In recent years, cyanobacteria received great attention as heterologous systems for the biosynthesis of industrially relevant compounds, given their abilities to fix carbon dioxide and to harness energy from light. Being a model organism, Synechocystis sp. PCC6803 (hereafter referred as Synechocystis) has been largely employed and engineered for this purpose. The aim of this study is the development of a whole-cell system for biocatalysis, using Synechocystis as a production platform. Two different transgenic strains, previously produced in our laboratory, were characterised and used for further analysis: one of them harbours a gene encoding a Baeyer-Villiger monooxigenase from Cyanidioschyzon merolae (CmBVMO), the other one an ene-reductase from Chroococcidiopsis thermalis (CtOYE). A liter-scale protein purification protocol was optimised; enzymatic activities on several substrates were tested to assess whether the enzymes were correctly synthesised and folded, as previously observed in E. coli. Regarding CmBVMO, two substrates were used to perform an in vivo toxicity assay, aiming at correlating different growth phenotypes to bio-transformations occurring into the cells. A substrate-related and a product-related toxicity were observed, and two different mechanisms are suggested. A putative metabolic pathway, which uses one of the substrates as a building block and culminates with the production of a molecule with high added value was hypothesised. In a parallel work, the feasibility of targeting enzymes to the external surface of the cyanobacterial membrane by means of fusion constructs was investigated. PETase and MHETase were chosen as proofs of concept enzymes, to open up the possibility of developing a two-strain consortium for PET degradation, but no transgenic strains capable of growth on the selective medium were obtained. Further investigations should be conducted to validate the putative metabolic pathway by detecting the final product in Synechocystis, from one side, and to prove the possibility of surface-displaying enzymes, for the other one.
Recentemente i cianobatteri hanno suscitato una grande attenzione come sistemi eterologhi per la biosintesi di composti d’interesse industriale grazie alla loro abilità di fissare l’anidride carbonica e ricavare energia dalla luce. Trattandosi di un organismo modello, Synechocystis sp. PCC6803 (in seguito abbreviato in Synechocystis) è stato largamente impiegato ed ingegnerizzato per questo scopo. L’obiettivo di questo studio è lo sviluppo di un sistema whole-cell per la biocatalisi, utilizzando Synechocystis come piattaforma di produzione. Sono stati caratterizzati due diversi ceppi transgenici, precedentemente sviluppati nel nostro laboratorio, ed utilizzati per le successive analisi: uno ospita un gene codificante una Baeyer-Villiger monoossigenasi proveniente da Cyanidioschyzon merolae (CmBVMO), l’altro una ene-reduttasi proveniente da Chroococcidiopsis thermalis (CtOYE). È stato perfezionato un protocollo per la purificazione di proteine ricombinanti da colture nell’ordine del litro; sono state misurate attività enzimatiche nei confronti di diversi substrati per verificare che gli enzimi venissero propriamente sintetizzati e foldati, come precedentemente osservato in E. coli. Riguardo la CmBVMO, due substrati sono stati utilizzati per effettuare un test di tossicità in vivo, con l’obiettivo di correlare diversi fenotipi di crescita alle bio-trasformazioni che avvengono all’interno delle cellule. Sono stati osservati casi di tossicità da substrato e da prodotto, e vengono proposti due diversi meccanismi. È stata ipotizzata una via metabolica putativa la quale, a partire da uno dei substrati, porta alla produzione di una molecola ad alto valore aggiunto. In un progetto parallelo è stata investigata la possibilità di indirizzare enzimi sulla superficie esterna della membrana cianobatterica utilizzando costrutti di fusione. Gli enzimi PETasi e MHETasi sono stati scelti come prova per aprire la possibilità di sviluppare un consorzio per la degradazione del PET, ma non sono stati ottenuti ceppi transgenici capaci di crescere in terreno selettivo. Sarà necessario condurre ulteriori esperimenti, da un lato per validare la via metabolica putativa attraverso la rilevazione del prodotto finale in Synechocystis, dall’altro per provare la possibilità di esporre enzimi sulla superficie del batterio.
Engineering of Synechocystis sp. PCC 6803 as a photosynthetic cell-factory for biocatalysis
CONDOLUCI, GIULIO
2021/2022
Abstract
In recent years, cyanobacteria received great attention as heterologous systems for the biosynthesis of industrially relevant compounds, given their abilities to fix carbon dioxide and to harness energy from light. Being a model organism, Synechocystis sp. PCC6803 (hereafter referred as Synechocystis) has been largely employed and engineered for this purpose. The aim of this study is the development of a whole-cell system for biocatalysis, using Synechocystis as a production platform. Two different transgenic strains, previously produced in our laboratory, were characterised and used for further analysis: one of them harbours a gene encoding a Baeyer-Villiger monooxigenase from Cyanidioschyzon merolae (CmBVMO), the other one an ene-reductase from Chroococcidiopsis thermalis (CtOYE). A liter-scale protein purification protocol was optimised; enzymatic activities on several substrates were tested to assess whether the enzymes were correctly synthesised and folded, as previously observed in E. coli. Regarding CmBVMO, two substrates were used to perform an in vivo toxicity assay, aiming at correlating different growth phenotypes to bio-transformations occurring into the cells. A substrate-related and a product-related toxicity were observed, and two different mechanisms are suggested. A putative metabolic pathway, which uses one of the substrates as a building block and culminates with the production of a molecule with high added value was hypothesised. In a parallel work, the feasibility of targeting enzymes to the external surface of the cyanobacterial membrane by means of fusion constructs was investigated. PETase and MHETase were chosen as proofs of concept enzymes, to open up the possibility of developing a two-strain consortium for PET degradation, but no transgenic strains capable of growth on the selective medium were obtained. Further investigations should be conducted to validate the putative metabolic pathway by detecting the final product in Synechocystis, from one side, and to prove the possibility of surface-displaying enzymes, for the other one.The text of this website © Università degli studi di Padova. Full Text are published under a non-exclusive license. Metadata are under a CC0 License
https://hdl.handle.net/20.500.12608/32759