优化Bacillus subtilis对异源β-1,4-内切木聚糖酶分泌表达的研究

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

摘要:

[目的] 基于信号肽和信号肽酶在分泌系统中的重要作用，探索短小芽孢杆菌来源中性β-1，4-内切木聚糖酶在Bacillus subtilis中的重组分泌表达与优化。[方法] 首先，从短小芽孢杆菌基因组DNA中扩增β-1，4-内切木聚糖酶全长基因，连接到pWB980载体P₄₃启动子下游，转化B.subtilis WB800构建重组菌NZ-X。之后，构建信号肽筛选载体，对23个从B.subtilis 168基因组DNA中扩增得到的信号肽进行筛选。最后，以B.subtilis WB800的xynA基因为整合位点，分别整合过表达SipS和SipT两个主要信号肽酶，考察其对融合不同信号肽异源蛋白分泌的影响。[结果] 重组菌NZ-X成功实现β-1，4-内切木聚糖酶的分泌表达，摇瓶发酵上清液酶活为5.33 U/mL，信号肽筛选结果发现YlaE、YfhK、EglS、YqxI、YpjP信号肽与β-1，4-内切木聚糖酶契合度较高，对应酶活依次为7.15、6.69、6.36、6.32、6.18 U/mL，其中SipS信号肽酶对融合YfhK信号肽的β-1，4-内切木聚糖酶的分泌促进作用最大，摇瓶发酵上清液酶活提高到10.64 U/mL，为NZ-X的1.99倍。[结论] 信号肽优化与信号肽酶过表达联用可有效提高B.subtilis中异源蛋白的分泌表达量。

关键词:枯草芽孢杆菌;中性β-1,4-内切木聚糖酶;信号肽;信号肽酶

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.

Key words:Bacillus subtilis;neutral endo-β-1,4-xylanase;signal peptide;signal peptidase