Abstract
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.
Highlights
Production of D-lactate (LA) and succinate (SA) from renewable carbohydrate feedstocks provides a sustainable and greener alternative to their petroleum-based production (Abdel-Rahman et al, 2013; Ahn et al, 2016; Es et al, 2018)
LA and SA serve as two important monomers in the production of biodegradable plastics, including poly(butylene succinate) (PBS) and poly(lactic acid) (PLA), respectively
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
Summary
Production of D-lactate (LA) and succinate (SA) from renewable carbohydrate feedstocks provides a sustainable and greener alternative to their petroleum-based production (Abdel-Rahman et al, 2013; Ahn et al, 2016; Es et al, 2018). While different strains have their own unique advantages/disadvantages (e.g., ease of genetic manipulation, product tolerance, and other physiological benefits), from a bioprocessing perspective, it is desirable that it should be capable of rapidly and simultaneously utilizing the substrate at high initial loadings (e.g., ≥ 100 g L−1 total sugar) Under such conditions, E. coli has proven to be a promising biocatalyst for the production of both LA and SA. Via a combination of engineering strategies, E. coli strains have been developed to produce both LA and SA at high yields (>90%) and titers (>90 g L−1) and maximum productivities (>1.0 g L−1 h−1) (Sawisit et al, 2015; Utrilla et al, 2016; Sievert et al, 2017) Despite these achievements, challenges still remain with respect to the efficient conversion of glucose–xylose mixtures. This strategy of “population-level” tuning helps to alleviate biosynthetic burden while circumventing technical difficulties that would otherwise accompany the more traditional optimization of multiple catabolic pathways in a single strain
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