Reconstitution Of The Peptidoglycan Cytoplasmic Precursor Biosynthetic Pathway In Cell-free System And The Inhibition Of The Pathway By Antisense Oligonucleotides

Cell-free protein system (CFPS) is widely used for in vitro protein synthesis due to its open nature. Without the constraint of cell structures, cell-free protein system has great advantages compared with traditional cell-based ones. The CFPS could be easily controlled without complicated operating and also could be combined with high-throughput technologies easily. In this research, we explored its potential applications in synthetic biology through co-expression the Mur enzymes and reconstitution of the peptidoglycan cytoplasmic precursor biosynthetic pathway in vitro.The enzymes involved in the peptidoglycan biosynthesis are important targets for new wide-spectrum antibiotics screening because of their highly conserved structures among bacteria. Due to the lack of cell penetration, the screening efficiency of inhibitors targeting peptidoglycan cytoplasmic precursor synthesis steps was very low. In present work, the genes encoding Mur enzymes from Escherichia coli were co-expressed in the cell-free protein synthesis (CFPS) system using PCR derived templates. The activities of the Mur enzymes were detected by HPLC after adding the substrates into this system. The synthesis of intermediates and the final product of this pathway UDP-MurNAc-pentapeptide were validated by HPLC-ESI-MS method.Antisense oligonucleotides is a valuable tool in bacteria metabolic regulation studies and drug target validation. The Mur biosynthetic pathway reconstituted in vitro was used to screen a panel of specific antisense oligonucleotides for MurA-MurF. Combined with computer assistant calculation technology,27 oligos targeting the 6 enzymes were designed. The Western blot results showed that the inhibition efficiency of 10 oligos was above 90%. Furthermore, the translation of murA or murB could be effectively repressed by adding 40μM oligo A281 and 40μM oligo B139, respectively. The subsequent enzyme activity assay analyzed by HPLC revealed that the biosynthesis of UDP-acetylmuramate was eliminated in the presence of oligos, indicating the inhibition of muramic acid biosynthetic pathway. This work not only developed a rapid method to reconstruct and regulate a biosynthetic pathway in vitro, but also may provide insight into the development of novel antibiotics targeting on peptidoglycan biosynthetic pathway.Protein expression efficiency and the lack of affordable kits restricted the widly use of cell-free synthesis system. In this study, we improved the preparation procedure of cell-free expression system based on a reconstructed E. coli strain which could co-express creatine kinase and T7 RNA polymerase. This economic batch-mode system could be developed into a new cell-free protein expression kit. Meanwhile, we constructed the continuous exchange cell-free system (CECF) and manufactured a mini bioreactor for it. The GFP and odr could both be expressed in this system with high expression level.In general, we constructed the Mur biosynthetic pathway in CFPS system by a new developed protein co-expression technology. And also this in vitro pathway was used to study the antisense oligonucleotides rationally design and high efficient screening method.