Tailoring Escherichia coli BL21 (DE3) for preferential xylose utilization via metabolic and regulatory engineering

Abstract Xylose is the most abundant pentose in nature. However, it is usually obtained in mixtures with glucose, leading to carbon catabolite repression in many microorganisms. Among E. coli lineages, significant metabolic and regulatory differences exist, requiring distinct metabolic engineering strategies to develop a xylose-selective phenotype in the strains W, K-12, and C. In this study, strain ES02 was engineered from Escherichia coli BL21 (DE3) as a xylose-selective strain by deleting the glk , ptsG , and manZ genes . However, when grown in a mixture of xylose and glucose, this strain’s specific growth rate and xylose consumption rate decreased by about 50% compared to cultures with only xylose. A modified version of the xylose-responsive transcriptional activator XylR Q31K was utilized to overcome this issue. The resulting strain ES04 (BL21 (DE3) Δglk , ΔmanZ , ΔptsG , xylR::Km r , lacZ::xylR C91A -Gm r ) efficiently used xylose as carbon source either alone or in a mixture with glucose, with a specific xylose consumption rate 75% higher than that of the wild-type strain BL21(DE3). Unexpectedly, strain ES04 partially recovers the ability to grow and consume glucose at a low rate, preferentially consuming xylose over glucose in sugar mixtures, revealing an altered carbon catabolite repression phenotype. Transcriptomics analysis suggested that glucose assimilation in this strain was related to the overexpression of the galactitol operon gatDCBAZY. Further inactivation of this operon confirmed its participation in glucose assimilation. Key points • XylR Q31K alleviates carbon catabolite repression in the xylose-selective strain ES04. • Galactitol operon overexpression in ES04 links to partial glucose utilization. • ES04 strain preferentially uses xylose over glucose, revealing altered CCR.