Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering

Abstract

Our group has used the marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of “difficult” proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium’s intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY+. This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY+ was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression.

Modelling hCDKL5 Heterologous Expression in Bacteria

“…Human CDKL5 was recombinantly expressed as an N-terminally His-tagged engineered construct to allow for easy Western blot detection and quantification. Its gene was expressed under the control of an IPTG-regulatable promoter [ 33 ] cloned in a high-copy-number plasmid, named pB40_79C-CDKL5 (average copy number equal to 100, manuscript in preparation), in a mutant version of PhTAC125-KrPl LacY+ capable of fast IPTG internalisation [ 33 ]. hCDKL5 synthesis was induced in the late exponential phase with 5 mM IPTG at 15 °C in bacteria grown in GG medium [ 34 ] for 8 h. Total production of the target protein was estimated to be 5.2 mg/L of the culture by Western blot using a commercial His-tagged calibrator with a similar MW as hCDKL5.…”

“…The pB40_79C-CDKL5 plasmid was mobilised from E. coli S17-1(λpir) to KrPL LacY+ [ 33 ] through standard conjugation techniques [ 41 ]. E. coli S17-1(λpir)—a strain possessing mob and tra genes for plasmid mobilisation [ 42 ]—was routinely grown in LB (10 g/L of bacto-tryptone, 5 g/L of yeast extract, 10 g/L of NaCl) at 37 °C with the supplementation of 34 μg/mL of chloramphenicol, if needed, for plasmid selection.…”

Modelling hCDKL5 heterologous expression in bacteria

“…Human CDKL5 was recombinantly expressed as an N-terminally His-tagged engineered construct to allow for easy Western blot detection and quantification. Its gene was expressed under the control of an IPTG-regulatable promoter [33] cloned in a high-copy number plasmid, named pB40_79C-CDKL5 (average copy number equal to 100, manuscript in preparation) in a mutant version of PhTAC125 - KrPl LacY+ - capable of a fast IPTG internalization [33]. hCDKL5 synthesis was induced in late exponential phase with 5 mM IPTG at 15 °C in bacteria grown in the GG medium [34] for 8 hours.…”

“…Plasmid pB40_79C-CDKL5 was mobilized from E. coli S17-1(λpir) to KrPL LacY+ [33] through standard conjugation techniques [42]. E. coli S17-1(λpir) - a strain possessing mob and tra genes for plasmid mobilization [43] - was routinely grown in LB (10 g/L bacto-tryptone, 5 g/L yeast extract, 10 g/L NaCl) at 37 °C with the supplementation of 34 μg/mL chloramphenicol if needed for plasmid selection.…”

“…E. coli S17-1(λpir) - a strain possessing mob and tra genes for plasmid mobilization [43] - was routinely grown in LB (10 g/L bacto-tryptone, 5 g/L yeast extract, 10 g/L NaCl) at 37 °C with the supplementation of 34 μg/mL chloramphenicol if needed for plasmid selection. KrPL LacY+ - a P. haloplanktis TAC125 strain engineered for improved IPTG uptake [33] - was grown at 15 °C in TYP (16 g/L bacto-tryptone, 16 g/L yeast extract, 10 g/L NaCl) for conjugational experiments and initial preinocula. Recombinant KrPL LacY+ was selected with 25 μg/mL and 12.5 μg/mL chloramphenicol in liquid and solid media, respectively.…”