Part:BBa_K5175033:Design
T7 promoter-lac operator-pelB-FAST-PETase-G4S-MHETase-Terminator-T7 promoter-fucO-aldA-T7 termina7 t
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1645
Illegal PstI site found at 1988
Illegal PstI site found at 3117
Illegal PstI site found at 4865
Illegal PstI site found at 5578 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2820
Illegal NheI site found at 2876
Illegal NheI site found at 5636
Illegal PstI site found at 1645
Illegal PstI site found at 1988
Illegal PstI site found at 3117
Illegal PstI site found at 4865
Illegal PstI site found at 5578 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1528
Illegal BamHI site found at 4403
Illegal XhoI site found at 2786 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1645
Illegal PstI site found at 1988
Illegal PstI site found at 3117
Illegal PstI site found at 4865
Illegal PstI site found at 5578 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1645
Illegal PstI site found at 1988
Illegal PstI site found at 3117
Illegal PstI site found at 4865
Illegal PstI site found at 5578
Illegal NgoMIV site found at 166
Illegal NgoMIV site found at 1327
Illegal NgoMIV site found at 1715
Illegal NgoMIV site found at 2078
Illegal AgeI site found at 3833
Illegal AgeI site found at 4034
Illegal AgeI site found at 4675 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
It is a composite component consisting of the T7 promoter, lac operator, target genes PETase-MHETase, fucO, aldA. It is responsible for enabling E.coli to degrade PET and increasing E.coli 's ability to efficiently utilise EG.
FAST-PETase is a machine-learning obtained PETase with properties suitable for in situ PET degradation at mild temperatures and moderate pH conditions.However, the main product of PETase degradation of PET is MHET, and the MHET intermediate tends to bind tightly to PET degrading enzyme in a non-catalytic pose, which leads to the inhibition of PET degrading enzyme. Therefore, an efficient MHET hydrolase is needed to degrade the intermediate product in time to further depolymerise MHET into its monomers TPA and 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.
Source
PETase, MHETase are from Ideonella sakaiensis 201-F6. fucO, aldA are from E.coli.
=References
[1] LU H, DIAZ D J, CZARNECKI N J, et al. Machine learning-aided engineering of hydrolases for PET depolymerization [J]. Nature, 2022, 604(7907): 662-7.
[2] ZHANG J, WANG H, LUO Z, et al. Computational design of highly efficient thermostable MHET hydrolases and dual enzyme system for PET recycling [J]. Communications Biology, 2023, 6(1): 1135.
[3] ZHANG Y, HESS H. Toward Rational Design of High-efficiency Enzyme Cascades [J]. ACS Catalysis, 2017, 7(9): 6018-27.
[4] MONTELLA C, BELLSOLELL L, PéREZ-LUQUE R, et al. Crystal structure of an iron-dependent group III dehydrogenase that interconverts L-lactaldehyde and L-1,2-propanediol in Escherichia coli [J]. J Bacteriol, 2005, 187(14): 4957-66.
[5] ZHU Y, LIN E C. Loss of aldehyde dehydrogenase in an Escherichia coli mutant selected for growth on the rare sugar L-galactose [J]. J Bacteriol, 1987, 169(2): 785-9.