The xylFGH gene encodes the ABC transporter involved in xylose uptake. However, when this gene is deleted via adaptive evolution, xylose is absorbed through GatC, an alternative transporter [ 28]. In another study, the uptake and metabolism of xylose was enhanced using cell culture techniques coupled with evolutionary engineering, and a xylR mutation was identified in a mutant strain that readily consumed D-xylose [ 29]. Cirino PC, Chin JW, Ingram LO. Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. Biotechnol Bioeng. 2006;95(6):1167–76. https://doi.org/10.1002/bit.21082. The difference between the wild-type and adaptively evolved strains was confirmed based on their organic acid and ethanol output during fermentation. When provided with both D-glucose and D-xylose, there was no significant difference in the amount of acetate, formate, and lactate produced by the bacterial strains. However, while the wild-type strain produced 3.2 mM of ethanol, the adaptively evolved strains produced 15.2 and 14.4 mM. Conversely, the JH001 and JH019 strains produced 5.3 and 4.8 mM of succinate, respectively, whereas the wild-type strain produced 12.2 mM. Jeong H, Barbe V, Lee CH, Vallenet D, Yu DS, Choi SH, et al. Genome sequences of Escherichia coli B strains REL606 and BL21(DE3). J Mol Biol. 2009;394(4):644–52. https://doi.org/10.1016/j.jmb.2009.09.052.

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. For clarity, the following clauses related to delivery are taken from the relevant section of our terms and conditions.Ni L, Tonthat NK, Chinnam N, Schumacher MA. Structures of the Escherichia coli transcription activator and regulator of diauxie, XylR: an AraC DNA-binding family member with a LacI/GalR ligand-binding domain. Nucleic Acids Res. 2013;41(3):1998–2008. https://doi.org/10.1093/nar/gks1207.

An increase in the transcription of xylose metabolizing genes leads to the expression of metabolic enzymes, which can lead to an increase in xylulose-5-phosphate in the pentose phosphate pathway, which further increases glyceraldehyde-3-phosphate metabolic pool. Moreover, substantially more NADPH can be generated in the pentose phosphate pathway compared to the NADH produced in glycolysis. Differences in the reoxidation process between NADPH and NADH may change the pattern of metabolite fermentation (e.g., a decrease in succinate and/or an increase in ethanol) in the evolved strains (Table 1). Additionally, we cannot rule out the possibility that changes in the intracellular concentration of metabolites and/or coenzymes such as NADPH may induce allosteric regulation of one or more enzymes in the lower part of glycolysis or acetyl-CoA metabolism, which might affect the pattern of metabolite fermentation.Gorke B, Stulke J. Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol. 2008;6(8):613–24. https://doi.org/10.1038/nrmicro1932. In the event of our carriers being unable to deliver, a calling card will be left. Failure to respond to this calling card in a timely manner, may result in an extra carriage charge being applied. Eiteman MA, Lee SA, Altman R, Altman E. A substrate-selective co-fermentation strategy with Escherichia coli produces lactate by simultaneously consuming xylose and glucose. Biotechnol Bioeng. 2009;102(3):822–7. https://doi.org/10.1002/bit.22103.

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. Studier FW, Daegelen P, Lenski RE, Maslov S, Kim JF. Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21(DE3) and comparison of the E. coli B and K-12 genomes. J Mol Biol. 2009;394(4):653–80. https://doi.org/10.1016/j.jmb.2009.09.021.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. 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. We then tested whether the carB gene mutation, which was additionally identified in the adaptively evolved JH019 strain, affected the xylose consumption rate. Notably, the JH001 strain exhibited growth delays during D-xylose consumption (Fig. 3D). However, when the carB gene was deleted in the JH001 strain, faster cell growth with higher OD values were observed in the D-xylose medium (Fig. S4). It is still unclear how the carB gene mutation is related to the anaerobic growth and fermentation profiles of JH019 cells, a strain evolved from BL21(DE3). Strain BL21(DE3) is known to have defects in anaerobic metabolism due to its mutation in the fnr gene. Specifically, the DE3 episome is inserted within the genes encoding the molybdenum transport system in this strain [ 36]. Moreover, carbamoyl phosphate, which is generated by carbamoyl phosphate synthetase (encoded by carB), is known to be required for hydrogenase maturation [ 37] and may be linked to redox metabolism alterations.