大肠杆菌产1，3丙二醇工业化菌株的构建及调控
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摘要
1,3-丙二醇英文缩写为1,3-PDO。是无色、无味的粘稠液体。可溶于水、醇、醚等多种有机溶剂。主要用于增塑剂、洗涤剂、防腐剂、乳化剂的合成,也用于食品、化妆品和制药等行业。其最主要的用途是合成苯二甲酸丙二醇酯(PTT)。PTT较PET(聚对苯二甲酸乙二酯)、PBT(聚对苯二甲酸丁二酯)具有更优良的性能,兼具PET的高性能和PBT的易加工性,具有广阔的应用前景,是目前合成纤维新品种开发的热点。目前用化学方法合成1,3—PD成本较高,设备投资大,技术难度高,产品分离纯化困难,特别是催化剂的制备较难,且产生CO等污染环境的废气。与化学合成方法相比,微生物生产1,3—PD具有生产成本低、转化率高、产物分离简单、无环境污染等优点。其纤维比普通纤维长15%,而且是生物可降解纤维。本实验室之前的研究证明由葡萄糖一步法合成1,3-PD是可行的,现在利用代谢调控和微生物发酵优化的方法构建可用于工业生产的基因工程菌。
已发现在酿酒酵母(Saccharamces cerevisiae)中存在甘油合成途径。当环境渗透压升高时,S.cerevisiae将启动高渗甘油应答途径(the high osmolarity glycerolresponse pathway,HOG途径),通过合成并在胞内累积甘油以便维持细胞内外渗透压的平衡。3-磷酸甘油脱氢酶(GPD1)和3-磷酸甘油磷酸化酶(GPP2)是甘油合成途径中的关键酶。目前发现能以甘油为底物发酵生产1,3-丙二醇的自然菌株如克雷伯肺炎杆菌(Klebsiella pneumoniae)、丁酸梭状芽孢杆菌(Clostridiumbutyricum)、弗氏柠檬酸菌(Citrobacter freundii)、乳酸杆菌等(Lactobacilli),其中以克雷伯肺炎杆菌和丁酸梭菌有较高的甘油耐受率和1,3-丙二醇得率。由于肺炎杆菌中1,3-丙二醇合成途径已研究的较为明白,且其生化特性与大肠杆菌(E.coli)非常接近,这就为菌种的基因改良和利用基因工程技术构建新的菌种提供了便利,故选择肺炎杆菌中1,3-丙二醇合成酶的基因作为在大肠杆菌中异源表达合成1,3-丙二醇酶的基因。克雷伯氏肺炎杆菌是兼性厌氧菌,在以甘油为底物和厌氧环境下启动1,3-PD合成系统dha途径。合成1,3-丙二醇有两个关键酶,甘油脱水酶和1,3-丙二醇氧化还原酶,近来自大肠杆菌中发现1,3-丙二醇氧化还原酶的同工酶,由基因yqhD编码。
本研究从酿酒酵母中克隆GPD1和GPP2基因,构建由单个启动子控制两个基因表达的多顺反子质粒;从肺炎杆菌中克隆dhaB和dhaT基因(或者yqhD基因),构建由同一启动子控制多个基因表达的重组质粒,然后将这两个重组质粒共转化导入大肠杆菌,成功实现了大肠杆菌由葡萄糖直接发酵生产1,3-丙二醇的策略。在此基础上,我们从代谢调控和微生物发酵优化的角度,分别在质粒改造,同工酶,菌种优化,代谢途径改造等几个方面对构建的基因工程菌进行了优化,最终我们构建了一种不需要昂贵的诱导剂而产量却更高的微生物发酵体系,这使1,3-丙二醇的微生物发酵工业生产向前迈进了一大步。本实验的结果对于其他同类型的微生物发酵体系也有一定的指导意义和现实价值
1,3-Propanediol(l,3-PDO) is a three-carbon diol. An emerging large-volume application of 1,3-PD is as a monomer in the synthesis of polyesters for use in carpet and textile fibers and so on.
A variety of chemical routes to 1,3-PDO is known . It is currently manufactured by synthetic processes beginning with petroleum derivatives such as acrolein or ethylene oxide. The chemical methods are expensive and generate waste streams containing environmental pollutants. Although 1,3-PDO is presently mainly produced by these chemical ways, there is a worldwide interest in producing this chemical by a biotechnological route. Metabolic engineering, the modification, design and construction of biochemical pathways, is an emerging discipline of potential importance to chemicals.
We have known that Saccharamces cerevisiae can produce glycerol by converting glucose through the fructose-1,6-bisphosphate pathway. When osmotic pressure rises, S.cerevisiae can start up the high osmolarity glycerol response pathway(HOG pathway), which synthesizes and accumulates glycerol to maintain the balance of osmotic pressure between the cell membrane. And the Klebsiella pneumoniae can convert glycerol to 1,3-PDO.
In Klebsiella pneumoniae, Citrobacter freundii,and Clostridium pasteurianum, the genes encoding the three structural subunits of glycerol dehydratase(dhaB1-3) and located adjacent to a gene encoding a specific 1,3-propanediol oxidoreductase(dhaT). Although the genetic organization differs somewhat among these microorganismd. these genes are clustered in a group which also comprises dhaB4 and orf2b(genes encoding a dehydratase reactivation factors for glycerol dehydratase). The specific 1,3-propanediol oxidoreductase (dhaT) of these microorganism are known to belong to the family of type III alcohol dehydrogenases. The gene (yqhD) in E.coli encodes a hypothetical oxidoreductase and contains two alcohol dehydrogenase signatures also found in the Citrobacter freundii and Klebsiella pneumoniae 1,3-propanediol dehydrogenase encoded by the dhaT gene. It's an isozyme of the 1,3-propanediol oxidoreductase (dhaT).
In this paper, metabolic pathway from glucose to 1,3-proapanediol was successfully constructed in recombinant Escherichia coli by expression of gpd1 and gpp2 genes from Saccharomyces cerevisiae EBY100 and dhaB and dhaT genes from Klebsiella pneumoniae, respectively. HPLC analysis confirmed that the conversion of glucose to 1,3-propanediol by this recombinant biocatalyst. we analysed the impacts of different type of plasmids in E.coli recombined strains and metablic flows on the titer of glycerol and 1,3-propanediol.
已发现在酿酒酵母(Saccharamces cerevisiae)中存在甘油合成途径。当环境渗透压升高时,S.cerevisiae将启动高渗甘油应答途径(the high osmolarity glycerolresponse pathway,HOG途径),通过合成并在胞内累积甘油以便维持细胞内外渗透压的平衡。3-磷酸甘油脱氢酶(GPD1)和3-磷酸甘油磷酸化酶(GPP2)是甘油合成途径中的关键酶。目前发现能以甘油为底物发酵生产1,3-丙二醇的自然菌株如克雷伯肺炎杆菌(Klebsiella pneumoniae)、丁酸梭状芽孢杆菌(Clostridiumbutyricum)、弗氏柠檬酸菌(Citrobacter freundii)、乳酸杆菌等(Lactobacilli),其中以克雷伯肺炎杆菌和丁酸梭菌有较高的甘油耐受率和1,3-丙二醇得率。由于肺炎杆菌中1,3-丙二醇合成途径已研究的较为明白,且其生化特性与大肠杆菌(E.coli)非常接近,这就为菌种的基因改良和利用基因工程技术构建新的菌种提供了便利,故选择肺炎杆菌中1,3-丙二醇合成酶的基因作为在大肠杆菌中异源表达合成1,3-丙二醇酶的基因。克雷伯氏肺炎杆菌是兼性厌氧菌,在以甘油为底物和厌氧环境下启动1,3-PD合成系统dha途径。合成1,3-丙二醇有两个关键酶,甘油脱水酶和1,3-丙二醇氧化还原酶,近来自大肠杆菌中发现1,3-丙二醇氧化还原酶的同工酶,由基因yqhD编码。
本研究从酿酒酵母中克隆GPD1和GPP2基因,构建由单个启动子控制两个基因表达的多顺反子质粒;从肺炎杆菌中克隆dhaB和dhaT基因(或者yqhD基因),构建由同一启动子控制多个基因表达的重组质粒,然后将这两个重组质粒共转化导入大肠杆菌,成功实现了大肠杆菌由葡萄糖直接发酵生产1,3-丙二醇的策略。在此基础上,我们从代谢调控和微生物发酵优化的角度,分别在质粒改造,同工酶,菌种优化,代谢途径改造等几个方面对构建的基因工程菌进行了优化,最终我们构建了一种不需要昂贵的诱导剂而产量却更高的微生物发酵体系,这使1,3-丙二醇的微生物发酵工业生产向前迈进了一大步。本实验的结果对于其他同类型的微生物发酵体系也有一定的指导意义和现实价值
1,3-Propanediol(l,3-PDO) is a three-carbon diol. An emerging large-volume application of 1,3-PD is as a monomer in the synthesis of polyesters for use in carpet and textile fibers and so on.
A variety of chemical routes to 1,3-PDO is known . It is currently manufactured by synthetic processes beginning with petroleum derivatives such as acrolein or ethylene oxide. The chemical methods are expensive and generate waste streams containing environmental pollutants. Although 1,3-PDO is presently mainly produced by these chemical ways, there is a worldwide interest in producing this chemical by a biotechnological route. Metabolic engineering, the modification, design and construction of biochemical pathways, is an emerging discipline of potential importance to chemicals.
We have known that Saccharamces cerevisiae can produce glycerol by converting glucose through the fructose-1,6-bisphosphate pathway. When osmotic pressure rises, S.cerevisiae can start up the high osmolarity glycerol response pathway(HOG pathway), which synthesizes and accumulates glycerol to maintain the balance of osmotic pressure between the cell membrane. And the Klebsiella pneumoniae can convert glycerol to 1,3-PDO.
In Klebsiella pneumoniae, Citrobacter freundii,and Clostridium pasteurianum, the genes encoding the three structural subunits of glycerol dehydratase(dhaB1-3) and located adjacent to a gene encoding a specific 1,3-propanediol oxidoreductase(dhaT). Although the genetic organization differs somewhat among these microorganismd. these genes are clustered in a group which also comprises dhaB4 and orf2b(genes encoding a dehydratase reactivation factors for glycerol dehydratase). The specific 1,3-propanediol oxidoreductase (dhaT) of these microorganism are known to belong to the family of type III alcohol dehydrogenases. The gene (yqhD) in E.coli encodes a hypothetical oxidoreductase and contains two alcohol dehydrogenase signatures also found in the Citrobacter freundii and Klebsiella pneumoniae 1,3-propanediol dehydrogenase encoded by the dhaT gene. It's an isozyme of the 1,3-propanediol oxidoreductase (dhaT).
In this paper, metabolic pathway from glucose to 1,3-proapanediol was successfully constructed in recombinant Escherichia coli by expression of gpd1 and gpp2 genes from Saccharomyces cerevisiae EBY100 and dhaB and dhaT genes from Klebsiella pneumoniae, respectively. HPLC analysis confirmed that the conversion of glucose to 1,3-propanediol by this recombinant biocatalyst. we analysed the impacts of different type of plasmids in E.coli recombined strains and metablic flows on the titer of glycerol and 1,3-propanediol.
引文
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