Part:BBa_K274100

CrtEBI with rbs

This Composite Biobrick is created by standard assembly of Part BBa_K118014 (CrtE with rbs), Part BBa_K118006 (CrtB with rbs) and Part BBa_K118005 (CrtI with rbs), which are submitted by previous iGEM teams. The whole cassette is on plasmid pSB1A2 (high copy, Ampicillin resistance).

Together, enzymes CrtE, CrtB and CrtI convert colourless farnesyl pyrophosphate to red lycopene (via intermediates geranylgeranyl pyroiphosphate and phytoene). The red lycopene pigment can then be used as a coloured signal output, e.g. for biosensors.

There is already individual ribosome binding site before each enzyme gene sequence. Internal restriction sites have been removed by previous iGEM teams. Please refer to Parts BBa_K118014, BBa_K118006 and BBa_K118005 for more information on individual Biobricks components.

This Composite Biobrick has been put under constitutive promoter R0011 (see Part BBa_K274110) and arabinose-inducible promoter I0500 (see Part BBa_274120), transformed and tested in E.coli strain MG1655.

Amount of lycopene produced can be measured by photospectrometer with absorbance at 475nm (lycopene extraction using acetone).

A related Composite Biobrick is Part BBa_K274200 (CrtEBIY with rbs), which "goes on" one step after lycopene, converting red lycopene to orange beta-carotene pigment.

Datasheet for Part BBa_K274100 and Part BBa_274110 in E. coli strain MG1655. You may also wish to refer to the "Experience" page.

For PDF version of this Datasheet: File:Lycopene.pdf

Reference

Hal Alper, et al. Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nature Biotechnology 23 (2005).

Nishizaki T, et al. Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis. Appl Environ Microbiol. 2007 Feb

Luke Z. Yuan, et al. Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metabolic Engineering 8 (2006).

Luan Tao, et al. Isolation of chromosomal mutations that affect carotenoid production in Escherichia coli: mutations alter copy number of ColE1-type plasmids. FEMS Microbiology Letters 243 (2005)

von Lintig J, et al. Filling the gap in vitamin A research. Molecular identification of an enzyme cleaving beta-carotene to retinal. J Biol Chem. 2000 Apr 21;275(16).

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

Assembly Compatibility:

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

  Illegal BamHI site found at 1974
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1510
  Illegal NgoMIV site found at 1640
  Illegal AgeI site found at 725
  
• 1000
  COMPATIBLE WITH RFC[1000]