Enhancing the production of xylitol in recombinant Escherichia coli BL21 by metabolic engineering

Abstract

Xylitol, a pentose sugar alcohol with sweetness comparable to sucrose, has advantageous properties such as low caloric value and anti-cariogenic characteristics, leading to its extensive applications in food, chemical, and pharmaceutical industries. Current industrial production predominantly employs chemical catalytic methods, which suffer from high cost and environmental concerns. In contrast, microbial fermentation through metabolic engineering enables direct conversion of xylose to xylitol via microbial metabolism, demonstrating superior advantages of cost-effectiveness, environmental friendliness, and mild reaction conditions. This study developed an engineered Escherichia coli strain for high-efficiency xylitol production by implementing three key strategies: 1) introducing the heterologous xylose reductase gene xyrB, 2) optimizing NADPH cofactor supply, and 3) CRISPR/Cas9-mediated knockout of the competing pathway gene xylA. Subsequent fermentation condition optimization achieved remarkable production performance. Under optimized conditions with 15 g/L xylose and 10 g/L glucose in a 1-L shake flask system, the engineered strain E. coli BL21(DE3)/ΔptsGΔxylA-pETDuet-1 -xyrB-zwf demonstrated maximum xylitol production of 13.8 g/L (0.92 g/g xylose), representing significant improvement in bioconversion efficiency. This study contributes to the industrial production of xylitol by engineered strain E. coli BL21(DE3) via metabolic engineering.