Engineering new-to-nature biochemical conversions by combining fermentative metabolism with respiratory modules

Anaerobic microbial fermentations provide high product yields and are a cornerstone of industrially established bio-based processes. However, the need for redox balancing limits the array of fermentable substrate-product combinations. With the aim to overcome this limitation, we designed an aerobic fermentative metabolism that allows the introduction of selected respiratory modules. Through these modules, oxygen can be used to re-balance otherwise unbalanced fermentations, hence allowing controlled respiro-fermentative growth. Following this design, we engineered and characterized an obligate fermentative Escherichia coli strain that aerobically ferments glucose to stoichiometric amounts of lactate. We then re-integrated the quinone-dependent glycerol 3-phosphate dehydrogenase and demonstrated glycerol fermentation to lactate while selectively transferring the surplus of electrons to the respiratory chain. To showcase the platform potential of this novel fermentation mode, we replaced lactate with isobutanol production and demonstrated its growth-coupled synthesis from glycerol. In summary, our design permits the use of oxygen with unprecedented selectivity for the re-balancing of fermentations. This concept is a groundbreaking advance for freeing highly efficient microbial fermentation from the limitations imposed by redox balancing.