Murata Yoshimitsu, Jitsukawa Tomotaka, Iida Maiko, Furuta Toshiaki, Haramoto Yoshikazu, Shigeri Yasushi, Fujisaki Shingo
Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
J Biosci Bioeng. 2023 May;135(5):382-388. doi: 10.1016/j.jbiosc.2023.02.003. Epub 2023 Mar 2.
Overproduction of isopentenyl diphosphate by the amplification of the genes for the methylerythritol 4-phosphate pathway, dxs and dxr, is known to be deleterious for the growth of Escherichia coli. We hypothesized that overproduction of one of the endogenous isoprenoids, in addition to isopentenyl diphosphate itself, might be the cause of the reported reduced growth rate and attempted to identify the causative agent. In order to analyze polyprenyl phosphates, they were methylated by the reaction with diazomethane. The resulting dimethyl esters of polyprenyl phosphates with carbon numbers from 40 to 60 were quantitated by high-performance liquid chromatography-mass spectrometric analysis detecting ion peaks of the sodium ion adducts. The E. coli was transformed by a multi-copy plasmid carrying both the dxs and dxr genes. Amplification of dxs and dxr significantly increased the levels of polyprenyl phosphates and 2-octaprenylphenol. The levels of Z,E-mixed polyprenyl phosphates with carbon numbers of 50-60 in the strain in which ispB was co-amplified with dxs and dxr were lower than those in the control strain where only dxs and dxr were amplified. The levels of (all-E)-octaprenyl phosphate and 2-octaprenylphenol in the strains in which ispU/rth or crtE was co-amplified with dxs and dxr were lower than those in the control strain. Although the increase in the level of each isoprenoid intermediate was blocked, the growth rates of these strains were not restored. Neither polyprenyl phosphates nor 2-octaprenylphenol can be determined to be the cause of the growth rate reduction seen with dxs and dxr amplification.
已知通过磷酸甲基赤藓糖醇途径的基因dxs和dxr的扩增导致异戊烯基二磷酸的过量产生，这对大肠杆菌的生长是有害的。我们推测，除了异戊烯基二磷酸本身外，一种内源性类异戊二烯的过量产生可能是报道的生长速率降低的原因，并试图确定致病因子。为了分析聚异戊二烯磷酸酯，它们通过与重氮甲烷反应进行甲基化。通过检测钠离子加合物的离子峰的高效液相色谱-质谱分析对所得的碳数为40至60的聚异戊二烯磷酸酯的二甲酯进行定量。用携带dxs和dxr基因的多拷贝质粒转化大肠杆菌。dxs和dxr的扩增显著增加了聚异戊二烯磷酸酯和2-辛戊烯基苯酚的水平。在ispB与dxs和dxr共扩增的菌株中，碳数为50 - 60的Z,E-混合聚异戊二烯磷酸酯的水平低于仅扩增dxs和dxr的对照菌株。在ispU/rth或crtE与dxs和dxr共扩增的菌株中，（全-E）-辛戊烯基磷酸酯和2-辛戊烯基苯酚的水平低于对照菌株。尽管每种类异戊二烯中间体水平的增加被阻断，但这些菌株的生长速率并未恢复。聚异戊二烯磷酸酯和2-辛戊烯基苯酚都不能被确定为dxs和dxr扩增导致生长速率降低的原因。
