Optimization of secretion of heterologous endo-β-1,4-xylanase in Bacillus subtilis

Abstract:

[Objective] We aimed to perform and optimize the recombinant secretory expression of neutral endo-β-1,4-xylanase derived from Bacillus pumilus in Bacillus subtilis WB800 on the basis of important role of signal peptide and signal peptidase.[Methods] We amplified the full-length neutral endo-β-1,4-xylanase gene from B. pumilus genomic DNA and then ligated to the downstream of the P₄₃ promoter in the pWB980 vector. Thereafter, the recombinant vector was transformed into B. subtilis WB800 to construct the recombinant strain NZ-X. Signal peptides were screened among 23 signal peptides that were amplified from B. subtilis 168 genomic DNA. On this basis, we constructed two strains that overexpress two signal peptidases (SipS and SipT), respectively. And the effect of these two signal peptidases on the secretion of endo-β-1,4-xylanase was investigated.[Results] Neutral endo-β-1,4-xylanase was successfully secreted from the recombinant strain NZ-X and the enzyme activity in supernatant was 5.33 U/mL via shake flask fermentation. The results of signal peptide screening indicated that five signal peptides (YlaE, YfhK, EglS, YqxI, YpjP) were effective, and the enzyme activities were 7.15, 6.69, 6.36, 6.32, 6.18 U/mL, respectively. Among these signal peptides, the secretion of endo-β-1,4-xylanase was promoted mostly by SipS signal peptidase when fusion with YfhK signal peptide. The enzyme activity could be increased to 10.64 U/mL, which was 1.99 times than that of NZ-X.[Conclusion]] The secretion of heterologous proteins in B. subtilis could be improved effectively through signal peptide optimization and signal peptidase overexpression.