Abstract
Background: Xylan is the second most abundant polysaccharide biomass on the earth, the polymers have a backbone of β-1,4-linked xylose residues with various side-chain substitutions, such as arabinose, acetic acid, glucuronic acid, and other esterified groups, thus, the removal of arabinose side groups by α-L-arabinofuranosidases is helpful in various industrial processes involving xylan treatment. Bacillus subtilis ATCC 6051a is known for its excellent capacity of secretory production of recombinant peptides, however, poor experience in genetic manipulation and lack of universal expression elements impede this strain for wider application.Results: Xylose inducible comK was integrated into the genome of B. subtilis ATCC 6051a, and the transformation efficiency of the engineered strain B. subtilis 164S was increased by more than 1000 folds. B. subtilis 164S was further modified to generate B. subtilis 164T7P which incorporates a D-xylose inducible T7 RNA polymerase. The recombinant GFP expressed by 164T7P is more than thirteen times that achieved by P43 promoter, representing the most efficient expression system that had been ever reported in B. subtilis . Subsequently, abfA1 , encoding a glycoside hydrolase (GH) family 51 enzyme was cloned and overexpressed in 164T7P. The activity of recombinant α-L-arabinofuranosidase (AbfA1) reached 90.6±2.0 U mL -1 in the fermentation broth. Using p NPA as a substrate, kinetic parameters of the crude enzyme were Km of 1.4±0.1 mM and kcat of 139.4 s −1 . The optimum temperature and pH of the recombinant AbfA1 towards p NPA were observed to be 45 °C and pH 6.5, respectively.Conclusion: With enhanced cellular competence and introduction of T7 RNA polymerase, B. subtilis ATCC 6051a was engineered as a versatile cell tool for recombinant production of heterologous peptides employing T7 promoter. The novel expression kit demonstrated a very low level of leaky expression of target genes. The efficiency and applicability of the system were demonstrated by high-level production of a bacterial type α-L-arabinofuranosidase.