CN114438000B - 一株铜绿假单胞菌及其构建方法与应用 - Google Patents
一株铜绿假单胞菌及其构建方法与应用 Download PDFInfo
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- CN114438000B CN114438000B CN202011222579.8A CN202011222579A CN114438000B CN 114438000 B CN114438000 B CN 114438000B CN 202011222579 A CN202011222579 A CN 202011222579A CN 114438000 B CN114438000 B CN 114438000B
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Abstract
本发明公开了一株铜绿假单胞菌及其构建方法与应用。本发明公开一株铜绿假单胞菌重组菌,其过表达NAD+合成酶。本发明为微生物利用甘油发酵生产鼠李糖脂提供了新的开发策略。
Description
技术领域
本发明属于基因技术领域,涉及一株铜绿假单胞菌及其构建方法与应用。
背景技术
鼠李糖脂是由假单胞菌产生的一种糖脂类的阴离子表面活性剂,它具有和化学表面活性剂相同的乳化、润湿、发泡等性能,同时兼具健康无毒、绿色环保等特性,是目前研究最成熟,最深入的生物表面活性剂之一。在石油开采、污水污泥治理、土壤修复、农业生产、日用洗涤产品、化妆品、食品医药等领域均具有广泛的应用。
NADH(还原型烟酰胺腺嘌呤二核苷酸),在维持细胞生长、分化和能量代谢以及细胞保护方面起着重要作用,而调节微生物胞内NADH的浓度,可以定向的提高微生物细胞整体代谢水平,增加对碳源的消耗,实现发酵产能的提高。
CN201510100736.0孙叶芳等人通过过表达鼠李糖脂合成酶RhlAB,鼠李糖脂产量提高接近10倍,CN201711021178.4朱坤等人通过分子手段降低铜绿假单胞菌多糖合成,提高鼠李糖脂合成水平,最终鼠李糖脂产量提高93%。目前尚无铜绿假单胞菌中nadE基因与鼠李糖脂生产相关性的研究。
发明内容
本发明的目的是通过在铜绿假单胞菌中过量表达NAD+合成酶基因nadE,以提高鼠李糖脂的产量。
在一个方面,本发明提供一株铜绿假单胞菌重组菌,其过表达NAD+合成酶;相对于出发菌株,所述铜绿假单胞菌重组菌能提高鼠李糖脂的产量。
在一些实施方案中,上述铜绿假单胞菌重组菌中,所述NAD+合成酶为如下a)或b)所示的蛋白质:
a)SEQ ID NO:4所示的蛋白质;
b)将SEQ ID NO:4所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且功能相同的蛋白质;
所述b)中的蛋白质的编码基因可通过将SEQ ID NO:3所示的DNA序列缺失或添加一个或几个氨基酸残基的密码子,和/或进行一个或几个碱基对的错义突变得到。
在一些实施方案中,上述任一所述的铜绿假单胞菌重组菌中,所述过表达NAD+合成酶是通过在出发菌株中过表达nadE基因实现的;
优选地,所述nadE基因为如下1)或2)或3)所示的基因:
1)SEQ ID NO:3所示的DNA分子;
2)在严格条件下与1)限定的DNA分子杂交且编码上述任一所述的NAD+合成酶的DNA分子;
3)与1)或2)限定的DNA分子具有90%以上的同一性且编码上述任一所述的NAD+合成酶的DNA分子;
本领域普通技术人员可以很容易地采用已知的方法,例如定向进化和点突变的方法,对本发明的编码NAD+合成酶的核苷酸序列进行突变;那些经过人工修饰的,具有与本发明的编码NAD+合成酶的核苷酸序列有一定同一性的核苷酸,只要编码NAD+合成酶且具有NAD+合成酶的功能,均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与核酸序列的序列相似性。“同一性”可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间的同一性可以用百分比(%)表示,其可以用来评价相关序列之间的同一性。
在一些实施方案中,上述铜绿假单胞菌重组菌中,所述出发菌株为铜绿假单胞菌(Pseudomonas aeruginosa)KT1115,其保藏编号为CCTCC M2016686。
在第二个方面,本发明提供上述任一所述的铜绿假单胞菌重组菌的构建方法,包括在出发菌株中过表达NAD+合成酶。
在一些实施方案中,上述方法中,所述NAD+合成酶为如下a)或b)所示的蛋白质:
a)SEQ ID NO:4所示的蛋白质;
b)将SEQ ID NO:4所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且功能相同的蛋白质。
在一些实施方案中,上述任一所述的方法中,所述过表达NAD+合成酶是通过在出发菌株中过表达nadE基因实现的;
优选地,所述nadE基因为如下1)或2)或3)所示的基因:
1)SEQ ID NO:3所示的DNA分子;
2)在严格条件下与1)限定的DNA分子杂交且编码上述任一所述的NAD+合成酶的DNA分子;
3)与1)或2)限定的DNA分子具有90%以上的同一性且编码上述任一所述的NAD+合成酶的DNA分子;
所述nadE基因是通过表达载体pUCP18-nadE导入所述出发菌株中实现NAD+合成酶的过表达的,所述pUCP18-nadE是通过将SEQ ID NO:3所示的DNA分子替换pUCP18的HindⅢ和EcoRⅠ酶切位点间序列,pUCP18的其余序列不变得到的。
在一些实施方案中,上述任一所述的方法中,所述出发菌株为铜绿假单胞菌(Pseudomonas aeruginosa)KT1115,其保藏编号为CCTCC M2016686。
在第三个方面,本发明提供一种制备鼠李糖脂的方法,包括在能使上述任一所述的铜绿假单胞菌重组菌合成鼠李糖脂的条件下发酵培养所述铜绿假单胞菌重组菌。
在一些实施方案中,上述方法中,发酵培养所用的发酵培养基组成如下:甘油40-100g/L,硝酸钠2-10g/L,蛋白胨2-10g/L,磷酸氢二钾2-10g/L,磷酸氢二钠2-10g/L,硫酸镁0.1-2g/L,氯化钙0.1-2g/L,余量为水,pH自然;优选为,甘油40-100g/L,硝酸钠7.5g/L,蛋白胨5.0g/L,磷酸氢二钾2.5g/L,磷酸氢二钠2.5g/L,硫酸镁0.5g/L,氯化钙0.5g/L,余量为水,pH自然;更优选为,甘油60g/L,硝酸钠7.5g/L蛋白胨5.0g/L,磷酸氢二钾2.5g/L,磷酸氢二钠2.5g/L,硫酸镁0.5g/L,氯化钙0.5g/L;培养条件可以为30℃、200rpm震荡培养;在发酵过程中,加入IPTG诱导NAD+合成酶表达。
在第四个方面,本发明提供上述任一所述的铜绿假单胞菌重组菌在制备鼠李糖脂中的应用;
鼠李糖脂可以用在石油开采、污水污泥治理、土壤修复、农业生产、日用洗涤产品、化妆品、食品医药等领域。
本发明采用分子生物学手段,在铜绿假单胞菌KT1115中过表达nadE基因获得重组菌KT1115-nadE;将重组菌KT1115-nadE在具有不同含量的甘油的发酵培养基中进行发酵,相比于出发菌株KT1115,重组菌KT1115-nadE发酵生产的鼠李糖脂产量提高,并且对高含量甘油的耐受性提高,甘油利用率最高提升22.29%,鼠李糖脂产量最高提升33.89%。说明该重组铜绿假单胞菌菌株提高了甘油利用速率并显著提高了鼠李糖脂的产量。
本发明的有益效果是:
通过分子生物学手段,在出发铜绿假单胞菌中过表达nadE基因得到重组菌,该重组菌显著提高了鼠李糖脂的产量;另外,在增加甘油含量的条件下发酵,重组菌的甘油利用率和鼠李糖脂产量也得到明显的提高,甘油利用率最高提升22.29%,鼠李糖脂产量最高提高33.89%。本发明为微生物利用甘油发酵生产鼠李糖脂提供了新的开发策略。
附图说明
图1为nadE基因表达载体pUCP18-nadE示意图。
图2为本发明实施例1中表达载体pUCP18-nadE的PCR验证,M为DNA Marker,1为含有nadE基因的PCR扩增片段。
图3为本发明实施例2中表达载体pUCP18-nadE转化入铜绿假单胞菌KT1115的菌落PCR验证,M为DNA Marker,1为含有nadE基因的PCR扩增片段。
具体实施方式
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。
以下结合具体实施例,对本发明作进一步说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
pUCP18为上海瑞楚生物科技有限公司产品,货号为523183975015。
铜绿假单胞菌(Pseudomonas aeruginosa)KT1115是保藏于中国典型培养物保藏中心CCTCC的菌株,保藏号为CCTCC M2016686。
实施例1:pUCP18-nadE载体的构建
1、以铜绿假单胞菌KT1115的基因组DNA为模板,以nadE-F和nadE-R为引物进行PCR扩增,得到含有nadE基因的片段,nadE基因如SEQ ID NO:3所示。nadE基因编码的NAD+合成酶的氨基酸序列如SEQ ID NO:4所示。
nadE-F:5’-acgacggccagtgccaagcttATGCAACAGATCCAACGCG-3’(SEQ ID NO:1)
SEQ ID NO:1中下划线所示序列为HindⅢ酶切识别位点;
nadE-R:5’-tatgaccatgattacgaattcTCAGGGCGCCTTCGGCAG-3’(SEQ ID NO:2)
SEQ ID NO:2中下划线所示序列为EcoRⅠ酶切识别位点。
SEQ ID NO:3:
ATGCAACAGATCCAACGCGACATCGCCCAAGCCCTGCAGGTCCAGCCGCCGTTCCAGTCGGAGGCCGACGTGCAGGCGCAGATCGCCCGGCGCATCGCCTTCATCCAGCAGTGCCTGAAGGATTCCGGGCTGAAGACCCTGGTGCTGGGGATCAGCGGCGGCGTCGACTCGCTCACCGCCGGCCTGCTGGCCCAGCGCGCCGTCGAGCAACTGCGCGAGCAGACCGGCGACCAGGCCTACCGCTTCATCGCCGTGCGCCTGCCCTACCAGGTGCAGCAGGACGAGGCCGATGCCCAGGCCTCGCTGGCGACCATCCGCGCCGACGAAGAGCAGACCGTCAACATCGGTCCGTCGGTGAAAGCCCTGGCGGAACAGCTGGAAGCCCTGGAAGGACTCGAGCCGGCGAAGAGCGACTTCGTCATCGGCAATATCAAGGCGCGCATCCGCATGGTCGCCCAGTACGCCATCGCCGGCGCCCGCGGCGGCCTGGTGATCGGCACCGACCATGCTGCCGAGGCGGTCATGGGGTTCTTCACCAAGTTCGGCGACGGCGCCTGCGACCTGGCTCCGCTCAGCGGCCTGGCCAAGCATCAGGTACGCGCCCTCGCCCGCGCCCTCGGCGCTCCGGAGAACCTGGTGGAGAAGATCCCCACCGCCGACCTCGAGGACCTGCGCCCCGGCCATCCGGACGAGGCTTCCCACGGCGTCACCTATGCCGAGATCGACGCCTTCCTGCACGGCCAGCCGCTGCGCGAGGAAGCTGCGCGGGTGATCGTCGACACCTACCACAAGACCCAGCACAAGCGCGAACTGCCGAAGGCGCCCTGA
SEQ ID NO:4:
MQQIQRDIAQALQVQPPFQSEADVQAQIARRIAFIQQCLKDSGLKTLVLGISGGVDSLTAGLLAQRAVEQLREQTGDQAYRFIAVRLPYQVQQDEADAQASLATIRADEEQTVNIGPSVKALAEQLEALEGLEPAKSDFVIGNIKARIRMVAQYAIAGARGGLVIGTDHAAEAVMGFFTKFGDGACDLAPLSGLAKHQVRALARALGAPENLVEKIPTADLEDLRPGHPDEASHGVTYAEIDAFLHGQPLREEAARVIVDTYHKTQHKRELPKAP
PCR反应体系如表1所示。
表1
PCR反应程序如下:
95℃预变性5min;94℃变性30s,58℃退火30s,72℃延伸1min,循环30次;72℃后延伸10min,4℃保存。
2、HindⅢ和EcoRⅠ双酶切步骤1得到的含有nadE基因的片段,得到基因片段;HindⅢ和EcoRⅠ双酶切pUCP18,得到载体片段;将基因片段与载体片段连接,得到nadE基因表达载体pUCP18-nadE。
pUCP18-nadE的示意图如图1所示。
以表达载体pUCP18-nadE为模板,以nadE-F和nadE-R为引物进行PCR验证,结果如图2所示。并将表达载体pUCP18-nadE进行测序,结果与预期一致。
实施例2:nadE基因过表达菌株KT1115-nadE的构建
1、铜绿假单胞菌KT1115感受态细胞的制备
(1)将铜绿假单胞菌KT1115接种于2mL的LB培养基中,30℃、200rpm过夜培养;
(2)按照1%的接种量,将步骤(1)得到的菌液转接至50mL LB培养基中,30℃、200rpm培养至OD600为0.6左右;
(3)将步骤(2)得到的菌液8000rpm、4℃离心10min,取上清;
(4)将上清用30mL提前预冷的300mM的蔗糖溶液重悬,洗涤2-3次;
(5)最后加入0.5mL的300mM的蔗糖溶液重溶,即完成铜绿假单胞菌KT1115感受态细胞的制备。
2、铜绿假单胞菌KT1115的电转化
(1)取约500ng的pUCP18-nadE载体与100μL约106个/mL浓度的步骤1得到的铜绿假单胞菌KT1115感受态细胞混合,冰上静置10min;
(2)将电转混合液转入电转杯中,电转条件25μF,200Ω,2.5kw,电击;
(3)电击过后加入1mL LB培养基,转移至离心管中,30℃、100rpm培养2h;
(4)将培养液涂布到含有50μg/mL氨苄青霉素的LB平板上,30℃过夜培养;
(5)挑选平板上的转化子进行菌落PCR验证(使用引物nadE-F和nadE-R),结果如图3所示,得到重组菌,即铜绿假单胞菌KT1115的nadE过表达菌株KT1115-nadE。
实施例3:KT1115-nadE发酵生产鼠李糖脂
发酵培养基组成:甘油40-100g/L,硝酸钠7.5g/L,蛋白胨5.0g/L,磷酸氢二钾2.5g/L,磷酸氢二钠2.5g/L,硫酸镁0.5g/L,氯化钙0.5g/L,余量为水。
将实施例2获得的重组菌KT1115-nadE挑取单菌落接种到5mL LB培养基中30℃、200rpm震荡培养12h获得种子液,然后按照6%的接种量接种到装有50mL具有不同含量的基础碳源的发酵培养基中,OD600为0.3,30℃、200rpm震荡培养2天后加入终浓度为1mM的IPTG诱导NAD+合成酶表达,继续发酵至第7天,结束发酵,8000rpm常温离心10min,取上清,采用蒽酮法测定上清中的鼠李糖脂含量,其中鼠李糖脂的含量以鼠李糖含量乘以系数3.4,并采用默克的甘油含量测定试剂盒测定发酵液中的残余甘油含量。结果如表2所示。发酵实验以铜绿假单胞菌KT1115作为对照菌株。
表2
由表2可以看出,相对于对照菌株KT1115,重组菌KT1115-nadE可以提高鼠李糖脂产量,甘油消耗能力最高提高了22.29%,相应地鼠李糖脂产量最高提高了33.89%。因此,在铜绿假单胞菌KT1115中过表达nadE基因,对产鼠李糖脂具有明显有益效果。
序列表
<110> 万华化学(四川)有限公司
万华化学集团股份有限公司
<120> 一株铜绿假单胞菌及其构建方法与应用
<130> DSP1F202907ZX
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
acgacggcca gtgccaagct tatgcaacag atccaacgcg 40
<210> 2
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tatgaccatg attacgaatt ctcagggcgc cttcggcag 39
<210> 3
<211> 828
<212> DNA
<213> 铜绿假单胞菌(Pseudomonas aeruginosa)
<400> 3
atgcaacaga tccaacgcga catcgcccaa gccctgcagg tccagccgcc gttccagtcg 60
gaggccgacg tgcaggcgca gatcgcccgg cgcatcgcct tcatccagca gtgcctgaag 120
gattccgggc tgaagaccct ggtgctgggg atcagcggcg gcgtcgactc gctcaccgcc 180
ggcctgctgg cccagcgcgc cgtcgagcaa ctgcgcgagc agaccggcga ccaggcctac 240
cgcttcatcg ccgtgcgcct gccctaccag gtgcagcagg acgaggccga tgcccaggcc 300
tcgctggcga ccatccgcgc cgacgaagag cagaccgtca acatcggtcc gtcggtgaaa 360
gccctggcgg aacagctgga agccctggaa ggactcgagc cggcgaagag cgacttcgtc 420
atcggcaata tcaaggcgcg catccgcatg gtcgcccagt acgccatcgc cggcgcccgc 480
ggcggcctgg tgatcggcac cgaccatgct gccgaggcgg tcatggggtt cttcaccaag 540
ttcggcgacg gcgcctgcga cctggctccg ctcagcggcc tggccaagca tcaggtacgc 600
gccctcgccc gcgccctcgg cgctccggag aacctggtgg agaagatccc caccgccgac 660
ctcgaggacc tgcgccccgg ccatccggac gaggcttccc acggcgtcac ctatgccgag 720
atcgacgcct tcctgcacgg ccagccgctg cgcgaggaag ctgcgcgggt gatcgtcgac 780
acctaccaca agacccagca caagcgcgaa ctgccgaagg cgccctga 828
<210> 4
<211> 275
<212> PRT
<213> 铜绿假单胞菌(Pseudomonas aeruginosa)
<400> 4
Met Gln Gln Ile Gln Arg Asp Ile Ala Gln Ala Leu Gln Val Gln Pro
1 5 10 15
Pro Phe Gln Ser Glu Ala Asp Val Gln Ala Gln Ile Ala Arg Arg Ile
20 25 30
Ala Phe Ile Gln Gln Cys Leu Lys Asp Ser Gly Leu Lys Thr Leu Val
35 40 45
Leu Gly Ile Ser Gly Gly Val Asp Ser Leu Thr Ala Gly Leu Leu Ala
50 55 60
Gln Arg Ala Val Glu Gln Leu Arg Glu Gln Thr Gly Asp Gln Ala Tyr
65 70 75 80
Arg Phe Ile Ala Val Arg Leu Pro Tyr Gln Val Gln Gln Asp Glu Ala
85 90 95
Asp Ala Gln Ala Ser Leu Ala Thr Ile Arg Ala Asp Glu Glu Gln Thr
100 105 110
Val Asn Ile Gly Pro Ser Val Lys Ala Leu Ala Glu Gln Leu Glu Ala
115 120 125
Leu Glu Gly Leu Glu Pro Ala Lys Ser Asp Phe Val Ile Gly Asn Ile
130 135 140
Lys Ala Arg Ile Arg Met Val Ala Gln Tyr Ala Ile Ala Gly Ala Arg
145 150 155 160
Gly Gly Leu Val Ile Gly Thr Asp His Ala Ala Glu Ala Val Met Gly
165 170 175
Phe Phe Thr Lys Phe Gly Asp Gly Ala Cys Asp Leu Ala Pro Leu Ser
180 185 190
Gly Leu Ala Lys His Gln Val Arg Ala Leu Ala Arg Ala Leu Gly Ala
195 200 205
Pro Glu Asn Leu Val Glu Lys Ile Pro Thr Ala Asp Leu Glu Asp Leu
210 215 220
Arg Pro Gly His Pro Asp Glu Ala Ser His Gly Val Thr Tyr Ala Glu
225 230 235 240
Ile Asp Ala Phe Leu His Gly Gln Pro Leu Arg Glu Glu Ala Ala Arg
245 250 255
Val Ile Val Asp Thr Tyr His Lys Thr Gln His Lys Arg Glu Leu Pro
260 265 270
Lys Ala Pro
275
Claims (6)
1.一株铜绿假单胞菌重组菌,其过表达NAD+合成酶;
所述过表达NAD+合成酶是通过在出发菌株中过表达nadE基因实现的;
所述NAD+合成酶为SEQ ID NO:4所示的蛋白质;
所述出发菌株为铜绿假单胞菌(Pseudomonas aeruginosa)KT1115,其保藏编号为CCTCC M2016686。
2.根据权利要求1所述的铜绿假单胞菌重组菌,其特征在于:所述nadE基因为SEQ IDNO:3所示的DNA分子。
3.权利要求1或2所述的铜绿假单胞菌重组菌的构建方法,包括在出发菌株中过表达NAD+合成酶;
所述过表达NAD+合成酶是通过在所述出发菌株中过表达nadE基因实现的;
所述NAD+合成酶为SEQ ID NO:4所示的蛋白质;
所述出发菌株为铜绿假单胞菌(Pseudomonas aeruginosa)KT1115,其保藏编号为CCTCC M2016686。
4.根据权利要求3所述的方法,其特征在于:所述nadE基因为SEQ ID NO:3所示的DNA分子。
5.一种制备鼠李糖脂的方法,包括在能使权利要求1或2所述的铜绿假单胞菌重组菌合成鼠李糖脂的条件下发酵培养所述铜绿假单胞菌重组菌。
6.权利要求1或2所述的铜绿假单胞菌重组菌在制备鼠李糖脂中的应用。
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