ASABE 2016 #304-11 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

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

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

Global Production of Fatty Alcohols 3500 3000 2500 2000 Market Demand, 2013 (Kilotons) 1500 1000 500 http://www.grandviewresearch.com/industry-analysis/fatty-alcohols-market Add numbers on axis Medium chain fatty alcohols large portion of global market

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

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

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

(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

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

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

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

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

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

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

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

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

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

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?