Coding

Part:BBa_K5184000

Designed by: Daniel Xie, Ziyan Zhou   Group: iGEM24_GreatBay-SCIE   (2024-09-10)


SltNPPS

Targeting both the prevention and extermination of spider mites, we aim to produce 7-epi-zingiberene (7epiZ),a sesquiterpene that is found to have repellent, fecundity-reducing, and fatal effects towards spider mites. In order to synthesize 7epiZ from glucose in E. coli, we introduce SltNPPS, a neryl diphosphate synthase which catalyzes the formation of neryl diphosphate (NPP) from dimethylallyldiphosphate (DMAPP) and isopentenyl diphosphate (IPP), products of the MVA pathway introduced in E. coli. Through exploring the usage of SltNPPS, we provide future iGEM teams with an insight into how to biosynthesize sesquiterpenes using E. coli as the chassis.

This part belongs to a collection used for the synthesis of 9HZ and 9H10epoZ, two sesquiterpenes with strong repellent effects towards spider mites. This part collection includes SltNPPS, Mvan4662, ShZIS, ShZPO, SlCPR2 and AtCPR1.

Essential Information

Sequences

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

"SltNPPS is a cis-prenyl transferase from Solanum lycopersium with its N-terminal 45 amino acid truncated that catalyzes the conversion of geranyl diphosphate into neryl diphosphate, which is an essential step in the biosynthesis of monoterpenes in plants. We constructed a novel sesquiterpene synthesis pathway in E. coli Using glucose as our raw material, we introduce the MVA pathway, which transforms glucose into dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). Afterwards, SltNPPS, a neryl diphosphate synthase catalyze the production of NPP from IPP and DMAPP. Mvan4662 is then introduced to catalyze the formation of Z,Z-FPP. In the end, ShZIS transforms Z,Z-FPP into 7epiZ. "

Characterization

7-epi-zingiberene (7epiZ), having repellent, fecundity-reducing, and toxic effects on the spider mites[1], is a crucial ingredient of our acaricide. To produce it, we introduced two plasmids to E. coli: pMVA, which introduces the enzymes of the Mevalonate pathway, and pW1-ZIS-NPPS-Mvan4662, which introduces the subsequent enzymes necessary for the production of 7epiZ [Fig1A], [Fig1B]. The enzymes, LA2167ZIS(ZIS), the zingiberene synthethase, Mvan4662Q93S(Mvan4662), a Z,Z-farnesyl diphosphate (Z,Z-FPP) synthase, and SltNPPS(NPPS) a neryl pyrophosphate synthase (NPP), are introduced to E. coli via plasmid expression [Fig1C]. The three enzymes are cloned into the vector pW1, giving pW1-ZIS-NPPS-Mvan4662. Coding sequences for ZIS is placed closest to the promoter to increase its expression level, for we believe it will likely be the rate-limiting enzyme of the three.

Figure 1: (A) The metabolic pathway of 7epiZ (B) Dual-plasmid E. Coli, containing pMVA and the zingiberene synthesis plasmid. pMVA consists of a total of 7 coding sequences divided between two promoters. Zingiberene synthesis plasmid consists of three coding sequences under one promoter. (C) Plasmid construct of zingiberene synthesis plasmid: pW1-ZIS-Mvan4662-SltNPPS

pW1-ZIS-Mvan4662-SltNPPS, via GoldenGate cloning, is transformed into E. coli strain DH5ɑ. After having sequence verified, plasmids are transformed into pMVA DH5ɑ competent cells. The resultant strain is thus capable of producing zingiberene using glucose as the sole carbon source.

Figure 2: (A) Colony PCR results of DH5ɑ strain harboring pW1-ZIS-Mvan4662-NPPS, the coding sequence is divided into four regions, denoted by A, B, C, and D (B) Alignment of colony PCR sequencing results against the designed plasmid

The pW1-ZIS-Mvan4662-SltNPPS+pMVA DH5ɑ strain is induced by IPTG and its fermentation is carried out with dodecane as the solvent. The products are collected, and GC-MS analysis and structure elucidation results [Fig3A], [Fig3B] suggest that the desired 7epiZ is produced.

Our products also underwent quantitative analysis using caryophyllene as an internal standard according to the method described in the study of Zhang, Suping, et al.[2] According to the GC-MS analysis, the production of 7epiZ is 740mg/L [Fig4&B].

Figure 3: (A) Gas-phase chromatography results for the pMVA+pW1-ZIS-Mvan4662-NPPS culture with dodecane as solvent (B) Mass spectrometry and structure elucidation results of the sample
Figure 4: (A) Standard curve of 7epiZ (B) Yield of 7epiZ of the DH5α harboring pW1-ZIS-Mvan4662-NPPS+pMVA strain

References

[1] Dawood, M. H., & Snyder, J. C. (2020). The alcohol and epoxy alcohol of zingiberene, produced in trichomes of wild tomato, are more repellent to spider mites than zingiberene. Frontiers in Plant Science, 11. https://doi.org/10.3389/fpls.2020.00035

[2]Zhang, Suping, et al. ‘De Novo Biosynthesis of Alpha-Zingiberene from Glucose in Escherichia Coli’. Biochemical Engineering Journal, vol. 176, Dec. 2021, p. 108188. DOI.org (Crossref), https://doi.org/10.1016/j.bej.2021.108188


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