Expression of thurincin H, ChiA74 and Cry proteins at the sporulation phase in Bacillus thuringiensis HD1.

AIMS: The objective of this study was to produce thurincin H, ChiA74 and Cry proteins together using Bacillus thuringiensis subsp. kurstaki HD1 as a heterologous host. METHODS AND RESULTS: pSTAB-ThurH and pSTAB-ChiA74 constructs were designed to produce thurincin H and chitinase, respectively, at the sporulation phase. They were transformed into Bt HD1 generating the recombinant strains HD1/pSTAB-ThurH and HD1/pSTAB-ThurH/pSTAB-ChiA74. Antimicrobial and chitinolytic activity tests were performed with recombinant strains. Both strains were able to produce thurincin H up to 72 h with antibacterial activity of ~4000 U mg-1 . The HD1/pSTAB-ThurH/pSTAB-ChiA74 strain also showed chitinolytic activity of ~23 mU mg-1 at 72 h. All B. thuringiensis strains exhibited crystal formation at 72, and 96 h. In addition, the application of thurincin H in corn seeds increased the germination percentage and root length by 7% and 10%, respectively. CONCLUSIONS: We showed that is possible to produce three proteins of biotechnological interest at the sporulation stage in B. thuringiensis, which two of them (thurincin H, and ChiA74) are naturally expressed in the vegetative stage. SIGNIFICANCE AND IMPACT OF THE STUDY: These results form the basis for developing of a biocontrol and biostimulator product that can be used as an alternative for chemical application.

Improving the yields of thurincin H in a native producer strain.

Thurincin H is a bacteriocin produced by Bacillus thuringiensis with activity against a wide range of bacteria, including Gram positive and Gram negative. Disadvantages of producing thurincin H in B. thuringiensis is the low production level in the native strain probably due to the highly regulated mechanism of biosynthesis. The present study aimed to increase the synthesis of thurincin H produced by the native strain (Btm) through the establishment of additional copies of the structural gene (i.e. thnA) and the genes responsible for the bacterial self-immunity (thnRDE). Here, three recombinant strains of Btm were generated, harboring three, six and nine additional copies of thnA, and three with one copy of thnRDE upstream to the thnA copies. Data showed that the highest yield was obtained at 16 h using three additional copies of thnA (Btm/pHT-One) with a bacteriocin activity of 20,000 U/mg compared with the parental strain which showed 10,000 U/mg, increase of 100% in the production of the bacteriocin. Also, the addition of the genes responsible for self-immunity showed that recombinant B. thuringiensis (Btm/pHT-TwoRDE) can support six additional copies of thnA with an increase of 60% compared with the parental strain.