Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74∆sp chitinase inclusions, Cry crystals and spores for applied use

Microbial Cell Factories

Research

Dennis K Bideshi¹  José Cristóbal Castañeda-Ramírez²  Luz Edith Casados-Vázquez²  Jorge Luis Delgadillo-Ángeles²  José Eleazar Barboza-Corona³  Ma Cristina del Rincón-Castro³  Uriel Eleazar Barboza-Pérez⁴ 

[1] Department of Natural and Mathematical Sciences, California Baptist University, 8432 Magnolia Avenue, 92504, Riverside, California, USA;Department of Entomology, University of California, 92521, Riverside, California, USA;División Ciencias de la Vida, Posgrado en Biociencias, Universidad de Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, México;División Ciencias de la Vida, Posgrado en Biociencias, Universidad de Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, México;División Ciencias de la Vida, Departamento de Alimentos, Universidad de Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, México;Tecnológico de Monterrey Campus Querétaro, Epigmenio González 500 Fracc. San Pablo Querétaro, 76130, Querétaro, México;

关键词: Bacillus thuringiensis;    Endochitinase ChiA74;    Cry proteins;    Inclusion bodies;   

DOI  :  10.1186/1475-2859-13-15

 received in 2013-11-20, accepted in 2014-01-20,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundThe endochitinase ChiA74 is a soluble secreted enzyme produced by Bacillus thuringiensis that synergizes the entomotoxigenecity of Cry proteins that accumulate as intracellular crystalline inclusion during sporulation. The purpose of this study was to produce alkaline-soluble ChiA74∆sp inclusions in B. thuringiensis, and to determine its effect on Cry crystal production, sporulation and toxicity to an important agronomical insect, Manduca sexta. To this end we deleted the secretion signal peptide-coding sequence of chiA74 (i.e. chiA74∆sp) and expressed it under its native promoter (pEHchiA74∆sp) or strong chimeric sporulation-dependent cytA-p/STAB-SD promoter (pEBchiA74∆sp) in Escherichia coli, acrystalliferous B. thuringiensis (4Q7) and B. thuringiensis HD1.ResultsBased on mRNA analyses, up to ~9-fold increase in expression of chiA74∆sp was observed using the cytA-p/STAB-SD promoter. ChiA74∆sp (~70 kDa) formed intracellular inclusions that frequently accumulated at the poles of cells. ChiA74∆sp inclusions were dissolved in alkali and reducing conditions, similar to Cry crystals, and retained its activity in a wide range of pH (5 to 9), but showed a drastic reduction (~70%) at pH 10. Chitinase activity of E. coli- pEHchiA74∆sp was ~ 150 mU/mL, and in E. coli- pEBchiA74∆sp, 250 mU/mL. 4Q7-pEBchiA74∆sp and 4Q7-pEHchiA74∆sp had activities of ~127 mU/mL and ~41 mU/mL, respectively. The endochitinase activity in HD1-pEBchiA74∆sp increased 42x when compared to parental HD1 strain. HD1-pEBchiA74∆sp and HD1 harbored typical bipyramidal Cry inclusions, but crystals in the recombinant were ~30% smaller. Additionally, a 3x increase in the number of viable spores was observed in cultures of the recombinant strain when compared to HD1. Bioassays against first instar larvae of M. sexta with spore-crystals of HD1 or spore-crystal-ChiA74∆sp inclusions of HD1- pEBchiA74∆sp showed LC50s of 67.30 ng/cm2 and 41.45 ng/cm2, respectively.ConclusionsAlkali-labile ChiA74∆sp inclusion bodies can be synthesized in E. coli and B. thuringiensis strains. We demonstrated for the first time the applied utility of synthesis of ChiA74∆sp inclusions, Cry crystals and spores in the same sporangium of HD1, a strain used successfully worldwide to control economically significant lepidopteran pests of agriculture. Our findings will allow to us develop strategies to modify expression of ChiA74∆sp while maximizing Cry crystal synthesis in commercial strains of B. thuringiensis.

【 授权许可】

CC BY   
© Barboza-Corona et al.; licensee BioMed Central Ltd. 2014