Rearrangement of Gene Order in the phaCAB Operon Leads to Effective Production of Ultrahigh-Molecular-Weight Poly[(R)-3-Hydroxybutyrate] in Genetically Engineered Escherichia coli
Rearrangement of Gene Order in the phaCAB Operon Leads to Effective Production of Ultrahigh-Molecular-Weight Poly[(R)-3-Hydroxybutyrate] in Genetically Engineered Escherichia coli
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DOI：
10.1128/aem.07715-11
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发表时间：
2012-05-01
影响因子：
4.4
通讯作者：
Tsuge, Takeharu
中科院分区：
文献类型：
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作者：
Hiroe, Ayaka;Tsuge, Kenji;Tsuge, Takeharu
由基因工程大肠杆菌合成的超高分子量聚[（R）-3-羟基丁酸酯] [β-P（3 HB）]是一种环境友好的生物塑料材料，可加工成高强度的薄膜或纤维。一个由三个基因组成的操纵子（组织为phaCAB）编码在天然生产者真养罗尔斯通氏菌中生产P（3 HB）所必需的蛋白质。phaCAB操纵子的三个基因是phaC，其编码聚羟基链烷酸（PHA）合酶，phaA，其编码3-酮硫解酶，和phaB，其编码乙酰乙酰辅酶A（乙酰乙酰-CoA）还原酶。在本研究中，在基因工程大肠杆菌中，研究了phaCAB操纵子基因顺序（phaABC、phaACB、phaBAC、phaBCA、phaCAB和phaCBA）对表达质粒的影响。对大肠杆菌进行了筛选，以确定产β-P（3 HB）的最佳组织。结果表明，P（3 HB）分子量和积累水平都依赖于PHA基因相对于启动子的顺序。在携带phaBCA表达质粒的菌株中实现了最平衡的生产结果。此外，P（3 HB）生物合成酶的表达水平和活性以及P（3 HB）分子量的分析表明，活性PHA合成酶的浓度与P（3 HB）分子量呈负相关，与细胞P（3 HB）含量呈正相关。这一结果表明，P（3 HB）合酶活性水平是产生β-P（3 HB）的限制因素，并对P（3 HB）的产生有显着影响。
Ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineered Escherichia coli is an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized as phaCAB) encodes the essential proteins for the production of P(3HB) in the native producer, Ralstonia eutropha. The three genes of the phaCAB operon are phaC, which encodes the polyhydroxyalkanoate (PHA) synthase, phaA, which encodes a 3-ketothiolase, and phaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of the phaCAB operon (phaABC, phaACB, phaBAC, phaBCA, phaCAB, and phaCBA) on an expression plasmid in genetically engineered E. coli was examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of the pha genes relative to the promoter. The most balanced production result was achieved in the strain harboring the phaBCA expression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production.