(425g) Conversion of Proteins Into Biofuels by Engineering Nitrogen Flux

Current biorefinery schemes are suboptimal for several reasons. For examples, all existing schemes, including sugar-based or cellulosic biorefining, lead to the accumulation of protein by-products, but there are no strategies to convert these by-products into liquid fuels. In additions, schemes based on algae have limited efficiency because cultures used for biofuel production must be starved so that they produce lipid feedstocks, resulting in less cell growth and less total CO₂ fixation. A heretofore unexplored solution to these limitations would be to use proteins as a feedstock. If proteins were deaminated and converted to fuel or chemicals, the reduced nitrogen could be recycled to achieve a nitrogen neutral biofuel production. More importantly, proteins are the major component of the photosynthesis apparatus, CO₂ fixation pathways and other biosynthetic and cell growth machinery. Proteins are the dominant fraction in fast-growing photosynthetic microorganisms and industrial fermentation residues. Thus, using proteins as feedstock might maximize growth and CO₂ fixation rates. However, current schemes aim to increase production of carbohydrates or lipids, rather than proteins. Proteins have not been used to synthesize fuels because of the difficulties of deaminating protein hydrolysates.

In this study, we apply metabolic engineering to generateEscherichia 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. With this nitrogen-centric metabolic engineering strategy, all amino acids can be converted to their corresponding keto acids, and can in turn be further converted to fuels or chemicals, directly or through key metabolites such as acetyl-CoA or succinate. The compounds that could be produced from proteins include bulk chemicals, monomers and pharmaceutical intermediates, in addition to fuels. 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.

For large-scale applications, the protein raw material could come from several sources. In the short term, waste proteins generated from the current fermentation, food processing and biofuel production industries could be used. In particular, genetically modified organisms used in fermentation cannot be disposed of as fertilizers or animal feed without additional treatment, and thus provide an excellent source material for protein-based biorefining. For long-term, large-scale applications, we envision using algal biomass, because proteins are the major component of fast-growing microalgae in open-pond cultures that are not artificially induced to accumulate lipids. Even with contamination by heterotrophic bacteria, the combined protein sources are still usable as raw material.