Part:BBa_K1653001
ispDF
ispDF is an operon which made of ispD and ispF in Escherichia coli. It was cloned by PCR. ispD catalyzes the third step in the methylerythritol phosphate pathway, the CTP-dependent conversion of 2-C-methyl-D-erythritol-4-phosphate to 4-(cytidine 5'-diphospho)-2-C-methyl-erythritol. The ispF catalyzes the Mn2+- or Mg2+-dependent conversion of 2-phospho-4-(cytidine 5'diphospho)-2-C-methyl-D-erythritol into 2-C-methyl-D-erythritol-2,4-cyclodiphosphate.
Contribution of 2024 AIS-China
Characterization
In our project, HMBPP is used to attract blood-feeding mosquitoes. Since HMBPP cannot be chemically synthesized, we selected E. coli as the chasis for HMBPP production, utilizing its inherent MEP pathway, which is similar to that of Plasmodium (Emami et al., 2017; Viktoria et al., 2021). To enhance HMBPP yield, we implemented dual metabolic engineering strategies: overexpression of the upstream genes in the MEP pathway and downregulating the expression of the downstream IspH enzyme.
To this end, we have strategically chosen DXS, DXR, IspD, IspF, and IspG to develop 4 distinct MEP overexpression cassettes (Figure 1a), aiming to identify the optimal set of rate-limiting enzymes in the MEP pathway. And the PCR and gel electrophoresis were carried out to prove the successful construction of these MEP overexpression cassettes (Figure 1c).
However, quantifying HMBPP requires LC-MS or GC-MS, equipment not currently available in our lab, making the process laborious and time-consuming. To assess the overexpression efficiency of our four cassettes, we introduced a lycopene expression cassette as reporter into the E. coli strain DH5a with these 4 cassettes, creating strains 1-4 (Figure 1a).
We measured the A470/A600 ratio of these strains to analyze lycopene production per cell unit. All strains 1-4 demonstrated a notable increase in lycopene yield relative to the control strain with the reporter cassette alone. Notably, strain 3, harboring the MEP overexpression cassette 3, outperformed with a 2.03-fold enhancement in overexpression efficiency, indicating that the combination of DXS, IspG, and IspDF is the most promising candidate. (Figure 1d)
Figure 1. Using lycopene as reporter, the best MEP overexpression cassette is selected for higher yield of HMBPP. (a) Various MEP pathway overexpression cassettes expression in E. coli strain DH5a (b) Production of lycopene via the endogenous MEP pathway in E. coli. (c) Gel electrophoresis analysis of transformed MEP pathway overexpression cassettes. (d) Relative lycopene production while using various MEP Overexpression Cassettes in E. coli.
Reference
Zhaobao W., JingXin S., Qun Y., Jianming Y. Metabolic Engineering Escherichia coli for the Production of Lycopene. MOLECULES. 2020, 25(14): 3136. https://www.mdpi.com/1420-3049/25/14/3136
Zhou, J., Yang, L., Wang, C., Choi, E. S., & Kim, S. W. Enhanced performance of the methylerythritol phosphate pathway by manipulation of redox reactions relevant to IspC, IspG, and IspH. J Biotechnol. 2017, 248, 1-8. https://doi.org/10.1016/j.jbiotec.2017.03.005
Emami, S. N., Lindberg, B. G., Hua, S., Hill, S. R., Mozuraitis, R., Lehmann, P., Birgersson, G., Borg-Karlson, A.-K., Ignell, R., & Faye, I. A key malaria metabolite modulates vector blood seeking, feeding, and susceptibility to infection. Sci. 2017, 355(6329): 1076-1080. https://doi.org/doi:10.1126/science.aah4563
Viktoria, E. S., Melika, H., Elizabeth, V., Raimondas, M. , S. Noushin, E. Plasmodium metabolite HMBPP stimulates feeding of main mosquito vectors on blood and artificial toxic sources. Commun. Biol. 2021, 4(1): 1161. https://www.nature.com/articles/s42003-021-02689-8
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1064
Illegal BamHI site found at 392 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |