Part:BBa_K4281003

M271-14685-14690-up-M271-14685-14690-down

M271-14685-14690-up-M271-14685-14690-down

Contribution

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 (Figure 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.

Figure 1. Two-component system schematic diagram..

Engineering Success

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.

I. Construction of double knockout plasmid experiment

1. PCR amplification of upstream and downstream homology arms gene of M271_14685/M271_14690. We designed the program by inserting the M271_14685/14690 upstream homology arm gene into HindIII and EcoRI sites of the pKC1139 vector. In order to build our plasmids, we amplified the gene fragments from the genome of Streptomyces rapamycinicus NRRL 5491 by PCR (Figure 2), double-enzyme digestion, and ligase to pKC1139 carrier.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 986
  Illegal NgoMIV site found at 1837
  Illegal NgoMIV site found at 1870
  Illegal NgoMIV site found at 2009
  Illegal AgeI site found at 1063
  Illegal AgeI site found at 1109
  Illegal AgeI site found at 1486
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 785