Applying NMN+ as non-canonical redox cofactor in Escherichia coli

Microbial biotechnology is a sustainable alternative for petrochemical industry. Unfortunately, many microbial production processes are not yet efficient enough to economically compete. A key parameter in microbial production is the product yield. Ideally, the yield should approach the maximum theoretical yield where all electrons of the substrate end up in the product. Therefore, we need to be able to control the electron flow to maximize the product yield.

Molecules that are key components in electron transfer are redox cofactors, such as NAD⁺/NADH and NADP⁺/NADPH. A great number of enzymes involved in biomass and by-product formation also compete for NADH and NADPH. This entanglement in metabolism complicates the regulation of the electron flow.

As a solution, Weusthuis et al. (2020) proposed to use non-canonical redox cofactors (NRC), such as NMN⁺. NMN⁺ is a moiety of the NAD⁺ molecule that is involved in electron transfer. NMN⁺ itself is not a natural redox cofactor. To implement NMN⁺ as NRC, the cofactor dependency of specific enzymes in glycolysis and a product formation pathway will be changed to NMN⁺. Consequently, NMN⁺ is reduced during glycolysis and can only be regenerated in the product formation pathway. Thus, these electrons will exclusively be directed from substrate to product.

NMN⁺ levels should be sufficient for the use as electron acceptor. In E. coli, NAD⁺ is degraded to NMN⁺ by DNA ligases. Natural cellular NMN⁺ levels in E. coli are too low to function as electron acceptor. Thus, the metabolic NMN⁺ pathway needs to be adjusted first.

Description

This challenging project aims to enhance cellular NMN⁺ levels in E. coli so that it can be used as NRC. First, we use metabolic engineering to increase NMN⁺ synthesis. Later in this project, all components required to implement NMN⁺ as NRC will be integrated in E. coli.

To achieve these objectives, heterologous genes will be introduced and some native genes knocked out. In addition, expression of certain genes needs to be fine-tuned for optimal functioning of the metabolic pathways.

The bacterial strains will be engineered using molecular biology techniques, including CRISPR-Cas guided engineering and Golden Gate assembly. The engineered strains will be characterized with different cultivation experiments. Finally, analytical methods will be used such as HPLC, UPLC-MS, but also biosensors to measure NAD⁺ and NMN⁺ levels.

Publications

• Weusthuis, R. A., Folch, P. L., Pozo-Rodríguez, A., & Paul, C. E. (2020). Applying Non-canonical Redox Cofactors in Fermentation Processes. IScience, 23(9), 101471.