1. galK gene inactivation

We firstly prepared the electrocompetent E. coli MG1655 cells, and used electroporation to introduce the plasmid harboring lambda red system and different CRISPR proteins. The resulting strains are EC85 and EC88, which grow on the plates with Chloramphenicol. Then, we prepared the electrocompetent EC85 and EC88 cells for the galK gene inactivation. During the competent cell preparation, we added 0.2% arabinose as an inducer for Cas system, and culture cells using 42 degree 15 min to induce the lambda red system before chilling the cells (the step starting the competent cell preparation). Then, we transformed the four editing plasmids into EC85 and EC88 using electroporation, and cultured the cells on the MacConkey Agar with 1% galactose.

The colonies observed as white color were the strains with the galK gene inactivation. The colonies observed as red color were the wild-type strains (Fig. 1). The editing efficiency was calculated using the formula as follows:

We compared the editing efficiency using different editing plasmid for the galK gene inactivation. When using the editing plasmid EC21, the editing efficiency in EC85 and EC88 are both lowest compared to other editing plasmids (Fig. 2). Thus, we use the plasmid EC21 as the template for the design and construction of the third plasmid of strainer system.

2. Construction and validation of the “strainer”

We ordered the second plasmid harboring the sacB gene with Trc inducible promoter. Then, we prepared the electrocompetent EC85 and EC88 cells and transformed the second plasmid into EC85 and EC88. We also ordered four DNA fragments contained the same spacer targeting on the second plasmid with the promoter of lexA, sula, umuD and recA genes. Then, we constructed these new editing plasmids by Gibson assembly using EC21 as template.

We firstly prepared the electrocompetent cells for the galK gene inactivation using the strain EC85 with sacB plasmid. During the competent cell preparation, we added 0.2% arabinose as an inducer for Cas system, and culture cells using 42 degree 15 min to induce the lambda red system before chilling the cells (the step starting the competent cell preparation). Then, we transformed the new editing plasmid (LexA) with the lexA promoter into the strain EC85 with sacB plasmid using electroporation, and cultured the cells on the MacConkey Agar with 1% galactose and different concentration of sucrose.

We firstly tested the new editing plasmid LexA using lexA gene promoter in EC85 to validate the “strainer” method. At a 0.01% sucrose concentration, the highest CFU/μg and the highest editing efficiency was found in the experiment. The editing efficiency using LexA plasmid is 25% higher than the control without “strainer” system. However, the CFU/μg decreased significantly compared to the control group that did not use “strainer”. In addition, we used the 0.01% sucrose to tested another three new editing plasmids. All of them showed that the editing efficiency is 25% higher than the control without “strainer” system (Fig. 3A).

Firstly, the “strainer” system significantly improves the gene editing efficiency compared to the control without “strainer” system. Secondly, the CFU/μg is still 96% lower than that of the control even using 0.01% sucrose (Fig. 3B). To this end, we should further improve CFU/μg by designing and modifying SacB protein to reduce its toxicity.

3. Protein modification

We followed the instruction of Mut Express II Fast Mutagenesis Kit V2 to design the primer. Then, we used the following PCR conditions to construct the SacB_S164T mutation. Thirdly, we followed the instruction of Mut Express II Fast Mutagenesis Kit V2 to construction the SacB_S164T mutant.

We prepared the electrocompetent cells for the galK gene inactivation using the strain EC85 and EC88. Then, we transformed the plasmid harboring SacB_S164T mutation into EC85 and EC88.

We prepared the electrocompetent cells for the galK gene inactivation using the strains EC85 and EC88 with SacB or SacB_S164T mutation. During the competent cell preparation, we added 0.2% arabinose as an inducer for Cas system, and culture cells using 42 degree 15 min to induce the lambda red system before chilling the cells (the step starting the competent cell preparation). Then, we transformed the editing plasmid (LexA) with the lexA promoter into the strain using electroporation, and cultured the cells on the MacConkey Agar with 1% galactose and different concentration of sucrose.

The results showed that the editing efficiency of SacB_S164T mutant (in EC85) is 25% higher than the control without “strainer” system. The CFU/μg of SacB_S164T mutant (in EC85) increased 3-fold compared to the original “strainer” system while still keep high editing efficiency (Fig. 4A).

We also used the same condition to test SacB_S164T mutant in EC88. The results showed that the editing efficiency using “strainer” system with SacB_S164T mutant is 4-fold higher than the control, although the CFU/μg of SacB_S164T mutant (in EC88) is still 71% lower than the control without “strainer” system (Fig. 4B).

We also used the same condition to test SacB_S164T mutant in EC88. However, the results showed that the toxicity of the modified SacB protein was low, and the screening effect served by the lower concentration of sucrose chosen at this point was no longer obvious, so we further optimized the sucrose concentration and found that testing the mutants at 0.1% concentration conditions, the CFU/μg using the "strainer" system with the SacB_S164T mutant was 2-fold higher than the control, although the SacB_ S164T mutant (in EC88) still had a lower CFU/μg than the control without the "strainer" system.

These results showed that our strainer system works as a good purification system to remove the unedited cells, and SacB_S164T mutant is less toxicity than the original SacB.

4. Insertion of the long gene fragment

We firstly constructed EC86 strain which is MG1655 with the plasmid harboring lambda red system and Cas9 protein. Then, we introduced the plasmid with sacB_S164T into the EC86. Thirdly, we introduced the gRNA_SS9 plasmid and the genome fragment of the IPA pathway into host strain using two methods. The strains after transformation were spread on the LB plates, and then using colony PCR to verify the efficiency of long gene fragment insertion.

We selected 30 colonies that were labeled and added to the reaction solution for colony PCR, and performed colony PCR under the reaction conditions shown in the table 2. Then we observed the running gel results, and a band at the 600 bp position was considered as a successful import.

Through the three repeated experiments of the long gene fragment insertion, 30 samples were detected by colony PCR each time, and the electrophoresis results were observed to calculate the proportion of the colonies that had inserted IPA pathway gene fragments. We found that the efficiency of successfully inserting long gene fragment using the CRISPR/Cas9 method was very low, and even there were cases where the introduction was not successful at all. In contrast, the use of the “strainer” method effectively improved the editing efficiency of the long gene fragment insertion, so that the insertion efficiency is about 30%. (Fig. 5)

5. Fermentation for IPA production

We added 24 mL of SD-8 medium, 1.25 mL of 400 g/L glucose, 20 μL of trace elements, and antibiotics to each shake flask (10 μL was taken to measure pH before inoculation to ensure that the pH range was around 6.0 before inoculation) in the reagent preparation phase of the shake flask fermentations. SD-8 medium (NH4Cl, 7.0g/liter; KH2PO4, 7.5g/liter; Na2HPO4, 7.5g/liter; K2SO4, 0.85g/liter; MgSO4·7H2O, 0.17g/liter; trace elements, 0.8 mL/liter; yeast extract; 10g/liter) containing 2% glucose was used for fermentations. The trace element solution contained the following (in grams per liter of 5 M HCl): FeSO4·7H2O, 40.0; MnSO4· H2O, 10.0; Al2(SO4)3, 28.3; CoCl2·6H2O, 4.0; ZnSO4·7H2O, 2.0; Na2MoO4·2H2O, 2.0; CuCl2·2H2O, 1.0; and H3BO4, 0.5. For antibiotic selection, the concentrations of antibiotics were 100 μg/mL (Ampicillin) and 34 μg/mL (Chloramphenicol). We took samples by shaking a dozen times after inoculation, centrifuged and kept the supernatant in the freezer for HPLC detection, measured pH, measured sugar concentration and recorded data. The shake flasks were incubated at 37°C and 200 rpm (after 12 h incubation, the speed could be slightly lowered at 180 rpm).

We found that the pH of EC65 decreased significantly after 12 h, so we added a small amount of 5M NaOH to keep the pH of the culture solution at about 6.0; when the result of our sugar measurement was less than 10 g/L, we added glucose to make the concentration of glucose in the shake flasks to around 20 g/L, and then shaked the flask for a dozen times after the sugar was added. Take the sample and store it in the freezer.

We stored the samples taken during the fermentation process in the freezer. After completing the 48 h fermentation process, we used HPLC to detect glucose and IPA concentrations, and finally calculated the IPA yield and productivity of the EC61 and EC65 fermentations.

We could see that the isopropanol titer of EC65 was 3-fold higher than that of EC61 from the result of HPLC (Fig. 6). In the process of strain modification for IPA production, the “strainer” method could significantly accelerate the construction speed of the strains.