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Comparative Transcriptomics as a Gene Discovery Tool in Solanum pennellii , a Potential Source of Biogasoline Tom McKnight, Sachi Mandal, Wang Ming Ji, Department of Biology. Photo by TRGC. xkcd.com. C1 Methane C2 Ethanol C4 Biobutanol C4 to C10 S. pennellii biogasoline

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  1. Comparative Transcriptomics as a Gene Discovery Tool in Solanum pennellii, a Potential Source of Biogasoline Tom McKnight, Sachi Mandal, Wang Ming Ji, Department of Biology Photo by TRGC

  2. xkcd.com

  3. C1 Methane C2 Ethanol C4 Biobutanol C4 to C10 S. pennellii biogasoline C16 & C18 Biodiesel C1 to C4 gases Fractions decreasing in density and boiling point C5 to C9 naphtha C5 to C10 gasoline Fractions increasing in density and boiling point C10 to C16 kerosene C14 to C20 diesel Crude oil C20 to C50 lubricating oil Heating C50 to C70 fuel oil > C70 residue

  4. Solanum pennellii is native to extremely arid regions of Peru

  5. Glucolipids are secreted by trichomes onto the leaf surface Fobes, J.F., Mudd, J.B. and Marsden, M.P.F. (1985) Plant Physiol. 77, 567-570.

  6. Glucolipids accumulate to over 20% of dry weight of the plant! mg/g extractable lipid % DW 200 150 100 50 20% 15% 10% 5% S. pennellii VF36 5 6 7 8 9 10 11 12 13 14 15 16 WeeksofGrowth Fobes, J.F., Mudd, J.B. and Marsden, M.P.F. (1985) Plant Physiol. 77, 567-570.

  7. The S. pennellii glucolipid has three short-chain fatty acids (C4 to C10) esterified to glucose CH2OH O O O OH CH3(CH2)n-C-O O O-C-(CH2)nCH3 O C-(CH2)nCH3

  8. Transesterification of triglycerides produces biodiesel H2C-OH O H2C-O-C-(CH2)n-CH3 HC-OH O H2C-OH NaOH HC-O-C-(CH2)n-CH3 O O CH3-O-C-(CH2)nCH3 H2C-O-C-(CH2)n-CH3 O CH3-O-C-(CH2)nCH3 + O CH3OH CH3-O-C-(CH2)nCH3 Vegetable oil + MeOH Glycerol + 3 long-chain fatty acid esters

  9. 2,3,4-tri-O-acylglucose + CH3OH Transesterification of glucolipid produces biogasoline CH2OH O OH OH HO NaOH OH O CH3-O-C-(CH2)nCH3 O CH3-O-C-(CH2)nCH3 O CH3-O-C-(CH2)nCH3 Glucolipid + MeOH Glucose + 3 short-chain fatty acid esters

  10. Predominant fatty acids in acylsugars of S. pennellii accessions t = Trace (<2%)measured. JosephA.Shapiroet al. (1993) BiochemicalSystematicsandEcology 22, 545-561.

  11. Advantages ofS. pennellii Not a food or feed crop Drought tolerant and can grow on marginal land Lipid is on leaf surface and can be extracted in the field with a simple ethanol rinse to rapidly yield a high-energy, high-value liquid without transporting large amounts of low-value biomass Glucolipid can be converted to gasoline with standard transesterification technology Resulting biogasoline should be compatible with existing fuel technology (transportation and engines) Potential Disadvantages of S. pennellii Not perennial (yet) Yield per acre is not known (yet)

  12. Biosynthetic Pathway for Glucolipid • Glucolipid is made from UDP-Glucose and short chain fatty acids. • Genes encoding the first two enzymes have been cloned and characterized. • Step 1 – Glucosyltransferase • Step 2 – Glucose acyltransferase • There are only two or three additional steps, making this short pathway a good candidate for moving into other plants. UDP Glucose Fatty acid Cloned 1-O-acyl-ß-glucose Cloned Glucose Not Cloned or Characterized 1,2-di-O-acyl-ß-glucose 2,3,4-tri-O-acyl-ß-glucose

  13. Information Flow DNA is the chemical stable genetic material. RNA is an unstable messenger that conveys information from DNA to ribosomes. Ribosomes read the genetic code on RNA to make proteins. Proteins do the bulk of work in cells. DNA makes RNA makes Protein Growing protein chain

  14. Generation of S. pennelliitranscriptome PurifiedmRNA (< 1% of total RNA) Total RNA isolated from different Solanumpennelliilines Oligo dT selection Reverse Transcription ~200million paired-endsreads Next-Gen DNA Sequencing cDNAlibrary QC&End Trimming AssembledSolanum pennelliitranscriptomeforfurtheranalysis Assembly Trimmedreads (101 or 125 nt long)

  15. Short reads (101 nt) mapped to Gene 1 genomic DNA region Intergenic region Promoter (switch) Protein coding region

  16. Identities of Putative Transcripts No Match (20,547) Matched to GO Term (32,909) Matched, No Info (8,313) CEGMA: 456 of 458 conserved genes represented (99.5%)

  17. Comparative Transcriptomics Four high and four low glucolipid-producing accessions High (>20% DW) Low (<5% DW) • 1911 • 1912 • 1920 • 1926 • 0716 • 1941 • 1946 • 1302 ~200 million reads (125 bp x 2 ends) for 8 accessions

  18. Transcriptomes ofdifferentSolanum pennellii accessions after Trinity assembly

  19. Mapping RUBISCO small subunit sequence reads for QC 1911_Low 1912_Low 1920_Low 1920_Low 1302_High 1941_High 1946_High 0716_High

  20. Expressionlevelofcontrol genesindifferentS. pennellii accessions 0716 1302 1941 1946 1911 1912 1920 1926 RPKM = Reads per kilobase of model (gene)

  21. Gene 1: sequence and expression levels 1911_Low 1912_Low 1920_Low 1926_Low 1302_High 1941_High 1946_High 0716_High

  22. ExpressionlevelofGene1 & 2indifferentS. pennellii accessions 0716 1302 1941 1946 1911 1912 1920 1926 RPKM = Reads per kilobase of model (gene)

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