1. ASABE 2016 # Metabolic Engineering of Saccharomyces cerevisiae to Produce Long Chain and Medium Chain Fatty Alcohols
Joseph Stevens
PI: Dr. Xueyang Feng
Virginia Tech
July 20, 2016
Currently: Graduate Research Assistant, Department of Biosystems and Agricultural Engineering, University of Kentucky

2. Fatty alcohols used in industry
Yeast
Natural Sources
Petroleum Sources
Environmental
Concerns
Declining Supply

3. Highest Titer Achieved: 1.1 g/L
Long chain fatty alcohol production in S. cerevisiae
Citrate
Rpd3
YlACL
G6P
Acetyl-CoA
Ino1
TaFAR
ACC1
Fatty Acids
Fatty Acyl-CoA
Fatty Alcohols
Malonyl-CoA
Add citation from paper, cite previous work
Peroxisome (ẞ-oxidation)
Feng et al., Metab. Eng. 2015
Highest Titer Achieved: 1.1 g/L

4. Global Production of Fatty Alcohols
3500
3000
2500
2000
Market Demand, 2013 (Kilotons)
1500
1000
500
Add numbers on axis
Medium chain fatty alcohols large portion of global market

5. Peroxisomal Protein Import Machinery
Moyersoen et al., FEMS Microbio. Rev. 2004

6. Medium Chain Fatty Alcohol Production
Add slide about tags, include table of tags
Sheng et al., Sci. Rep. 2016

7. Production possible, move forward with optimization and screening
Medium Chain Fatty Alcohol Confirmation
Production possible, move forward with optimization and screening

8. (R/K)-(L/V/I)-xxxxx-(H/Q)-(L/A)
Peroxisomal Import Tags
PTS1 pathway uses tripetide sequence
Elgersma et al., Journ. Biol. Chem. 1996
PTS2 pathway uses nonapetide sequence
(R/K)-(L/V/I)-xxxxx-(H/Q)-(L/A)
Lazarow, Biochim. Biophys. Acta 2006

9. Import Pathway Tag Screening
PTS1 pathway more efficient
Combining PTS1 and PTS2 tags resulted in 15% increase
Change words to focus more on screening

10. TaFAR targeting confirmation
PEX14-GFP used to image peroxisome membrane
KIQL-mCherry-SKL used to image peroxisome cytosol

11. Cargo Protein Optimization
Sheng et al., Sci. Rep. 2016

12. Cargo Protein Optimization
Further peroxisome engineering increased fatty alcohol production
To reduce metabolic stress, PTS2+PEX7 strain selected further experimentation

13. Fatty Acyl-CoA Synthesis Optimization
Cargo proteins optimization, etc
Sheng et al., Sci. Rep. 2016

14. Fatty Acyl-CoA Synthesis Engineering
C14-OH production detected for first time

15. Fed Batch Fermentation, Medium Optimization
Highest producing strain tested under fed batch conditions
Medium optimization tested
C:N, K+ concentration
Resulted in highest titer reported in yeast, ~1.3 g/L
Nitrogen limited medium
Highest titer reported in s cereviaise

16. Summary of medium chain fatty alcohol production in yeast
“Pathway compartmentalization”
For second bullet:
Cargo proteins
Optimization, different levels or pathways
Keep 3rd the same
Pathway compartmentalization results in first reported medium chain fatty alcohol production in Saccharomyces cerevisiae
Production optimization
Cargo protein engineering increases production
Pathway optimization further increases production
Medium optimization increases final titer to ~1.3 g/L fatty alcohol: highest reported

17. Future Work
TaFAR used in study, shown to favor production of 1-hexadecanol
AdFAR previously shown to favor production of medium chain fatty alcohols
Merge with future work slides
Use different enzymes, AdFAR
More peroxisome engineering
Bioprocess optimization
Further peroxisome engineering
Optimizing growth vs. production

18. Questions? Acknowledgements Dr. Xueyang Feng Dr. Jiayuan Sheng
Group Members
Funding from Virginia Tech
Use logos, ASABE logo, thank the session chair, BSE logo, funding from VT, VT logo
Questions?