Part:BBa_K1462230

Gal1+Crt+cyc1

Characterization by 2022 iGEM Team East_China

pLY15-opt

Profile

Name: pLY15-opt

Base Pairs: 1227 bp

Origin: pIB184-vector

Properties: A protein expression plasmid

Usage and Biology

This composite part is the recombinant plasmid constructed by Pcrt-crt-ter-hbd-Pthl-thl-opt fusion DNA fragment (BBa_K4515012) and pIB184-vector (BBa_K4515010) [1-6]. This plasmid could be transferred into Lactobacillus Brevis ATCC367 to produce N-butanol (Figure 1).

Figure 1. The metabolic pathway of N-butanol synthesis in L. Brevis ATCC367.

Experimental approach

1. Construct the pLY15-opt plasmid

To build the plasmid, firstly amplified the thlA, crt, hbd, and ter genes fragments from the Clostridium acetylbutyrate 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 transforming them into Lactobacillus Brevis ATCC367 competent cells.

Proof of function

1. Measure the growth curve of Streptococcus Brevis transformants

The constructed plasmid (containing 4 codon-optimized genes) was transformed into the Lactobacillus Brevis ATCC367 by electroporation method and incubated at 37℃ for 24-48 hours. After identifying the successfully transformed Lactobacillus Brevis strain, we inoculated it in the MRS medium, incubated it in the anaerobic chamber, and measured the growth rate (Figure 2).

Figure 2. After the butanol expression plasmid pLY15-opt was transferred into L. Brevis, the growth curve of the strain was measured at different times (48h, 69h, 95h, and 159h).

2. Functional test

To confirm if the Pcrt-crt-ter-hbd-Pthl-thl-opt system worked well in the host strain L. Brevis ATCC367, we also measured the yield of N-butanol through gas chromatography. As shown in Figure 3, the yield of N-butanol is increasing with an increased time of fermenting.

Figure 3. After pLY15-opt was transformed into L. Brevis, N-butanol production of pLY15-opt strain was measured at different times (48h, 69h, 95h, and 159h).

In Figure 2 and Figure 3, we can find that L. Brevis ATCC367 behaves well in N-butanol tolerance and could be used for N-butanol production in the future.

Improvement of an existing part

Our composite part BBa_K4515014 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. And our functional test 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.

References

1.Hickman AB, Dyda F. The casposon-encoded Cas1 protein from Aciduliprofundum boonei is a DNA integrase that generates target site duplications. Nucleic Acids Res. 2015 Dec 15;43(22):10576-87. doi: 10.1093/nar/gkv1180. PMID: 26573596

2.Krupovic M, Shmakov S, Makarova KS, Forterre P, Koonin EV. Recent Mobility of Casposons, Self-Synthesizing Transposons at the Origin of the CRISPR-Cas Immunity. Genome Biol Evol. 2016 Jan 13;8(2):375-86. doi:10.1093/gbe/evw006. PMID: 26764427; PMCID: PMC4779613.

3.Béguin P, Charpin N, Koonin EV, Forterre P, Krupovic M. Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems. Nucleic Acids Res. 2016 Dec 1;44(21):10367-10376. doi: 10.1093/nar/gkw821. PMID: 27655632; PMCID: PMC5137440.

4.Krupovic M, Béguin P, Koonin EV. Casposons: mobile genetic elements that gave rise to the CRISPR-Cas adaptation machinery. Curr Opin Microbiol. 2017 Aug; 38:36-43. doi: 10.1016/j.mib.2017.04.004. PMID: 28472712; PMCID: PMC5665730.

5.Béguin P, Chekli Y, Sezonov G, Forterre P, Krupovic M. Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei. Nucleic Acids Res. 2019 Jul 9;47(12):6386-6395.doi:10.1093/nar/gkz447.PMID:31114911; PMCID: PMC6614799.

6.Wang X, Yuan Q, Zhang W, Ji S, Lv Y, Ren K, Lu M, Xiao Y. Sequence specific integration by the family 1 casposase from Candidatus Nitrosopumilus koreensis AR1. Nucleic Acids Res. 2021 Sep 27;49(17):9938-9952. doi: 10.1093/nar/gkab725. PMID: 34428286; PMCID: PMC8464041.

The n-butanol pathway in the cytoplasm is constructed as a control group to identify the effectiveness of the pathway directed to the mitochondria. Crt,originated from Clostridium beijerinckii,expresses crotonase, which converts HbCoA to CrCoA, the third step of the n-butanol pathway.This part is constructed in pUC57 individually at the beginning and it is assembled with Hbd(BBa_K1462220),Erg10 (BBa_K1462210) in plasmid 181 ,which is leucine auxotroph.

Figure 1. The n-butanol biosynthetic pathway. Enzymes are from these organisms: Erg10, S. cerevisiae; Hbd, Crt, AdhE2, Clostridium beijerinckii; Ccr, Streptomyces collinus.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 150
  
• 1000
  COMPATIBLE WITH RFC[1000]