Construction Of Food-grade Expression Systems And Study Of Protein Secretion In Bacillus Subtilis

The recombinant microbial expression systems constructed based on food-grade microorganisms are name Food-grade Expression Systems. In contrast to the general expression systems, such systems are distinguished by the requirements that all the elements used should only be derived from food-safe microorganisms. Due to the safe status, the food-grade expression systems have great potential to be used directly in food fermentation and processing. Bacillus subtilis is a kind of food-grade microorganism which can be developed into food-grade expression systems. A major property of B. subtilis is that it can secret a lot of proteins to culture supernatants, and the study on signal peptides and secretion pathway of B. subtilis may help to develop this strain into a cell factory for production of heterologous proteins.This thesis takes the B. subtilis as a research material. Two food-grade expression systems were constructed for B. subtilis and the growth property and stability of the expression systems were also studied. The signal peptide of twin-agarginine secretion pathway was used for study of the secretion of a cytoplasmic enzyme in B. subtilis and its secretion pathway was genetically modified to probe the enhancement of secretion of heterologous protein. The detailed works are described as following.The alanine racemase gene dal was cloned from B. subtilis and was confirmed to be able to complement the D-alanine auxotrophic phenotype of B. subtilis QB928. This gene was used as both selection marker and homologous region for integration and construction of the integrative food-grade expression system. The thermostableβ-galactosidase BgaB from Geobacillus stearothermophilus was used as a reporter for expressional test of the expression system. The growth speed of the recombinant strain was similar to that of the host. The maximum BgaB enzymatic activity in the shaking-flask fermentation was 0.16 U/(mg protein). Stability examination of the food-grade strain showed it was 98% stable after 100 generations of successive cultivation without selection pressure.Food-grade expression system which based on replicative plasmids was also constructed. The dal locus on the chromosome of B. subtilis 168 was knocked out to generate the food-grade host FG01. The food-grade expressional plasmids pXFGT03 and pXFGT05 were constructed by the following elements: the gene dal was used as selection marker; the new theta-type replicon was used as Bacillus backbone; and the promoter P43 was used for expression of heterologous gene. Results showed deletion of ORF-3 and ORF-4 from the theta-replicon has no impact on replicative function of plasmid pXFGT05.The gene bgaB was cloned into plasmids pXFGT03 and pXFGT05 to generate food-grade plasmids pXFGT03-bgaB and pXFGT05-bgaB. These two plasmids was transformed into host FG01 to generate recombinant strains FG01(pXFGT03-bgaB) and FG01(pXFGT05-bgaB). The BgaB enzymatic activities of these two strains were 1.90 U/(mg protein) and 2.01 U/(mg protein) respectively at 13 h of cultivation, which were 10 times higher than that obtained from the integrative food-grade B. subtilis expression system. Plasmids pXFGT03-bgaB and pXFGT05-bgaB were 100% stable after 100 generations of successive cultivation without selection pressure.The cytoplasmic protein BgaB was used as a reporter for seretion and the coding gene bgaB was fused to the signal peptide coding sequence of B. subtilis NprB to generate plasmid pMKSigbgaB01. Results showed this Sec-type signal peptide can not mediate the secretion of BgaB in B. subtilis. While fused to the Tat-type signal peptide coding sequence of B. subtilis PhoD, the plasmid pMKSigbgaB03 was constructed and results showed this Tat-type signal peptide can mediate the secretion of BgaB in B. subtilis in phosphate starvation medium. The plasmid pMKSigbgaB05 was constructed for secretion of BgaB in rich medium. This plasmid was transformed into host 168 and the recombinant strain was albe to secret BgaB. At 8-18 h of cultivation, the extracellular BgaB activity obtained maximally accounted for 40% of the total enzymatic activity and averagely 27.5%.The coding sequence of molecular motor SecA and the cytoplasmic chaperom SecB was cloned from Escherichia coli and integrated into the chromosome of B. subtilis. Inducible expression (by 0.5% xylose) of SecA and SecB can not increase the secretion level of the reporter penicillin G acylase in B. subtilis.