Fundamental Establishment Of The Bacillus Genetic Manipulation System And Molecular Improvement Of Engineered Bacillus Strains

Bacillus spp. are gram-positive bacteria that are capable of secreting proteins into the culture medium. Without endotoxin, Bacillus spp. are used to produce a variety of important industrial enzymes. Along with the rapid development of molecular biology and genetic engineering, Bacillus as an expression system has been developed and shows excellent prospects for industrial application. The aims of the present study are to optimize the Bacillus subtilis expression system, to improve the genetic manipulation systems of three wild-type industrial Bacillus strains for high-level expression, and to establish a Bacillus expression system with application potentials.To establish a B. subtilis expression system, a series of vectors were firstly screened, including vectors containing different promoters(i.e. constitutive promoter P43, lactose inducible promoter Pgrac, xylose inducible promoter Pxyl, and amylase promoter PamyQ), cloning vectors harboring different selective markers(i.e. kanamycin, erythromycin, chloramphenicol, and tetracycline), and integrative vectors of these elements. Transformation methods for B. subtilis expression were also optimized to improve the transformation efficiency. Thus a B. subtilis system was successfully established for high level expression of heterologous proteins. By using this platform, the aminopeptidase gene lapB was highly expressed in B. subtilis, showing the enzyme activity of 58.8 U/mL in shake flask level.The genetic transformation method for production of mesophilic α-amylase in industrial Bacillus amyloliquefaciens BF.7658 was established through methylation of transformed plasmids and optimization of preparation conditions of competent cells. The alkaline protease AprE from Bacillus clausii was successfully expressed in B. amyloliquefaciens BF.7658. By increasing the copy numbers of expression vector pUB110-aprEA in B. amyloliquefaciens BF.7658, a recombinant strain, B. amyloliquefaciens AprEA, was constructed. After 72 h fermentation in shake flasks, the alkaline protease activity reached 7800 U/mL.B. amyloliquefaciens K11 is a wild-type strain conserved in our laboratory, which has high neutral protease-producing ability. Its neutral protease activity in shake flasks was 2700 U/mL. To further improve the expression level of neutral protease, the coding gene K11 npr was cloned from B. amyloliquefaciens K11, and two expression vectors pKan300-K11 npr and pUB110-K11 npr were constructed in B. subtilis. Multi-copy plasmids carrying the K11 npr were then transformed into competent cells of B. amyloliquefaciens K11 to construct recombinant strains F20 and 110N-6. Protease activity assays on milk plates indicated that strains F20 and 110N-6 showed higher protease activities than wild-type strain K11. By optimizing the fermentation conditions, the protease activity of strain 110N-6 was increased to 8995 U/ml in shake flasks and 24,000-28,000 U/mL in 15-l fermentor.Instability of recombinant plasmids has limited the widespread application of recombinant Bacillus strains containing multi-copy episomal plasmids. The distinguished difference between expression vectors pKan300-K11 npr and pUB110-K11 npr is that pKan300-K11 npr contains the replication element pBR322-ori of Escherichia coli and pUB110-K11 npr has the replication element of Bacillus. Genetic stability analysis indicated that the genetic stability of strain F20 is far less than strain 110N-6. Strain F20 lost all recombinant plasmids after 8 generations, whereas strain 110N-6 contained 90% recombinant plasmids even after 100 generations. Harboring two replication elements simultaneously might account for the plasmid instability in Bacillus. The recombinant plasmid pUB110-K11 npr in strain 110N-6 is very stable, which can meet the requirements of industrial production.B. subtilis AS.1398 is an industrial neutral protease-producing strain widely used in China, which has been developed by traditional mutation breeding approaches(UV treatment and chemical mutagenesis). By supplementing different concentrations of ampicillin into medium to suppress cell wall synthesis, the genetic transformation platform of strain AS.1398 was established. By introducing the recombinant expression vector pUB110-K11 npr into strain AS.1398, the recombinant strain B. amyloliquefaciens Z5-1 was constructed. The neutral protease activity of B. amyloliquefaciens Z5-1 reached 9295 U/mL in shake flask, which was much higher than that of original strain AS.1398.In summary, a high-level B. subtilis expression system was improved from multiple aspects, several transformation methods were tested, and a few genetic transformation system specific for wild-type Bacillus spp. were established. The bottleneck of plasmid instability in Bacillus was solved. This study provides a new insight into the development of new genetic expression system.