Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum.
Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum.
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DOI：
10.1186/s12934-015-0254-0
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发表时间：
2015-05-17
影响因子：
6.4
通讯作者：
Liu SJ
中科院分区：
文献类型：
--
作者：
Zhang B;Zhou N;Liu YM;Liu C;Lou CB;Jiang CY;Liu SJ
莽草酸(SA)途径是合成芳香族化合物的基本途径，用于细胞生长和代谢过程，以及各种芳香族化合物的发酵生产。编码SA途径酶的基因在基因组上是不连续的，它们受到不同的调控。本研究致力于构建受同一PTAC启动子调控的SA途径的连续遗传模块。首先，从谷氨酸棒杆菌中获得了aro基因[arg(NCgl2098)、arb(NCgl1559)、arod(NCgl0408)和aroe(NCgl1567)]和为上述基因量身定制的核糖体结合位点(RBS)文库，并对4个文库中每个RBS的强度进行了量化。其次，从RBS文库中构建了9个遗传模块，它们分别是核酶绝缘子(Ribozyme绝缘子)、转录启动子(PTAC)和终止子(PTAC)，以及AROG、AROB、ARORD和AROE。通过SA合成的测定，对构建的遗传模块在谷氨酰胺中的功能和效率进行了评价。结果表明，携带遗传模块的谷氨酸杆菌RES167ΔAROK在250mL摇瓶发酵过程中，SA产量为4.3g/L，是RES167ΔAROK菌株(80 mg/L，不含遗传模块)的54倍。同一菌株在5-L分批发酵和补料分批发酵中的SA摩尔产量分别为7.4g/L和11.3g/L，对应的SA摩尔产量分别为0.39和0.24/摩尔蔗糖消耗。这些结果表明，所构建的SA途径模块能有效地促进谷氨酸菌合成SA，并可用于发酵生产SA途径衍生的芳香族化合物。
The shikimic acid (SA) pathway is a fundamental route to synthesize aromatic building blocks for cell growth and metabolic processes, as well as for fermentative production of various aromatic compounds. Genes encoding enzymes of SA pathway are not continuous on genome and they are differently regulated. In this study, efforts were made to construct continuous genetic modules of SA pathway that are regulated by a same Ptac promoter. Firstly, aro genes [aroG (NCgl2098), aroB (NCgl1559), aroD (NCgl0408) and aroE (NCgl1567)] from Corynebacterium glutamicum and ribosome binding site (RBS) libraries that were tailored for the above genes were obtained, and the strength of each RBS in the 4 libraries was quantified. Secondly, 9 genetic modules were built up from the RBS libraries, a previously characterized ribozyme insulator (RiboJ) and transcriptional promoter (Ptac) and terminator, and aroG, aroB, aroD and aroE. The functionality and efficiency of the constructed genetic modules were evaluated in C. glutamicum by determination of SA synthesis. Results showed that C. glutamicum RES167ΔaroK carrying a genetic module produced 4.3 g/L of SA, which was 54 folds higher compared to that of strain RES167ΔaroK (80 mg/L, without the genetic module) during fermentation in 250-mL flasks. The same strain produced 7.4, and 11.3 g/L of SA during 5-L batch and fed-batch fermentations, respectively, which corresponding to SA molar yields of 0.39 and 0.24 per mole sucrose consumption. These results demonstrated that the constructed SA pathway modules are effective in increasing SA synthesis in C. glutamicum, and they might be useful for fermentative production of aromatic compounds derived from SA pathway.
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DOI：
10.1126/science.1191652
发表时间：
2010-10-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Ajikumar PK;Xiao WH;Tyo KE;Wang Y;Simeon F;Leonard E;Mucha O;Phon TH;Pfeifer B;Stephanopoulos G
通讯作者：
Stephanopoulos G
影响因子：
2.9
作者：
Chandran, SS;Yi, J;Frost, JW
通讯作者：
Frost, JW
影响因子：
2.8
作者：
Amador, E;Castro, JM;Martín, JF
通讯作者：
Martín, JF
影响因子：
6.4
作者：
Escalante A;Calderón R;Valdivia A;de Anda R;Hernández G;Ramírez OT;Gosset G;Bolívar F
通讯作者：
Bolívar F
影响因子：
4.4
作者：
Krause, Felix S.;Blombach, Bastian;Eikmanns, Bernhard J.
通讯作者：
Eikmanns, Bernhard J.