Different species of Streptomyces produce different antibiotics, so the modification of Streptomyces is of great significance for improving the production process of antibiotics. However, the experimental operation method of Streptomyces is not as mature as E. coli and yeast. Many commonly used biotechnologies do not work well with Streptomyces.
In the past iGEM teams, there is no very detailed and complete characterization of Streptomyces. For this reason, our team provides some valuable information for future iGEM teams.
We submit information on the Streptomyces and E. coli shuttle plasmid-backbone. If a future team is committed to genetic engineering related to Streptomyces, the plasmid-backbone can be used. Moreover, we provide a set of practical solutions for knocking out Streptomyces genome genes. Please refer to the experimental methods page for details. If there is a future team dedicated to knocking out the gene of Streptomyces, they can refer to the knockout scheme we used. In addition, we have also sorted out 10 groups of two-component systems in S. rapamycinicus that affect the metabolic pathway of rapamycin, of which group 10 is the gene knocked out in this project. The results show that the production of rapamycin is greatly improved after knockout. If a future team is also committed to improving the rapamycin production of S. rapamycinicus, they can start with these two-component systems and try to knock out or modify them to increase the production.
pKC1139-backbone is one of the most commonly used Streptomyces gene knock-out vectors, which can shuttle in E. coli and S. rapamycinicus, and it is thermosensitivity. The vector is a high-copy-number plasmid. When expressed in the prokaryotic system, the Apramycin+ resistance can be used to screen the right colony, and the strain should be cultured at 30℃, while transformed into the S. rapamycinicus, the strain should be cultured at 37℃. This plasmid backbone can be used to express different proteins in the future.
Take the gene M271_14685/M271_14690 knocked out by our project as an example, M271-14685-14690-up-M271-14685-14690-down is the upstream and downstream homologous arm of gene M271_14685/M271_14690 gene, and it is cloned into pKC1139 plasmid to knock out both M271_14685 and M271_14690 genes through recombination. Knockout strains can be successfully obtained by screening single-exchange strains and double-exchange strains in turn. The design of these composite parts and the experimental protocol are useful for future iGEMers committed to knocking out the genome gene of S. rapamycinicus.
Histidine kinases are an ancient conserved family of enzymes that are found in bacteria, archaebacteria, and S. rapamycinicus. They are activated by a wide range of extracellular signals and transfer phosphate moieties to aspartates found in response regulators. It was regulated by a PhoB family transcriptional regulator (M271_14685). Our project has shown that when knocked out the histidine kinase M271_14690, the yield of Rapamycin increased. That means the two-component signal transduction system can be used to regulate in improve the yield of Rapamycin.
We also identified other kinds of the two-component signal transduction system (Table 1), such as the M271_02725 histidine kinase was under the regulation of the LuxR family transcriptional regulator, which is possessing the typical LuxR-type helix–turn–helix (HTH)-DNA binding motif. In the table below, we show the two-component signal transduction system in the form of grouping.
| Part Number | Protein Number | Function | System |
|---|---|---|---|
| BBa_K4281005 | M271_00895 | LuxR family transcriptional regulator | group 1 |
| BBa_K4281006 | M271_00900 | histidine kinase | |
| BBa_K4281007 | M271_02035 | histidine kinase | group 2 |
| BBa_K4281008 | M271_02040 | transcriptional regulator | |
| BBa_K4281009 | M271_02720 | LuxR family transcriptional regulator | group 3 |
| BBa_K4281010 | M271_02725 | histidine kinase | |
| BBa_K4281011 | M271_03140 | histidine kinase | group 4 |
| BBa_K4281012 | M271_03145 | LuxR family transcriptional regulator | |
| BBa_K4281013 | M271_06210 | transcriptional regulator | group 5 |
| BBa_K4281014 | M271_06215 | histidine kinase | |
| BBa_K4281015 | M271_06710 | histidine kinase | group 6 |
| BBa_K4281016 | M271_06715 | transcriptional regulator | |
| BBa_K4281017 | M271_09110 | response regulator, OmpR type | group 7 |
| BBa_K4281018 | M271_09115 | histidine kinase | |
| BBa_K4281019 | M271_09415 | histidine kinase | group 8 |
| BBa_K4281020 | M271_09420 | LuxR family transcriptional regulator | |
| BBa_K4281021 | M271_14200 | histidine kinase | group 9 |
| BBa_K4281022 | M271_14205 | PhoB family transcriptional regulator | |
| BBa_K4281023 | M271_14685 | transcriptional regulator | group 10 |
| BBa_K4281024 | M271_14690 | histidine kinase | |
