Part:BBa_K5184015
SltNPPS-Mvan4662-ShZIS
In order to manufacture zingiberene from glucose, we incorporate SltNPPS (part number), 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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, 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.
pW1-ZIS-Mvan4662-SltNPPS, via golden gate 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.
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.[1] According to the GC-MS analysis, the production of 7epiZ is 740mg/L [Fig4&B].
References
[1]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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