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.

厌氧微生物发酵具备产物得率高的优势，是工业规模化生物基工艺的核心支柱之一。然而，氧化还原平衡的刚性需求限制了可发酵底物-产物组合的范围。为突破这一局限，我们设计了一种需氧发酵代谢途径，可引入定制化的呼吸模块。借助这些模块，氧气可用于重构原本失衡的发酵体系，从而实现可控的呼吸-发酵协同生长。基于该设计，我们构建并表征了一株专性发酵型大肠杆菌（Escherichia coli），该菌株可在需氧条件下将葡萄糖发酵为化学计量比当量的乳酸。随后我们重新整合了醌依赖型甘油3-磷酸脱氢酶，实现了甘油发酵产乳酸的同时，将多余电子选择性传递至呼吸链。为验证该新型发酵模式的平台化应用潜力，我们将乳酸合成路径替换为异丁醇合成路径，并实现了以甘油为底物的生长偶联型异丁醇合成。综上，本设计可通过前所未有的选择性利用氧气来重构发酵体系的氧化还原平衡。该理念为高效微生物发酵摆脱氧化还原平衡的束缚提供了突破性进展。