Organ transplantation faces the problem of immune rejection while giving patients a second life. Rapamycin is a new type of macrolide antibiotic, and it is also a widely used clinical drug for the treatment of immune rejection, which can greatly improve the survival rate of transplanted organs after surgery. But the drug is produced at a low level and is expensive.
In our project we optimized the metabolic regulation network of rapamycin biosynthesis by knocking out the two-component system encoding gene M271_14685/M271_14690 in Streptomyces rapamycinicus, thus bringing greater value to clinical treatment and bringing good news to patients with organ transplantation.
In this experiment, we amplified the 1000bp upstream and downstream gene fragments of M271_14685/M271_14690, which are two-component systems encoding genes in Streptomyces rapamycinicus, ligated them into the plasmid pKC1139, and then transformed it into the host strain Streptomyces rapamycinicus. Through homologous recombination, single-crossover strains and double-crossover strains were obtained, and the target gene M271_14685/M271_14690 was knocked out to finally obtain mutant strain ΔM271_14685/M271_14690 (Fig1).
Figure 1. The two-component systems working diagram
A gene knockout plasmid (pKCM271_14685/M271_14690) was constructed (Fig. 2). The plasmid is used to knock out the two-component system encoding gene M271_14685/M271_14690 in Streptomyces rapamycinicus. The construction was confirmed by DNA sequencing.
Figure 2. Schematic map of the plasmid
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 3), double-enzyme digestion, and ligase to pKC1139 carrier.
Figure 3: Gel electrophoresis diagram. In Figure 3, a clear and single DNA band at 1kp can be seen, indicating that the upstream and downstream homology arms of M271_14685/M271_14690 were successfully amplified by PCR.
Figure 4: Gel electrophoresis diagram. To verify if the recombinant plasmid is correct, we did double-enzyme-digestion. It can be seen from the Figure 4 that the size of the plasmid we constructed is correct, and the identification results of double enzyme digestion are also correct.
We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region, and the plasmid was successfully constructed.
To construct the engineering strain, we firstly transferred the recombinant plasmid into ET12567/pUZ8002 competent cells and screened the correct strain through 3 antibodies, and cultured it in the liquid medium. Then co-cultured the E.coli with Streptomyces rapamycinicus and screened for the cross-over strain.
It can be seen from Figure 5 that the gene fragments of the engineered bacteria we constructed were successfully exchanged compared with the negative control. That is, the engineered bacteria that knocked out the M271_14685/M271_14690 gene were successfully obtained.
Figure 5. Gel electrophoresis diagram. Line 1: double cross-over strain, 326bp, correct. Co-cultured Streptomyces rapamycinicus NRRL 5491 with ΔM271_14685/M271_14690 in the fermentation medium, we collected the sample respectively at 5d,7d,9d, and 11d. We mixed 0.5 mL samples with the same volume of methanol and tested the yield of rapamycin after centrifuge. To identify the correct peek of rapamycin, we use the standard rapamycin as positive control. It can be seen from Figure 8 that the △M271_14685/m271_14690 detected more rapamycin at the same time as standard rapamycin.
Figure 6. △M271_14685/m271_14690 and NRRL 5491 have produced rapamycin in 11 days.
It can be seen from the above figure that the rapamycin produced by our knockout △M271_14685/m271_14690 is much higher than that produced by NRRL 5491 (Figure 6). And the amount of rapamycin produced on the ninth day reached 120mg/L.
This indicates that the knockout of the target gene M271_14685/M271_14690 is more conducive to the secretion of rapamycin by Streptomyces rapamycinicus. This suggests that the subject is feasible to improve metabolic pathways by knocking out the two-component system, which can be applied to factories in the near future.
