Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine.
Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine.
复制标题
DOI：
10.1186/1475-2859-6-30
复制
发表时间：
2007-09-19
影响因子：
6.4
通讯作者：
Gosset G
中科院分区：
文献类型：
--
作者：
Báez-Viveros JL;Flores N;Juárez K;Castillo-España P;Bolivar F;Gosset G
通过结合不同的遗传策略，如磷酸烯醇式丙酮酸：磷酸转移酶运输系统(PTS)的失活和关键基因(DAHP合成酶、转酮醇酶和分支酸变位酶-预苯酸脱水酶)的过度表达，成功地实现了L-苯丙氨酸(L-苯丙氨酸)高产微生物的合理设计，产量达到了0.33g-Phe/g-Glc，相当于理论最大产量的60%。尽管引入细胞以产生高产生物体的基因修饰是专门针对特定途径的，但这些修饰可以触发几个基因的意想不到的转录反应。在目前的工作中，利用实时定量聚合酶链式反应对L-Phe高产菌株和非工程菌株进行了代谢转录分析，从而能够检测到对PTS缺失的转录反应和中心碳代谢相关基因的质粒存在。编码糖酵解、糖异生、磷酸戊糖、三羧酸循环、发酵和芳香族氨基酸途径等酶的86个基因。此外，还分析了30个编码芳香族化合物和碳水化合物的调节蛋白和转运蛋白的基因。MTA分析表明，PTS失活后，一组编码糖类转运蛋白(Galp、mglB)、糖异生(PPSA、pck A)和发酵酶(LDHA)的基因被显著诱导，而其他一些基因如PPC、pflB、PTA和ackA表达下调。最相关的发现之一是在最好的PTS-L-Phe高产菌株(PB12-EV2)中，几个专门糖异生的基因(fBP、ppSA、pck A、maeB、sfca和乙醛分流)协同上调。此外，值得注意的是，大多数TCA基因在存在多拷贝质粒时以一种未知的机制显示出强烈的上调。一组基因对PTS失活和质粒的存在都表现出转录反应。例如，acs-ackA，如ABCD和sdhABCD操纵子在PB12(携带arcB突变的PTS突变体)中上调。这些操纵子的诱导被pB12-EV2中的质粒的存在进一步增加。在L-Phe高产菌株中，参与莽草酸途径和特定芳香族氨基酸途径的一些基因表达下调，可能导致莽草酸途径的代谢限制。对高产微生物中潜在限速步骤的识别和对转录反应的检测可能会为进一步改良L-苯丙氨酸生产菌株提供“反向工程”策略。
The rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed. MTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt) in the best PTS- L-Phe overproducing strain (PB12-ev2). Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway. The identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains.
登录
查看更多内容
影响因子：
17.3
作者：
Berry, A
通讯作者：
Berry, A
影响因子：
2.9
作者：
Chandran, SS;Yi, J;Frost, JW
通讯作者：
Frost, JW
影响因子：
1.2
作者：
Flores, N;de Anda, R;Bolivar, F
通讯作者：
Bolivar, F
新型克隆载体的构建和特性分析 2. 多用途克隆系统
影响因子：
3.5
作者：
BOLIVAR, F;RODRIGUEZ, RL;FALKOW, S
通讯作者：
FALKOW, S
影响因子：
3.9
作者：
ARORA, KK;PEDERSEN, PL
通讯作者：
PEDERSEN, PL