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

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

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

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

8. The S. pennellii glucolipid has three short-chain fatty acids (C4 to C10) esterified to glucose O C-(CH 2 ) n CH 3 O O O-C-(CH 2 ) n CH 3 O CH 3 (CH 2 ) n -C-O OHCH 2 OH O

9. O CH 3 -O-C-(CH 2 ) n CH 3 O O + CH 3 OH Transesterification of triglycerides produces biodiesel H 2 C-OH HC-OH H 2 C-OH H 2 C-O-C-(CH 2 ) n -CH 3 HC-O-C-(CH 2 ) n -CH 3 O O O Vegetable oil + MeOHGlycerol + 3 long-chain fatty acid esters NaOH

10. O OH CH 2 OH HO OH O CH 3 -O-C-(CH 2 ) n CH 3 O O 2,3,4-tri-O-acylglucose + CH 3 OH Glucose + 3 short-chain fatty acid estersGlucolipid + MeOH NaOH Transesterification of glucolipid produces biogasoline

11. Predominant fatty acids in acylsugars of S. pennellii accessions Fatty acidLA 0716 (n=6) LA 1941 (n=6) LA 1946 (n=6) LA 1912 (n=6) 2-methylpropanoate (C4)41.8 (0.4)42.2 (1.6)41.6 (0.8)t 2-methylbutanoate (C5)10.8 (0.2)9.9 (0.7)9.0 (0.3)- 3-methylbutanoate (C5)4.0 (0.2)8.5 (2.2)13.0 (0.5)t 5-methylhexanoate (C7)-t2.0 (0.2)- 6-methylheptanoate (C8)---t 7-methyloctanoate (C9)ttt- n-octanoate (C8)---- 8-methylnonanoate (C10)26.3 (0.6)19.7 (2.3)9.9 (0.3)t n-decanoate (C10)10.7 (0.4)10.6 (1.1)14.8 (0.4)- 9-methyldecanoate (C11)tttt N-dodecanoate (C12)4.9 (0.2)5.7 (0.6)7.1 (0.6)t t = Trace (<2%) measured. Joseph A. Shapiro et al. (1993) Biochemical Systematics and Ecology 22, 545-561.

12. Advantages of S. 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)

13. 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. Biosynthetic Pathway for Glucolipid Cloned UDP GlucoseFatty acid 1-O-acyl- ß- glucose 1,2-di-O-acyl- ß- glucose Cloned 2,3,4-tri-O-acyl- ß- glucose Glucose Not Cloned or Characterized

14. DNA makes RNA makes ProteinInformation 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. Growing protein chain

15. Total RNA isolated from different Solanum pennellii lines Purified mRNA (< 1% of total RNA) cDNA library Next-Gen DNA Sequencing Oligo dT selection ~200 million paired-ends reads Generation of S. pennellii transcriptome QC & End Trimming Trimmed reads (101 or 125 nt long) Assembly Assembled Solanum pennellii transcriptome for further analysis Reverse Transcription

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

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

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

19. Transcriptomes of different Solanum pennellii accessions after Trinity assembly Solanum pennellii lines Number of contigs 0716 Hi Reference 57242 0716 Hi70749 1302 Hi67214 1941 Hi61207 1946 Hi60790 1911 Lo66962 1912 Lo68316 1920 Lo62045 1926 Lo62908

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

21. Expression level of control genes in different S. pennellii accessions RPKM = Reads per kilobase of model (gene) 0716 1302 1941 1946 1911 1912 1920 1926

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

23. Expression level of Gene1 & 2 in different S. pennellii accessions RPKM = Reads per kilobase of model (gene) 0716 1302 1941 1946 1911 1912 1920 1926

24. Alpha/beta hydrolase family Thaumatin family Glutathione S-transferase, N- terminal domain Initiation factor 2 subunit family Cyclophilin type peptidyl-prolyl cis- trans isomerase/CLD Fatty acid desaturase hypothetical protein Plant invertase/pectin methylesterase inhibitor hypothetical protein Xylanase inhibitor N-terminal Prephenate dehydratase hypothetical protein Plant invertase/pectin methylesterase inhibitor AP2 domain EamA-like transporter family Glycosyl hydrolases family 17 Chitinase class I Dirigent-like protein Uncharacterised protein family (UPF0041) hypothetical protein Cytochrome P450 Glycosyl hydrolases family 16 Light regulated protein Lir1 Potato inhibitor I family Sugar (and other) transporter alpha/beta hydrolase fold Pathogenesis-related protein Bet v I family Major intrinsic protein Thaumatin family Subtilase family Protein of unknown function (DUF_B2219) hypothetical protein UDP-glucoronosyl and UDP-glucosyl transferase hypothetical protein No apical meristem (NAM) protein Peptidase family M20/M25/M40 BURP domain Cytochrome P450 Patatin-like phospholipase Cytochrome P450 Serine carboxypeptidase Transmembrane amino acid transporter protein Leucine Rich repeats (2 copies) hypothetical protein F-box associated Cytochrome P450 Mycolic acid cyclopropane synthetase hypothetical protein Cytochrome P450 Reverse transcriptase (RNA-dependent DNA polymerase) Rieske (2Fe-2S) domain Gibberellin regulated protein AP2 domain Putative lysophospholipase Pathogenesis-related protein Bet v I family Glycosyl hydrolases family 32 N-terminal domain DnaJ domain Plastocyanin-like domain hypothetical protein Yippee zinc-binding/DNA-binding /Mis18, centromere assembly Leucine rich repeat non-haem dioxygenase in morphine synthesis N-terminal Domain associated at C-terminal with AAA Alpha/beta hydrolase family K-box region Chitinase class I Pectinesterase Terpene synthase family, metal binding domain Papain family cysteine protease non-haem dioxygenase in morphine synthesis N-terminal hypothetical protein EF hand Chitinase class I Gibberellin regulated protein Myb-like DNA-binding domain hypothetical protein Translationally controlled tumour protein Glutathione S-transferase, N-terminal domain Plant mobile domain Uncharacterised protein family (UPF0113) Protein of unknown function (DUF1298) Helix-loop-helix DNA-binding domain PA domain Cytochrome P450 hypothetical protein Xylanase inhibitor C-terminal MOSC N-terminal beta barrel domain gag-polypeptide of LTR copia-type Reverse transcriptase (RNA- dependent DNA polymerase) hypothetical protein Protein kinase domain Cytochrome P450 Glycosyl hydrolases family 17 Protein kinase domain Leucine Rich Repeat Xylanase inhibitor C-terminal Glutathione S-transferase, N-terminal domain