JBEI - Exploring Non-Canonical Terpene Space With a Heterologous Lepidopteran Mevalonate Pathway

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ChrisEiben15-trimmed_2019-02-12_11-23-30.sky.zip2019-02-13 17:13:3915818126315
Exploring Non-Canonical Terpene Space With a Heterologous Lepidopteran Mevalonate Pathway

  • Organism: Escherichia coli
  • Instrument: 6460 Triple Quadrupole LC/MS
  • SpikeIn: No
  • Keywords: Mevalonate Pathway, Terpenes, Juvenile Hormones, homoisopentenyl pyrophosphate, HIPP, homodimethylallyl pyrophosphate, HDMAPP
  • Lab head: Chris Petzold
Lepidoptera (butterflies and moths) make the six-carbon compounds homoisopentenyl pyrophosphate (HIPP) and homodimethylallyl pyrophosphate (HDMAPP) that are incorporated into sixteen, seventeen and eighteen carbon farnesyl pyrophosphate (FPP) analogues. In this work we heterologously expressed the lepidopteran modified mevalonate pathway, a propionyl-CoA ligase, and terpene cyclases in E. coli to produce several novel terpenes containing sixteen carbons. Changing the terpene cyclase generated different novel terpene product profiles. To further validate the new compounds we confirmed 13C propionate was incorporated, and that the masses and fragmentation patterns were consistent with novel sixteen carbon terpenes by GC-QTOF. Based on the available FPP analogues lepidoptera produce, this approach should greatly expand the reachable biochemical space with applications in areas where terpenes have traditionally found uses.
Experiment Description
Lysis of E. coli cell pellets combined with protein precipitation was achieved by the addition of 80 µL of methanol, 20 µL of chloroform, and 60 µL of water, with vortexing. The samples were centrifuged at 20,817 x g for 1 minute for phase separation. The methanol and water (top) layer was removed, then 100 µL of methanol was added and the sample was vortexed briefly followed by centrifugation for 1 minute to isolate the protein pellet. The protein pellet was resuspended in 100 mM ammonium bicarbonate with 20% methanol and quantified by the Lowry method. A total of 100 µg of protein was reduced by adding tris 2-(carboxyethyl) phosphine (TCEP) to a final concentration of 5 mM, alkylated by adding iodoacetamide at a final concentration of 10 mM, and digested overnight at 37 ºC with trypsin at a ratio of 1:50 (w/w) trypsin:total protein. Peptides were analyzed using an Agilent 1290 liquid chromatography system coupled to an Agilent 6460QQQ mass spectrometer (Agilent Technologies, Santa Clara, CA) operating in MRM mode. Peptide samples (10 µg) were separated on an Ascentis Express Peptide ES-C18 column (2.7 µm particle size, 160 Å pore size, 50 mm length x 2.1 mm i.d., 60 ºC; Sigma-Aldrich, St. Louis, MO) by using a chromatographic gradient (400 µL/min flow rate) with an initial condition of 95% Buffer A (99.9% water, 0.1% formic acid) and 5% Buffer B (99.9% acetonitrile, 0.1% formic acid) then increasing linearly to 65% Buffer A/35% Buffer B over 5.5 minutes. Buffer B was then increased to 80% over 0.3 minutes and held at 80% for two minutes followed by ramping back down to 5% Buffer B over 0.5 minutes where it was held for 1.5 minutes to re-equilibrate the column for the next sample. The data were acquired using Agilent MassHunter, version B.08.02, processed using Skyline version 4.1, and peak quantification was refined with mProphet in Skyline.
Created on 2/13/19, 5:13 PM

This data is available under the CC BY 4.0 license.