Development and in silico evaluation of an expression platform based in E. coli fo...
| Grant number: | 10/08089-2 |
| Support type: | Research Grants - Young Investigators Grants |
| Duration: | October 01, 2010 - September 30, 2015 |
| Field of knowledge: | Engineering - Chemical Engineering - Chemical Process Industries |
| Principal Investigator: | Sindelia Freitas Azzoni |
| Grantee: | Sindelia Freitas Azzoni |
| Home Institution: | Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia, Inovações e Comunicações (Brasil). Campinas , SP, Brazil |
| Assoc. researchers: | Jose Geraldo da Cruz Pradella ; Marcelo Brocchi ; Roberto Ruller |
Abstract
Biomass conversion by enzymatic hydrolysis still represents a high cost step for production of second generation ethanol. To overcome this drawback it is important increase the enzyme efficiency, obtaining balanced cocktails tailored made for each pretreated biomass and decrease production costs by using genetics tools for enzyme engineering and by production process optimization. The objective of this project is study and develops a recombinant protein expression platform based on modified strains of E. coli by using genetic engineering and new vectors and strains recently available. Two major drawbacks of E. coli large-scale protein production will be addressed: 1) the intracellular expression; and 2) the need of antibiotic as a selection marker. To reach this objective, E. coli mutants strains, BL21DE3 and SE1 will be modified in order to achieve membrane permeability. The mutation will be done by gene knock out of the lpp gene which codifies to a Braun´s lipoprotein. The efficacy of these systems will be evaluated by performing high cell densities fermentations for the production of three model enzymes (endoglicanase, xylanase, feruloyl esterase). Critical fermentation parameters will be obtained in bioreactors such as cellular growth rate and enzyme biosynthesis rate. Special attention will be dedicated to the induction phase by optimizing parameters such as inducer type and concentration, inducer feeding rate, carbon and nitrogen sources concentration by using a factorial design. We expect develop a robust and economic process to enzymes production in order to make enzymatic complex tailored made for several pretreated biomass in study in the CTBE. (AU)