An improved integrative GFP-based vector for genetic engineering of Parageobacillus thermoglucosidasius facilitates the identification of a key sporulation regulator.

Autor: Millgaard M; The Novo Nordisk Foundation Center for Biosustainability, Building 220, Kemitorvet, 2800, Kgs. Lyngby, Denmark., Bidart GN; The Novo Nordisk Foundation Center for Biosustainability, Building 220, Kemitorvet, 2800, Kgs. Lyngby, Denmark., Pogrebnyakov I; The Novo Nordisk Foundation Center for Biosustainability, Building 220, Kemitorvet, 2800, Kgs. Lyngby, Denmark., Nielsen AT; The Novo Nordisk Foundation Center for Biosustainability, Building 220, Kemitorvet, 2800, Kgs. Lyngby, Denmark. atn@biosustain.dtu.dk., Welner DH; The Novo Nordisk Foundation Center for Biosustainability, Building 220, Kemitorvet, 2800, Kgs. Lyngby, Denmark. diwel@biosustain.dtu.dk.
Jazyk: angličtina
Zdroj: AMB Express [AMB Express] 2023 May 08; Vol. 13 (1), pp. 44. Date of Electronic Publication: 2023 May 08.
DOI: 10.1186/s13568-023-01544-9
Abstrakt: Parageobacillus thermoglucosidasius is a thermophilic Gram-positive bacterium, which is a promising host organism for sustainable bio-based production processes. However, to take full advantage of the potential of P. thermoglucosidasius, more efficient tools for genetic engineering are required. The present study describes an improved shuttle vector, which speeds up recombination-based genomic modification by incorporating a thermostable sfGFP variant into the vector backbone. This additional selection marker allows for easier identification of recombinants, thereby removing the need for several culturing steps. The novel GFP-based shuttle is therefore capable of facilitating faster metabolic engineering of P. thermoglucosidasius through genomic deletion, integration, or exchange. To demonstrate the efficiency of the new system, the GFP-based vector was utilised for deletion of the spo0A gene in P. thermoglucosidasius DSM2542. This gene is known to be a key regulator of sporulation in Bacillus subtilis, and it was therefore hypothesised that the deletion of spo0A in P. thermoglucosiadius would produce an analogous sporulation-inhibited phenotype. Subsequent analyses of cell morphology and culture heat resistance suggests that the P. thermoglucosidasius ∆spo0A strain is sporulation-deficient. This strain may be an excellent starting point for future cell factory engineering of P. thermoglucosidasius, as the formation of endospores is normally not a desired trait in large-scale production.
(© 2023. The Author(s).)
Databáze: MEDLINE
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