Strains and culture conditions
The strains and plasmids used in this study are listed in Supplementary Table 1. Bacterial seed cultures were grown in 5 ml of LB broth (Cat. No. LB-05, LPS solution, Daejeon, Korea) at 37 °C with constant mixing at 180 rpm. One milliliter of seed culture was used to inoculate a 125 ml serum vial with a butyl rubber stopper containing 100 ml of fermentation medium, as described previously [38]. The medium contained the following components (per liter): yeast extract = 5 g; NaHCO3 = 10 g; NaH2PO4·H2O = 8.5 g; K2HPO4 = 15.5 g. Yeast extract (Cat. No. 212750) was purchased from Becton Dickinson (Sparks, MD, United States). NaHCO3 (Cat. No. S6014), NaH2PO4·H2O (Cat. No. S9638), and K2HPO4 (Cat. No. P3786) were purchased from Sigma-Aldrich (St. Louis, MO, United States). The headspace of the fermentation bottles was filled with nitrogen gas, and sodium sulfide (final concentration 1 mM) was added to quench the dissolved oxygen, thus yielding strictly anaerobic conditions. Bacterial cells were grown anaerobically at 37 °C with constant mixing at 180 rpm. Additionally, 25 mM D-glucose (Cat. No. 64220S0650, JUNSEI, Tokyo, Japan), 25 mM D-xylose (Cat. No. 25190S0401, JUNSEI, Tokyo, Japan), or 12.5 mM D-glucose + 12.5 mM D-xylose were incorporated as carbon sources.
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
Sugar and metabolite concentrations were measured using high-performance liquid chromatography (Waters 410 RI Monitor, Waters; MA, United States) using an Aminex HPX-87H column (300 mm × 7.8 mm, BioRad, Hercules, CA, United States) as described previously [39]. The cell culture broth was then centrifuged, after which the supernatant was passed through a 0.2 μm syringe filter. The column was isocratically eluted at 47 °C with a flow rate of 0.5 mL min− 1 using 0.01 N H2SO4 (Cat. No. 258105-500 ml, Sigma-Aldrich, St. Louis, MO, United States). Cell growth was monitored by measuring the optical density of the culture media at 600 nm using an Ultraspec 8000 spectrophotometer (GE Healthcare, Uppsala, Sweden). The cell cultures were diluted using phosphate buffered saline to measure the optical density. The maximum sugar consumption rate was calculated as the amount of sugar consumed divided by the fermentation time (mM/h) in the section where sugar was consumed most rapidly.
Genome sequencing
The genomic DNA of different Escherichia coli strains was purified with the Wizard Genomic DNA Purification Kit (Cat. No. A1120, Promega, Madison, WI, United States). The genome sequences of the adaptively evolved strains were obtained with an Illumina HiSeq 2500 sequencer. Pretreatment of the reads, reference mapping, and variant detection were carried out using the Genome Analysis Tool Kit (GATK). Reads shorter than 50 nt were filtered out after quality trimming using Trimmomatic Version 0.36 (Table 2). The genome sequences of E. coli BL21(DE3) (CP001509.3) were used for reference mapping. Genome sequencing data were deposited in the NCBI BioProject database under accession number PRJNA689415. Sanger sequencing was conducted to confirm the xylR sequence. xylR was amplified using the xylR_250F and xylR_100R primer pairs in Supplementary Table 2.
Genome editing
Mutations were transferred to other strains via standard P1 transduction [40]. To obtain the ΔxylR mutant strain, P1 vir phage lysates of kanamycin-resistant strain BW25113 ΔxylR (JW3541) from the KEIO collection were used to transduce the BL21(DE3) strain to generate JH003 strain.
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.
To introduce a ΔcarB mutation, P1 vir phage lysates of kanamycin-resistant strain BW25113 ΔcarB (JW0031) from the KEIO collection were used to transduce strains BL21(DE3) and JH001 as recipient cells to generate JH042 and JH044, respectively.
Transcript analyses
The transcription of the xylA and xylF genes was confirmed using quantitative real-time PCR (qRT-PCR). The BL21(DE3) wild type strain and adapted strains were grown for 8 h under anaerobic conditions in fermentation media containing 12.5 mM D-glucose and 12.5 mM D-xylose or 25 mM D-xylose at 37 °C, and RNA was isolated using the RNeasy® Mini Kit (Cat. No. 74104, Qiagen, Hilden, Germany). qRT-PCR primer sequences for target genes were designed using the IDT PrimerQuest®Tool (Supplementary Table 2). qRT-PCR was conducted using a CFX Connect system (BioRad, Hercules, California, United States) using the RealHelix™ qRT-PCR Kit (Cat. No. QRT-S500, Nanohelix, Daejeon, Korea). Five Nanograms of total RNA was used in qRT-PCR reactions under the following conditions: cDNA synthesis (50 °C, 40 min); denaturation (95 °C, 12 min); amplification for 40 cycles (95 °C, 20 s; 60 °C, 1 min). The raw fluorescence data were normalized against the expression level of the 16S ribosomal RNA and their corresponding expression levels in the BL21(DE3) wild-type strain.