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498
- 1. Metabolic Engineering ofYeast to IncreaseYield of Fatty Alcohols Dr. Stuart’s Lab Supervisor: Bonnie McNeil
- 2. Metabolic Engineering • Microorganisms treated like cellular factories to produce valuable compounds from low value starting material Simple sugars Product • Use techniques from synthetic biology and genetic engineering Optimize the cellular processes so that levels of metabolites used in product synthesis are increased C Relieve constraints in a metabolic pathway or increase enzymatic reactions that improve yield of products by increasing flux in pathway A B Product x x
- 3. Goal for my project: Engineer yeast to produce greater yields of fatty alcohol Hexadecanol Octadecanol AKA Cetyl alcohol, Palmityl alcohol, C16:0 OH Stearyl alcohol, C18:0 OH Simple sugars
- 4. Palm oil derivatives found in most shampoos, detergents and cosmetics Palmityl/Cetyl Alcohol, Isopropyl Palmitate, Sodium Laureth Sulfate, Sodium Dodecyl Sulfate, etc. Refinement Palm oil found in many packaged foods C16/C18 Fatty alcohol from Palm Oil refinement is a major component of many household products
- 5. Deforestation of rainforests in countries like Indonesia and Malaysia. The Issue with High Demand for Palm Oil For Oil Palm Plantations Unsustainable practice with negative environmental and social consequences: •Contributes to global warming •Displacement of species from their habitat •Affects the livelihood of the inhabitants in these regions
- 6. Using S. Cerivisiae as a Production Host for Fatty Acid Derived Alcohols Malonyl-CoA + • Yeast synthesize long-chain acyl-CoA via the fatty acid synthesis pathway. ACC1 FAS1/FAS2 Simple sugars Hexadecanol Octadecanol mFAR • By expressing the mouse FAR gene in yeast, we can make yeast convert C16 and C18 acyl-CoAs into alcohols. • Initial strain for my project was engineered with a constitutive promoter for FAS1, these cells overexpress FAS1 and so more acyl-CoA is made.
- 7. •Acyl-CoA synthesis is part of a complex metabolic network •Limitations on the amount of acyl-CoA produced by yeast •What are other potential targets for genetic engineering that will increase the amount of acyl-CoA (and thus fatty alcohol)? mFARC16-OH C18-OH
- 8. Malonyl-CoA ACC1 FAS1/FAS2+ Simple sugars Glycolysis pxa1/ pxa2 •Peroxisomal bound import proteins. Function as a heterodimer •Acyl-CoA brought into peroxisome and degraded back into acetyl-CoA •Knockout either one to prevent degradation of acyl-CoA? Lipid Synthesis Pathway is Complex and Tightly Regulated gpt2/sct1 TAG synthesis DHAP G-3-P gpt2/ sct1 •acyltransferase proteins •attach acyl-CoA to molecules with glycerol backbone •Knockout one of these genes to prevent acyl-CoA from being diverted to TAG synthesis? More acyl-CoA in the cytosol More substrate for mFAR Increased yields of fatty alcohol
- 9. LEU2 5’ 5’3’ 3’ Using PCR, amplify a segment of DNA from a plasmid corresponding to an auxotrophic marker pUG73 ClonNat ClonNat ClonNat LEU2 Plate on -leu + dex Inoculate at 30° for 48-72 hours To select for yeast cells that have been transformed and are able to grow on plates without leucine Transformation protocol LEU2 gpt2 Through homologous recombination events, the PCR knockout cassette is inserted in place of the gpt2 ORF in the yeast genome 5’ 5’ 3’ 3’ gpt2 Prom. gpt2 Term.. Constructing a gpt2 Knockout Strain Inoculate yeast strain Spin down yeast (FAS1 overexpressing)
- 10. Confirmation that gpt2 has been knocked out with LEU2 LEU2 5’ 5’ 3’ 3’ gpt2 Prom. ~500 bp gpt2 term. LEU2 5’ 3’ gpt2 Prom. ~500 bp gpt2 term. Isolate genomic DNA from candidate yeast colonies on plate 5’ 3’ fwd primer complementary to gpt2 promoter rev. primer complementary to sequence in LEU2 fwd. primer complementary to sequence in LEU2 rev. primer complementary to gpt2 terminator and PCR verification Run PCR rxns on gel gpt2 gene - 2232 bp LEU2 gene - 1095 bp If gpt2 gene has been replaced/knocked out with LEU2 gene, a band at ~500 bp will be present on gel (due to primer design) A. B. Run PCR rxns with:
- 11. Confirmation that gpt2 has been knocked out with LEU2 100 200 300 400 1650 12000 500 650 2000 3000 bp DNALadder Colony1A Colony1B Colony2A Colony2B Colony3A Negativecontrol Positivecontrol 850 1000 Colony3B Colony4A Colony4B PCR rxns run with extracted genomic DNA from 4 different colonies show band at ~500bp Have a gpt2∆ KO strain (fas1::pPYK1-FAS1-HIS3 gpt2::LEU2) Gel 1 DNALadder Gel 2
- 12. ClonNat 5’ 5’3’ 3’ Using PCR, amplify a segment of DNA from a plasmid corresponding to a resistance marker pAG25 ClonNat ClonNat ClonNat ClonNat Plate onYEPD:ClonNat Incubate at 30° for 48-72 hours To select for yeast cells that have been transformed and contain resistance to drug Transformation protocol ClonNat pxa2 Through homologous recombination events, the PCR knockout cassette is inserted in place of the pxa2 ORF in the yeast genome 5’ 5’ 3’ 3’ pxa2 Prom. Constructing a pxa2 Knockout Strain Inoculate yeast strain Spin down yeast (FAS1 overexpressing)
- 13. Confirmation that pxa2 has been knocked out with ClonNat pxa2 gene - 2562 bp Sequence amplified - ~400 bp Isolate genomic DNA from candidate yeast colonies on plate ClonNat 5’ 5’ 3’ 3’ pxa2 Prom. Run PCR with forward primer for pxa2 promoter and reverse primer for sequence in ClonNat ~400 bp Run the PCR sample on a gel If ClonNat has replaced/knocked out the pxa2 gene in yeast cells, then a band at around 400bp position will appear on gel 100 200 300 400 1650 12000 DNALadder pxa2::ClonNat pxa2::ClonNat pxa2::ClonNat PCR verification bp Successfully engineered a pxa2∆ (fas1::pPYK1-FAS1-HIS3 pxa2::ClonNat) Now test the effects on fatty alcohol synthesis/yieldControl Control Control
- 14. Inoculate FAS1 overexpressing strain Transforming FAS1 Starting Strain with an EmptyVector Transformation Procedure To make a negative control group Plate on -ura plates Incubate at 30° for 48-72 hours To select for yeast cells that have been transformed with these URA containing plasmids URA3
- 15. Inoculate KO yeast strains + FAS1 overexpressing strain tesA Transformation Procedure Plate on separate -ura plates Incubate at 30° for 48-72 hours To select for yeast cells that have been transformed with these URA containing plasmids Transform each strain with plasmid containing tesA, mFAR and URA3 marker Expression of mFAR in Strains to Convert Acyl-CoA to Fatty Alcohol AhdI(8203) BsrFI(8118) NmeAIII(8056) AatII(7284) ZraI(7282) ApaI*(6604) PspOMI*(6600) BsmBI(6352) BspDI* - ClaI*(6064) BmeT110I(5011) AvaI - BsoBI(5010) HpaI(4718) Bsu36I (698) EcoNI (1083) BstXI (1180) PasI (1696) Acc65I KpnI (2 PmlI ( PflMI BsrGI ( AscI - BssHII (324 BlpI (3725) BamHI (4226) XbaI (4239) EagI - NotI (4250) YEplac195bb-FBA1-tesA-PYK1-mFAR1 9217 bp URA3 YEplac195 w/ tesA, mFAR genes and constitutive promoters inserted mFAR tesA
- 16. x3 FAS1 + tesAmFAR x3 Inoculate colonies from -ura plates... spin down cells... Initial OD600 of 0.05 50 mL -ura+dex medium Grow 3 mL -ura medium Inoculate Take 10 mL aliquots FAS1::pxa2 + tesAmFAR FAS1 + empty vector FAS1::gpt2 + tesAmFAR Method 1 for extracting lipids and fatty alcohols: 96 hrs Add inoculate wash... then...
- 17. Add glass beads Vortex samples Incubate at 80°C Add 1 mL hexane w/ internal stds (0.1mg/mL of C15:0 - OH and C19:0 ME) + 1 mL hexane w/o internal stds Incubate at 30° Spin down samples Extract hexane phase Add MSTFA to derivatize fatty alcohols Extract Intracellular Lipids + Fatty Alcohols Fatty alcohol and Fatty acid standards so that we can quantify fatty alcohol yield and determine changes in lipid substrate pool using GC-FID analysis } Break open cells to release contents. Fatty acids are derivatized to fatty acid methyl esters during incubation with 3N methanolic HCl Resuspend cells in 3N Methanolic HCl Derivatization of fatty acids and fatty alcohols necessary for detection using GC-FID
- 18. Method 2: Dodecane overlay x3 FAS1 + tesAmFAR 50 mL -ura+dex medium Grow 3 mL -ura medium Inoculate FAS1::pxa2 + tesAmFAR FAS1 + empty vector FAS1::gpt2 + tesAmFAR 5 mL dodecane overlay 3 days Initial OD600 of 0.05 Add 1 mL Grow cells with dodecane overlay Cells release fatty alcohols into dodecane Spin down solution Cell Pellet Liquid Media Dodecane Layer Extract dodecane layer •Add internal std •Derivatize fatty alcohols •Sample 100 µL with GC-FID
- 19. Analyzing fatty alcohol yields using GC-FID FAS1 with empty vector •No mFAR being expressed •See no fatty alcohol peaks FAS1 with tesAmFAR vector •mFAR expressed •Get peaks for hexadecanol and octadecanol Fatty alcohol internal std Fatty acid internal std Hexadecanol Octadecanol
- 20. Compare the yield of fatty alcohol from the knockout strains to the FAS1 overexpressing strain we started with (control) Expect fatty alcohol to be produced in greater amounts Simple sugars Malonyl-CoA ACC1 FAS1/FAS2+ Glycolysis TAG synthesis DHAP G-3-P gpt2/ sct1 pxa1/ pxa2 x x Hexadecanol Octadecanol mFAR
- 21. Results using extraction method 1 (no dodecane overlay) No increase in intracellular fatty alcohol yield for gpt2 knockout strain compared to control Significant increase in fatty alcohol yield for pxa2 knockout strain for both C16 and C18 alcohol compared to control Comparing intracellular fatty alcohol levels
- 22. Results using extraction method 2 (dodecane overlay) •Yield of C16 alcohol appears to increase significantly for gpt2 knockout strain using this extraction method •Possibly outliers? Data from only two samples Data for pxa2 knockout shows increased yield of C16 alcohol Comparing External fatty alcohol yields
- 23. Conclusions and future directions • Target other genes for knock out or overexpression based on our understanding of the lipid synthesis pathways • Knocking out the pxa2 gene encoding for the acyl-CoA peroxisomal import protein results in yeast cells that synthesize more fatty alcohol compared to the starting strain • More tests with gpt2 to determine the effects of knockout on fatty alcohol synthesis • Scale up the cultivation and extraction process