초록
With the increasingly serious global environment problem caused by slather use of fossil fuels, numerous efforts have been made in developing renewable alternatives. Converting lignocellulose to bioenergy has accomplished by cellulases but the production is limited, therefore the recombinant expression platforms in E. coli have been established. Since E. coli is partly restricted by its inability to secrete enzymes to extracellular membrane, we constructed a synthetic circuit to coexpress cellulases and colicin E7 lysis to solve this problem. The pBAD-RBS-lysis-TT (BBa_K117010) sequence from iGEM was added to form a chimeric plasmid based on pET system, which harboring cellulases from Bacillus subtilis or Thermobifida fusca. The presence of arabinose for the promoter pBAD, leading to induce lysis and further breakdown of the host membrane to release cellulase to extracellular membrane. The extracellular activities increase to 48.1 and 55.5% under lysis gene functioned on Cel5 and CD1, respectively. This is the first attempt to show that cellulases have successfully expressed and released to extracellular membrane without cumbersome steps. Finally, this synthetic circuit simplifies and achieves the simultaneous release and heterologous expression of the cellulases as well as obtain a long-term stable enzyme in E. coli system.