Secretion of the cytoplasmic and high molecular weight [beta]-galactosidase of Paenibacillus wynnii with Bacillus subtilis

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. In this study, the cytoplasmic and 120 kDa [beta]-galactosidase of Paenibacillus wynnii ([beta]-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 [beta]-gal-Pw gene led to an increase in extracellular [beta]-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular [beta]-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 [+ or -] 6 [micro]kat/L.sub.culture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the P.sub.AprE promoter. Production of extracellular [beta]-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 [+ or -] 10 [micro]kat/L.sub.culture with secretion efficiencies of more than 80%. For the first time, the [beta]-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.