Adaptation of hydroxymethylbutenyl diphosphate reductase enables volatile isoprenoid production

  1. Mareike Bongers  Is a corresponding author
  2. Jordi Perez-Gil
  3. Mark P Hodson
  4. Lars Schrübbers
  5. Tune Wulff
  6. Morten OA Sommer
  7. Lars K Nielsen
  8. Claudia E Vickers  Is a corresponding author
  1. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark
  2. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Australia
  3. Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Campus UAB Bellaterra, Spain
  4. Metabolomics Australia, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Australia
  5. School of Pharmacy, The University of Queensland, Australia
  6. CSIRO Synthetic Biology Future Science Platform, Australia
4 figures, 2 tables and 1 additional file

Figures

Simplified scheme of the plastidic MEP pathway, important volatile isoprenoids, and their atmospheric reactions.

The MEP pathway makes IPP and DMAPP simultaneously through the action of HDR (pink box), and produces the bulk of volatile isoprenoids, contributing >80 % of total BVOCs (Sindelarova et al., 2014) . Non-volatile isoprenoids are essential and synthesised by all organisms, while volatile isoprenoid production is non-essential and highly species-dependent. The cytosolic MVA pathway contributes most sesquiterpenes (<3 % of BVOCs), but is omitted here for clarity. Emitted volatile isoprenoids are rapidly oxidised, resulting in complex atmospheric photochemistry impacting aerosol and cloud condensation nuclei formation, extension of methane residence time, ozonolysis as well as surface-level ozone formation in the presence of mono-nitrogen oxide (NOx) pollutants (Wennberg et al., 2018). BVOCs, biogenic organic volatile compounds; DMAPP, dimethylallyl pyrophosphate; DXS, deoxyxylulose synthase; IDI, isopentenyl diphosphate isomerase; IPP, isopentenyl pyrophosphate; IspS, isoprene synthase; HDR, hydroxymethylbutenyl diphosphate reductase.

Figure 2 with 3 supplements
DMAPP:IPP ratio and isoprene production with different HDR enzymes.

(a) In vivo ratio of DMAPP:IPP measured via LC-MS/MS in E. coli overexpressing HDR genes from different species, in the genetic context of dxs and lycopene biosynthetic pathway overexpression. Filled circles and squares indicate that the HDR source species natively emits C5 or C10 isoprenoids. Open symbols indicate no emission, and no symbol indicates no data or conflicting data. (b) Isoprene production in E. coli when the HDR enzymes shown in panel (a) are overexpressed with dxs and an isoprene synthase. (c) Comparison of DMAPP:IPP ratios between selected HDRs co-expressed with dxs and with expression of either lycopene or isoprene as the metabolic sink. (d) Comparison of DMAPP:IPP ratios in E. coli overexpressing Picea sitchensis (Ps) HDR1 or HDR2 in the context of dxs and lycopene biosynthetic pathway overexpression. (e) Isoprene production in E. coli overexpressing P. sitchensis HDR1 or HDR2 along with dxs and an isoprene synthase. (f) The maximum specific growth rate (µmax) of E. coli expressing selected HDRs in the context of dxs and lycopene biosynthetic pathway overexpression, with or without induction of HDR expression by addition of IPTG. All data shown as mean ± SD from > 3 biological replicates; (-) indicates the control strain without HDR overexpression.

Figure 2—source data 1

Raw data for metabolomics, proteomics and isoprene measurements shown in Figure 2 and supplements.

https://cdn.elifesciences.org/articles/48685/elife-48685-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Complementation of lethal knockout of ispH in E. coli using different HDRs, and associated DMAPP toxicity.

(a) An E. coli strain with a ∆ispH knockout and a heterologously expressed lower mevalonate (MVA) pathway depends on mevalonate for survival. When a functional ispH/HDR gene is expressed from a plasmid, growth can be restored in the absence of mevalonate. (-), empty vector negative control; (+), plasmid expressing E. coli ispH as a positive control. E. grandis HDR2 and P. trichocarpa HDR2 did not complement the ∆ispH knockout. MVA, mevalonate. (b) Toxicity of P. sitchensis HDR2 in E. coliispH. Plasmid-encoded HDR genes are expressed from the trc promoter which can be induced with IPTG or partially repressed with glucose. P. sitchensis HDR2-associated toxicity can be alleviated by adding glucose to repress HDR expression, and is strongest under full IPTG induction. Arabinose, which induces the genomically encoded lower MVA pathway, including a heterologous idi gene, partly alleviates IPTG-induced toxicity. (-), empty vector negative control; (+), plasmid expressing E. coli HDR as a positive control. IPTG, Isopropyl β-D-1-thiogalactopyranoside; MVA, mevalonate.

Figure 2—figure supplement 2
Protein quantification of IDI, HDR and the lycopene biosynthetic pathway.

Relative protein abundance in E. coli overexpressing HDR genes from different species, in the genetic context of dxs and lycopene biosynthetic pathway overexpression. Proteins were quantified using untargeted proteomics via LC-MS/MS, and data represent the three most abundant peptide counts from each protein, normalized across all samples. (a) E. coli native Idi shows no significant difference between strains (one-way ANOVA, p = 0.536). (b) E. coli native HDR quantification. Only the EcHDR overexpression strain is significantly different to the ‘no HDR overexpression’ (-) control (Welch’s one-way ANOVA, p = 0.0048), no significant difference were observed between the other strains (p ≥ 0.743). (c) Overexpressed, heterologous HDR proteins. Comparison of protein abundance between different HDR proteins is not possible due to a lack of shared tryptic peptides across all HDRs. Unique peptides for all overexpressed HDR proteins were detected with high abundance in the respective strains. (d) Plasmid-encoded lycopene biosynthetic pathway proteins. No significant differences were detected across strains for CrtI and CrtB (ordinary or Welch’s ANOVA, respectively, p ≥ 0.05). For Idi and CrtE, differences with p < 0.05 were detected between selected strains (e.g. E. grandis vs. R. communis CrtE p = 0.034); however, no significant differences were observed when comparing any of the strains to the negative control (p ≥ 0.78 for Idi, Welch’s one-way ANOVA; p ≥ 0.46 for CrtE, ordinary one-way ANOVA). For Idi and CrtE, a total of only 3 peptides each were detected in our proteomics analysis, indicating low protein abundance and potentially explaining the higher variability between strains. Data represent means +/- SD from n ≥ 3. All p-values were corrected for multiple hypothesis testing using Dunnett’s method.

Figure 2—figure supplement 3
Quantification of DMAPP and IPP using LC-MS/MS.

(a) Absolute quantification of intracellular DMAPP and IPP in E. coli overexpressing HDR genes from different species, in the genetic context of dxs and lycopene biosynthetic pathway overexpression. The difference in product ratio between HDRs at opposite ends of the graph is driven by both an increase in DMAPP and a decrease in IPP concentration. Data represent means +/- SD from n ≥ 3. (b) Representative chromatograms of matrix-matched calibration standards for DMAPP and IPP, including mevalonate-5-phosphate as internal standard (IS). Due to a slow, consistent drift in retention time over the HPLC column lifetime, no fixed retention times are given for DMAPP and IPP; however, with the presented method the analytes remain baseline-separated as shown here.

Phylogenetic tree of HDR proteins from land plants, the cyanobacterium Synechococcus and Escherichia coli.

Where known, each species’ C5 (isoprene) and C10 (monoterpenes) emission spectra are shown (Wiedinmyer et al., 2020). High DMAPP-producing HDR proteins (from P. trichocarpa, R. communis and P. persica) cluster together based on high sequence similarity. Homologues within species, such as P. trichocarpa, tend to be highly similar; except for in gymnosperms where two separate groups of likely paralogous HDRs exist. Proteins analysed in this study are highlighted in bold. The Asterids clade is collapsed for clarity. Tree generated from BLAST sequence alignment with A. thaliana HDR against all land plants, using maximum likelihood phylogeny. Empty symbol, no volatile emission; filled symbol, volatile emission; no symbol, no or conflicting data available.

Author response image 1

Tables

Table 1
Genetic information and volatile isoprenoid emission profiles for species studied in this work.

Key: blank cell indicates species has not been tested, or genome sequence (or other information) not available; Y indicates significant emissions of isoprene or isoprenoids have been detected, or gene/transcript has been identified; N indicates significant emissions of isoprene or isoprenoids have NOT been detected, or gene/transcript has NOT been identified; MTs, monoterpenes; IspS, isoprene synthase; TPS, terpene synthase.

EmissionsGene/transcript*
KingdomPhylum/CladeCladeGenus, speciesCommon NameHDR protein accession numberE. coli construct Genbank IDComplements?Isoprene (C5)MTs (C10)IspSShort chain TPSReference
PlantaeAngiospermsEudicotsRicinus communiscastor bean plantXP_002519102.1MH605331yesNYNYWiedinmyer et al., 2020Kadri et al., 2011Xie et al., 2012)
PlantaeAngiospermsEudicotsPopulus trichocarpablack cottonwood1ACD70402MH605329yesYYYYWiedinmyer et al., 2020Tuskan, 2006)
2PNT41333.1MH605330no
PlantaeAngiospermsEudicotsPrunus persicapeachXP_007199828.1MH605326yesNYNYWiedinmyer et al., 2020Verde et al., 2013)
PlantaeAngiospermsEudicotsEucalyptus grandisflooded gum1XP_010028563.1MH605323yesYYYYWiedinmyer et al., 2020Myburg et al., 2014
 2XP_010047332.1MH605324no
PlantaeAngiospermsEudicotsTheobroma cacaocacao treeXP_007042717.1MH605333yesNYNYWiedinmyer et al., 2020Argout et al., 2008
PlantaeAngiospermsEudicotsArabidopsis thalianathale cressAEE86362.1MH605322yesNYNYSharkey et al., 2005Chen et al., 2004Bohlmann et al., 2000
PlantaeAngiospermsMonocotsElaeis guineensisoil palmXP_010909277.1MH605325yesYYWiedinmyer et al., 2020; Wilkinson et al., 2006
PlantaeGymnospermsPinophytaPicea sitchensisSitka spruce1ACN40284.1MH605327yesYYYWiedinmyer et al., 2020Hayward et al., 2004
 2ACN39959.1MH605328yes – toxic
BacteriaCyanobacteriaSynechococcus sp. PCC 7002SynechococcusACA98524.1MH605332yesNNN
  1. * Identified from data/genomes available on NCBI (https://www.ncbi.nlm.nih.gov/) and literature search (references noted).

    † Whether protein expression was able to functionally complement an E. coli ΔispH knockout in this study.

  2. ‡ Also known as Populus balsamifera ssp. trichocarpa.

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Escherichia coli)ispH/HDRNCBI ‘Gene’Gene_ID:944777; EcoGene:EG11081; ECK0030; lytBhydroxymethylbutenyl diphosphate reductase
Strain, strain background (Escherichia coli)Escherichia coli WATCCATCC:9637obtained from L. Nielsen lab, Australia
Genetic reagent (Escherichia coli)E. coli W∆cscR, lacZ::PtDXS, arsB::PaISPSThis paper and PMID: 21782859 (Arifin et al., 2011)knockout of cscR, knock-in of PtDXS and PaISPS
Genetic reagent (Escherichia coli)E. coli WΔcscR, lacZ::MVA, ∆ispHThis paper and PMID: 11115399 (Campos et al., 2001)knock-in of MVA pathway, knockout of ispH
Genetic reagent (Populus trichocarpa)DXSNCBI ‘Reference Sequence’XP_006378082.1Deoxyxylulose phosphate synthase, gene was truncated for expression in E. coli
Genetic reagent (Populus alba)ISPS(del2-52,A3T,L70R,S288C)Patent WO2012058494 (Beck et al., 2011)Isoprene synthase (Genbank:EF638224) variant, truncated and mutated
Recombinant DNA reagentpLacZ-KIKO(cm) plasmidPMID: 23799955 (Sabri et al., 2013)Addgene:46764used to integrate PtDXS into the genome
Recombinant DNA reagentpArsBKIKO(cm) plasmidPMID: 23799955 (Sabri et al., 2013)Addgene:46763used to integrate PaISPS into the genome
Recombinant DNA reagentpT-HDR plasmidsThis paperderived from pTrc99aall HDR genes were cloned into this expression vector
Recombinant DNA reagentpAC-LYC04PMID: 7919981 (Cunningham et al., 1994)
Recombinant DNA reagentRicinus communis HDR expression plasmidGenbankMH605331HDR protein XP_002519102.1
Recombinant DNA reagentPopulus trichocarpa HDR 1 expression plasmidGenbankMH605329HDR protein ACD70402
Recombinant DNA reagentPopulus trichocarpa HDR 2 expression plasmidGenbankMH605330HDR protein PNT41333.1
Recombinant DNA reagentPrunus persica HDR expression plasmidGenbankMH605326HDR protein XP_007199828.1
Recombinant DNA reagentEucalyptus grandis HDR 1 expression plasmidGenbankMH605323HDR protein XP_010028563.1
Recombinant DNA reagentEucalyptus grandis HDR 2 expression plasmidGenbankMH605324HDR protein XP_010047332.1
Recombinant DNA reagentTheobroma cacao HDR expression plasmidGenbankMH605333HDR protein XP_007042717.1
Recombinant DNA reagentArabidopsis thaliana HDR expression plasmidGenbankMH605322HDR protein AEE86362.1
Recombinant DNA reagentElaeis guineensis HDR expression plasmidGenbankMH605325HDR protein XP_010909277.1
Recombinant DNA reagentPicea sitchensis HDR 1 expression plasmidGenbankMH605327HDR protein ACN40284.1
Recombinant DNA reagentPicea sitchensis HDR 2 expression plasmidGenbankMH605328HDR protein ACN39959.1
Recombinant DNA reagentSynechococcus sp. PCC 7002 HDR expression plasmidGenbankMH605332HDR protein ACA98524.1
Commercial assay or kitAstec Cyclobond I2000 chiral HPLC columnSigma Aldrich20024ASTHPLC column used for IPP/DMAPP separation
Chemical compound, drugIsopreneSigma AldrichCat. # I19551
Chemical compound, drugIsopentenyl pyrophosphateSigma AldrichCat. # I0503
Chemical compound, drugDimethylallyl pyrophosphateSigma AldrichCat. # D4287
Chemical compound, drug(±)-Mevalonic acid 5-phosphateSigma AldrichCat. # 79849
Chemical compound, drugMevalonolactoneSigma AldrichCat. # M4667
Software, algorithmCLC Main WorkbenchQiagenRRID:SCR_000354
Software, algorithmiTOLPMID: 27095192 (Letunic and Bork, 2016)https://itol.embl.de/Interactive Tree of Life

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  1. Mareike Bongers
  2. Jordi Perez-Gil
  3. Mark P Hodson
  4. Lars Schrübbers
  5. Tune Wulff
  6. Morten OA Sommer
  7. Lars K Nielsen
  8. Claudia E Vickers
(2020)
Adaptation of hydroxymethylbutenyl diphosphate reductase enables volatile isoprenoid production
eLife 9:e48685.
https://doi.org/10.7554/eLife.48685