- Poster Presentation
- Open Access
Cell engineering of Pseudoalteromonas haloplanktis TAC125: construction of a mutant strain with reduced exo-proteolytic activity
© Parrilli et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Suicide Vector
- Intergeneric Conjugation
- Single Recombination Event
- Minimum Solid Medium
- Phenylalanine Analog
We have already shown that using cold-adapted bacteria as host vectors, some "intractable" proteins can be efficiently produced at temperature as low as 4°C [1, 2]. Furthermore, we set up a "cold" gene-expression system implemented for the secretion of recombinant proteins in the Antarctic Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 (Ph TAC125). Such a system could effectively conjugate the positive effect of low temperature on the recombinant product solubility with the obvious advantages linked to extra-cellular protein targeting. This novel system makes use of the psychrophilic α-amylase from Ph TAB23  as secretion carrier. Several chimerical proteins were produced and used to test the versatility and efficiency of the novel secretion system. All the chimerical proteins were efficiently produced and secreted (Cusano AM, Ph. D thesis 2005 Università di Napoli "Federico II"). However, bacteria belonging to Pseudoalteromonas genus are reported to secrete a wide range of exo-proteins, especially proteases. This feature could hamper both applicability and efficiency of the cold-adapted secretion system, due to the possible recombinant product degradation.
The Ph TAC125 genome sequence  was recently determined. The in silico genome analysis highlighted the presence of a putative Type II secretion system (T2SS), while the extra-cellular targeting of the cold α-amylase depends on a still uncharacterized secretion pathway .
Construction of the Ph TAC125 suicide vector
Construction of a Ph TAC125 gsp- [ΔgspCN] strain
Phenotypic characterization of Ph TAC125 gsp- strain
We report here a cell engineering approach to the construction of a Ph TAC125 strain with reduced exo-protease activity. By applying a gene-placements strategy, we obtained a mutant strain in which the gene cluster encoding the T2SS was almost totally deleted. While the growth behavior and some physiological features of the gsp- mutant are indistinguishable from the wild type ones, the deleted strain displays a remarkable reduction in the protease content in the culture supernatant. This aspect makes the Ph TAC125gsp- mutant a promising host for the recombinant secretion into the host extra-cellular medium of proteins with biotechnological potential.
This work was supported by grants from Ministero dell'Università e della Ricerca Scientifica (Progetti di Rilevante Interesse Nazionale 2003; FIRB 2001), of Programma Nazionale di Ricerche in Antartide 2004 and of Regione Campania L.R. 05/03. Support from the National Center of Excellence in Molecular Medicine (MIUR – Rome) and from the Regional Center of Competence (CRdC ATIBB, Regione Campania – Naples) is gratefully acknowledged.
- Duilio A, Tutino ML, Marino G: Recombinant protein production in Antarctic Gram negativebacteria. Methods Mol Biol. 2004, 267: 225-237.Google Scholar
- Duilio A, Marino G, Mele A, Sannia G, Tutino ML: Ufficio Italiano Brevetti e Marchi n. RM2003/A000155.Google Scholar
- Tutino ML, Duilio A, Parrilli E, Remaut E, Sannia G, Marino G: A novel replication element from an Antarctic plasmid as a tool for the expression of proteins at low temperature. Extremophiles. 2001, 5: 257-264. 10.1007/s007920100203.View ArticleGoogle Scholar
- Médigue C, Krin E, Pascal G, Barbe V, et al.: Coping with cold: the genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. Genome Research. 2005, 15: 1325-35. 10.1101/gr.4126905.View ArticleGoogle Scholar
- Kast P: pKSS – a second-generation general purpose cloning vector for efficient positive selection of recombinant clones. Gene. 1994, 138: 109-14. 10.1016/0378-1119(94)90790-0.View ArticleGoogle Scholar
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