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

Abstract

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.

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

“…Fermentations of 120 g/L glucose–xylose–arabinose mixtures by LY180 and AF25 were performed using a modified version of AM1 mineral salts medium with 2‐fold (NH 4 ) 2 HPO 4 and (NH 4 )H 2 PO 4 relative to the original recipe (Martinez et al, ). All batch fermentations were performed in fermentation vessels with 300 ml working volume as previously described (Flores et al, ; Miller et al, ). Briefly, an initial inoculum of 0.022 g cell dry weight/L was used for all fermentation tests, and fermentations were maintained at 37°C and pH 7.0 by automatic addition of base (2.0 and 6.0 M KOH for LY180 and W derived strains, respectively).…”

“…Cell growth during fermentation was monitored by measuring OD 550 nm using a UV/Vis spectrophotometer (Beckman Coulter DU‐730, Brea, CA). All quantification of sugars and ethanol was performed using HPLC as previously described (Flores et al, ). Compounds were separated and quantified with an UltiMateTM 3000 (Thermo Fisher, Waltham, MA) using an Aminex HPX‐87H column (Bio‐Rad, Hercules, CA) and 4 mM sulfuric acid as the mobile phase.…”

“…Specific sugar catabolic rates (g sugar ·g biomass −1 ·hr −1 ) were calculated using sugar utilization rates in the initial 24 hr divided by biomass concentrations (g cell dry weight/L). Cell dry weight was calculated from the measured OD 550 nm as previously described (Flores et al, ). Experimental data represent mean values of three or more biological replicates with standard deviations unless otherwise indicated.…”

“…To test if xylR* was able to effectively release CCR in an extensively engineered bioproduction host, an ethanologenic E. coli LY180 derived from E. coli W was selected since CCR remains a bottleneck limiting its fermentation performance when using lignocellulosic sugar mixtures (Flores, Ayla, Nielsen, & Wang, ; Miller et al, ). Using a λ‐red recombinase‐mediated two‐step method (Sievert et al, ), the wild‐type copy of xylR was replaced by xylR * in LY180, resulting in the strain AF25.…”

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

“…This general strategy was later expanded upon by others to develop a three-member community of E. coli specialists to co-utilize a mixture of glucose, galactose, and mannose (∼7.5 g L −1 total sugars) (Chappell and Nair, 2017). Most recently, meanwhile, our group engineered two different catabolically orthogonal co-culture systems (derived from wild-type E. coli W or ethanologenic E. coli LY180), each capable of co-utilizing 100 g L −1 of a glucose-xylose mixture (2:1 by weight) in mineral salt media by simple batch fermentation (Flores et al, 2019). In this work, we further explore the utility of this strategy by applying analogous principles to engineer two unique co-culture systems composed of catabolically orthogonal E. coli strains for the production of LA and SA from glucose-xylose mixtures.…”