Strains and culture media
An Escherichia coli strain BW38029 (F− ∆(araD-araB)567 lacZp4105(UV5)-lacY λ− hsdR514, an independent isolate of strain BW38028 (Conway et al. 2014) was kindly provided by Dr. Hirotada Mori (Division of Biological Science, Nara Institute of Science and Technology) and was used as a parental strain for S-adenosylmethionine (AEC)-resistant mutants and a host strain for expressing the ThrA variants. BW38029-derived strains were cultured in M9 medium (4 g/L glucose, 65 mM sodium/potassium phosphate, 8.6 mM NaCl, 18.7 mM ammonium chloride, and 1 mM MgSO4), unless otherwise stated. E. coli strains DH5α (F− λ− Φ80lacZ∆M15 ∆(lacZYA argF)U169 deoR recA1 endA1 hsdR17(rk−mk+) supE44 thi-1 gyrA96) and BL21 (DE3) (F− ompT hsdS(rB− mB−) gal dcm λ(DE3) (λ(DE3):lacI, lacUV5-T7 gene1 ind1 sam7 nin5) were used for construction of expression plasmids and for expression of the recombinant ThrA, respectively. These E. coli strains were cultured in Luria–Bertani (LB) medium (5 g/L yeast extract, 10 g/L tryptone, and 5 g/L NaCl) containing appropriate antibiotics or in M9CA medium (M9 medium supplemented with 20 g/L casamino acid) containing 100 μg per mL ampicillin.
Isolation of lysine analogue-resistant mutants
The wild-type (WT) strain BW38029 was randomly mutagenized by treatment with 2% of ethyl methanesulfonate (EMS) in phosphate-buffered saline (PBS; pH7.4) at 37 °C for 45 min. Mutagenized cells were washed with 10% (w/w) sodium thiosulfate in PBS twice and then suspended in PBS. Approximately 5 × 106 cells were spread onto an M9 medium containing 100 μg per mL of AEC. After being cultivated at 37 °C for 2 days, the resulting colonies were collected, and then AEC resistance and lysine production were analyzed.
AEC sensitivity of E. coli cells
BW38029-derived strains were pre-cultured at 37 °C. After 24 h of cultivation, E. coli cells were harvested, washed by PBS twice, and suspended in PBS. The suspension was serially diluted to an optical density at 600 nm (OD600) of 10−1 to 10−5, then spotted onto M9 agar medium without or with 100 μg/mL of AEC, and incubated at 37 °C for 24 h.
Measurement of intracellular amino acids contents
BW38029-derived strains were pre-cultivated at 37 °C overnight and then inoculated to a new medium at an OD600 of 0.05. After cultivation at 37 °C for 24 h, E. coli cells were collected by centrifugation and washed twice with sterilized water. Harvested cells were resuspended in sterilized water, and the suspension was adjusted to an OD600 of 40. Consequently, intracellular amino acids in an aliquot (0.15 mL) of the cell suspension were extracted by boiling at 100 °C for 10 min. Cell debris was removed by centrifugation, and each supernatant was subsequently quantified with an UF-amino station (Shimadzu, Kyoto, Japan) with pre-column derivatization using 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (Wako Pure Chemical, Osaka, Japan). The content of each amino acid was represented as μmol per gram dry cell weight (DCW).
Whole genome sequence analysis
The extracted genomic DNAs from the parent strain (BW38029) and the AEC-resistant strain (AEC28) were quantified with Qubit (Thermo Fisher Scientific, Waltham, MA). A next-generation sequencing library was constructed for each genome using the Nextera DNA Library Preparation Kit (Illumina, San Diego, CA) according to the manufacturer’s instructions. The genome libraries were sequenced using MiSeq (Illumina) with MiSeq Reagent Kit v2 or v3 (Illumina). Sequencing data processing of BW38029 and AEC28, as well as sequencing data from the Sequence Read Archive (SRA), was performed with CLC Genomics Workbench v 10.1.1 (Qiagen, Hilden, Germany). This process included trimming, mapping, and variants calling against the reference genome of E. coli BW25113 (GCA_000750555). Reads bases not matching in the alignment were scored as variants. The coverage table files and the variants table files were exported from Genomics Workbench and retained for further analysis. These files were converted into a FASTA file of synthetic sequences with custom scripts. These scripts generate the sequences of homozygous SNPs from the data of coverage and variants. The sequencing data were deposited to DNA Data Bank of Japan (DDBJ) sequence read archive (DRA). The accession numbers of strains BW38029 and AEC28 are SAMD00324911 and SAMD00324912, respectively.
Construction of E. coli strains expressing the ThrA variants
To construct strains expressing the G474D and C554Y ThrA variants, an open reading frame (ORF) of thrA in the genomic DNA was replaced with the mutant genes, thrAG474D and thrAC554Y (corresponding to G474D and C554Y substitutions, respectively), using λ-red recombination system (Datsenko and Wanner 2000). The thrAG474D and thrAC554Y genes were amplified using the genomic DNA of AEC28 and AEC106 as templates with primers thrA_in_fusion_fw (5′-TCG AAT TCA AAG GAG GTA CCC ACC ATG CGA GTG TTG AAG TTC GG-3′) and rv (5′-GAG ACA ACT TCT AGA TCA GAC TCC TAA CTT CCA TGA GAG GG-3′). The amplified DNA fragments were sub-cloned into pSF-OXB1 vector (OXGENE, Oxford, UK) using by In-Fusion HD Cloning Kit (Takara Bio, Shiga, Japan). After the nucleotide sequences were verified, the DNA fragments including the thrA ORF were obtained by digesting with BamHI and EcoRI and then ligated into the same sites of pK18mobSacB vector (Kvitko and Collmer 2011) resulted in pKMS_thrAG474D and pKMS_thrAC554Y. These plasmids were linearized by digestion of SspI for pKMS_thrAG474D and MunI for pKMS_thrAC554Y, respectively. BW38029 harboring pKD46 (Datsenko and Wanner 2000) was cultured in LB medium including arabinose to induce the gene expression of λ-red recombinase and then transformed with the aforementioned linearized DNA. Single crossover strains, which harbor the full length of pKMS_thrAG474D and pKMS_thrAC554Y in the thrA locus by homologous recombination, were selected by growth phenotype (kanamycinr and sucroses) and confirmed by PCR analysis. To induce second-time recombination, single-crossover strains were grown to an OD600 of 0.8, and then harvested cells were spread onto LB medium (without NaCl) containing 100 g/L sucrose. Double crossover strains were picked up by growth phenotype (kanamycins and sucroser), and objective strains, which harbor thrAG474D and thrAC554Y in the thrA locus, were selected by PCR analysis from double crossover strains (G474D and C554Y, respectively). Replacement of the thrA ORF to thrAG474D and thrAC554Y was confirmed by DNA sequencing.
Construction of plasmids for expressing the recombinant ThrA
To construct plasmids for expressing the recombinant ThrA enzymes, the WT and the mutant thrA genes were amplified from the genomic DNA of WT, AEC28, and AEC106 by PCR with the primers thrA_gateway_Fw (5′-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT AAT GCG AGT GTT GAA GTT CGG-3′) and Rv (5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG GAC TCC TAA CTT CCA TGA GAG G-3′). The PCR-amplified DNA fragment was introduced into the pDONR221 vector (Thermo Scientific, Waltham, MA) using BP clonase II (Thermo Scientific, Waltham, MA), resulting in pDONR221_thrA, pDONR221_thrAG474D, and pDONR221_thrAC554Y. The nucleotide sequences of the thrA genes were verified, and they were transferred to the pET53-dest expression vector (Thermo Scientific, Waltham, MA) using LR clonase II (Thermo Scientific, Waltham, MA), resulting in pET53_thrA, pET53_thrAG474D, and pET53_thrAC554Y.
Expression and purification of the recombinant ThrA
E. coli BL21 (DE3) cells harboring pET53_thrA, pET53_thrAG474D, and pET53_thrAC554Y were cultivated in M9CA medium containing ampicillin and grown at 37 °C to an OD600 of 0.8. The cells were cooled on ice for 5 min, and isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM. After 20 h of cultivation at 18 °C, the cells were harvested by centrifugation and suspended in buffer A (50 mM Tris–HCl (pH 7.4), 500 mM NaCl, and 20% (w/w) glycerol). The cell suspension was homogenized under cooling and then centrifuged to remove the insoluble fraction. The supernatant was filtrated by a 0.45-μm filter and subsequently applied onto a nickel affinity column (Ni Sepharose™ 6 Fast flow, GE Healthcare Life Sciences, Chicago, IL). After the column was washed with buffer A containing 40 mM imidazole, the recombinant proteins were eluted by buffer A supplemented with 500 mM imidazole. The elution fraction was dialyzed twice with buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 10% (w/w) glycerol at 4 °C. Proteins were quantified using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) and subjected to SDS–polyacrylamide gel electrophoresis.
Enzymatic analysis of the recombinant ThrA
AK activity was measured by the production of ADP in an enzyme-coupled system with pyruvate kinase (PK) and lactate dehydrogenase (LDH) (Wampler and Westhead 1968; Chassagnole et al. 2001; James and Viola 2002). The reaction mixture (final volume, 1 mL) contained the following: 100 mM HEPES–NaOH (pH7.5), 100 mM KCl, 10 mM MgCl2, 1 mM phosphoenolpyruvate, 0.25 mM NADH, 15 U of PK/LDH (Sigma-Aldrich, St. Louis, MO), 2 μg of purified ThrA, and various concentrations of aspartate and ATP. The reaction mixture except for aspartate was pre-equilibrated for 3 min at 37 °C, and then the reaction was initiated by the addition of aspartate. ThrA-dependent oxidation of NADH was monitored at 340 nm with a DU-800 spectrophotometer (Beckman Coulter, Brea, CA) and maintained at 37 °C. For steady-state kinetics, when the concentration of aspartate was kept at 10 mM, the concentrations of ATP were varied (0.5–10 mM). With a fixed concentration of 10 mM ATP, the concentration of aspartate was 0.5–10 mM. In order to examine the feedback inhibition sensitivity of ThrA, the concentration of aspartate and ATP was fixed at 6 and 10 mM, respectively, and threonine was added to the reaction mixture at a concentration of 0.05–1 mM. The reaction rate was calculated with the extinction coefficient of NADH, 6220 M−1‧cm−1. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of ADP per min.
HSDH activity (reverse direction) was measured by the synthesis of NADPH. The reaction mixture (final volume, 1 mL) contained 100 mM HEPES–NaOH (pH7.5), 100 mM KCl, 5 mM NADP+, 30 μg of purified ThrA, and 1–5 mM of homoserine. The reaction mixture except for homoserine was pre-equilibrated for 3 min at 37 °C, and then the reaction was initiated by the addition of homoserine. ThrA dependent reduction of NADP+ was monitored at 340 nm with a DU-800 spectrophotometer and maintained at 37 °C. The reaction rate was calculated with the extinction coefficient of NADPH, 6220 M−1‧cm−1. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of NADPH per min. Kinetic parameters of each enzyme were calculated with GraphPad Prism version 9 (GraphPad Software, San Diego, CA) using nonlinear regression analysis.