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Synthetic Protein Scaffolds for Enhanced Carotenoid Biosynthesis
Başlık:
Synthetic Protein Scaffolds for Enhanced Carotenoid Biosynthesis
Yazar:
Qu, Jiale, author.
ISBN:
9780438147874
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (130 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-11(E), Section: B.
Advisors: Jiang Xia.
Özet:
Biosynthesis of chemical compounds in living organisms often requires long cascade reactions involving multiple enzymes, in which the local concentration and the transportation of intermediates are of paramount importance. Nature develops the strategy of forming protein complex with delicate structure to achieve substrate channeling effect, a process where intermediates are directly transported from the active site of one enzyme to another without being diffused into the bulk phase, to regulate the biosynthesis pathway.
Inspired by these, we designed and constructed covalent enzyme complexes based on the synthetic protein scaffold and applied it into the carotenoid biosynthesis pathway. SpyCatcher and SpyTag, originated from a split bacteria adhesin protein, retain their parental activity to reconstitute an isopeptide bond between them when mixed in solution. These two parts exhibit scaffolding effect that are capable of bringing enzymes together through genetic fusion to form a larger protein complex. We made use of the SpyCatcher/SpyTag scaffold partners and another similar, yet orthogonal pair SnoopCatcher/SnoopTag to build stable covalent protein complex linking various enzymes firstly in vitro. In addition, as a proof of principle, we also engineered protein complexes consisting of a phosphatase and a protease to perform a dephosphorylation/proteolysis cascade reaction, where the substrate channeling effect was evaluated by fluorescence label dilution and iodoacetamide inhibition experiment.
We further employed the synthetic protein scaffold in the biosynthesis of lycopene and astaxanthin, two important carotenoids of great scientific and pharmaceutical interest, inside bacterial cells. The upstream mevalonate pathway, which is crucial in providing enough precursors for the production of carotenoids, is however constrained by a rate-limiting enzyme. A protein complex tethering three cascade enzymes together through the synthetic Catcher/Tag scaffold smoothed the metabolic flux of the pathway by precisely controlled the enzymes arrangement and stoichiometry. Spatial closeness thereby overcomes the rate-limiting step. Utilization of the protein scaffold in vivo enhanced the lycopene and astaxanthin production yield by five folds and two folds respectively, proving the generalizability of this method. Characterization of the protein complex through TEM and cryo-EM revealed an interesting globular structure with diameter around 25nm with a hollow interior. Also another scaffold structure was able to alter the products composition in astaxanthin biosynthesis process, providing comprehensive control over different aspects of the carotenoid biosynthesis pathway.
Taken together, this work explores the use of site-specific covalent protein reaction to assembly multienzyme complexes. It demonstrates the first construction of a protein nanostructure mimicking pyruvate dehydrogenase complex with a superior catalytic function in vivo.
Notlar:
School code: 1307
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(697033.1) | 697033-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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