Part:BBa_K5175003
fucO
fucO is the gene for L-1,2-propanediol oxidoreductase, which is an iron-dependent group III dehydrogenase and can convert Ethylene glycol(EG) to glycolaldehyde (GLA).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 167
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 167
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 167
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 167
Illegal AgeI site found at 883
Illegal AgeI site found at 1084 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The final products of PET degradation by the two-enzyme system are TPA and EG. However, wild-type E.coli cannot rapidly utilise these substances for various life activities.In order to increase the efficiency of E. coli in utilising the PET degradation products and to improve its viability, we overexpressed L-1,2-propanediol oxidoreductase and aldehyde dehydrogenase A.This modification was able to increase E.coli 's ability to efficiently utilise EG. We chose fucO as the gene for L-1,2-propanediol oxidoreductase and aldA as the gene for aldehyde dehydrogenase A. L-1,2-propanediol oxidoreductase is an iron-dependent group III dehydrogenase, and aldehyde dehydrogenase A is an enzyme with a relatively broad substrate specificity for small hydroxyaldehyde substrates. EG is first converted in E.coli to glycolaldehyde (GLA) by L-1,2 -propylene glycol oxidoreductase, which is subsequently converted to glycolic acid (GA) by aldehyde dehydrogenase A. GA can be metabolised by condensation with acetyl coenzyme A via the glyoxalate shunt to form malic acid. GA can also enter the metabolic pathway of H. coli by condensing with succinate via isocitrate lyase (encoded by the aceA gene) , forming isocitrate.
Molecular cloning
Initially, we transformed the company-synthesized plasmids containing designed sequences into E. coli DH5α for amplification, allowing us to obtain a sufficient quantity of plasmid DNA for subsequent experiments. Following this, colony PCR was performed to confirm successful transformation, and the required plasmids were subsequently extracted for further experimentation. Subsequently, we employed PCR to obtain the target fragments, which were then integrated into the requisite plasmids for our study.
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