Part:BBa_K1950003

ispC (dxr : 1-deoxy-D-xylulose 5-phosphate reductoisomerase)

ispC catalyzes the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into the dedicated MEP pathway intermediate 2-C-methyl-D-erythritol-4-phosphate (MEP). This reaction is NADPH-dependent, and required a bivalent metal cofactor.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 142
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 142
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 142
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 142
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 142
  Illegal AgeI site found at 441
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

DXR is a gene that encodes the enzyme 1-deoxy-D-xylulose 5-phosphate(DXP) reductoisomerase, which plays a pivotal role in the methylerythritol phosphate (MEP) pathway. This enzyme catalyzes the initial and rate-determining step in isoprenoid biosynthesis, specifically the conversion of DXP to MEP. The gene can be amplified via PCR from the E. coli strain DH5a. Recent research underscores the importance of overexpressing key enzymes within the MEP pathway to enhance isoprenoid production. Notably, DXR, along with DXS (BBa_K3166061), is identified as a major rate-limiting enzyme, and its overexpression is particularly highlighted for enhancing the efficiency of isoprenoid synthesis.

Usage and biology

DXR is a gene that encodes the enzyme 1-deoxy-D-xylulose 5-phosphate(DXP) reductoisomerase, which plays a pivotal role in the methylerythritol phosphate (MEP) pathway. This enzyme catalyzes the initial and rate-determining step in isoprenoid biosynthesis, specifically the conversion of DXP to MEP. The gene can be amplified via PCR from the E. coli strain DH5a.
Recent research underscores the importance of overexpressing key enzymes within the MEP pathway to enhance isoprenoid production. Notably, DXR, along with DXS (BBa_K3166061), is identified as a major rate-limiting enzyme, and its overexpression is particularly highlighted for enhancing the efficiency of isoprenoid synthesis.

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