The Q31K and A247V mutations obtained in our study belong to subdomain 1 of XylR protein [ 33], and it is assumed that these mutations might be involved in XylR dimerization (Fig. S2). The transcriptional expression level of xylA and xylF genes was higher in the xyl operon of the JH001 and JH019 strains, indicating that the xylR mutations were responsible for the accelerated uptake and metabolism of D-xylose (Fig. 4). Kremling A, Geiselmann J, Ropers D, de Jong H. Understanding carbon catabolite repression in Escherichia coli using quantitative models. Trends Microbiol. 2015;23(2):99–109. https://doi.org/10.1016/j.tim.2014.11.002. Kim D, Woo HM. Deciphering bacterial xylose metabolism and metabolic engineering of industrial microorganisms for use as efficient microbial cell factories. Appl Microbiol Biotechnol. 2018;102(22):9471–80. https://doi.org/10.1007/s00253-018-9353-2. Anaerobically-adapted BL21(DE3) cells were obtained through short-term adaptive evolution and xylR mutations responsible for faster D-xylose consumption were identified, which may aid in the improvement of microbial fermentation technology.

Ammar EM, Wang X, Rao CV. Regulation of metabolism in Escherichia coli during growth on mixtures of the non-glucose sugars: arabinose, lactose, and xylose. Sci Rep. 2018;8(1):609. https://doi.org/10.1038/s41598-017-18704-0.

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Beisel CL, Afroz T. Rethinking the hierarchy of sugar utilization in bacteria. J Bacteriol. 2016;198(3):374–6. https://doi.org/10.1128/JB.00890-15. Shomura Y, Higuchi Y. Structural basis for the reaction mechanism of S-carbamoylation of HypE by HypF in the maturation of [NiFe]-hydrogenases. J Biol Chem. 2012;287(34):28409–19. https://doi.org/10.1074/jbc.M112.387134. Mutations were transferred to other strains via standard P1 transduction [ 40]. To obtain the Δ xylR mutant strain, P1 vir phage lysates of kanamycin-resistant strain BW25113 Δ xylR (JW3541) from the KEIO collection were used to transduce the BL21(DE3) strain to generate JH003 strain.

Martinez-Carrillo BE, Rosales-Gomez CA, Ramirez-Duran N, Resendiz-Albor AA, Escoto-Herrera JA, Mondragon-Velasquez T, et al. Effect of chronic consumption of sweeteners on microbiota and immunity in the small intestine of young mice. Int J Food Sci. 2019;2019:9619020. https://doi.org/10.1155/2019/9619020.

Varsity Jackets

For serial transfer, 1 mL of seed cultures were added to a 100 mL fermentation medium containing 12.5 mM D-glucose and 12.5 mM D-xylose. Cells were grown anaerobically at 37 °C with constant mixing at 180 rpm. Cell growth and residual D-glucose and D-xylose were monitored throughout the experiment. Once D-glucose and D-xylose were fully depleted, serial passages were performed by diluting the culture to a 1:100 ratio in 100 mL of a fermentation medium containing 12.5 mM D-glucose and 12.5 mM D-xylose. To obtain adapted strains, the cultures were spread on LB plates to obtain pure isolates from fermentation broth. Analytical procedures

The xylFGH and xylAB genes, which respectively encode xylose uptake and metabolism-related enzymes, are co-regulated by the XylR transcriptional factor, as well as the intracellular cyclic AMP (cAMP) concentration. XylR is a transcriptional activator that directly regulates the xylose operon by binding to the promoter of the regulatory region in the presence of D-xylose (i.e., the inducer) [ 14]. However, D-xylose metabolism is inhibited by glucose in many microorganisms including E. coli, a phenomenon known as carbon catabolite repression (CCR) [ 15, 16]. In the presence of D-glucose, CCR inhibits the uptake of other sugars such as D-xylose or lactose. This results in a phenomenon referred to as diauxic growth, whereby other sugars are consumed once D-glucose is fully depleted [ 17, 18, 19, 20]. In the absence of D-glucose (i.e., the preferred carbon source), sugars such as lactose, L-arabinose, and D-xylose are consumed sequentially, depending on the sugar preference [ 21]. Inhibition of the consumption of other phosphotransferase system (PTS)-sugars by glucose can also be interpreted as an inducer exclusion mechanism [ 22]. In the presence of glucose, the phosphate group of glucose-specific enzyme IIA [EIIA (glc)] is transferred to the incoming sugar, and EIIA exists in an unphosphorylated form and binds to non-PTS sugar permeases. Therefore, the transport of non-PTS sugars is inhibited [ 16, 23]. Co-utilization studies of xylose and glucose in E. coli have also been performed by co-culturing a xylose transporter-deficient strain and a strain in which glucose transport-related genes (e.g., ptsG, glk, and manZ) were deleted [ 24, 25]. Additionally, other studies have reported the use of a cyclic AMP-independent CRP mutant to avoid catabolic repression [ 26, 27].To introduce xylR C91A or C361T point mutation, oligo-directed mutagenesis was performed, and negative selection was carried out using the CRISPR-Cas9 system, as described in a previous study [ 30]. The genomic point mutations were confirmed via Sanger sequencing. Next, the CRISPR-Cas9 gene in the genome of the edited E. coli cells was removed through P1 transduction, and temperature-sensitive sgRNA plasmids were removed by incubating the cells at 42 °C. Pinske C, Bonn M, Kruger S, Lindenstrauss U, Sawers RG. Metabolic deficiences revealed in the biotechnologically important model bacterium Escherichia coli BL21(DE3). PLoS One. 2011;6(8):e22830. https://doi.org/10.1371/journal.pone.0022830.