Part:BBa_K5184001

Mvan4662

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. After the synthesis of NPP using SltNPPS, we incorporate Mvan4462 for the production of Z,Z-farnesyl diphosphate (Z,Z-FPP) from neryl pyrophosphate (NPP) and isopentenyl pyrophosphate (IPP) in E. coli. Our incorporation of this enzyme provide future iGEM teams with insight into novel enzyme capable of catalyzing the formation of longer-chain isoprenoid diphosphates.

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

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. Mvan4662 requires Mg2+ or Mn2+ for activity. The product of this reaction is an intermediate in the synthesis of decaprenyl phosphate, which plays a central role in the biosynthesis of most features of the mycobacterial cell wall, including peptidoglycan, linker unit galactan and arabinan. Neryl diphosphate can also act as substrate. 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 Tetranychidae 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 enzyme Mvan4662Q93S(Mvan4662), a Z,Z-farnesyl diphosphate (Z,Z-FPP) synthase introduced in this part is introduced with LA2167ZIS(ZIS), the zingiberene synthethase and SltNPPS(NPPS) a neryl pyrophosphate synthase (NPP), 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 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.

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.[1] According to the GC-MS analysis, the production of 7epiZ is 740mg/L. [Fig4A&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

Reference

[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.