Novel expression system based on enhanced permeability of Vibrio natriegens cells induced by D,D- carboxypeptidase overexpression

Abstract

Vibrio natriegens is a fast-growing, non-pathogenic marine bacterium with promising features for biotechnological applications such as high-level recombinant protein production or fast DNA propagation. A remarkable short generation time (< 10 min), robust proteosynthetic activity and versatile metabolism with abilities to utilise wide range of substrates contribute to its establishment as a future industrial platform for fermentation processes operating with high productivity.D,D-carboxypeptidases are membrane-associated enzymes involved in peptidoglycan biosynthesis and cell wall formation. This study investigates the impact of overexpressed D,D-carboxypeptidases on membrane integrity and the increased leakage of intracellular proteins into the growth medium in V. natriegens. Our findings confirm that co-expression of these enzymes can enhance membrane permeability, thereby facilitating the transport of target proteins into the extracellular environment, without the need for secretion signals, tags, or additional permeabilization methods. Using only a single step IMAC chromatography, we were able to purify AfKatG, MDBP or Taq polymerase in total yields of 117.9 ± 56.0 mg/L, 36.5 ± 12.9 mg/L and 26.5 ± 6.0 mg/L directly from growth medium, respectively. These results demonstrate the feasibility of our V. natriegens based system as a broadly applicable extracellular tag-less recombinant protein producer.

Keywords: D,D-carboxypeptidases; Extracellular production; Recombinant proteins; Vibrio natriegens.

Fig. 1

Determination of PBP4 or PBP5/6 D,D-carboxypeptidase overexpression on cell growth and biomass formation (dashed lines). Growth curves for V. natriegens Vmax and PF strains were determined according to DCW increase. D,D-carboxypeptidase overexpression was not present in control cultivations (Vmax and PF – solid lines). PF – V. natriegens Prophage-free; Vmax – V. natriegens Vmax; DCW - dry cell weight

Fig. 2

A - Outer membrane permeability of V. natriegens strains was determined by a fluorescence increase resulting from NPN uptake into the hydrophobic area of outer membrane. B - Inner membrane permeability of V. natriegens strains was determined by a fluorescence increase resulting from PI uptake into intracellular area of the cell. The cultivation without overexpression of PBP4 or PBP5/6 D,D-carboxypeptidases was used as a control. A p-value of less than 0.05 was considered statistically significant. PF – V. natriegens Prophage-free, Vmax – V. natriegens Vmax

Fig. 3

Effect of the D,D-carboxypeptidase PBP4 or PBP5/6 overexpression under T7-promoter on intracellular production of GFP as well as its extracellular transport into growth media in V. natriegens Vmax and PF A – Fluorescence intensity of intracellular and extracellular GFP with PBP4 or PBP5/6 co-expression after induction with 1 mM IPTG in V. natriegens Vmax; B – Fluorescence intensity of intracellular and extracellular GFP in medium after PBP4 or PBP5/6 co-expression after induction with 1 mM IPTG in V. natriegens PF; Control cultivations were lacking permeabilization plasmids for PBP4 and PBP5/6 overexpression. IP – Intracellular protein; EP – Extracellular protein; GFP - Green fluorescent protein; A p-value less than 0.05 was considered statistically significant. PF – V. natriegens Prophage-free; Vmax – V. natriegens Vmax

Fig. 4

A - SDS-PAGE analysis of AfKatG production and its transport into growth medium in V. natriegens PF with co-expression of PBP5/6 under T5-lac promoter; B – SDS-PAGE analysis of AfKatG after IMAC purification from growth media. MS – protein molecular weight standard; IP – intracellular protein; EP – extracellular protein; FT – flow through; W- wash fraction; E – elution fraction; PF – V. natriegens Prophage-free; the numbering of lanes on SDS-PAGE or Western blot corresponds to the time in hours elapsed after induction

Fig. 5

A - SDS-PAGE analysis of MDBP production and its extracellular transport into growth medium in V. natriegens PF with co-expression of PBP5/6 under T5-lac promoter; B – SDS-PAGE analysis of MDBP production after IMAC purification from growth medium. Control cultivation was lacking plasmid for D,D-carboxypeptidase overexpression. MS – protein molecular weight standard; IP – intracellular protein; EP – extracellular protein; the numbering of lanes on SDS-PAGE or Western blot corresponds to the time in hours elapsed after induction

Fig. 6

A - SDS-PAGE analysis of Taq polymerase production and its extracellular transport into growth medium in V. natriegens Vmax with co-expression of PBP5/6 under T7 promoter; B – Western blot analysis of Taq polymerase production after IMAC purification from growth medium. Control cultivations were lacking plasmid for PBP5/6 overexpression. MS – protein molecular weight standard; FT – flow through; W- wash fraction; E – elution fraction, IP – intracellular protein; EP – extracellular protein; Vmax – V. natriegens Vmax; the numbering of lanes on SDS-PAGE or Western blot corresponds to the time in hours elapsed after induction

Fig. 7

A - SDS-PAGE analysis of MutTaq polymerase production and its extracellular transport into growth media in V. natriegens Vmax with co-expression of PBP5/6 under T7 promoter; B – Western blot analysis of MutTaq polymerase production after IMAC purification from growth medium. Control cultivations were lacking plasmid for D,D-carboxypeptidase PBP5/6 overexpression. MS – protein molecular weight standard; P – insoluble fraction; S – soluble fraction of MutTaq polymerase after 24 h intracellular production; FT – flow through; W- wash fraction; E – elution fraction, IP – intracellular protein; EP – extracellular protein; Vmax – V. natriegens Vmax; the numbering of lanes on SDS-PAGE or Western blot corresponds to the time in hours elapsed after induction