Pyrococcus yayanosii L-天冬酰胺酶在枯草芽孢杆菌中分泌途径的鉴定及其分泌能力的提高

Secretion Pathway Identification of Pyrococcus yayanosii L-Asparaginase in Bacillus subtilis and the Secretion Level Improvement

摘要:

食品安全（GRAS）菌株枯草芽孢杆菌由于其具有良好的分泌表达能力及易于基因工程操作等特性被广泛用作外源蛋白质的表达菌株。枯草芽孢杆菌中蛋白质的分泌大多依赖于信号肽介导的Sec分泌途径（General secretion pathway）和Tat分泌途径（Twin-arginine translocation pathway）。在本研究中，通过信号肽预测、蛋白质N端测序等手段发现来自于Pyrococcus yayanosii L-天冬酰胺酶在枯草芽孢杆菌中的分泌并不依赖信号肽，该酶通过非经典蛋白质分泌途径（non-classical protein secretion pathway）进行分泌。通过信号肽筛选，发现最适合该酶在枯草芽孢杆菌中表达的信号肽为Tat分泌途径信号肽SP_phoD，并通过共表达分子伴侣PrsA的方式将该L-天冬酰胺酶的分泌量提高了72.11%。

Abstract:

Bacillus subtilis, generally recognized as safe (GRAS),waswidely used as host strain to express heterologous proteins due to its good secretion ability and easy geneticmanipulations. In B. subtilis, most of the signal peptide-mediated secretion was depended on Sec-pathway(General secretion pathway) or Tat-pathway(Twin-arginine translocation pathway). In this study, through signal peptide prediction and protein N terminal sequencing, the secretion of Pyrococcus yayanosii L-asparaginase in B. subtilis was confirmedto depend onnon-classical protein secretion pathway. By signal peptidescreening, the Tat-pathway signal peptide SP_{phoD was found to bebeneficial for the L-asparaginase secretion in B. subtilis. With the co-expression of SPphoD and molecular chaperone PrsA, the L-asparaginase secretion was increased by 72.11% compared with that of the original one.}