Improvement
Overview
Our composite part BBa_K4515012 is the N-butanol pathway we used in Streptococcus Brevis ATCC367. It is
improved based on the existing part BBa_K1462230. In 2014, group iGEM14_SCUT designed a basic part BBa_K1462230,
Gal1+Crt+cyc1, and intended to produce N-butanol in Clostridium beijerinckii. However, the tolerance of
Clostridium bacteria to N-butanol is not good enough for large-scale production. Based on this problem, by reading
literature and consulting experts in related fields, we chose Streptococcus Brevis ATCC367, a lactobacillus with
better N-butanol tolerance that has been isolated by researchers, as our host strain in this project.
In order to prove the function of our new composite part
Pcrt-crt-ter-hbd-Pthl-thl-opt, we transferred the recombinant plasmid into Streptococcus Brevis ATCC367 to
establish an N-butanol-producing platform and measured the yield of N-butanol. Then, by detecting the growth curve
of Streptococcus Brevis ATCC367 transformants, it was further confirmed that Streptococcus Brevis ATCC367 has
better tolerance for N-butanol and could be used to produce N-butanol in factories in the future.
Introduction
Genes thlA, crt, hbd, and ter, play important roles in the N-butanol biosynthesis
pathway. Gene thlA is coding for acetyl-CoA acetyltransferase and converts Acetyl-CoA into Acetoacetyl-CoA in the
N-butanol biosynthesis pathway. Gene hbd, encodes β-Hydroxybutyryl-CoA dehydrogenase and converts Acetoacetyl-CoA
into 3-Hydroxybutyryl-CoA. Gene crt encodes 3-Hydroxybutyryl-CoA dehydratase, which converts 3-Hydroxybutyryl-CoA
to Crotonyl-CoA, the third step of the N-butanol pathway. According to the references, we constructed a
gene-optimized Pcrt-crt-ter-hbd-Pthl-thl-opt in one plasmid, transferred the recombinant plasmid into
Streptococcus Brevis ATCC367, and used this platform to produce N-butanol.
a) Construction of expression plasmids
We design a recombinant plasmid: The DNA sequences of the Pcrt-crt-ter-hbd-Pthl-thl-opt
was inserted into the ApaI and BglII sites of the pIB184 vector (Figure 1), respectively. The recombinant plasmid
was transferred into Streptococcus Brevis ATCC367.
Figure 1. The map of the recombinant plasmid
To build the plasmid, firstly amplified the thlA, crt, hbd, and ter genes fragments from the Lactobacillus Brevis
ATCC824 genomic DNA. 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.
We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 2). Thus, our plasmid was successfully constructed. And the last step was extracting the recombinant plasmids from E. coli DH5α and transferring them into Streptococcus Brevis ATCC367 competent cells, so that can be used to produce N-butanol.
We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 2). Thus, our plasmid was successfully constructed. And the last step was extracting the recombinant plasmids from E. coli DH5α and transferring them into Streptococcus Brevis ATCC367 competent cells, so that can be used to produce N-butanol.
Figure 2. The results of the sequencing data mapped to the plasmids
b) Measure the growth curve of Streptococcus Brevis transformants
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. After identifying the
successfully transformed Streptococcus Brevis strain, we inoculated it in the MRS medium, incubated it in the
anaerobic chamber, and measured the growth rate (Figure3).
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)
c) Functional test
To confirm if the Pcrt-crt-ter-hbd-Pthl-thl-opt system worked well in the host strain
Streptococcus Brevis ATCC367, we also measured the yield of N-butanol through gas chromatography. As shown in
Figure 4, the yield of N-butanol is increasing with an increased time of fermenting.
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)
In Figure 3 and Figure 4, we can find that Streptococcus Brevis ATCC367 behaves well in
N-butanol tolerance and could be used for N-butanol production in the future.