One-round-per-day CRISPR genome editing of E. coli for engineering green-chemical overproducer

Isobutanol, a representative higher alcohol, offers many advantages as a substitute for gasoline due to its higher energy content and higher hydrophobicity. It is both a transportation fuel and a platform chemical targeting a billion-dollar-per-year market. The green production of isobutanol relies on engineered microbial cell factories (MCF) with increased titer, yield, and productivity. However, the de novo rational construction of an MCF with dozens of designed mutations is time consuming and labor intensive. Here, we demonstrated that designed DNA fragments could be assembled into replicable and functional vectors inside the target cells to accomplish CRISPR-dependent gene editing and positive-strain selection in a one-pot manner in Escherichia coli, leaving the whole strain editing process relies only on polymerase chain reactions (PCR) and several other liquid mixing steps. The time and labor cost of rational strain construction could then be minimized and all handling is doable by routine automation equipment. Using this plasmid construction-less (PCL) strategy, we engineered an existing isobutanol overproducer and increased its production titer by 28.47 %, reaching 48.37 g/L in a 3L bioreactor. Our strategy could accomplish one round of strain engineering in one day and is compatible with routine automated platforms, having the potential to significantly speed up the MCF construction with biofoundries in the field of green production.