Joshua Demeo , Yin Wong, Cuiwen He, and Yang Liu May 31, 2012

1. Joshua Demeo, Yin Wong, Cuiwen He, and Yang Liu May 31, 2012

2. Background • Biofuels are currently produced from carbohydrates and lipids in feedstock • Problems: • The algae based schemes have limited efficiency due to feedstock cultures having to be starved • Results in lipid feedstocks with less cell growth and less total CO2 fixation • All current schemes result in the accumulation of protein by-products • There are currently no ways in which to convert proteins into liquid fuels • Normally used as animal feed • Feed markets lack the infrastructure to absorb the increasing number of protein-by-products • Reduced nitrogen (ammonia) is not recycled • Increase in the amount of nitrous oxide produced (from bacteria in waste, fertilizer, or cultivating soil) • Future crops must be supplemented with nitrogen (ammonia) • Requires the energy-intensive and environmentally unfriendly Haber-Bosch process

3. Ethanol Fermentation (Corn) • Corn is the main feedstock for producing ethanol in the United States • Issues related to ethanol production: • High area of land usage and land nutrient depletion • Requires vehicles that use fossil fuels to harvest the crops • Large excess of biomass that is unusable (insoluble carbohydrates, proteins, etc.)

4. Haber-Bosch Process • Nitrogen fixation reaction of nitrogen gas and hydrogen gas to produce ammonia • Catalyzed by enriched iron or ruthenium • Performed under 150-250 bar and temperatures between 300 and 550 oC • Forms CO2 during conversion of CH4 to H2 • Removes nitrogen from the environment • Sustains one-third of the earth’s population from the fertilizer created

5. Protein as a feedstock to make biofuels? • Importance: • Deamination of amino acids would complete the nitrogen loop • Problem: limited by thermodynamic reversibility and biological regulation that favors anabolism • Amino acids could act as a carbon source to create biofuels in the form of alcohols • Problem: biological regulation and competing metabolic pathways • Proteins are the dominate fraction in industrial fermentation residues and fast-growing photosynthetic microorganisms • Maximize growth and CO2 fixation in feedstocksvs lipid and carbohydrate production

6. Summary • Applied metabolic engineering to generate E. coli that can deaminate protein hyrdrolysates • Completed the nitrogen loop • Created an irreversible metabolic force to drive deamination reactions to completion • Ammonia can be harvested/recycled • Created biofuels from amino acids • Createdthree exogenous transamination and deamination cycles • Enabled conversion of proteins to C4 and C5alcohols at 56% of theoretical yield • Able to produce as high as 4,035 mg/l of alcohols from biomass containing ~22g/l of amino acids

7. Wild-type E. coli w/ Isobutanol synthesis pathway Yield: 2.3% of theoretical yield E. Coli can grow well in yeast extract or mixtures of 20 amino acids, but the utilization of amino acids is incomplete.

8. Improvement on amino acid utilization NTG NTG NTG Wild Type (JCL16) Mutant (YH19) …

9. Inhibition of AI-2 re-uptake luxS AI-2 Met lsrABCD

10. Redirect the nitrogen flux

11. Release carbon skeletons

12. YH83 has the highest biofuel production

13. Biofuel production by YH83 Identified by GC-MS and quantified by GC-FID C2: Ethanol (3%) C4: Isobutanol (50%) C5: C5 alcohols (47%)

14. What happen to the rest of the amino acids? Pseudomonas can grow on the six left over AA: Lys, Tyr, Phe, Trp, Met and His, and convert them to 20 amino acids.

15. Proposed protein-based biorefinery scheme

16. Using algal and bacterial proteins as a feedstock Algal biomass mixture includes C. vulgaris, P. purpureum, S.platensis and S.elongatus. C. vulgaris- green algae P. purpureum-red algae S. platensis-green blue algae S.elongatus-cyanobacterium Algae and bacteria were grew and collected, digested with protease and used as feedstock for biofuel production with YH83.

17. Significance • Engineering strategies focus  carbon flux (previous) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels ShotaAtsumi, TaizoHanai & James C. Liao Nature 451, 86-89(3 January 2008)

18. Significance • Engineering strategies focus  carbon flux (previous) Metabolic engineering of Saccharomycescerevisiaefor the production of n-butanol Steen et al.Microbial Cell Factories 2008 7:36

19. Significance • Engineering strategies focus  carbon flux (previous) Microbial production of fatty-acid-derived fuels and chemicals from plant biomass Eric J. Steen, Yisheng Kang, Gregory Bokinsky, ZhihaoHu, Andreas Schirmer, Amy McClure, Stephen B. del Cardayre & Jay D. Keasling Nature 463, 559-562(28 January 2010)

20. Significance • Engineering strategies focus  carbon flux (previous) Synthesis of Transportation Fuels from Biomass:  Chemistry, Catalysts, and Engineering George W. Huber, Sara Iborra, and AvelinoCorma Chem. Rev., 2006, 106 (9), pp 4044–4098

21. Significance • Engineering strategies focus  nitrogen flux (this paper) This paper: Conversion of proteins into biofuels by engineering nitrogen flux Yi-XinHuo, KwangMyung Cho, Jimmy G Lafontaine Rivera, Emma Monte, Claire R Shen, Yajun Yan & James C Liao Nature Biotechnology 29, 346–351 (2011)

22. Proteins readily available from difference sources Energy-intensive Haber-Bosch process limits biofuel efficiency Removal of dependence Biofuels from protein Jonathan R Mielenz Nature Biotechnology 29, 327–328 (2011)

23. Significance • More advantages of this approach: • Higher theoretical yield (long chain alcohol production) • Protein more readily hydrolyzes • Oligopeptides but not oligosaccharides can be utilized • No microbial growth impeding by-products generated • Can produce a liquid fuel/bulk chemicals/pharmaceutical intermediates • Using fast growing microbes with high protein content X photobioreactors • Nitrogen can be recycled  for other usage

24. Challenges • Large scale algal production and harvesting • Product purification • Nitrogen recycling