Engineering
Introduction
N-butanol, an important chemical raw material, is expected to become one of the new generations of biofuels. At present, the domestic industrial synthesis of N-butanol mainly adopts the low-pressure carbonyl synthesis method, from the production process, propylene, CO, H2, and carbonyl are the main production materials. However, this production method relies on non-renewable petroleum products as essential raw materials, which is not friendly to the environment. Therefore, it is necessary to design an environmentally friendly production method for N-butanol to meet the demand of environmental protection.
It has been found that N-butanol can be synthesized naturally in Clostridium, but the tolerance of Clostridium bacteria is not good enough for large-scale production. Recently, Lactobacillus Brevis with better N-butanol tolerance has been isolated by researchers. Therefore, this project develops an engineering N-butanol-producing bacteria to improve the yield of N-butanol, meet the needs of industrial production, and lay a foundation for subsequent improvement.
How we design our plasmid
The N-butanol pathway we developed according to the pathway in Lactobacillus Brevis ATCC824. To achieve this, we designed the DNA sequences of thlA, crt, hbd, and ter gene into the ApaI and BglII sites of the pIB184 vector (Figure 1.), and transferred the recombinant plasmid into Streptococcus Brevis ATCC367 for fermentation.
Figure 1. The map of recombinant plasmid
How we build our plasmid
To build the plasmid, firstly amplified the thlA, crt, hbd, and ter genes fragments from the Lactobacillus Brevis ATCC824 genomic DNA (Figure 2). The second step was to obtain the linear carrier. The third step was to ligate the genes and linearized vector and transfer the ligation product into E. coli DH5α competent.
Figure 2. The PCR gel electrophoresis picture of gene fragments.
Line M: DNA marker; Line 1: The gene fragment of gene crt ; Line 2: The gene fragment of gene hbd ; Line 3: The gene fragment of gene ter; Line 4: The gene fragment of gene thlA.

We send the constructed recombinant plasmid to a sequencing company for sequencing, and the results proved that the plasmid was successfully constructed (Figure 3). And the last step was extracting the recombinant plasmid from E. coli DH5α and transferring it into Streptococcus Brevis ATCC367, so that can be used to produce N-butanol.
Figure 3. The results of the sequencing data mapped to the plasmids
How we produce N-butanol
a) Identification of transformed Streptococcus Brevis ATCC367
The constructed plasmid (containing 4 codon-optimized genes) was transformed into the Streptococcus Brevis ATCC367 by electroporation method and incubated on MRS solid medium plate at 37℃ for 24-48 hours. Next, we used colony PCR to identify transformants (Figure 4). At this point, we got the engineered strain we wanted.
Figure 4. Verification of transformant Streptococcus Brevis ATCC367
Line M: DNA marker; Line 1: Plasmid positive control; Line 2-7: Different transformants; Line 8: negative control; Line9: blank control without any DNA template.
b) Measure the yield of N-butanol
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. 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 5). As shown in the result, we successfully detected a higher yield of N-butanol. This result is really encouraging.
Figure 5. 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)
How we learn from our projectHow we learn from our project
We have already collected the figures from our experiments. We developed an N-butanol-producing pathway in Streptococcus Brevis ATCC367 and successfully increase the yield of N-butanol. With the development of society, people pay more and more attention to the impact of environmental problems while improving their own lives. Nowadays the greenhouse effect is serious, one of the important factors is the overuse of fossil energy.
Because N-butanol is environmentally friendly energy, we believe that if we can use N-butanol in the future and reduce our dependence on oil, it will relieve the pressure of resource shortage and make our environment better.