(253b) Exploiting Inter-Strain Metabolic Cooperation for CO2 Recycling and Increased Carbon Conversion Efficiency during Biofuel Production

Regardless of the of source of lignocellulosic sugars used, significant carbon loss (~50% weight of the feedstock) in the form of CO₂, a potent greenhouse gas, accompanies biofuel production, limiting the economic viability and environmental sustainability of this approach. This problem is not unique to just conventional ethanol biofuel, since many other advanced biofuel compounds are also derived from precursors produced as a result of pyruvate decarboxylation (e.g. acetaldehyde for ethanol; acetolactate for isobutanol; acetyl-CoA for n-butanol, farnesene and fatty acids) which directly releases CO₂. To overcome this intrinsic limitation we developed a modular, catabolically-orthogonal coculture-coproduction system capable of capturing and fixing CO₂ evolved during biofuel production through inter-strain ‘metabolic cooperation’. Orthogonal sugar catabolism was achieved by disabling xylose catabolism (ΔxylR; a transcriptional activator required for xylose catabolism) in a ‘glucose-to-ethanol’ Escherichia coli ‘specialist’ strain (G2E) while also blocking glucose catabolism in a complementary ‘xylose-to-succinate’ E. coli ‘specialist’ strain (X2S) by inactivating all major glucose uptake systems. In this coculture system, CO₂ emitted by G2E is captured and reutilized by X2S via its fixation by anaplertoic reactions to coproduce succinate. To confirm and characterize the extent of inter-strain metabolic cooperation,¹³C-labeling experiments were used to determine both the carbon fixation capacity and overall carbon utilization efficiency, as well as to elucidate other possible exchanges of central carbon metabolism intermediates. Further demonstrating the modularity of this approach, another ‘xylose-to-C4-dicarboxylate’ E. coli ‘specialist’ strain was also engineered and used to coproduce with ethanol. This flexible and robust CO₂-fixing platform allows for (i) co-utilization of glucose-xylose mixtures without carbon catabolite repression, (ii) reduction in CO₂ emissions during biofuel production (iii) increased overall carbon utilization efficiency, (iv) the potential for facile production of different biofuels and coproducts using modular and interchangeable coculture members. Together, these features provide promising avenue for carbon management and support the prospect of developing carbon neutral bioprocesses.