Difference between revisions of "Part:BBa K5186009"

Latest revision as of 10:01, 2 October 2024

PTac-riboJ-DXS-IspG-DXR-IspDF-B0015

PTac-riboJ-DXS-IspG-DXR-IspDF-B0015 is an expression cassette in E. coli expressing DXS (BBa_K3166061), IspG (BBa_K1088004), DXR(BBa_K1950003) and IspDF (BBa_K1653001) used to overexpress methylerythritol phosphate(MEP) pathway. With this expression cassette, DXS, IspG, DXR and IspDF can be IPTG-inducibly expressed under the control of pTac-RiboJ (BBa_K3552015) in all strains of E. coli.

This is a part of a part collection where we enable the overproduction of HMBPP. The part collection includes sRNA(IspH)s (BBa_K5186001, BBa_K5186002, BBa_K5186003, BBa_K5186004, BBa_K5186005) for the downregulation of downstream gene IspH expression and various MEP overexpression cassettes (BBa_K5186006, BBa_K5186007, BBa_K5186008, BBa_K5186009). This collection can help and inspire other iGEM teams and researchers to achieve higher yield of HMBPP or other isoprenoids in E. coli.

DXS(BBa_K3166061), IspG(BBa_K1088004), DXR(BBa_K1950003) and IspDF(BBa_K1653001) encode the 1-deoxy-D-xylulose 5-phosphate(DXP) synthase, HMBPP synthase, DXP reductoisomerase, CDP-ME cytidylyltransferase and MEC synthase respectively.

Among them, DXS and DXR catalyzes the first, rate-limiting step in the MEP pathway of isoprenoid biosynthesis. However, considering IspDF also catalyzes the rate-limiting step in the MEP pathway, it has been proved that the co-overexpression of IDI, DXS, IspDF in the MEP pathway leads to a 6-fold increase in lycopene production(Yan Z. et al, 2013).

All of the DXS, DXR, IspGand IspDF can be amplified from E. coli and assembled into the multiple cloning site of pET28a, which is located downstream of PTac and upstream of B0015, thereby facilitating the acquisition of this composite part.

In our efforts to enhance the production of HMBPP this year, we have successfully engineered this overexpression cassette for DXS, DXR, IspG and IspDF in E. coli DH5a. By co-expressing this cassette with the lycopene expression cassette (BBa_K274100), we have demonstrated about a 1.69-fold increase in overexpression efficiency with the help of the lycopene reporter. (Zhaobao W. et al, 2020; Zhou et al.,2017)

DXS(BBa_K3166061), IspG(BBa_K1653001), DXR(BBa_K1950003) and IspDF(BBa_K1653001) are from E. coli.

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