A coculture-coproduction system designed for enhanced carbon conservation through inter-strain CO2 recycling.

Carbon loss in the form of CO2 is an intrinsic and persistent challenge faced during conventional and advanced biofuel production from biomass feedstocks. Current mechanisms for increasing carbon conservation typically require the provision of reduced co-substrates as additional reducing equivalents. This need can be circumvented, however, by exploiting the natural heterogeneity of lignocellulosic sugars mixtures and strategically using specific fractions to drive complementary CO2 emitting vs. CO2 fixing pathways. As a demonstration of concept, a coculture-coproduction system was developed by pairing two catabolically orthogonal Escherichia coli strains; one converting glucose to ethanol (G2E) and the other xylose to succinate (X2S). 13C-labeling studies reveled that G2E + X2S cocultures were capable of recycling 24% of all evolved CO2 and achieved a carbon conservation efficiency of 77%; significantly higher than the 64% achieved when all sugars are instead converted to just ethanol. In addition to CO2 exchange, the latent exchange of pyruvate between strains was discovered, along with significant carbon rearrangement within X2S.

Catabolic Division of Labor Enhances Production of D-Lactate and Succinate From Glucose-Xylose Mixtures in Engineered Escherichia coli Co-culture Systems.

Although biological upgrading of lignocellulosic sugars represents a promising and sustainable route to bioplastics, diverse and variable feedstock compositions (e.g., glucose from the cellulose fraction and xylose from the hemicellulose fraction) present several complex challenges. Specifically, sugar mixtures are often incompletely metabolized due to carbon catabolite repression while composition variability further complicates the optimization of co-utilization rates. Benefiting from several unique features including division of labor, increased metabolic diversity, and modularity, synthetic microbial communities represent a promising platform with the potential to address persistent bioconversion challenges. In this work, two unique and catabolically orthogonal Escherichia coli co-cultures systems were developed and used to enhance the production of D-lactate and succinate (two bioplastic monomers) from glucose-xylose mixtures (100 g L-1 total sugars, 2:1 by mass). In both cases, glucose specialist strains were engineered by deleting xylR (encoding the xylose-specific transcriptional activator, XylR) to disable xylose catabolism, whereas xylose specialist strains were engineered by deleting several key components involved with glucose transport and phosphorylation systems (i.e., ptsI, ptsG, galP, glk) while also increasing xylose utilization by introducing specific xylR mutations. Optimization of initial population ratios between complementary sugar specialists proved a key design variable for each pair of strains. In both cases, ∼91% utilization of total sugars was achieved in mineral salt media by simple batch fermentation. High product titer (88 g L-1 D-lactate, 84 g L-1 succinate) and maximum productivity (2.5 g L-1 h-1 D-lactate, 1.3 g L-1 h-1 succinate) and product yield (0.97 g g-total sugar-1 for D-lactate, 0.95 g g-total sugar-1 for succinate) were also achieved.

The XylR variant (R121C and P363S) releases arabinose-induced catabolite repression on xylose fermentation and enhances coutilization of lignocellulosic sugar mixtures.

Microbial production of fuels and chemicals from lignocellulosic biomass provides a promising alternative to conventional petroleum-derived routes. However, the heterogeneous sugar composition of lignocellulose prevents efficient microbial sugar co-fermentation due to carbon catabolite repression, which negatively affects production metrics. We previously discovered that a mutant copy of the transcriptional regulator XylR (P363S and R121C; denoted as XylR*) in Escherichia coli has a higher DNA-binding affinity than wild-type XylR, leading to a stronger activation of the d-xylose catabolic genes and a release from glucose-induced repression on xylose fermentation. Here, we showed that XylR* also releases l-arabinose-induced repression on xylose fermentation through altered transcriptional control, enhancing co-fermentation of arabinose-xylose sugar mixtures in wild-type E. coli. Integrating xylR* into an ethanologenic E. coli resulted in the coutilization of 96% of the provided glucose-xylose-arabinose mixtures (120 g/L total sugars supplied) with an ethanol yield higher than 90% of the theoretical maximum by simple batch fermentations.

Engineering a Synthetic, Catabolically Orthogonal Coculture System for Enhanced Conversion of Lignocellulose-Derived Sugars to Ethanol.

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. A series of synthetic, catabolically orthogonal coculture systems were systematically engineered, as derived from either wild-type Escherichia coli W or ethanologenic LY180. Net catabolic activities were effectively balanced by simple tuning of the inoculum ratio between specialist strains, which enabled coutilization (98% of 100 g L-1 total sugars) of glucose-xylose mixtures (2:1 by mass) for both culture systems in simple batch fermentations. The engineered ethanologenic cocultures achieved ethanol titer (46 g L-1), productivity (488 mg L-1 h-1), and yield (∼90% of theoretical maximum), which were all significantly increased compared to LY180 monocultures.