Self-regulated 1-butanol production in Escherichia coli based on the endogenous fermentative control

As a natural fermentation product secreted by Clostridium species, bio-based 1-butanol has attracted great attention for its potential as alternative fuel and chemical feedstock. Feasibility of microbial 1-butanol production has also been demonstrated in various recombinant hosts.

In this work, we constructed a self-regulated 1-butanol production system in Escherichia coli by borrowing its endogenous fermentation regulatory elements (FRE) to automatically drive the 1-butanol biosynthetic genes in response to its natural fermentation need. Four different cassette of 5' upstream transcription and translation regulatory regions controlling the expression of the major fermentative genes ldhA, frdABCD, adhE, and ackA were cloned individually to drive the 1-butanol pathway genes distributed among three plasmids, resulting in 64 combinations that were tested for 1-butanol production efficiency. Fermentation of 1-butanol was triggered by anaerobicity in all cases. In the growth-decoupled production screening, only combinations with formate dehydrogenase (Fdh) overexpressed under FRE demonstrated higher titer of 1-butanol anaerobically. In vitro assay revealed that 1-butanol productivity was directly correlated with Fdh activity under such condition. Switching cells to oxygen-limiting condition prior to significant accumulation of biomass appeared to be crucial for the induction of enzyme synthesis and the efficiency of 1-butanol fermentation. With the selection pressure of anaerobic NADH balance, the engineered strain demonstrated stable production of 1-butanol anaerobically without the addition of inducer or antibiotics, reaching a titer of 10 g/L in 24 h and a yield of 0.25 g/g glucose under high-density fermentation.

Here, we successfully engineered a self-regulated 1-butanol fermentation system in E. coli based on the natural regulation of fermentation reactions. This work also demonstrated the effectiveness of selection pressure based on redox balance anaerobically. Results obtained from this study may help enhance the industrial relevance of 1-butanol synthesis using E. coli and solidifies the possibility of strain improvement by directed evolution.