Difference between revisions of "Part:BBa K1088004"

Latest revision as of 11:48, 2 October 2024

IspG (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase)

1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG) is part of the bacterial MEP (methylerythritol phosphate) pathway used for production of isoprenoids.

From ecocyc: "IspG catalyzes the conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. IspG activity depends on as-yet unidentified additional proteins, most likely involved in the oxidation portion of the reaction" [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10370 (Link)]

Usage and Biology

Characterized by SEFLS_SHANGHAI

Construction of the plasmids pET-IAY, pET-IAY-CN, pET-HIAY and pMEP-DG

1. Obtain the target fragments YSS, DXS, ispH, ispG and IIA(rbs-idi-rbs-ispA)

Gene dxs, ispH and ispG were obtained by PCR from E. coli, and IIA fragment was obtained by PCR using plasmid p35151 as template. The following figure shows the fragment YSS, ispH, DXS, ispG and IIA after gel recycling.

2. Conduct double enzyme digestion on the skeleton vectors pBBR1MCS-2, pETDuet-1 and pET-YN, respectively. pBBR1MCS-2 is the skeleton of pMEP-DG, pETDuet-1 is the skeleton of pET-IAY and pET-HIAY, and pET-YN is the skeleton of pET-IAY-CN.

3. The plasmid skeleton recovered by enzyme digestion and the target fragment recovered by gel were verified by electrophoresis. The results are shown below:

4. Connect the homologous recombinant fragment with the corresponding backbone, the ligation reaction system is as follows:

pET-IAY： The enzyme digestion product of pET Duet-1 +IIA+YSS

pET-HIAY： The enzyme digestion product of pET Duet-1 +ispH+IIA+YSS

pET-IAY-CN： The enzyme digestion product of pET-YN + IIA+YSS

pMEP-DG： The enzyme digestion product of pBBR1MCS-2 +dxs+ispG

During transformation, we encountered some difficulties, for example, there were many clones on the plate, but none of them is the positive transformant. Later, we solved this problem by adding DpnI enzyme to the PCR product and speculated that the concentration of plasmid template added was too high when preparing the PCR reaction solution, resulting in excessive negative transformant growing on the plate. We encountered situation where there was no clone on the plate. After increasing the concentration of products from plasmid enzyme digestion and decreasing the concentration of products from gel recycling, this problem is resolved. We speculated that the problem may be due to the low concentration of products from plasmid enzyme digestion and the high concentration of fragment from gel recycling, which affected the accuracy of the ligation reaction system. The addition of plasmids and ligated fragments that are not in the proper range may lead to the failure of ligation reactions.

5. Monoclones on the transformation plate were selected for bacterial solution PCR verification, and the verification results were as follows, indicating that plasmids pET-IAY, pET-IAY-CN, pET-HIAY and pMEP-DG were successfully constructed.

The genes dxs and ispG were placed on the backbone of low-copy plasmid pBBR1MCS-2, and the promoter was medium strength lac promoters, to construct plasmid pBBR1MCS-2-dxs-ispG (pMEP-DG).

Genes ispH, idi, and ispA were placed on the backbone of high-copy plasmid pETDuet-1, and the promoter was strong promoter T7 to construct plasmid pETDuet-1-1T7-IspH-Idi-IspA-Yss (pET-HIAY).

When Dxs and IspG were overexpressed, the squalene yield increased by 4.3 times. We speculated that the overexpressed gene ispG was constructed on low-copy plasmids and was controlled by promoters of medium strength, so it didn’t produce large amounts of harmful intermediate HMBPP. And overexpressing Dxs promoted the metabolic flow, increasing the squalene yield.

H3: p35151/pET-HIAY

H5: p35151/pMEP-DG/pET-HIAY

Contribution of 2024 AIS-China

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.

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