaa gaagttggcg ctgactctga taacaactcc 540
gtagacttcg agactggagc tccaactcca acgtcctcgg gaggatccgc tcctgttgac 600
ccaggcgagc cagaaactcc agtaaaccct ggggaaacag tctccatcgc acaaatccaa 660
ggaaccggtc tcgctacccc actcgagggt cagaccgtca ccaccgaagg tattgtcact 720
gccgtttacg cagaaggtgg cttcaacggt tactacatcc agacacctgg atctggtact 780
gcaccaaagg ttgctggcga cgcatccgac ggcatcttcg tctacgtggg aagcaatggt 840
tcctacccag agctcggcgc atctgtcacc gtcactggca aggccaccga acactacgag 900
atgactcagc taggcaactc ctccttcacc gtttcggaca ccgcattcga gccagtaacc 960
ccactcgaac tggacaccgt tcctactggc gatgacattc gcgaagcata cgaaggcatg 1020
ctgctgaagc caaccggcgc tcacaccgtg accaacaact acgcaaccaa caccttcggt 1080
gaaattgccc tcgccccagg taacgagcct ttgtaccagg ccactcaaat ggtggcaccg 1140
ggagccgaag cgattgcgta cgaggcggaa aacgtcgcaa agcaaattac gctggatgac 1200
ggacgctccg gcaactacac tcgcggcgac tccagcacgc ctatggcatg gcttgtgcag 1260
gacggtggcg agaccatcaa gtccatccgc accggcgacc aggtggaatt ccaggcacca 1320
gtaatcttcg attaccgcta cgacctgtgg aaattccagc caaccacccc tgtcaccggc 1380
aacaccgcaa gctccgacct tcctatcacc tgggatgaca cccgcgcggc tgagctagct 1440
tcaatcaatg acgttgctgg cgaattccac atcgcaagct tcaacgtgct caactacttc 1500
acctctctcg gcgaagatga accaggctgc agcgcataca gggatatcaa caacacccca 1560
gtcaccgcca acaactgtaa cgtccgtggc gcttacaccg aagaagcact cgaagatcag 1620
cagagcaaga tcgtcgaagc aatcaaccgc cttgacgtcg atgttcttgg acttgaagaa 1680
atcgaaaaca ccgcgaccgt caccggcgac gtctcccgtc gcgatgacgc actcaatacc 1740
ctcgtcgcag cactcaacga agcagttgga tccgatcgct gggcggccgt cgaatctcca 1800
gaacaattgg gcaccgatga agactacatc cgcgtcgcct tcatctacga ccaaaccacc 1860
gtcaagcccg tcggcgaatc ccgaatcttc gacgacgcag ccttcaccgg caccgcacgc 1920
cagccactcg cacaggaatt ccagccactc aacgacagcg agaaatcctt cgtcggcgta 1980
gtcaaccact tcaagtccaa gggctctgtc actcgtggag acgccgacac cggcgacggc 2040
caaggcaaca acgccaacgt tcgcgtcgca caggcacagg cactcatcga ccacctggaa 2100
aaccaggacg actgggcatc caagccaatc ttcatcctcg gcgacaccaa ctcctacgcc 2160
aaggaaaccg cgatgaccac cctttacggc gctggctaca ccaacatcgc caccgaattc 2220
gacgctggct acagctacca gttctccggc cgcattggca gcctcgacca cgcactcggc 2280
aacgaagcag ccatgaagca cgtcatcgac gccgaggtct gggacatcaa cgctgacgaa 2340
gcaatcgcat tcgaatactc ccgtcgactc aacaacacct ccgacgtatt cgagaacaac 2400
gtcttccgct cctccgacca cgacccgatc aaggtcggat tcaacctcag cgagaccact 2460
gagcccacca ttccggtaga gcccactgat cctgcagaac ctaccgatcc aactacccca 2520
gttaagccaa ctgatccggt agagaccacg gatccatctg agccaaccga ccctgcagaa 2580
cctactgatc cagctgaacc aactgaccct gaggaaacga agaagccaga ggagccgaag 2640
aaccctggtt cctccaacgg aagctcccaa tacgccacca ttgcagcaat catcgcagca 2700
atcctaggtg ccattgcttt ggccttccag ttcttcccat tcaagttcta a 2751
<210> 3
<211> 1751
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tcaacgcaaa gaagttggcg ctgactctga taacaactcc gtagacttcg agactggagc 60
tccaactcca acgtcctcgg gaggatccgc tcctgttgac ccaggcgagc cagaaactcc 120
agtaaaccct ggggaaacag tctccatcgc acaaatccaa ggaaccggtc tcgctacccc 180
actcgagggt cagaccgtca ccaccgaagg tattgtcact gccgtttacg cagaaggtgg 240
cttcaacggt tactacatcc agacacctgg atctggtact gcaccaaagg ttgctggcga 300
cgcatccgac ggcatcttcg tctacgtggg aagcaatggt tcctacccag agctcggcgc 360
atctgtcacc gtcactggca aggccaccga acactacgag atgactcagc taggcaactc 420
ctccttcacc gtttcggaca ccgcattcga gccagtaacc ccactcgaac tggacaccgt 480
tcctactggc gatgacattc gcgaagcata cgaaggcatg ctgctgaagc caaccggcgc 540
tcacaccgtg accaacaact acgcaaccaa caccttcggt gaaattgccc tcgccccagg 600
taacgagcct ttgtaccagg ccactcaaat ggtggcaccg ggagccgaag cgattgcgta 660
cgaggcggaa aacgtcgcaa agcaaattac gctggatgac ggacgctccg gcaactacac 720
tcgcggcgac tccagcacgc ctatggcatg gcttgtgcag gacggtggcg agaccatcaa 780
gtccatccgc accggcgacc aggtggaatt ccaggcacca gtaatcttcg attaccgcta 840
cgacctgtgg aaattccagc caaccacccc tgtcaccggc aacaccgcaa gctccgacct 900
tcctatcacc tgggatgaca cccgcgcggc tgagctagct tcaatcaatg acgttgctgg 960
cgaattccac atcgcaagct tcaacgtgct caactacttc acctctctcg gcgaagatga 1020
accaggctgc agcgcataca gggatatcaa caacacccca gtcaccgcca acaactgtaa 1080
cgtccgtggc gcttacaccg aagaagcact cgaagatcag cagagcaaga tcgtcgaagc 1140
aatcaaccgc cttgacgtcg atgttcttgg acttgaagaa atcgaaaaca ccgcgaccgt 1200
caccggcgac gtctcccgtc gcgatgacgc actcaatacc ctcgtcgcag cactcaacga 1260
agcagttgga tccgatcgct gggcggccgt cgaatctcca gaacaattgg gcaccgatga 1320
agactacatc cgcgtcgcct tcatctacga ccaaaccacc gtcaagcccg tcggcgaatc 1380
ccgaatcttc gacgacgcag ccttcaccgg caccgcacgc cagccactcg cacaggaatt 1440
ccagccactc aacgacagcg agaaatcctt cgtcggcgta gtcaaccact tcaagtccaa 1500
gggctctgtc actcgtggag acgccgacac cggcgacggc caaggcaaca acgccaacgt 1560
tcgcgtcgca caggcacagg cactcatcga ccacctggaa aaccaggacg actgggcatc 1620
caagccaatc ttcatcctcg gcgacaccaa ctcctacgcc aaggaaaccg cgatgaccac 1680
cctttacggc gctggctaca ccaacatcgc caccgaattc gacgctggct acagctacca 1740
gttctccggc c 1751
<210> 4
<211> 3000
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tcacttcgcg acctcaactc ccaccacatc gaagtcttcg atgacggtgt ttgcgaggag 60
ggtttcagca attttcttta ggtcagcttc ggtgacggaa tcatctacct caagctcgaa 120
gcgctttccc tgacggacat cggaaacgcc agaaactccg atacgtccga gggcgcggtg 180
taccgcctgc ccctggggat ccagaatctc agccttaggc atgacattga caactacacg 240
ggccacggta ttttccctta ctcaagaaat ggggaggaca atgttttacg agcacaagtg 300
taactgttgc cactggtcaa acctagccag cccttagata gggagattct cctcgattgc 360
ttccactacc tcagctgcag atggttccgt ccgaggagca aagcgcttaa tcgtattacc 420
ttctgcatct accaggaatt tctcaaaatt ccactcgatt tcgctaccat cagttgcctc 480
tttgagcacc ttgtacaggg ggtgggcacc ctccccattc acctcggttt tgctcaagag 540
cgggaaggtg acgtcgtact gattttgcgc gaaagcacac acctcagcgt cggttccagg 600
ttcctggccg ttgaattgat tgcagggcac gccaatgaca aagaagcctc gatcttggta 660
ttcctcatac agtttttgaa gcccttcata ctgtggcgtg agtccgcact tggatgccac 720
gttcacgatg agcaaaaggt ggcccgccca atccgccatg gtggtttctg tgccgtcgtt 780
gagagttacg ctgatgtcat gaatagaagt cataatcgca accctagttg agggggagga 840
tttagtgcat catctaaata aaggtcagct aataggtgaa ctttggtgag accaaaggtg 900
aactgccagg tcgaccaaat tgctcgccaa gcagactccg aaaaacacgg gtaattcata 960
tggcttgtat ctaatccata ctgaacagag gacctctcct atgtctcgca tttctgcgcg 1020
cactctggca atcgcacttg ccggtgcaac cgcggccagc ctggcagttg ttccagcagc 1080
aacagctaat cctgccggaa ccgctcctgt catcaacgaa atctacggag gcggtggaaa 1140
cagcggatcg ttgttctcca acgacttcat tgagctctac aacccaacct caggggacat 1200
ttccctcgac ggttggagcg ttacctacta cgcagccaac ggtaactccg gcggaaccac 1260
aaacctgacc ggaaacatcc ctgccaacgg ttactacctc atccagcaac gcgcaggcag 1320
caacaacacc ggcgctctgc ctaccccaga cgccaccggt aacttggcaa tgggtgcctc 1380
ccaaggatca gttgcactga ccgacaactc tggcctaacc gctgaccttg tcggattcgg 1440
tggcacgtcc atgtttgaag gaacagctgc tgcacctgag accagcaaca aattgtctgt 1500
gcattggcag cctcgaccac gcactcggca acgaagcagc catgaagcac gtcatcgacg 1560
ccgaggtctg ggacatcaac gctgacgaag caatcgcatt cgaatactcc cgtcgactca 1620
acaacacctc cgacgtattc gagaacaacg tcttccgctc ctccgaccac gacccgatca 1680
aggtcggatt caacctcagc gagaccactg agcccaccat tccggtagag cccactgatc 1740
ctgcagaacc taccgatcca actaccccag ttaagccaac tgatccggta gagaccacgg 1800
atccatctga gccaaccgac cctgcagaac ctactgatcc agctgaacca actgaccctg 1860
aggaaacgaa gaagccagag gagccgaaga accctggttc ctccaacgga agctcccaat 1920
acgccaccat tgcagcaatc atcgcagcaa tcctaggtgc cattgctttg gccttccagt 1980
tcttcccatt caagttctaa ttaactctta gggagtaatc ccctgccctc gcattacgcg 2040
agtgcagggg attttgcatt tcggggcctc cgcgttcctg cgtcttgaaa acagcgattt 2100
aagaggtcgt gttttcagaa tgcctatcac cttggggagg gtgccttgag gcgcttagaa 2160
gcgaatctga gagggcactt tttgcagaac cggggtttga aatccggcat ttcttgaagc 2220
gaccagacag gcgcgacaag attggcgaaa aagtcaaagt tttggcacgt gtgtccggtt 2280
tccagtggtt caaaccagac aggcgtgcca aaagctcgca agatgtcaga tttttgtcac 2340
gcctgtctgg tctctcccaa ttgggtgccc cgaacctccc caactaggcc agttgtgcgg 2400
ccgcctgaga ataggtttgc acctgagcac ccaaatccaa ggcagctaat gcagcttccc 2460
ggaagtcagc tgcgacttta gggttggtga gatttcgcgc cgatgcggac aagcgcactg 2520
tatttccttt ttccgctcca cgagtgacag ccttgatgac attcttgcgc caccattttg 2580
cagcaccaaa accttcaccc acggagaggt ttctacacgc caagaattcg ccggtgtcga 2640
ggttatgtaa tcccctggtc gaggcggcga cgttgaaatc aaattctggg agtaccgcga 2700
aggtgccttc tgacattctc caacgaggtg gggcaaagat ttggaattcg aagccaattt 2760
tctgcatttg cctaatggca ccggtaaggc gaagacgtgc ttcgtgccgt tcaaggttgg 2820
cgaattctga gcgacgtccc tgaactgctt ggtcgaaacc gttgaggatg agttcgtggc 2880
cgcgttcgcg ttgttgttcc atccacgcga gggtcccttt gtctttggcg agacgccagt 2940
ccccatcgat gcggggtgcg acaagtaatg agacctcgat gccgtcggct cgcaggtctg 3000
<210> 5
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
aattcgagct cggtacccgg ggatcctcac ttcgcgacct caactccc 48
<210> 6
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
acagacaatt tgttgctggt ctcagg 26
<210> 7
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cctgagacca gcaacaaatt gtctgtgcat tggcagcctc gaccacg 47
<210> 8
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gcctgcaggt cgactctaga ggatcccaga cctgcgagcc gacgg 45
Claims (4)
1.敲除胞外核酸酶ExeR的谷氨酸棒杆菌在发酵制备脯氨酸中的应用,其特征在于,该菌株中胞外核酸酶ExeR基因失活,所述胞外核酸酶ExeR失活后的基因序列如SEQ ID NO:3所示,所述谷氨酸棒杆菌为谷氨酸棒状杆菌ATCC13032。
2.根据权利要求1所述的应用,其特征在于,所述的敲除胞外核酸酶ExeR的谷氨酸棒杆菌的构建方法如下:
(1) 构建ExeR基因打靶片段,所述打靶片段的核苷酸序列如SEQ ID NO:1所示;
(2) 将步骤(1)得到的ExeR基因打靶片段构建到质粒上,得到重组质粒;
(3) 将重组质粒转化谷氨酸棒状杆菌,通过基因重组得到敲除胞外核酸酶ExeR的谷氨酸棒杆菌。
3.根据权利要求2所述的应用,其特征在于,步骤(2)中,所述质粒为pk18mobsacB。
4.根据权利要求2所述的应用,其特征在于,步骤(3)中,所述谷氨酸棒状杆菌为谷氨酸棒状杆菌ATCC13032。
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