Difference between revisions of "Part:BBa K5175033:Design"

Revision as of 11:51, 2 October 2024

T7 promoter-lac operator-pelB-FAST-PETase-G4S-MHETase-Terminator-T7 promoter-fucO-aldA-T7 termina7 t

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 fromIdeonella 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.