Conversion of proteins into biofuels by engineering nitrogen flux

Biofuels are currently produced from carbohydrates and lipids in feedstock. Proteins, in contrast, have not been used to synthesize fuels because of the difficulties of deaminating protein hydrolysates. Here we apply metabolic engineering to generate Escherichia coli that can deaminate protein hydrolysates, enabling the cells to convert proteins to C4 and C5 alcohols at 56% of the theoretical yield. We accomplish this by introducing three exogenous transamination and deamination cycles, which provide an irreversible metabolic force that drives deamination reactions to completion. We show that Saccharomyces cerevisiae, E. coli, Bacillus subtilis and microalgae can be used as protein sources, producing up to 4,035 mg/l of alcohols from biomass containing ∼22 g/l of amino acids. These results show the feasibility of using proteins for biorefineries, for which high-protein microalgae could be used as a feedstock with a possibility of maximizing algal growth and total CO2 fixation.

[1]  L. Ingram,et al.  Genetic engineering of ethanol production in Escherichia coli , 1987, Applied and environmental microbiology.

[2]  B. Bassler,et al.  Regulation of Uptake and Processing of the Quorum-Sensing Autoinducer AI-2 in Escherichia coli , 2005, Journal of bacteriology.

[3]  James C. Liao,et al.  Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes , 2009, Applied Microbiology and Biotechnology.

[4]  A. Zeng,et al.  Microbial production of 1,3-propanediol , 1999, Applied Microbiology and Biotechnology.

[5]  J. Pronk,et al.  The Ehrlich Pathway for Fusel Alcohol Production: a Century of Research on Saccharomyces cerevisiae Metabolism , 2008, Applied and Environmental Microbiology.

[6]  A. Wolfe,et al.  Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids , 1994, Journal of bacteriology.

[7]  T. Ohshima,et al.  The purification, characterization, cloning and sequencing of the gene for a halostable and thermostable leucine dehydrogenase from Thermoactinomyces intermedius. , 1994, European journal of biochemistry.

[8]  J. Melillo,et al.  Indirect Emissions from Biofuels: How Important? , 2009, Science.

[9]  J. Liao,et al.  Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels , 2008, Nature.

[10]  Hong Wang,et al.  Substrate Specificity and Thermostability of the Dehairing Alkaline Protease from Bacillus pumilus , 2009, Applied biochemistry and biotechnology.

[11]  F. Neidhart.  Escherichia coli and Salmonella. , 1996 .

[12]  W. Winiwarter,et al.  How a century of ammonia synthesis changed the world , 2008 .

[13]  R. Wijffels,et al.  An Outlook on Microalgal Biofuels , 2010, Science.

[14]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[15]  James C. Liao,et al.  Expanding metabolism for biosynthesis of nonnatural alcohols , 2008, Proceedings of the National Academy of Sciences.

[16]  James C. Liao,et al.  3-Methyl-1-butanol production in Escherichia coli: random mutagenesis and two-phase fermentation , 2010, Applied Microbiology and Biotechnology.

[17]  J. Liao,et al.  Pentanol isomer synthesis in engineered microorganisms , 2009, Applied Microbiology and Biotechnology.

[18]  William E Bentley,et al.  Cyclic AMP (cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 in Escherichia coli , 2005, Journal of bacteriology.

[19]  Shelie A. Miller,et al.  Minimizing land use and nitrogen intensity of bioenergy. , 2010, Environmental science & technology.

[20]  L. Reitzer,et al.  Nitrogen assimilation and global regulation in Escherichia coli. , 2003, Annual review of microbiology.

[21]  James C. Liao,et al.  Engineering Corynebacterium glutamicum for isobutanol production , 2010, Applied Microbiology and Biotechnology.

[22]  Alexander J. Ninfa,et al.  Activation of the glnA, glnK, and nac Promoters as Escherichia coli Undergoes the Transition from Nitrogen Excess Growth to Nitrogen Starvation , 2002, Journal of bacteriology.

[23]  J. Benemann,et al.  Look Back at the U.S. Department of Energy's Aquatic Species Program: Biodiesel from Algae; Close-Out Report , 1998 .

[24]  James J. Valdes,et al.  Mapping Stress-Induced Changes in Autoinducer AI-2 Production in Chemostat-Cultivated Escherichia coli K-12 , 2001, Journal of bacteriology.

[25]  J. Keasling,et al.  Microbial production of fatty-acid-derived fuels and chemicals from plant biomass , 2010, Nature.

[26]  James C Liao,et al.  Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde , 2009, Nature Biotechnology.

[27]  Keith A. Smith,et al.  N 2 O release from agro-biofuel production negates global warming reduction by replacing fossil fuels , 2007 .

[28]  E. Becker.  Micro-algae as a source of protein. , 2007, Biotechnology advances.