title: Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature.
abstract: BACKGROUND
  In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in Pseudoalteromonas haloplanktis TAC125. This system makes use of the psychrophilic alpha-amylase from P. haloplanktis TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins. However, Pseudoalteromonales are reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The aim of this study is the construction of a P. haloplanktis TAC125 mutant strain with reduced extra-cellular proteolytic activity.
  
  
  RESULTS
  P. haloplanktis TAC125 culture medium resulted to contain multiple and heterogeneous proteases. Since the annotation of the Antarctic bacterium genome highlighted the presence of only one canonical secretion machinery, namely the Type II secretion pathway (T2SS), we have inactivated this secretion system by a gene insertion strategy. A mutant strain of P. haloplanktis TAC125 in which the gspE gene was knocked-out, actually displayed a remarkable reduction of the extra-cellular protease secretion. Quite interestingly this strain still retained the ability to secrete the psychrophilic amylase as efficiently as the wild type. Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-beta-lactamase chimera.
  
  
  CONCLUSIONS
  Here we report a cell engineering approach to the construction of a P. haloplanktis TAC125 strain with reduced extra-cellular protease activity. The improved strain is able to secrete the psychrophilic alpha-amylase (the carrier of our recombinant secretion system), while it displays a significant reduction of protease content in the culture medium. These features make the gspE mutant an improved host with a remarkable biotechnological potential in recombinant protein secretion at low temperature. Moreover this work demonstrates that P. haloplanktis TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach. To the best of our knowledge, this is the first described example of a strain improvement strategy applied to an Antarctic bacterium.
authors.affiliation: Dipartimento di Chimica Organica e Biochimica, Università degli studi di Napoli Federico II - Complesso Universitario M,S, Angelo via Cinthia 4, 80126, Napoli Italia. tutino@unina.it.
authors.forename: Ermenegilda
authors.initials: E
authors.lastname: Parrilli
authors.forename: Daniela
authors.initials: D
authors.lastname: De Vizio
authors.forename: Claudia
authors.initials: C
authors.lastname: Cirulli
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Tutino