Construction Of Shuttle Vectors For Bacillus Subtilis

Bacillus is an attractive host for the expression of heterologous proteins as a gram-positive bacteria. 50% of the industrial enzymes were produced by Bacillus strains. In industrial processes, they applied a constitutive-expression system, in which a promoter was used to guide the growth-coupled expression of native proteases or amylases. However, that constitutiveexpression was mostly used in producing native proteins, which is inconvenient to producing certain proteins sunch as toxic ones. As a result, new expression strategies should be developed to exploit the application areas for Bacillus hosts.Inducilbe mechenism can control the growth and expression of the strain. Among the induction expression systems studied, xylose-inducible promoters was shown to have advantages both in expression efficiency and economics.Three inducible expression plasmids were constructed under xylose promoters, which were choosed from Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis. These inducible expression systems could realize the expression of MTSase and MTHase from Sulfolobus acidocaldarius between Escherichia coli and B. subtilis. The results shows that xylose promoter from B. subtilis has the best efficiency of expression. Furthermore, the conditions for the production of trehalose synthase in B. subtilis were optimized. The best carbon, nitrogen source and concentration were glycerol with 5 g·L-1 and tryptone with 24 g·L-1. After culturing 8 hours, 6 g·L-1 xylose was added and further cultivited 16 hours under 37 °C, the trehalase activity was improved from 1.66 U·mL-1 to 4.09 U·mL-1.We also constructed an expression plasmid harbored trehalose synthase encoding gene, which was named TF-Tre from Thermomonospora curvata, which was expressed under the control of xylose operons from Bacillus subtilis. Furthermore, three constitutive plasmids were constructed harbored MTSase, MTHase and trehalose synthase encoding gene separately. The results showed that the recombinant MTSase and MTHase converted most into trehalose, which expressed by adding xylose.