Part:BBa_K5186006

PTac-riboJ-DXS-IspG-B0015

Description

PTac-RiboJ-DXS-IspG-B0015 is an expression cassette in E. coli expressing DXS (BBa_K3166061) and IspG (BBa_K1653001) used to overexpress methylerythritol phosphate(MEP) pathway. With this expression cassette, DXS and IspGcan 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.

Usage and Biology

DXS(BBa_K3166061) and IspG(BBa_K1088004) encode the 1-deoxy-D-xylulose 5-phosphate(DXP) synthase and HMBPP synthase respectively. Among them, DXS catalyzes the first, rate-limiting step in the MEP pathway of isoprenoid biosynthesis.

Both DXS and IspG 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 both DXS and IspG in E. coli DH5a. By co-expressing this cassette with the lycopene expression cassette (BBa_K274100), we have demonstrated about a 1.64-fold increase in overexpression efficiency with the help of the lycopene reporter. (Zhaobao W. et al, 2020; Zhou et al.,2017)

Source

DXS(BBa_K3166061) and IspG(BBa_K1653001) are from E. coli.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4325
  Illegal BamHI site found at 3653
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2102