E. coli strains

Molecular cloning and manipulation of plasmids were done with E. coli DH5α (TransGen, Beijing). BW25113-T7 strains were used for CRISPR/Cas9-induced Double Strain Break and recombination. All E. coli strains were cultured routinely in standard LB medium when not mentioned otherwise.

Growth conditions

LB medium (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl, pH 7.2) was used in all DNA manipulations. During cultivation and fermentation, the modified M9 medium (M9YE) was used that contained 1 g/L NH4Cl, 0.5 g/L NaCl, 3 g/L KH2PO4, 17.1 g/L Na2HPO4·12H2O, 2 mM MgSO4, 0.1 mM CaCl2, 2 g/L yeast extract and 10 g/L glucose. Glycine (2 g/L) was added as indicated to serve as the substrate for the C4 pathway. Ampicillin (100 mg/mL), chloramphenicol (25 mg/mL) and kanamycin (50 mg/mL) were added to provide selective pressure for E. coli during cultivation when necessary. To induce expression of plasmid-borne genes, Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to cultures which resulted in a final concentration of 0.1 mM. Considering cell growth and ALA stability, the pH was measured by a glass electrode and controlled at 6.5 ± 0.3 with 4 M NaOH.

Selection of integration site and design of homologous recombination

The function and detailed message of the hemF gene (and other genes) was verified in NCBI and BioCyc Database. Sequence of the hemF gene (and other genes) in the BW25113-T7 genome was confirmed in NCBI. As the recognition site for sgRNA, N20 site directs the Cas9 protein to enable site-specific induction of a DSB. The N20 site was found by BROAD international design tool, which is available at: http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design.

Plasmid construction for CRISPR and preparation of linear donor dsDNA by PCR

All primer pairs we designed for gene cloning and intermediate plasmid construction are listed in Table S2. Plasmid pCas (Fig. S1A) and pTarget were prepared in our laboratory. Plasmid pTarget-gene was constructed by Reverse-PCR and T4-Ligation (T4 DNA Ligase, NEB, England) to replace the N20 fragment (Fig. S1B) with specific primers (Table S2). Plasmid pACYCD-gene and pACYCD-Donor-Gene were both constructed for preparing Donor DNA (Fig. S2) by In-Fusion® HD Cloning Kit (Takara, Japan). Donor DNA was cloned from pACYCD-Donor-Gene by Hi-Fi PCR (Phusion® High-Fidelity PCR Master Mix, NEB, England). All plasmids used in this research are listed in Table 1.

Electroporation, cell recovery, and plating

For transformation, the plasmid or linear DNA were electroporated into competent cells in the pre-chilled cuvette (0.1 cm) using Bio-Rad MicroPulser (1.8 kV, time constant > 5.0 ms). For selection, 25 μg/mL chloramphenicol (Chl) or 50 μg/mL kanamycin (Kan) were used alone or in combination. For induction of λ-Red proteins and lac operator, 1 mM arabinose and 1 mM IPTG were used.

To prepare cells harboring pCas, cells cultured at 37℃ (OD600 = 0.45–0.55) were made competent, mixed with pCas (100 ng) and subjected to electroporation, after which the cells were recovered in SOC medium (1 mL) for 1 h at 30℃, plated onto the Kan plate, and cultured at 30 ℃ for 18–24 h.

For CRISPR/Cas9-mediated homologous recombination, cells harboring pCas were cultured at 30℃ in medium containing Kan and Arabinose and made competent. After co-electroporation of Donor DNA (400 ng) with pTarget-Gene (100 ng), cells were recovered in SOC (1 mL) medium for 1 h at 30℃, plated onto Chl/Kan plate, and cultured at 30℃ for 18–24 h.

For elimination of pTarget-Gene, cells harboring both pCas and pTarget were cultured at 30℃ in medium containing Kan and IPTG for 2 h. Cells were plated onto Kan plates and cultured at 30℃ for 18–24 h.

For elimination of pCas, cells harboring pCas were cultured at 37℃ in the medium without any antibiotic for 12–16 h. Then the cells were plated onto non-antibiotic plates and cultured at 37℃ for 12–16 h.

Confirmation of CRISPR/Cas9-mediated gene knock-in in BW25113

The targeted genetic modifications were rationally engineered by CRISPR/Cas9 in E. coli BW25113-T7. The six targeted genes (hemF, ybdK, gadB, gdhA, dppA and mppA) were cloned from E. coli BW25113-T7 to prepare for Donor DNA. The process of hemF gene knock-out is shown here as example. The map of hemF gene knock-out in BW25113-T7 in ideal condition is shown in Fig. S3. The homologous left arm (HRL) and the homologous right arm (HRR) were set near site of N20. Both homologous arms were approximately 400 bp, which could have a high efficiency of recombination [43]. After CRISPR/Cas 9 mediated gene knock-out, it would make a deletion of 100 bp DNA in targeted gene.

To make CRISPR/Cas9-mediated homologous recombination in BW25113-T7, we electroporated pCas (encoding both Cas and λ-Red proteins) into E. coli BW25113-T7, followed by Arabinose (Ara) induction of pCas-encoded λ-Red proteins Gam, Bet and Exo. After preparing competent cells, pTarget-gene (such as pTarget-hemF) and Donor DNA were co-electroporated into cells (Fig. S4).

To verify deletion in target locus, all mutated bacterial strains were selected for colony PCR (Fig. S5). The primer pairs for colony PCR are shown in Table S2. Characteristics of those mutant strains are shown in Table 1.

Growth of bacteria in different medium

All E. coli strains were grown at 37℃ in conical flasks (250 mL) containing M9YE medium or LB medium with Kanamycin (50 μg/mL). Growth was measured by monitoring optical density at 600 nm (OD600) using a spectrophotometer.

The growth rate was fitted with Sigmoidal-4PL curves. The OD of the stationary phase (ODsp) and the time required to reach it are calculated from this fitted curve. The ratio of the ODsp to the time required to reach it was set as growth rate (hr-1) of the strain in medium.

Analytical procedures

Flask cultivations were carried out in 100 mL conical flasks supplied with 30 mL modified minimal medium at 37℃ with agitation of 200 rpm. A 1% (v/v) inoculum from an overnight culture (12) was used. Cells were cultured in M9YE at 37℃ until OD600 reached 0.7. Then IPTG with a final concentration of 0.1 mM was added to the induced group. To analyze ALA production, culture (30 mL) after inducing for 24 h was centrifuged (12,000 × g for 2 min at 4℃). The supernatant was analyzed for extracellular ALA concentration. ALA concentration was analyzed using modified Ehrlich’s reagent [44]. Specifically, standard or sample (2 ml after diluted) was mixed with 1 ml 1.0 M sodium acetate (pH 4.6) in a cuvette, and 0.5 ml acetylacetone (2,4-pentanedione) was added to each cuvette. Then the mixtures were heated to 100 ℃ for 15 min. After cooling for 15 min, the reaction mixture (1 ml) and freshly prepared modified Ehrlich's reagent (1 ml) were mixed together. After 30 min, the absorbance at 554 nm was measured. Standard plot for ALA measurement is shown in Fig. S6.

For analyzing glucose, 1 mL of culture was centrifuged (12,000 g for 2 min at 4 ℃) and the supernatant was then filtered through a 0.22 mm syringe filter for analysis. The HPLC system was equipped with a cation exchange column (HPX-87 H, BioRad Labs), and a differential refractive index (RI) detector (Shimadzu RID-10 A). A 0.5 mL/min mobile phase using 5 mM H2SO4 solution was applied to the column. The column was operated at 65 ℃.

Data analysis

Data for ALA production of were subjected to analysis of variance (ANOVA) by GraphPad Prism (version 7.00). Error bars indicate standard error of the mean (SEM). P values were calculated using Dunnett's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). The mean of each column was compared with the mean of a control column. BW25113-T7 (D0) was set as control column.