LAUSR

Biotechnological production of butanol in heterologous hosts has recently attracted many interests. Of the heterologous hosts investigated to date, engineered Escherichia coli has shown a superior butanol yield than the natural butanol-producing clostridial strains. However, all reported butanol-producing E. coli strains contain vectors and inducible promoters, which means antibiotics and inducers are required in the fermentation. The aim of this study was to develop a completely chromosomally engineered E. coli strain capable of producing butanol efficiently in the absence of vectors, antibiotics, and inducers. The challenges are the expression strength of chromosomally engineered genes under constitutive promoters is much weaker than the vector engineered genes under inducible promoters. To address these challenges, the butanol pathway was engineered into the chromosome in the first place, then the host and the butanol pathway was iteratively engineered through rational and non-rational strategies to develop an efficient butanol producer where the heterologous butanol pathway fits the host well. Finally, a systematically chromosomally engineered E. coli strain EB243, in which 33 native genes were deleted and 5 heterologous genes were introduced, was developed. Strain EB243 could produce 20g/L butanol with a yield of 34% (w/w, 83% of theoretical yield) in batch fermentation without any antibiotics and inducers, thus showed great potential for industrial application. This work also demonstrated a procedure on how to integrate the existing knowledge to engineer a strain with industrial application potential.