Difference between revisions of "Part:BBa K5184015"

Latest revision as of 21:41, 1 October 2024

SltNPPS-Mvan4662-ShZIS

In order to manufacture zingiberene from glucose, we incorporate SltNPPS (BBa_K5184000), an neryl diphosphate synthase with its N-termial 45 amino acid-trucated. Originally found in Solanum lycopersium, it is used for transformation of glucose into IPP and DMAPP. Mvan4662 (part number), a farnesyl diphosphate synthase facilitating the formation of longer-chain isoprenoid diphosphates. In our context, it is included for synthesis of Z,Z-FPP from NPP. Lastly, ShZIS, a zingiberene synthase derived from Solanum habrochaites, catalyzes the formation of sesquiterpenes, a compound with 15 carbons. Specifically, we us ShZIS to produce 7-epi-zingiberene from its substrate Z,Z-FPP. Through incorporation of the three enzymes, we can obtain our desired product from glucose in a straightforward pathway.

Abstract

SltNPPS-Mvan4662-ShZIS is a pathway of enzymes used for the synthesis of 7epiZ in E. coli. SltNPPS transforms IPP and DMAPP from the MVA pathway to NPP. Mvan4662 catalyzes the formation of Z,Z-FPP from NPP. Lastly, ShZIS produces 7epiZ from Z,Z-FPP.

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. Mvan4662 is a cis-farnesyl diphosphate synthase that catalyzes the transfer of an isopentenyl group from isopentenyl pyrophosphate (IPP) to prenyl diphosphates, facilitating the formation of longer-chain isoprenoid diphosphates. ShZIS is a sesquiterpene synthase derived from Solanum habrochaites, and is present and expressed in the tomato's glandular trichomes. By utilizing three isoprenoid units, ShZIS catalyzes the formation of sesquiterpenes, a compound with 15 carbons. Intermediates that are commonly used include isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), and (E,E)-α-farnesyl diphosphate (FPP).

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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 ===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