Secretion of the cytoplasmic and high molecular weight β-galactosidase of Paenibacillus wynnii with Bacillus subtilis

Abstract

Background: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa.

Results: In this study, the cytoplasmic and 120 kDa β-galactosidase of Paenibacillus wynnii (β-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the β-gal-Pw gene led to an increase in extracellular β-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular β-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/L_culture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the P_AprE promoter. Production of extracellular β-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/L_culture with secretion efficiencies of more than 80%.

Conclusion: For the first time, the β-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.

Keywords: Bacillus subtilis; β-galactosidase; Bioreactor; Protein secretion; Recombinant enzyme production.

Fig. 1

Schematic overview of generated plasmids with a modular expression cassette. Plasmids were generated to test different combinations of expression and secretion elements. Symbols in brackets refer to the abbreviation used for recombinant strain designation

Fig. 2

Comparison of native and codon-optimized β-gal-Pw gene sequence for extracellular production. Expression of the native (Bs43Y1 and BsAY1) or codon-optimized (Bs43Y2 and BsAY2) gene was under control of the P₄₃ (blue) and P_AprE (grey) promoter. Shake flask cultivation was done for 73 h with sampling after 32, 56 and 73 h of cultivation. The star (*) indicates significance (p < 0.05)

Fig. 3

Shake flask cultivation of Bs43Y2 at 37 and 30 °C. Samples were taken after 24, 47, 73 and 96 h of cultivation

Fig. 4

Combinatorial screening of different promoters and signal peptides for β-gal-Pw production. Different promoters (BsA = P_AprE and Bs43 = P₄₃) and signal peptides (Y = YoaW; A = AprE; G = GlmU; P = PhoD; nc = negative control, empty vector) were tested. The β-galactosidase activity was determined in the supernatant after 73 h of cultivation. Significance analyses between the activities of all strains were performed and significance (p < 0.05) is indicated by: * = significance to Bs43Y2; Φ = significance to BsAP2; # = significance to BsAA2. n.d. = activity not detected

Fig. 5

Cultivation of BsAP2 in 1 L bioreactors for secretory β-gal-Pw production. Cultivation was done in duplicates at 30 °C, without pH control and with a working volume of 0.8 L

Fig. 6

SDS PAGE of (A) secretome and (B) partial β-gal-Pw purification from the supernatant of BsAP2 bioreactor cultivation. (A) The culture supernatant was analyzed after 1 = 22 h, 2 = 28 h, 3 = 38 h, 4 = 44 h, 5 = 51 h and 6 = 68 h of cultivation. (B) 1 = Supernatant of cultivation; 2 = Concentrated supernatant; 3 = Flow-through fraction IMAC; 4–9 = Fractions of IMAC purification. An amount of 8 µg of protein was loaded in each lane