Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature

Abstract

BackgroundIn 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 α-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.ResultsP. 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-β-lactamase chimera.ConclusionHere 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 α-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.

PssA is required for α-amylase secretion in Antarctic Pseudoalteromonas haloplanktis

“…This secretion system is not involved in recombinant a-amylase secretion in P. haloplanktis TAC125 (Parrilli et al, 2008b). Indeed, a P. haloplanktis TAC125 mutant strain in which the T2SS was knocked out was able to specifically secrete the cold-adapted a-amylase like the wild-type strain (Parrilli et al, 2008b), thus suggesting the occurrence of an as yet uncharacterized secretion pathway.…”

“…P. haloplanktis TAC125 wild-type and P. haloplanktis TAC125-DpssA mutant strains were grown at 15 uC in standard conditions and the zymographic assay was performed as previously reported (Parrilli et al, 2008b).…”

“…Secreted proteins provide the means by which micro-organisms interact with and modify their surrounding environment, also a crucial aspect necessary for survival in hostile extreme habitats, such as Antarctica. Although several cold-adapted exoenzymes have been characterized, the small amount of information available on the mechanisms responsible for their extracellular addressing has come from our laboratory (Cusano et al, 2006a, b;Parrilli et al, 2008bParrilli et al, , 2009Tutino et al, 2001). The development of a cold-adapted expression system (Parrilli et al, 2008a) paved the way to investigate the cellular machineries devoted to protein secretion in Antarctic bacterial hosts.…”

“…a-Amylase secretion was studied in P. haloplanktis TAC125. Several reasons prompted us to use this bacterium as recombinant host: (i) it is the first Antarctic micro-organism whose genome has been sequenced and annotated (Médigue et al, 2005); (ii) in this bacterium, recombinant a-amylase secretion occurs in a way similar to that in the source strain (Tutino et al, 2001(Tutino et al, , 2002; (iii) P. haloplanktis TAC125 is devoid of endogenous a-amylase activity (Tutino et al, 2001); and (iv) the cold-adapted a-amylase was successfully used as secretion carrier in a cold gene-expression system for secretion of heterologous proteins in P. haloplanktis TAC125 (Cusano et al, 2006b;Parrilli et al, 2008b).…”

Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories: production of human alpha-galactosidase A

“…In addition, as was established for E. 75 coli expression system, mutation of genes coding for proteases greatly reduces proteolysis of the recombinant 76 protein (Parrilli et al, 2008). The system has demonstrated to be especially useful in improving protein 77 solubility in relation to the widely used E. coli expression system and gives higher protein yield for secreted 78 proteins (Cusano et al, 2006;Giuliani et al, 2014;Vigentini et al, 2006).…”