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_K1462040, this is a biological part submitted by iGEM14_SCUT in 2014, with only DNA sequence information and simple text description information. Because the tolerance of Clostridium bacteria to N-butanol is not good enough for large-scale production. Based on this problem, 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. Our team carried out a comprehensive characterization of this part in the laboratory, adding data from fermentation testing to dedicate its function of producing N-butanol. This information can be a good reference for future iGEM teams working on improving the yield of N-butanol.
In addition, through literature research, we developed an N-butanol biosynthesis pathway, Pcrt-crt-ter-hbd-Pthl-thl-opt and constructed these genes in the plasmid. What's more, 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. We upload the DNA sequence information and basic introduction information in the registry of standard biological parts to provide more choices of N-butanol-producing for future iGEM teams.

Gene crt encodes 3-Hydroxybutyryl-CoA dehydratase, which converts 3-Hydroxybutyryl-CoA to Crotonyl-CoA, the third step of the N-butanol pathway.

Gene ctr was promoted by the Pcrt promoter and other related genes, thlA, hbd, and ter, were all amplified from the Lactobacillus Brevis ATCC824 genomic DNA through PCR. The DNA sequences of the Pcrt-crt-ter-hbd-Pthl-thl-opt was inserted into the ApaI and BglII sites of the pIB184 vector, respectively. The certificate of recombinant plasmid sequencing results is as Figure 1.

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 2, the yield of N-butanol is increasing with an increased time of fermenting.

Genes thlA, crt, hbd, and ter, play important roles in the N-butanol biosynthesis pathway. Those genes were codon-optimized. 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. In this part, genes crt, ter and hbd were promoted by Pcrt promoter, gene thl was promoted by Pthl promoter, and these DNA fragments were ligated in order into pIB184 vector.

pIB184-vector is an E. coli - Streptococci shuttle plasmid for gene expression in streptococci with P23 promoter. This plasmid is a low-copy plasmid and Erythromycin resistance can be used to screen the correct colony in bacteria. This vector contains MCS-A. The backbone of this vector is based on pOri23.

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). This plasmid could be transferred into Streptococcus Brevis ATCC367 to produce N-butanol.
Above all, we look forward to the future iGEM team making new additions, explorations, and explanations to our biological components.

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