CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium

Butanolproduction from agricultural residues is the most promising alternative for fossil fuels. To reach the economic viability of biobutanol production, both glucose and xyloseshould be utilized and converted into butanol. Here, we engineered a dual-operon-based synthetic pathway in the genome of E. coli MG1655 to produce n-butanolusing CRISPR/ Cas9technology. Further deletion of competing pathway followed by fed-batch cultivation of the engineered strain in a bioreactor with glucose-containing complex medium yielded 5.4 g/L n-butanolalong with pyruvate as major co-product, indicating a redox imbalance. To ferment xyloseinto butanolin redox-balanced manner, we selected SSK42, an ethanologenic E. coli strain engineeredand evolved in our laboratory to produce ethanol from xylose, for integrating synthetic butanolcassette in its genome via CRISPR/ Cas9after deleting the gene responsible for endogenous ethanol production. The engineered plasmid- and marker-free strain, ASA02, produced 4.32 g/L butanolin fed-batch fermentation in completely defined AM1– xylosemedium.