Research On The Substrate Characteirstics Of Sec Pathway And Non-classical Protein Secretion Pathway In Bacillus Subtilis

Protein secretion generically refers to the movement of a protein from its site ofsynthesis, the cytoplasm, to the exterior of the cell. Because the protein secreted to the culturemedium facilitates downstream processing, protein folding and enabling the production ofsoluble and biologically active proteins at a reduced process cost, secretory expression is anideal expression for the heterologous proteins. Bacillus subtilis is recognized as a food-gradebacterium with Generally Recognized As Safe (GRAS) status, with high ability to secreteproteins, amenability to medium and genetic engineering, and excellent fermentationcapacities, so it has been regarded as attractive hosts for the production of heterologousproteins. A great number of foreign protein genes from different organisms have been clonedand expressed in Bacillus subtilis, but the low yield of the secreted proteins limits its use asmajor “cell factories” for secreted heterologous proteins of interest.In order to explore the reason for the low yield of some secreted proteins in Bacillussubtilis. First, we analyzed the amino acid composition of241predicted exported proteins and457cytoplasmic proteins of Bacillus subtilis using bioinformatics methods. On this basis, wefurther studied the secretion of an intrinsically disordered protein with different secretionsignals. Then, the secretion of specific protein was attempted by changing the properties ofthis protein. Finally, we explored non-classical protein secretion pathway and tried to utilizethis secretion pathway secrete heterologous proteins. The main results are described asfollows:(1) In this lab, we have tried to utilize different Sec-type signal peptides to lead thesecretion of thermal enzyme BgaB, but no success yet. It is not clear that why the cytoplasmicproteins, like BgaB can not be transported with the different signal peptides and whichproperties hinder their secretion. To answer the above questions, the amino acid compositionof241predicted exported proteins and457cytoplasmic proteins of Bacillus subtilis wasexplored using the programs written in the Python programming language and found that thefirst14residues of the mature domains have more disorder-promoting amino acids, but theNH2-terminal region of cytoplasmic proteins have more order-promoting amino acids. Thepresence of disordered domains in the NH2-terminal regions of mature domains may providethe binding sites with different proteins. This region in cytoplasmic proteins containing moreorder-promoting amino acids may easily form the structure, hindering its secretion.(2) The protein leading by different signal peptides has different secretion efficiency,current research mainly focus on seeking the suitable signal peptide for the target protein bysystematic screening of lots of signal peptides, but the prediction of “good” signal peptidesfor special heterologous proteins is unable to be fulfilled and the reason that the protein withdifferent signal peptides has different secretion efficiency is unknown. In the third chapter ofthis thesis, a naturally unsecretory intrinsically disordered domain of nucleoskeletal-likeprotein (Nsp) was attempted to be secreted with different types of secretion signals in Bacillussubtilis. The results showed that Nsp can be hypersecreted efficiently by all selected Sec-typesignal peptides, but Nsp could not be detected in the cytoplasm. When fused to Tat-type signal peptides or non-classical extracellular proteins, Nsp was successfully exported but lessefficient than Sec-type, and the fusion proteins can be detected in the cytoplasm.(3) Thermostable β-galactosidase BgaB from Geobacillus stearothermophilus hasthermal stability properties, favorable optima temperature activity and neutral pH activity, soit has potential for industrial application. With the help of different Sec-type signal peptides,BgaB can not be secreted to the exterior of the cell in our previous research. In the fourthchapter, we want to secrete BgaB by deleting the order-promoting amino acids in the NH2terminus of BgaB, or inserting the rare codons in the encoding sequence of residues3to11or35to44from BgaB. Unfortunately, all changes can not lead the secretion of BgaB. Furemore,we used alkaline phosphatase PhoA from Escherichia coli as the reporter protein to explorethe reason for the block in translocation and revealed multiple regions in BgaB contribute toits failure to be translocated.(4) Non-classically secreted proteins that lack classical and cleavable signal peptides aregenerally released into the growth medium in large amounts during the stationary phase. Inthe fifth chapter, we found that the secretion of these non-classically secreted proteins is not atime-dependent phenomenon, when overexpressed, they also can be detected in the mediumduring the exponential phase. The non-classically secreted proteins can lead the secretion ofintrinsically disordered Nsp, alkaline phosphatase PhoA and thermostable β-galactosidaseBgaB when used as secretion signals. Then, we used Nsp as the reporter protein to explore thesecretion signals of non-classically secreted proteins and found the first50residues of15non-classically secreted proteins and the first48residues of PdhB sufficiently lead the Nsp tothe exterior of the cell.