Results
I. Construction of double knockout plasmid experiment
1. PCR amplification of codon-optimized gene thlA, hbd, crt, and ter.
We designed the program by thlA, crt, hbd, and ter genes into ApaI and BglII sites of the pIB184 vector. In order to build our plasmids, we firstly amplified the gene fragments from the Lactobacillus Brevis ATCC824 genomic DNA (Figure 1), double-enzyme digestion, and ligase to pIB184 carrier.
Figure 1. Fragments of crt, hbd, ter, and thlA amplicons. A. crt gene, B. hbd gene, C. ter gene, D. thlA gene.
In Figure 1, a clear and single DNA band for each gene was shown, indicating that the genes were successfully amplified by PCR.
Then, the PCR products were purified by gel extraction, four gene fragments were ligated to the pIB184 carrier by the one-step cloning method. Finally, recombinant plasmid pLY15-opt was transformed into E. coli DH5α competent cells and coat on LB plate containing Erythromycin and incubated.
2. Transform plasmid pLY15-opt into Streptococcus Brevis ATCC367.
Streptococcus Brevis was cultured on a solid MRS plate, then a single colony was taken and cultured overnight in the MRS medium (Figure 2A), and remained 100 μL was mixed with an equal volume of 50% glycerin and stored at -80℃. Then the constructed plasmid (containing 4 codon-optimized genes) was transformed into the Streptococcus Brevis ATCC367 by electroporation method and incubated at 37℃ for 24-48 hours. We selected 7 monoclonal colonies, 1 was positive control, 8 was negative control (without any gene fragment or vector), 9 was blank control without any template (only enzyme, without 4 gene fragments), and 4 failed (Figure 2B).Streptococcus Brevis was cultured on a solid MRS plate, then a single colony was taken and cultured overnight in the MRS medium (Figure 2A), and remained 100 μL was mixed with an equal volume of 50% glycerin and stored at -80℃. Then the constructed plasmid (containing 4 codon-optimized genes) was transformed into the Streptococcus Brevis ATCC367 by electroporation method and incubated at 37℃ for 24-48 hours. We selected 7 monoclonal colonies, 1 was positive control, 8 was negative control (without any gene fragment or vector), 9 was blank control without any template (only enzyme, without 4 gene fragments), and 4 failed (Figure 2B).
Figure 2. A. Streptococcus Brevis Monoclonal colony on MRS plate. B.1: colony PCR verification of correct transformed colony. 1. Plasmid positive control, 2-7: Different transformants, 8: negative control, 9: blank control without any DNA template.
3. Growth curve measurement and Functional test
The identified single colony was cultured in MRS overnight, and antibiotics were added and put into the anaerobic chamber (this step can convert glucose into N-butanol) overnight, and we also measured its growth curve (Figure 3). N-Butanol was detected by gas chromatography. Finally, we analyzed all data using ultra-performance liquid chromatography, UPCL, and Q Exactive Orbitrap Mass spectrometer to measure the yield of N-butanol (Figure 4).
Figure 3. After the butanol expression plasmid pLY15-opt was transferred into Streptococcus Brevis, the growth curve of the strain was measured at different times (48h, 69h, 95h, and 159h)
Figure 4. After pLY15-opt was transformed into Streptococcus Brevis, N-butanol production of ply15-opt strain was measured at different times (48h, 69h, 95h, and 159h)
As shown in Figure3, after culturing for 48h, the growth of the strain reached the stable phase, and after being cultured for 95h, it turned into the decay phase, which means we improved the strain’s tolerance of N-butanol.
In Figure4, with the growth of the Streptococcus Brevis, the yield of N-butanol is also increasing. Although this yield of N-butanol is still needed to be improved, it can be inferred from the above result that with the engineered strain, we can finally optimize the workflow and produce enough N-butanol.In Figure4, with the growth of the Streptococcus Brevis, the yield of N-butanol is also increasing. Although this yield of N-butanol is still needed to be improved, it can be inferred from the above result that with the engineered strain, we can finally optimize the workflow and produce enough N-butanol.