ETD system

Electronic theses and dissertations repository

 

Tesi etd-11192019-205952


Thesis type
Tesi di laurea magistrale
Author
POMA, MELISSA
URN
etd-11192019-205952
Title
Establishment of the reductive glycine pathway in Vibrio natriegens
Struttura
SCIENZE AGRARIE, ALIMENTARI E AGRO-AMBIENTALI
Corso di studi
BIOTECNOLOGIE VEGETALI E MICROBICHE
Supervisors
relatore Prof. Forzan, Mario
relatore Dott. Wenk, Sebastian
controrelatore Prof. Bernardi, Rodolfo
Parole chiave
  • Vibrio natriegens
  • formate
  • reductive glycine pathway
  • bioeconomy
Data inizio appello
09/12/2019;
Consultabilità
Secretata d'ufficio
Data di rilascio
09/12/2089
Riassunto analitico
The current production of value-added chemicals mainly relies on depletable forms of energy and carbon, such as fossil carbon fuels, and simple sugars, the production of which undermines food and biodiversity security. In such scenario, formate has been proposed as a promising renewable feedstock, that could be converted into value-added chemicals through microbial assimilation and conversion. Formate could be an alternative for depletable feedstocks, since it can be produced by electrochemical reduction of CO2, which could be considered an “unlimited” resource. Hence, on an industrial scale, formate could be use as sole carbon source to feed microorganisms for the production of value-added chemicals.
Natural and synthetic formate-assimilation pathways have been taken into account to be engineered in host industrial microorganisms. Since, natural formate-assimilation pathways have either ATP inefficiency or a high metabolic complexity, an effective way to realize formatotrophic growth of model organisms would be the establishment of a synthetic pathway. To this purpose, the reductive glycine pathway has been suggested as a promising metabolic route for formate assimilation in model microbes, due to its ATP-efficiency and its metabolic simplicity.
In this thesis, it was studied whether the emerging model organism Vibrio natriegens could serve as a biotechnological platform for the establishment of the reductive glycine pathway. To establish the reductive glycine pathway in V. natriegens, firstly, a serine auxotroph strain was created,. This strain was then used to test for synthetic serine production from formate via the reductive glycine pathway. To this end, formate assimilation enzymes from Methylobacterium extorquens that assimilate formate into 5,10-methylene-THF were expressed on a plasmid. However plasmid overexpression of these genes combined with native expression of the glycine cleavage system did not recover the growth of the auxotroph strain. Hence, we assumed that the overexpression of the endogenous glycine cleavage system might be crucial for assimilation of formate and CO2 into glycine and serine. Therefore, we attempted to overexpress the glycine cleavage system on a second plasmid in the strain expressing the M. extorquens genes, but we did not manage to obtain a strain carrying both plasmids. In parallel, different synthetic promoters combined with different ribosome binding sites were tested in V. natriegens to later perform a promoter exchange aimed at overexpress the glycine cleavage system in the genome.
File