(785b) Engineered Consortium Overcomes Substrate-Induced Inefficiencies in Hexose Catabolic Pathways

Escherichia coli is an important commercial species used for production of biofuels, organic acids, sugar alcohols, biopolymers, and natural compounds. The growth media and time necessary for production of value added products are important factors for determining the cost effectiveness of a commercial process. Processed biomass and agroindustrial byproducts serve as low cost nutrient sources that contain a variety of carbon sources. However, bioconversion of mixtures of carbon sources by E. coli is inefficient due to carbon catabolite repression (CCR), where the transport and catabolic activity of one or more carbon sources is repressed and inhibited by the transport and catabolism of another carbon source. CCR can also result in incomplete bioconversion of carbon sources. Attempts to mitigate the inefficiencies of CCR can be categorized as i) identification or development of a single strain deficient of CCR ii) development of a microconsortia of strains that each catabolize a single sugar, and most work has focused on co-utilization of glucose and pentoses. However, the hexoses mannose and galactose can be found in high concentrations in certain biomass derived nutrient sources as well. We will present a new microconsortium of different E. coli strains, each engineered to preferentially catabolize a different hexose - glucose, galactose, or mannose. We modified the specificity and preference of carbon source using a combination of rational strain design and adaptive evolution. The modifications ultimately resulted in strains that preferentially catabolized their specified sugar, overcoming CCR mechanisms (inducer exclusion, transcriptional repression, the phosphorylation state of the adenylate cyclase activator EIIA^Glc ), substrate nonspecific transporters, and complications of resulting from the shared metabolic pathway for hexose catabolism. Finally, we compared the performance of the microconsortium to an existing CCR mutant strain. The microconsortium was capable of catabolizing the total available sugars much faster than a CCR mutant and resulted in much higher final cell density. Further, by including different selectable markers in each strain, we were able to show that by changing the relative sugar concentration in the media and strain composition of the inoculant, we can alter the strain composition of the culture over time. The results indicate that not only have we created a more efficient method for bioconversion of mixtures of glucose, galactose, and mannose, but we have also developed a method to control populations of constituent strains of a microconsortia by simply modifying the sugar composition of the media.