title: Ag43-mediated display of a thermostable β-glucosidase in Escherichia coli and its use for simultaneous saccharification and fermentation at high temperatures.
abstract: BACKGROUND
  The autotransporter (AT) system can potentially be used in the secretion of saccharolytic enzymes for the production of lignocellulosic biofuels and chemicals using Escherichia coli. Although ATs share similar structural characteristics, their capacity for secreting heterologous proteins widely varies. Additionally, the saccharolytic enzyme selected to be secreted should match the cell growth or cell fermentation conditions of E. coli.
  
  
  RESULTS
  In the search for an AT that suits the physiological performance of the homo-ethanologenic E. coli strain MS04, an expression plasmid based on the AT antigen 43 (Ag43) from E. coli was developed. The β-glucosidase BglC from the thermophile bacterium Thermobifida fusca was displayed on the outer membrane of the E. coli strain MS04 using the Ag43 system (MS04/pAg43BglC). This strain was used to hydrolyze and ferment 40 g/L of cellobiose in mineral media to produce 16.65 g/L of ethanol in 48 h at a yield of 81% of the theoretical maximum. Knowing that BglC shows its highest activity at 50°C and retains more than 70% of its activity at pH 6, therefore E. coli MS04/pAg43BglC was used to ferment crystalline cellulose (Avicel) in a simultaneous saccharification and fermentation (SSF) process using a commercial cocktail of cellulases (endo and exo) at pH 6 and at a relatively high temperature for E. coli (45°C). As much as 22 g/L of ethanol was produced in 48 h.
  
  
  CONCLUSIONS
  The Ag43-BglC system can be used in E. coli strains without commercial β-glucosidases, reducing the quantities of commercial enzymes needed for the SSF process. Furthermore, the present work shows that E. coli cells are able to ferment sugars at 45°C during the SSF process using 40 g/L of Avicel, reducing the gap between the working conditions of the commercial saccharolytic enzymes and ethanologenic E. coli.
authors: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A. P. 510-3, 62250, Cuernavaca, Mor, México. ivan@post.tau.ac.il.|Present address: Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel. ivan@post.tau.ac.il.
authors: Iván
authors: I
authors: Muñoz-Gutiérrez
authors: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A. P. 510-3, 62250, Cuernavaca, Mor, México. cessna_lma@hotmail.com.
authors: Cessna
authors: C
authors: Moss-Acosta
authors: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A. P. 510-3, 62250, Cuernavaca, Mor, México. berenice.trujillo@probiomed.com.mx.
authors: Berenice
authors: B
authors: Trujillo-Martinez
authors: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A. P. 510-3, 62250, Cuernavaca, Mor, México. gosset@ibt.unam.mx.
authors: Guillermo
authors: G
authors: Gosset
authors: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A. P. 510-3, 62250, Cuernavaca, Mor, México. alfredo@ibt.unam.mx.
authors: Alfredo
authors: A
authors: Martinez
mesh_terms: D039903:Actinobacteria / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D002482:Cellulose / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002482:Cellulose; D001617:beta-Glucosidase