CN114381412A - 一种合成3-羟基丙酸的重组菌及其构建方法与应用 - Google Patents
一种合成3-羟基丙酸的重组菌及其构建方法与应用 Download PDFInfo
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Abstract
一种合成3‑羟基丙酸的重组菌及其构建方法与应用,属于基因工程技术领域。针对3‑羟基丙酸的生物合成,本申请提供了一种以丙二酸为底物生物合成3‑羟基丙酸的重组菌,是将编码丙二酸单酰辅酶A合酶的基因matB、编码丙二酸单酰辅酶A还原酶的基因mcr、编码丙二酸跨膜蛋白的基因madL和编码丙二酸跨膜蛋白的基因madM导入到大肠杆菌获得的。经酶活验证,本发明制备的重组菌实现了3‑羟基丙酸的生物合成,为3‑羟基丙酸的合成提供新的思路,有助于3‑羟基丙酸的推广应用。
Description
技术领域
本发明涉及一种合成3-羟基丙酸的重组菌及其构建方法与应用,属于基因工程技术领域。
背景技术
3羟基丙酸是近些年来新兴的一种三碳平台化合物,该分子具有两种官能基团—羟基和羧基,以它为出发原料可用于合成涂料、胶黏剂、水处理化学品和个人护理用品等多种化工产品及日用品。3羟基丙酸还可通过还原作用生成1,3-丙二醇,1,3-丙二醇也是一种重要的化工原料,可作为保护剂、溶剂等,也可用于合成聚氨酯和聚对苯二甲酸丙二醇酯等。3-羟基丙酸聚合形成的3-羟基丙酸聚合物合成的材料具有熔点高、强度高、生物可降解的优势,可作为原料合成多种高附加值的材料,具有广泛的应用前景。
目前,3-羟基丙酸的生产以化学合成为主,主要以丙烯酸、3-丙内酯、3-羟丙腈、醋酸乙烯酯和1,3-丙二醇为前体物质,通过一系列化工路线进行转化。化学合成方法具有生产成本较高而产率较低等缺点;同时,用于化学合成的前体物质大都具有毒性大、致癌性的特点,严重制约了产业化应用。生物转化法合成3-羟基丙酸的相关研究起步于近二十年,一些学者利用重组大肠杆菌(Escherichia coli)或肺炎克雷伯菌(Klebsiellapneumoniae)以甘油或葡萄糖为底物进行微生物转化。为有效提高3-羟基丙酸产量,转化技术路线也各有不同,研究主要集中于工程菌的构建和发酵工艺的改进。
丙二酸是一种有机酸,目前应用非常广泛,主要用于香料和医药中间体,其下游产品覆盖面非常大,涉及塑料、染料、医药、农药、电镀等行业。目前,丙二酸主要通过丙二酸酯水解法获得,操作简单,经济方便。丙二酸分子结构中兼有羧基和活性亚甲基两种官能团,能参加各种化学反应,是非常重要的有机合成中间体。然而,目前尚无利用丙二酸合成3-羟基丙酸的报道。
发明内容
本发明的目的在于提供一种以丙二酸为底物生物合成3-羟基丙酸的重组菌及其制备方法与应用,具体技术方案如下:
一种合成3-羟基丙酸的重组菌,所述重组菌是将编码丙二酸单酰辅酶A合酶的基因matB、编码丙二酸单酰辅酶A还原酶的基因mcr、编码丙二酸跨膜蛋白的基因madL和编码丙二酸跨膜蛋白的基因madM导入到大肠杆菌获得的。
在本发明的一个实施例中,所述的编码丙二酸单酰辅酶A合酶的基因matB来源于沼泽红假单胞菌Rhodopseudomonaspalustris;所述编码丙二酸单酰辅酶A还原酶的基因mcr来源于嗜热光全绿丝菌Chloroflexus aurantiacus;所述编码丙二酸跨膜蛋白的基因madL和编码丙二酸跨膜蛋白的基因madM来源于假单胞菌pf5Pseudomonasfluorescens。
在本发明的一个实施例中,丙二酸单酰辅酶A合酶在NCBI的蛋白质编号为WP_011155789.1;丙二酸单酰辅酶A还原酶在NCBI的蛋白质编号为AAS20429.1;丙二酸跨膜蛋白MadL在NCBI的蛋白质编号为WP_015637351.1;丙二酸跨膜蛋白MadM在NCBI的蛋白质编号为WP_011063986.1。
在本发明的一个实施例中,所述编码丙二酸单酰辅酶A合酶的基因matB经过密码子优化,序列如SEQ NO.1所示;所述编码丙二酸单酰辅酶A还原酶的基因mcr经过密码子优化,序列如SEQ NO.2所示;所述编码丙二酸跨膜蛋白的基因madL和编码丙二酸跨膜蛋白的基因madM拼接后经过密码子优化,序列如SEQ NO.3所示。
本发明还提供了上述重组菌的构建方法,步骤如下:
1)分别合成所述编码丙二酸单酰辅酶A合酶的基因matB和编码丙二酸单酰辅酶A还原酶的基因mcr,将合成所得的基因连接到质粒pACYC dute-1中,然后转入E.coli DH5α感受态细胞中,筛选阳性克隆,获得重组载体pACYC dute-matB-mcr;
2)分别合成所述编码丙二酸跨膜蛋白的基因madL和编码丙二酸跨膜蛋白的基因madM,将合成所得的基因连接到质粒pBAD18中,然后转入E.coli DH5α感受态细胞中,筛选阳性克隆,获得重组载体pBAD-madLM;
3)将步骤1)与步骤2)所获得的重组载体,转化到宿主Escherichia coliBL21(DE3)感受态细胞中,获得大肠杆菌重组菌。
本发明还提供了上述重组菌在发酵生产3-羟基丙酸中的应用。
在本发明的一个实施例中,所述发酵生产3-羟基丙酸的方法,步骤如下:
1)活化重组菌,获得种子液;
2)将步骤1)所得的种子液接种到含有氨苄青霉素与氯霉素的LB培养基中培养至OD600在0.6~0.8,获得培养液;
3)向所得的培养液中加入异丙基-β-D-硫代半乳糖苷IPTG,阿拉伯糖和丙二酸,继续培养48h。
在本发明的一个实施例中,步骤2)所述培养条件为:37℃,220rpm。
在本发明的一个实施例中,步骤2)中所述种子液按体积比为2%的接种量接种到含终浓度为100μg·mL-1氨苄青霉素和终浓度为100μg·mL-1氯霉素的LB培养基中,37℃、180rpm条件下振荡培养,待OD600达到0.6时,温度调节至30℃,加入终浓度0.5mM IPTG,终质量浓度0.02%阿拉伯糖和终浓度50mM丙二酸或丙二酸衍生物,20-30℃继续培养48小时后终止发酵。
本发明还提供了一种检测上述重组菌中酶活性的方法,步骤如下:
1)活化重组菌,获得种子液;
2)将步骤1)所得的种子液接种到含有氨苄青霉素与氯霉素的LB培养基中培养至OD600在0.6~0.8,获得培养液;
3)向所得的培养液中加入异丙基-β-D-硫代半乳糖苷IPTG至终浓度为0.5mM,阿拉伯糖至终质量浓度0.02%,然后继续培养;
4)将所得的培养液于4℃离心,收集得到菌体;然后加入100mM Tris缓冲液重悬,将菌悬液用高压破碎机破碎,然后离心,得到上清即为粗酶液;
5)然后,向反应体系中加入丙二酸,步骤4)得到的粗酶液,NADPH,ATP,pH为7.8的Tris缓冲液和水,30℃摇床,220rpm,反应12个小时。
在本发明的一个实施例中,所述继续培养的条件为20℃,220rpm条件下继续培养20小时。
在本发明的一个实施例中,反应体系为1mL,其中丙二酸终浓度30mM,粗酶液300uL,NADPH终浓度1mM,ATP终浓度1mM,pH为7.8的Tris缓冲液终浓度100mM,30℃摇床,200rpm,反应12个小时。
在本发明的一种实施方式中,LB培养基的配方为:胰蛋白胨10.0g/L,酵母提取物5.0g/L,NaCl 10.0g/L。
本发明所获得的有益效果如下:
本发明以Escherichia coli BL21(DE3)为宿主菌株,通过引入3-羟基丙酸合成的关键基因,构建获得工程菌,首次以丙二酸为底物,实现了3-羟基丙酸的生物合成。丙二酸价格低廉,约为1.6万/吨,利用丙二酸通过两步酶反应合成出3-羟基丙酸,理论产率可达86.5%,将大幅降低3-羟基丙酸合成成本,并有效与现有化工体系对接,更有助于3-羟基丙酸的推广应用。
定义和缩写
在本文中使用下列的缩写或简称:
丙二酸单酰辅酶A合酶基因:matB;
丙二酸单酰辅酶A还原酶基因:mcr;
丙二酸跨膜蛋白基因:madL以及madM,实施例中为方便命名,将两基因名称合并为madLM;
大肠杆菌(Escherichia coli):E.coli;
3-羟基丙酸:3-HP。
附图说明
图1.利用丙二酸合成3-羟基丙酸的途径。
具体实施方式
下面通过实例来进一步阐明本发明,但本发明并不限于以下实施例。
下述实施例中所使用的实验方法若无特殊说明,均为常规方法。
下述实施例中所使用的材料、试剂等若无特殊说明,均可从商业途径获得。
所用酶试剂购自莫纳生物公司,提取质粒所用的试剂盒和纯化试剂盒购自SparkJade公司,相应的操作步骤按照产品说明书进行;所有培养基如无特别说明均用去离子水配制。
培养基配方:
1)培养基
LB培养基:5g/L酵母粉,10g/LNaCl,10g/L蛋白胨,其余为水,121℃,20min灭菌。
在实际培养过程中,可向上述培养基中添加一定浓度的抗生素以维持质粒的稳定性,如100mg/L的氨苄青霉素和100μg·mL-1氯霉素。
实施例1.合成3-羟基丙酸的重组菌的构建。
1.载体pACYC dute-matB-mcr的构建。
本实施例中,来源于沼泽红假单胞菌的丙二酸单酰辅酶A合酶的基因matB(matB在NCBI的蛋白质编号为WP_011155789.1)和来源于Chloroflexus aurantiacus(嗜热光全绿丝菌)的丙二酸单酰辅酶A还原酶的基因mcr(mcr在NCBI的蛋白质编号为AAS20429.1)是通过全基因合成,matB经密码子优化后合成的序列SEQ ID No.1所示;mcr经密码子优化后合成的序列如SEQ ID No.2所示;分别获得含有MtaB的质粒和mcr的质粒,载体为pACYCdute-1。
将MtaB质粒用BgIⅡ酶切,mcr质粒用BamHⅠ和BgIⅡ酶切,pACYC dute-1质粒用NcoⅠ,EcorⅤ和KpnⅠ酶切,纯化酶切产物,再进行连接,载体(pACYC dute-1质粒)与插入片段(MtaB、mcr)按照摩尔比1:2的比例,50℃连接30分钟,连接产物转化E.coli DH5α,然后涂布在加有100μg·mL-1氯霉素的LB固体平板上,PCR筛选阳性克隆。从阳性克隆中提取重组质粒pACYC dute-matB-mcr后,再通过限制性酶酶切和测序鉴定。
2.载体pBAD-madLM的构建。
本实施例中,来源于假单胞菌pf5的丙二酸跨膜蛋白的基因madLM(madLM在NCBI的蛋白质编号为WP_015637351.1和WP_011063986.1)是经密码子优化后通过全基因合成获取,合成的序列如SEQ ID No.3所示。
将madLM质粒用BgIⅡ和XbaⅠ酶切,pBAD18质粒用EcoRⅠ,XbaⅠ和HindⅢ酶切,纯化酶切产物,再进行连接,载体(pBAD18质粒)与插入片段(madLM)按照摩尔比1:2的比例,50℃连接30分钟,连接产物转化E.coli DH5α,然后涂布在加有100μg·mL-1氨苄青霉素的LB固体平板上,PCR筛选阳性克隆。从阳性克隆中提取重组质粒pBAD-madLM后,再通过限制性酶酶切和测序鉴定。
3.将pACYC dute-matB-mcr和pBAD-madLM导入E.coli BL21(DE3)并验证酶的活性。
1)将步骤1中获得的载体pACYC dute-matB-mcr与步骤2中获得的载体pBAD-madLM导入E.coli BL21(DE3)感受态细胞,涂布在含有100μg·mL-1氨苄青霉素和100μg·mL-1氯霉素的LB固体平板;涂布后的平板置于37℃恒温培养箱,继续培养至长出单克隆,将获得的工程菌株单克隆在LB培养中进行活化,获得种子液;
2)活化后的菌株按1:50的比例接种到含有50mL LB培养基的250mL摇瓶中(内含100μg·mL-1氨苄青霉素和100μg·mL-1氯霉素),37℃、220rpm条件下振荡培养,至OD600达到0.6左右,获得培养液;
3)向培养液中加入诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)至终浓度为0.5mM,阿拉伯糖至终质量浓度0.02%,然后转入20℃,220rpm条件下继续培养20小时。
4)将所得的培养液8000rpm,4℃,离心5分钟收集得到菌体;然后加入100mM Tris(pH7.8)缓冲液重悬,将菌悬液用高压破碎机破碎,将破碎后的菌液10000rpm,4℃,离心45min,得到上清即为粗酶液。
5)建立1mL反应体系:丙二酸终浓度30mM,粗酶液300uL,NADPH终浓度1mM,ATP终浓度1mM,Tris缓冲液(pH7.8)终浓度100mM,同时设计无酶空白实验作为对照,30℃摇床,220rpm,反应12个小时。将反应液4℃,10000rpm离心5min,取上清,用高效液相色谱-质谱联用检测产物。
经检测,3羟基丙酸的出峰时间为2.5分钟左右,m/z为89.0,与3-羟基丙酸标准品一致。与无酶空白对比,反应体系中检测到产物3羟基丙酸,表明了重组菌中的酶具有催化生成3羟基丙酸的活性。
实施例2.将pACYC dute-matB-mcr和pBAD-madLM导入E.coli BL21(DE3)合成3-HP。
1)将实施例1中获得的载体pACYC dute-matB-mcr与pBAD-madLM导入E.coli BL21(DE3)感受态细胞,涂布在含有100μg·mL-1氨苄青霉素和100μg·mL-1氯霉素的LB固体平板;涂布后的平板置于37℃恒温培养箱,继续培养至长出单克隆。将获得的工程菌株单克隆在LB培养中进行活化,获得种子液;
2)活化后的菌株按1:50的比例接种到含有50mL LB培养基的250mL摇瓶中(内含100μg·mL-1氨苄青霉素和100μg·mL-1氯霉素),37℃、180rpm条件下振荡培养至OD600达到0.6左右,获得培养液;
3)温度调节至30℃,向所得的培养液中加入终浓度0.5mM IPTG和终质量浓度0.02%的阿拉伯糖进行诱导,并加入终浓度50mM的底物丙二酸,继续培养48h终止发酵。
取1mL发酵液,4℃,10000rpm离心5min,取上清,用高效液相色谱-质谱联用检测产物。
3羟基丙酸的出峰时间为2.5分钟左右,m/z为89.0,与3-羟基丙酸标准品一致。反应体系中检测到产物3-羟基丙酸,产量为0.45g/L,表明了重组菌可以合成3-羟基丙酸。
合成基因序列:
matB:
AGATCTTAATTTTGTTTAACTTTAATAAGGAGATATACCatgaatgcaaatctgtttgcacgcttatttgataagcttgacgatcctcataaacttgcgatcgagacagcggctggagacaagatcagttatgccgaattagtggcacgcgcgggacgtgtagcgaatgtcttagtagcacgtggtttgcaggtaggtgaccgtgtcgctgcccaaacggaaaagagtgtagaagcattagttttgtacctggcgacggttcgcgcagggggcgtatatcttccgttaaacacggcgtacacgttgcatgagcttgattattttatcacggacgcagagcctaaaattgtcgtttgtgacccgtcaaagcgtgacggaatcgccgcgattgcagccaaggtaggggctaccgtagaaacacttggccctgatggccgtggcagtttgacagacgcagcagcaggggcttccgaggcgtttgccacaattgaccgtggtgctgacgatttagccgcaattctttacacaagcggaacgaccggtcgctctaaaggtgctatgctgagccacgacaaccttgcgagtaactccttgacgcttgtcgactattggcgctttactccagatgacgtattaattcacgctttaccgatctatcacactcacggattattcgtggcttccaacgttaccttatttgcacgcgggtccatgatctttttaccgaaatttgatcctgacaaaattctggaccttatggcacgcgctacagtcttgatgggtgtcccgactttctacacgcgtttactgcagagtccgcgtttgacgaaggaaacgaccggtcacatgcgcttgtttatcagtggcagtgctcctctgttagctgacacccatcgcgaatggtctgcaaagacaggtcacgctgtacttgaacgctatggtatgactgagacaaacatgaatacatctaatccttatgacggagaccgtgtgccgggcgcggtgggaccggcgttaccgggagtatctgcgcgcgttacagacccagaaacaggcaaagagttacctcgtggggatatcgggatgatcgaggtcaaaggtccgaacgtgttcaagggatactggcgcatgccagagaagaccaagtcggagttccgcgacgatggattcttcattactggtgatttggggaagattgatgagcgtggttatgtccatattttagggcgcggcaaagatttggtaattacaggtgggttcaacgtttacccaaaagaaattgaatctgaaattgatgcgatgccgggcgtcgtagagagtgcagttattggtgtccctcacgcggattttggggaaggggtaacagcagttgtagtacgcgataaaggagccaccatcgatgaggcccaagtccttcacggtctggacgggcagcttgcgaaatttaagatgcctaagaaagtgatctttgtagacgatttgcctcgcaatacaatgggaaaggtgcaaaagaatgtacttcgtgagacctacaaagacatctacaaataaggatccaggaggtaaataatgagtgAGATCTmcr:
GGATCCaggaggtaaataATGAGTGGCACTGGTCGGCTTGCCGGTAAGATTGCTCTGATCACGGGTGGGGCGGGCAATATCGGGTCCGAACTGACACGCCGCTTCCTGGCTGAAGGTGCCACGGTAATTATCTCTGGGCGCAATCGCGCAAAGCTCACGGCTTTAGCCGAACGGATGCAAGCTGAAGCAGGCGTCCCTGCCAAGCGGATTGATCTGGAAGTAATGGATGGTAGCGACCCAGTAGCCGTTCGCGCAGGTATTGAAGCAATTGTAGCGCGGCATGGTCAAATTGACATTCTTGTTAATAATGCTGGTAGTGCCGGTGCACAACGGCGCCTGGCTGAGATTCCGTTAACTGA
AGCGGAATTGGGGCCAGGCGCCGAAGAAACGTTGCACGCTTCCATCGCAAATTTACTCG
GCATGGGGTGGCACTTGATGCGGATCGCCGCGCCTCATATGCCGGTCGGCAGCGCAGTT
ATTAATGTTTCGACCATCTTTAGTCGTGCGGAGTATTACGGGCGTATTCCGTATGTCACGC
CGAAAGCAGCTCTGAATGCTTTGTCCCAACTCGCAGCCCGCGAACTGGGGGCACGTGG
CATTCGCGTTAACACGATCTTTCCAGGGCCTATTGAGAGTGATCGTATCCGTACGGTTTTC
CAACGGATGGATCAGCTGAAGGGTCGTCCAGAAGGGGACACGGCGCACCACTTCTTAA
ATACCATGCGTCTCTGTCGGGCCAATGACCAGGGTGCGTTGGAACGTCGCTTCCCTAGT
GTGGGGGACGTGGCCGATGCAGCCGTATTTTTAGCGTCTGCGGAGTCTGCCGCACTTTC
AGGTGAGACAATCGAGGTAACGCATGGTATGGAATTGCCTGCCTGCAGTGAAACCAGCT
TGCTTGCCCGCACAGATTTGCGCACCATTGATGCTTCTGGTCGGACAACGCTTATTTGCG
CAGGCGATCAAATCGAAGAAGTGATGGCTTTAACGGGCATGCTCCGCACGTGTGGGTCT
GAGGTCATTATCGGCTTTCGTTCCGCGGCAGCTTTAGCGCAATTTGAACAGGCAGTTAAC
GAGTCGCGGCGTCTTGCGGGGGCGGACTTCACGCCTCCTATCGCACTTCCGTTAGACCC
TCGTGACCCGGCAACAATCGATGCCGTGTTCGACTGGGGCGCGGGTGAAAACACTGGC
GGGATTCATGCAGCGGTGATTCTCCCAGCCACGTCACACGAACCAGCCCCATGCGTTATT
GAGGTGGATGACGAACGCGTCTTAAACTTTCTGGCGGATGAAATCACGGGCACAATCGT
TATCGCCTCTCGCCTCGCACGGTACTGGCAAAGCCAGCGTTTGACCCCTGGCGCCCGCG
CGCGTGGTCCTCGTGTGATTTTTCTCAGTAATGGCGCAGATCAGAACGGGAATGTGTATG
GGCGTATCCAGTCGGCTGCTATCGGCCAGCTCATTCGGGTCTGGCGGCATGAGGCGGAG
TTAGATTATCAGCGCGCAAGTGCGGCCGGGGATCACGTATTGCCACCAGTCTGGGCCAA
CCAGATTGTGCGCTTCGCGAATCGCTCGTTGGAAGGCTTGGAATTCGCATGCGCATGGA
CTGCACAATTATTACACAGCCAGCGTCATATTAACGAGATTACTTTAAATATCCCTGCCAA
TATTTCGGCTACAACGGGTGCGCGTTCCGCAAGCGTGGGGTGGGCTGAGTCCCTCATTG
GGCTTCATCTGGGGAAAGTCGCTCTCATCACGGGGGGTAGCGCAGGTATTGGGGGGCAG
ATTGGGCGGCTCTTAGCGCTTTCAGGTGCACGGGTTATGTTGGCAGCCCGCGATCGGCAT
AAGTTAGAACAAATGCAAGCCATGATTCAGTCCGAGCTGGCAGAGGTAGGCTATACTGA
CGTCGAGGATCGCGTTCATATCGCCCCAGGCTGTGACGTATCGAGTGAGGCCCAGTTAG
CAGACCTCGTCGAACGGACATTGTCTGCCTTCGGCACTGTCGATTACCTTATTAATAATG
CGGGCATTGCAGGGGTTGAGGAGATGGTTATCGATATGCCTGTAGAGGGGTGGCGGCAT
ACGCTTTTTGCGAACCTGATCTCGAACTATAGTCTTATGCGGAAATTGGCACCGCTTATG
AAAAAGCAGGGCAGTGGTTATATCCTCAATGTGTCCTCGTACTTCGGCGGCGAAAAAGA
CGCAGCAATTCCATACCCGAATCGCGCAGACTATGCAGTTTCAAAAGCCGGTCAACGGG
CGATGGCGGAAGTTTTTGCACGGTTTCTTGGCCCGGAAATTCAGATTAACGCTATCGCCC
CGGGTCCGGTTGAAGGGGATCGTTTGCGGGGTACTGGTGAACGGCCTGGTCTGTTCGCG
CGCCGTGCACGCTTAATCCTTGAGAATAAGCGTCTCAACGAATTACACGCTGCATTAATC
GCGGCGGCCCGTACTGATGAGCGGTCGATGCACGAGCTGGTCGAACTCTTACTTCCTAA
TGACGTCGCGGCACTGGAGCAAAATCCTGCGGCTCCTACTGCGCTGCGCGAGCTGGCA
CGGCGCTTCCGTTCAGAGGGCGACCCGGCGGCATCGAGCTCCTCGGCATTGCTTAACCG
TTCCATCGCCGCTAAATTACTTGCGCGGTTACATAACGGTGGCTACGTGCTTCCAGCCGA
CATTTTTGCAAACTTGCCTAACCCGCCAGACCCTTTTTTTACTCGGGCCCAAATCGATCG
TGAGGCTCGTAAGGTGCGGGACGGGATCATGGGTATGCTCTATTTACAGCGCATGCCAA
CGGAGTTCGACGTTGCAATGGCTACAGTTTACTACCTGGCGGACCGGAATGTCTCAGGG
GAGACCTTTCATCCATCAGGGGGTCTTCGCTATGAACGCACGCCTACAGGCGGCGAATT
GTTCGGCCTCCCGAGCCCAGAGCGGCTTGCCGAACTCGTGGGGAGCACGGTTTACTTGA
TCGGTGAGCACCTTACCGAACATCTCAACCTTCTCGCTCGTGCATACTTAGAACGGTATG
GGGCCCGCCAAGTCGTAATGATTGTTGAAACGGAAACGGGCGCAGAGACGATGCGGCGCCTCCTGCACGATCACGTCGAGGCGGGTCGGCTCATGACTATCGTTGCGGGCGACCAGATTGAGGCCGCTATCGATCAAGCCATCACCCGGTACGGCCGCCCTGGTCCTGTTGTGTGTACCCCGTTTCGTCCTTTACCGACAGTACCATTGGTTGGTCGCAAGGATAGTGATTGGAGCACGGTTCTTTCAGAGGCGGAATTCGCAGAACTCTGTGAACATCAACTTACCCACCATTTTCGGGTCGCCCGCAAGATTGCGCTTAGTGATGGGGCAAGCCTGGCCCTTGTTACTCCTGAGACCACTGCCACTTCAACTACCGAACAGTTCGCCTTAGCTAATTTTATCAAGACTACCTTGCACGCCTTTACCGCGACAATCGGCGTCGAATCTGAGCGCACTGCGCAGCGCATCCTGATTAATCAAGTAGATTTGACGCGGCGGGCTCGGGCTGAGGAACCACGGGACCCGCATGAGCGCCAACAAGAGCTGGAACGTTTCATTGAAGCTGTTCTGTTAGTAACAGCTCCTCTTCCTCCTGAGGCTGATACACGCTACGCGGGTCGCATCCACCGCGGCCGCGCCATTACGGTCTAAACCCTCGAGTCTGGTAAAGAAACCGCTGCAGATCT
madLM:
Agatcttttttttgggctagcgaattcgagctcgaaggagatatacaaatgaggagggaagccgcgaccgatagagcaccgactcaggctgacgaagcacaagaacaacaacttcgacgatgcactttgaggactacgacaatgattatctacggtgtggcattgctggcgatctgtacgctagcgggtgtgattatcggcgacatgctcggcgtgttgctgggggtgaaatccaatgtcggcggagtcggcatcgccatgatcctgctgatctgcgcccgtttgtggatgcacaggcacggcggcatgaccaaggattgcgagatgggcgtgggtttctggggcgccatgtacattccggtggtggtggccatggccgcccagcagaacgtggtgacggcgctgcacggcgggccggtggcggtgctggcggccatcggttcggtggtgctctgcggatgcaccatcgcccttatcagccgcacccacaagggcgagccactgccggccgagccgctggagccgggtgtgaaagccgcaccggcaggagggcgctgagatgtgggatctgatcgagaaaggcctggagcacaacggtctggtcaccgccttcgccttcgtcggagtgatcatgtgggtctcggtgctgctgtccaagcgcctgacgttcggccgggtacacggatcggccattgccattgtcatcggcctggtcctggcctgggttggcggcaccctgaccggtggtcagaagggcctggcggacctggcgctgttttccggcatcggcctgatggggggcgccatgctgcgggacttcgccatcgtcgccacggcctttgaagtacaagccaccgaggcgcgcaaggccgggatgatcggtgccgtggcgctgctgctgggtacggtactgccgttcattgtcggcgcgagcatcgcctgggccttcggctatcgcgatgcggtgagcatgaccaccattggtgccggcgcggtgacttatatcgtcggcccggtgaccggcgcagccattggtgccagctccgacgtggtggccctgtcgattgccacgggcctgatcaaggcgatcctggtgatggtgggtacgcccatggccgcgcgctggatgggcctggacaatccgcgttcggcgatggtcttcggtggcctggccggcaccgtcagcggcgtgaccgcggggctggcagccacggaccggcggctggtgccctacggcgccctgaccgccaccttccacaccgggctgggctgcctgctggggccatcgctgctgttcttcatcgtccgcggcctggtgggctaaaagcttggctgttttggcggattct
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 中国科学院青岛生物能源与过程研究所
<120> 一种合成3-羟基丙酸的重组菌及其构建方法与应用
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1582
<212> DNA
<213> 编码丙二酸单酰辅酶A合酶的基因matB
<400> 1
agatcttaat tttgtttaac tttaataagg agatatacca tgaatgcaaa tctgtttgca 60
cgcttatttg ataagcttga cgatcctcat aaacttgcga tcgagacagc ggctggagac 120
aagatcagtt atgccgaatt agtggcacgc gcgggacgtg tagcgaatgt cttagtagca 180
cgtggtttgc aggtaggtga ccgtgtcgct gcccaaacgg aaaagagtgt agaagcatta 240
gttttgtacc tggcgacggt tcgcgcaggg ggcgtatatc ttccgttaaa cacggcgtac 300
acgttgcatg agcttgatta ttttatcacg gacgcagagc ctaaaattgt cgtttgtgac 360
ccgtcaaagc gtgacggaat cgccgcgatt gcagccaagg taggggctac cgtagaaaca 420
cttggccctg atggccgtgg cagtttgaca gacgcagcag caggggcttc cgaggcgttt 480
gccacaattg accgtggtgc tgacgattta gccgcaattc tttacacaag cggaacgacc 540
ggtcgctcta aaggtgctat gctgagccac gacaaccttg cgagtaactc cttgacgctt 600
gtcgactatt ggcgctttac tccagatgac gtattaattc acgctttacc gatctatcac 660
actcacggat tattcgtggc ttccaacgtt accttatttg cacgcgggtc catgatcttt 720
ttaccgaaat ttgatcctga caaaattctg gaccttatgg cacgcgctac agtcttgatg 780
ggtgtcccga ctttctacac gcgtttactg cagagtccgc gtttgacgaa ggaaacgacc 840
ggtcacatgc gcttgtttat cagtggcagt gctcctctgt tagctgacac ccatcgcgaa 900
tggtctgcaa agacaggtca cgctgtactt gaacgctatg gtatgactga gacaaacatg 960
aatacatcta atccttatga cggagaccgt gtgccgggcg cggtgggacc ggcgttaccg 1020
ggagtatctg cgcgcgttac agacccagaa acaggcaaag agttacctcg tggggatatc 1080
gggatgatcg aggtcaaagg tccgaacgtg ttcaagggat actggcgcat gccagagaag 1140
accaagtcgg agttccgcga cgatggattc ttcattactg gtgatttggg gaagattgat 1200
gagcgtggtt atgtccatat tttagggcgc ggcaaagatt tggtaattac aggtgggttc 1260
aacgtttacc caaaagaaat tgaatctgaa attgatgcga tgccgggcgt cgtagagagt 1320
gcagttattg gtgtccctca cgcggatttt ggggaagggg taacagcagt tgtagtacgc 1380
gataaaggag ccaccatcga tgaggcccaa gtccttcacg gtctggacgg gcagcttgcg 1440
aaatttaaga tgcctaagaa agtgatcttt gtagacgatt tgcctcgcaa tacaatggga 1500
aaggtgcaaa agaatgtact tcgtgagacc tacaaagaca tctacaaata aggatccagg 1560
aggtaaataa tgagtgagat ct 1582
<210> 2
<211> 3716
<212> DNA
<213> 编码丙二酸单酰辅酶A还原酶的基因mcr
<400> 2
ggatccagga ggtaaataat gagtggcact ggtcggcttg ccggtaagat tgctctgatc 60
acgggtgggg cgggcaatat cgggtccgaa ctgacacgcc gcttcctggc tgaaggtgcc 120
acggtaatta tctctgggcg caatcgcgca aagctcacgg ctttagccga acggatgcaa 180
gctgaagcag gcgtccctgc caagcggatt gatctggaag taatggatgg tagcgaccca 240
gtagccgttc gcgcaggtat tgaagcaatt gtagcgcggc atggtcaaat tgacattctt 300
gttaataatg ctggtagtgc cggtgcacaa cggcgcctgg ctgagattcc gttaactgaa 360
gcggaattgg ggccaggcgc cgaagaaacg ttgcacgctt ccatcgcaaa tttactcggc 420
atggggtggc acttgatgcg gatcgccgcg cctcatatgc cggtcggcag cgcagttatt 480
aatgtttcga ccatctttag tcgtgcggag tattacgggc gtattccgta tgtcacgccg 540
aaagcagctc tgaatgcttt gtcccaactc gcagcccgcg aactgggggc acgtggcatt 600
cgcgttaaca cgatctttcc agggcctatt gagagtgatc gtatccgtac ggttttccaa 660
cggatggatc agctgaaggg tcgtccagaa ggggacacgg cgcaccactt cttaaatacc 720
atgcgtctct gtcgggccaa tgaccagggt gcgttggaac gtcgcttccc tagtgtgggg 780
gacgtggccg atgcagccgt atttttagcg tctgcggagt ctgccgcact ttcaggtgag 840
acaatcgagg taacgcatgg tatggaattg cctgcctgca gtgaaaccag cttgcttgcc 900
cgcacagatt tgcgcaccat tgatgcttct ggtcggacaa cgcttatttg cgcaggcgat 960
caaatcgaag aagtgatggc tttaacgggc atgctccgca cgtgtgggtc tgaggtcatt 1020
atcggctttc gttccgcggc agctttagcg caatttgaac aggcagttaa cgagtcgcgg 1080
cgtcttgcgg gggcggactt cacgcctcct atcgcacttc cgttagaccc tcgtgacccg 1140
gcaacaatcg atgccgtgtt cgactggggc gcgggtgaaa acactggcgg gattcatgca 1200
gcggtgattc tcccagccac gtcacacgaa ccagccccat gcgttattga ggtggatgac 1260
gaacgcgtct taaactttct ggcggatgaa atcacgggca caatcgttat cgcctctcgc 1320
ctcgcacggt actggcaaag ccagcgtttg acccctggcg cccgcgcgcg tggtcctcgt 1380
gtgatttttc tcagtaatgg cgcagatcag aacgggaatg tgtatgggcg tatccagtcg 1440
gctgctatcg gccagctcat tcgggtctgg cggcatgagg cggagttaga ttatcagcgc 1500
gcaagtgcgg ccggggatca cgtattgcca ccagtctggg ccaaccagat tgtgcgcttc 1560
gcgaatcgct cgttggaagg cttggaattc gcatgcgcat ggactgcaca attattacac 1620
agccagcgtc atattaacga gattacttta aatatccctg ccaatatttc ggctacaacg 1680
ggtgcgcgtt ccgcaagcgt ggggtgggct gagtccctca ttgggcttca tctggggaaa 1740
gtcgctctca tcacgggggg tagcgcaggt attggggggc agattgggcg gctcttagcg 1800
ctttcaggtg cacgggttat gttggcagcc cgcgatcggc ataagttaga acaaatgcaa 1860
gccatgattc agtccgagct ggcagaggta ggctatactg acgtcgagga tcgcgttcat 1920
atcgccccag gctgtgacgt atcgagtgag gcccagttag cagacctcgt cgaacggaca 1980
ttgtctgcct tcggcactgt cgattacctt attaataatg cgggcattgc aggggttgag 2040
gagatggtta tcgatatgcc tgtagagggg tggcggcata cgctttttgc gaacctgatc 2100
tcgaactata gtcttatgcg gaaattggca ccgcttatga aaaagcaggg cagtggttat 2160
atcctcaatg tgtcctcgta cttcggcggc gaaaaagacg cagcaattcc atacccgaat 2220
cgcgcagact atgcagtttc aaaagccggt caacgggcga tggcggaagt ttttgcacgg 2280
tttcttggcc cggaaattca gattaacgct atcgccccgg gtccggttga aggggatcgt 2340
ttgcggggta ctggtgaacg gcctggtctg ttcgcgcgcc gtgcacgctt aatccttgag 2400
aataagcgtc tcaacgaatt acacgctgca ttaatcgcgg cggcccgtac tgatgagcgg 2460
tcgatgcacg agctggtcga actcttactt cctaatgacg tcgcggcact ggagcaaaat 2520
cctgcggctc ctactgcgct gcgcgagctg gcacggcgct tccgttcaga gggcgacccg 2580
gcggcatcga gctcctcggc attgcttaac cgttccatcg ccgctaaatt acttgcgcgg 2640
ttacataacg gtggctacgt gcttccagcc gacatttttg caaacttgcc taacccgcca 2700
gacccttttt ttactcgggc ccaaatcgat cgtgaggctc gtaaggtgcg ggacgggatc 2760
atgggtatgc tctatttaca gcgcatgcca acggagttcg acgttgcaat ggctacagtt 2820
tactacctgg cggaccggaa tgtctcaggg gagacctttc atccatcagg gggtcttcgc 2880
tatgaacgca cgcctacagg cggcgaattg ttcggcctcc cgagcccaga gcggcttgcc 2940
gaactcgtgg ggagcacggt ttacttgatc ggtgagcacc ttaccgaaca tctcaacctt 3000
ctcgctcgtg catacttaga acggtatggg gcccgccaag tcgtaatgat tgttgaaacg 3060
gaaacgggcg cagagacgat gcggcgcctc ctgcacgatc acgtcgaggc gggtcggctc 3120
atgactatcg ttgcgggcga ccagattgag gccgctatcg atcaagccat cacccggtac 3180
ggccgccctg gtcctgttgt gtgtaccccg tttcgtcctt taccgacagt accattggtt 3240
ggtcgcaagg atagtgattg gagcacggtt ctttcagagg cggaattcgc agaactctgt 3300
gaacatcaac ttacccacca ttttcgggtc gcccgcaaga ttgcgcttag tgatggggca 3360
agcctggccc ttgttactcc tgagaccact gccacttcaa ctaccgaaca gttcgcctta 3420
gctaatttta tcaagactac cttgcacgcc tttaccgcga caatcggcgt cgaatctgag 3480
cgcactgcgc agcgcatcct gattaatcaa gtagatttga cgcggcgggc tcgggctgag 3540
gaaccacggg acccgcatga gcgccaacaa gagctggaac gtttcattga agctgttctg 3600
ttagtaacag ctcctcttcc tcctgaggct gatacacgct acgcgggtcg catccaccgc 3660
ggccgcgcca ttacggtcta aaccctcgag tctggtaaag aaaccgctgc agatct 3716
<210> 3
<211> 1349
<212> DNA
<213> 编码丙二酸跨膜蛋白的基因madLM
<400> 3
agatcttttt tttgggctag cgaattcgag ctcgaaggag atatacaaat gaggagggaa 60
gccgcgaccg atagagcacc gactcaggct gacgaagcac aagaacaaca acttcgacga 120
tgcactttga ggactacgac aatgattatc tacggtgtgg cattgctggc gatctgtacg 180
ctagcgggtg tgattatcgg cgacatgctc ggcgtgttgc tgggggtgaa atccaatgtc 240
ggcggagtcg gcatcgccat gatcctgctg atctgcgccc gtttgtggat gcacaggcac 300
ggcggcatga ccaaggattg cgagatgggc gtgggtttct ggggcgccat gtacattccg 360
gtggtggtgg ccatggccgc ccagcagaac gtggtgacgg cgctgcacgg cgggccggtg 420
gcggtgctgg cggccatcgg ttcggtggtg ctctgcggat gcaccatcgc ccttatcagc 480
cgcacccaca agggcgagcc actgccggcc gagccgctgg agccgggtgt gaaagccgca 540
ccggcaggag ggcgctgaga tgtgggatct gatcgagaaa ggcctggagc acaacggtct 600
ggtcaccgcc ttcgccttcg tcggagtgat catgtgggtc tcggtgctgc tgtccaagcg 660
cctgacgttc ggccgggtac acggatcggc cattgccatt gtcatcggcc tggtcctggc 720
ctgggttggc ggcaccctga ccggtggtca gaagggcctg gcggacctgg cgctgttttc 780
cggcatcggc ctgatggggg gcgccatgct gcgggacttc gccatcgtcg ccacggcctt 840
tgaagtacaa gccaccgagg cgcgcaaggc cgggatgatc ggtgccgtgg cgctgctgct 900
gggtacggta ctgccgttca ttgtcggcgc gagcatcgcc tgggccttcg gctatcgcga 960
tgcggtgagc atgaccacca ttggtgccgg cgcggtgact tatatcgtcg gcccggtgac 1020
cggcgcagcc attggtgcca gctccgacgt ggtggccctg tcgattgcca cgggcctgat 1080
caaggcgatc ctggtgatgg tgggtacgcc catggccgcg cgctggatgg gcctggacaa 1140
tccgcgttcg gcgatggtct tcggtggcct ggccggcacc gtcagcggcg tgaccgcggg 1200
gctggcagcc acggaccggc ggctggtgcc ctacggcgcc ctgaccgcca ccttccacac 1260
cgggctgggc tgcctgctgg ggccatcgct gctgttcttc atcgtccgcg gcctggtggg 1320
ctaaaagctt ggctgttttg gcggattct 1349
Claims (7)
1.一种合成3-羟基丙酸的重组菌,其特征在于,所述重组菌是将编码丙二酸单酰辅酶A合酶的基因matB、编码丙二酸单酰辅酶A还原酶的基因mcr、编码丙二酸跨膜蛋白的基因madL和编码丙二酸跨膜蛋白的基因madM导入到大肠杆菌获得的。
2.根据权利要求1所述的重组菌,其特征在于,所述的编码丙二酸单酰辅酶A合酶的基因matB来源于沼泽红假单胞菌Rhodopseudomonas palustris;所述编码丙二酸单酰辅酶A还原酶的基因mcr来源于嗜热光全绿丝菌Chloroflexus aurantiacus;所述编码丙二酸跨膜蛋白的基因madL和编码丙二酸跨膜蛋白的基因madM来源于假单胞菌pf5 Pseudomonasfluorescens。
3.根据权利要求1所述的重组菌,其特征在于,丙二酸单酰辅酶A合酶在NCBI的蛋白质编号为WP_011155789.1;丙二酸单酰辅酶A还原酶在NCBI的蛋白质编号为AAS20429.1;丙二酸跨膜蛋白MadL在NCBI的蛋白质编号为WP_015637351.1;丙二酸跨膜蛋白MadM在NCBI的蛋白质编号为WP_011063986.1。
4.根据权利要求1所述的重组菌,其特征在于,所述编码丙二酸单酰辅酶A合酶的基因matB经过密码子优化,序列如SEQ NO.1所示;所述编码丙二酸单酰辅酶A还原酶的基因mcr经过密码子优化,序列如SEQ NO.2所示;所述编码丙二酸跨膜蛋白的基因madL和编码丙二酸跨膜蛋白的基因madM拼接后经过密码子优化,序列如SEQ NO.3所示。
5.一种权利要求1-4任意一项所述的重组菌的构建方法,其特征在于,步骤如下:
1)分别合成所述编码丙二酸单酰辅酶A合酶的基因matB和编码丙二酸单酰辅酶A还原酶的基因mcr,将合成所得的基因连接到质粒pACYC dute-1中,然后转入E.coli DH5α感受态细胞中,筛选阳性克隆,获得重组载体pACYC dute-matB-mcr;
2)分别合成所述编码丙二酸跨膜蛋白的基因madL和编码丙二酸跨膜蛋白的基因madM,将合成所得的基因连接到质粒pBAD18中,然后转入E.coli DH5α感受态细胞中,筛选阳性克隆,获得重组载体pBAD-madLM;
3)将步骤1)与步骤2)所获得的重组载体,转化到宿主Escherichia coli BL21(DE3)感受态细胞中,获得大肠杆菌重组菌。
6.一种权利要求1-4任意一项所述的重组菌在发酵生产3-羟基丙酸中的应用。
7.根据权利要求6所述的应用,其特征在于,所述发酵生产3-羟基丙酸的方法,步骤如下:
1)活化重组菌,获得种子液;
2)将步骤1)所得的种子液接种到含有氨苄青霉素与氯霉素的LB培养基中培养至OD600在0.6~0.8,获得培养液;
3)向所得的培养液中加入异丙基-β-D-硫代半乳糖苷IPTG,阿拉伯糖和丙二酸,继续培养48h。
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