(28b) Engineering Synthetic, Catabolically-Orthogonal Co-Culture Systems for Enhanced Conversion of Lignocellulose-Derived Sugars to Fuels and Chemicals

Fermentation of lignocellulosic sugar mixtures is often suboptimal due to inefficient xylose catabolism and sequential sugar utilization caused by carbon catabolite repression. Unlike in conventional applications employing a single engineered strain, the alternative development of synthetic microbial communities facilitates the execution of complex metabolic tasks by exploiting the unique community features, including modularity, division of labor and facile tunability. In this work, a series of synthetic, catabolically-orthogonal co-culture systems (composed of two unique specialist strains, each capable of efficiently metabolizing only either glucose or xylose) were systematically engineered, as derived from either wild-type Escherichia coli W, ethanologenic LY180 or lactogenic TG114. Specifically, glucose specialist strains were engineered by deleting xylR (encoding xylose-specific transcriptional activator XylR) to disable xylose catabolism, whereas a xylose specialist strains were engineered by deleting several components of glucose transport systems (i.e., ptsI, ptsG, galP, and glk) while also increasing xylose consumption by introducing specific xylR mutations. Net catabolic activities were then readily balanced by simple tuning of the inoculum ratio between specialist strains, ultimately enabling improved co-utilization (98% of 100 g L^-1 total sugars) of glucose-xylose mixtures (2:1 by mass) under simple batch fermentation conditions. Engineered ethanologenic and lactatogenic co-cultures achieved respective titer (~46 g L^-1 ethanol and ~90 g L^-1 lactate), productivity (~0.5 g L^-1 h^-1 ethanol and >1 g L^-1 h^-1 lactate) and yields (~90% of theoretical maximum) each significantly increased compared to traditional LY180 and TG114 monocultures. Meanwhile, ¹³C-fingerprinting experiments have been performed to improve understanding of the flow and distribution of labeled substrates between co-cultures of specialist strains. Lastly, this synthetic co-culture engineering strategy has also been extended for use in novel co-production systems with the potential to achieve higher carbon utilization efficiencies. Holistically, this work contributes to an improved understanding of the dynamic behavior of synthetic microbial consortia as enhanced bioproduction platforms for renewable fuels and chemicals from non-food carbohydrates.