Difference between revisions of "Part:BBa K3866021"

Revision as of 10:48, 29 September 2021

ParaBAD:RBS-Superpart 1-double terminator

The production of butyrate by bacteria can be possible with a set of genes. This part includes the promoter ParaBAD with RBS(baba), the superpart 1(baba) and the double terminator(baba). The superpart 1 has only four of the nine genes that catalyze the formation of butyric acid. Those are PhaA, PhaB, Crt and Ter genes.

Fig.2:The alpha2 vector that includes the transcriptional unit of the Operon 1 with the ParaBAD promoter and a double terminator. The operon 1 consists of the PhaA, PhaB, Crt and Ter genes, responsible for the butyrate production.

Usage and Biology

First, PhaA and BktB are two β-ketothiolases that carry out the homo-fusion of two acetyl-CoA moieties into acetoacetyl-CoA. Then, the acetoacetyl-CoA is reduced to (S)-3-hydroxybutyryl-CoA (3-HB-CoA) by NADH-dependent Hbd or (R)-3-HB-CoA by NADPH-dependent PhaB. Both (R)-3-HB-CoA and (S)- 3-HB-CoA can be converted to (R)-3-HB and (S)-3-HB, respectively, by a CoA-removing enzyme, such as Pct, TesB, or Ptb/Buk. Alternatively, (R)-3-HB-CoA and (S)-3-HB-CoA can be converted to crotonyl-CoA by PhaJ and Crt, respectively. Crotonyl-CoA is then reduced to butyryl-CoA by NADH-dependent Ter, and subsequently converted to butyrate by one of the above CoA-removing enzymes.

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in alpha 2 vector BBa_K3505009 and has overhangs compatible for GoldenBraid cloning.

Sequence and Features

Source

Synthesized by Twist Biosciences.

References

• Miscevic D, Srirangan K, Kefale T, Abedi D, Moo-Young M, Chou CP. Production of cellulosic butyrate and 3-hydroxybutyrate in engineered Escherichia coli. Appl Microbiol Biotechnol. 2019 Jul;103(13):5215-5230. doi: 10.1007/s00253-019-09815-x. Epub 2019 May 2. PMID: 31049621.