Organ transplantation is the best choice for patients with organ failure. The number of kidney transplants in my country ranks second in the world. About 300,000 patients need organ transplantation each year. However, immune exclusion reactions will affect the long-term survival of transplant organs. The use of immunosuppressive agents can prevent immune rejection so that the long-term survival of transplant organs can reduce their adverse reactions to ensure the long-term high-quality life of transplant recipients. Rapamycin is a widely used clinical drug for the treatment of immune rejection, which can greatly improve the survival rate of transplanted organs after surgery. The traditional physical and chemical mutagenesis screening and fermentation process optimization enable the fermentation level of rapamycin to be obtained.
Rapamycin is a new type of macrolide antibiotic, and it is a compound isolated from Streptomyces rapamycinicus through its antifungal activity that inhibits the growth of Candida albicans, Cryptococcus neoformans, Penicillium, and Mucosococcus. Because of its complex chemical structure, it is difficult to synthesize it by chemical methods, so this medicine is low productivity and it is also expensive. However, there are few studies on improving the fermentation yield of rapamycin through metabolic engineering. We tried to improve the fermentation yield of rapamycin by modifying the two-component signal transduction system in Streptomyces rapamycinicus.
The two-component system is a basic control system for organisms to sense external stimuli and regulate various physiological metabolism and cell behaviors (Fig.1). It consists of histidine kinases and response regulatory proteins. The main type of signal transduction system is used. The two-component system is important for primary and secondary metabolism, morphological differentiation, osmotic pressure, and cell wall integrity of Streptomyces rapamycinicus.
Fig.1. Two-component system schematic diagram
To construct the engineered strain, we amplified the upstream and downstream homologous arm of gene M271_14685/M271_14690, cloned it into pKC1139 plasmid, and then transfer it into ET12567/pUZ8002 competent cell. Screen the correct strain and co-culture with Streptomyces rapamycinicus, choose the double cross-over strain and test the fermentation yield of rapamycin by HPLC.
You can see the whole process of our experiment. We constructed plasmids in E. coli and then transferred them into Streptomyces rapamycinicus and successfully detected the yield of rapamycin was improved by creating the engineered strain. Moreover, we developed a deeper understanding of complex lab processes and learned lots of experimental skills, such as PCR, DNA extraction, plasmids assembly, and HPLC. We hope our result is useful for future scientific research and even be applied in clinical treatment.
