Taking Control of Metabolism in Microbial Cell Factories by Applying Noncanonical Redox Cofactors

Background: Microbial cell factories are microorganisms engineered to produce valuable compounds ranging from biofuels to fine chemicals in a sustainable way. Product pathways are introduced in such microbial cell factories to produce the desired compound.

Challenge: Since yield is the most important industrial parameter to optimise, and the maximum theoretical yield can only be approached when all electrons present in the substrate end up in the product, control over the flow of electrons is a key engineering challenge. A product pathway typically involves several electron-transfer steps, some oxidizing, some reducing. Redox cofactors such as NAD+/NADH play a crucial role as they mediate electron transfer. Ideally, to approach the maximum yield all electrons transferred to and from NAD+/NADH should be contained within the product pathway. NAD+/NADH is however a central redox cofactor and involved in electron transfer in over a hundred redox reactions. This drains electrons from the product pathway and results in the formation of by-products. Moreover, the involvement of NAD+/NADH in both the product pathway and the cellular metabolic network interconnect product formation and other microbial processes to a high level. Introducing and tuning the product pathway can therefore cause uncontrolled pleiotropic effects that lead to strong negative selection pressures and unstable production strains.

Aim: We aim to reduce the interconnection of product pathways and the assimilatory metabolic network by equipping both with their own redox cofactor: keeping NAD+/NADH for assimilation and applying the non-canonical redox cofactor NMN+/NMNH for product formation. We aim to realize this breakthrough in the two industrial workhorses Escherichia coli and Saccharomyces cerevisiae.

Approach: Initially we will focus on the production of lactic acid and ethanol from glucose, because their production pathways are well characterised. They contain an NAD+-reducing glyceraldehyde-3-phosphate dehydrogenase and an NADH-oxidizing lactate dehydrogenase or alcohol dehydrogenase, respectively. The cofactor dependency of these enzymes will be changed to NMN+/NMNH and intracellular levels of NMN+/NMNH will be increased to support the activities of these enzymes. To allow NMN+/NMNH-independent assimilation for growth, we will introduce an additional parallel glycolytic pathway, e.g. based on the Calvin-cycle shunt or methylglyoxal bypass. By equipping this pathway with controllable promotors we want to be able to control product formation and growth independently. Laboratory evolution will be used to enhance the performance of the microbial cell factories and to study their fitness and stability. Based on the obtained results a blueprint will be made on how to rollout non-canonical redox cofactors in other microbial cell factories, for a wider range of products.

Impact. Implementing noncanonical redox cofactors in microbial cell factories is an exciting and potentially revolutionary new research field. It has the potential to improve productivity, reduce by-product formation and achieve near-optimal product yields and thereby result in a major breakthrough in microbial biotechnology. We will simultaneously gain deeper fundamental insights in the complexity and links between metabolism, growth and product formation, en route to completely new opportunities to tame the complexity of metabolism and realize important progress towards the biobased economy.