Biosynthesis of optically pure D-lactic acid from mixed sugars and undetoxified corn stover hydrolysate by metabolically engineered Escherichia coli consortium

The conversion of lignocellulosic biomass into high value-added bioproducts is a sustainable approach for valorizing agricultural wastes. Optically pure D -lactic acid ( D -LA) derived from such feedstocks has attracted great interest due to its applications in biodegradable polymers. However, carbon catabolite repression (CCR) restricts mixed sugar utilization and consequently limits the efficiency and yield of D -LA biosynthesis from lignocellulosic hydrolysates. In this work, based on our previous study, we successfully constructed a bacterial consortium using an engineered xylose-specific utilizing Escherichia coli strain XL15 with a previously constructed glucose-utilizing strain GL10. Strain XL15 did not utilize glucose in mixed sugars but efficiently consumed 88.64% of xylose, which was 5.43-fold higher than that of the parental strain E. coli BL21(DE3). XL15 achieved a D -LA titer of 2.37 g/L with 100% optical purity in shake flask experiments. D -LA accumulation significantly deteriorated XL15 activity in the bacterial consortium. With the addition of neutralizer, the bacterial consortium achieved 100% glucose utilization, 95.49% xylose utilization, and 6.85 g/L D -LA production. The optical purity was maintained at 100%. The bacterial consortium was capable of consuming mixed sugars in undetoxified corn stover hydrolysate and producing 6.9 g/L D -LA, and the yield was 0.79 g/g. The scaled-up fermentation in 5 L bioreactor resulted in D -LA titer reached 72.95 g/L, with a productivity of 1.35 g/L/h and the yield was 0.84 g/g. This metabolic division of labor consortium alleviates the inhibitory effect of CCR on xylose metabolism, provides a viable strategy for highly optical purity D -LA production from lignocellulosic hydrolysates.