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Development of an Orthogonal Cofactor System

  • Author(s): Black, William
  • Advisor(s): Li, Han
  • et al.
No data is associated with this publication.
Creative Commons 'BY' version 4.0 license

Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP+), but these cofactors are expensive to use in vitro and difficult to control in vivo. Recently, the use of noncanonical redox cofactors, or cofactor mimics, has emerged as a less expensive alternative to native cofactors in enzymatic biotransformation. While these noncanonical cofactors have shown promising results with reductive biotransformation, regeneration of spent cofactor and integration of these cofactors in biological systems has been met with limited success. In this work, we demonstrate the development and application of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN+).

First, using a computationally designed glucose dehydrogenase with high cofactor specificity towards NMN+, we construct an NMN+-cycling system to support diverse redox chemistries in vitro with a high total turnover number (~39,000) and temporal stability. Then we introduced this system into Escherichia coli whole cells, demonstrating the ability of this system to channel reducing power specifically in vivo from glucose to levodione, a pharmaceutical intermediate.

Subsequently, we applied the NMN(H)-cycling system for the production of the terpenoid aldehyde citronellal in crude lysate- and whole cell-based biotransformation. By specifically delivering reducing power to a recombinant enoate reductase, but not to endogenous ADHs, we convert citral to citronellal with minimal byproduct formation (97−100% product purity in crude lysate-based biotransformation). Using knowledge gained from rapidly prototyping the crude-lysate system expression ratios, we translated the system into whole cells, achieving a product purity of 83%. Remarkably, this was achieved without the need to disrupt any of the endogenous alcohol dehydrogenases.

Ultimately, the ability to perform crude lysate- and whole cell-based biotransformation without the need to supplement NMN+ and to generate NMN+ renewably for purified protein-based biotransformation is desired. We next engineered E. coli cells to biosynthesize NMN+. First, we developed a growth-based screening platform to identify effective NMN+ biosynthetic pathways in E. coli. Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~ 1.5 mM NMN+, a 130-fold increase over the cell’s basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN+ accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor.

Finally, we expanded the breadth of our orthogonal NMN(H)-cycling system by integrating it with Saccharomyces cerevisiae. Using the aforementioned production of citronellal as a model system, we demonstrated that our orthogonal redox cofactor system translates well across organisms, producing 28 mg/L of citronellal while the activity of endogenous alcohol dehydrogenases in the cellular background remained low. Furthermore, we engineered S. cerevisiae cells to accumulate 188 μM of NMN+ by integrating the NMN+ synthetase, NadE*, and phosphoribosyltransferase, NadV, from Francisella tularensis. Due to the cell wall of S. cerevisiae being resistant to the free diffusion of NMN+ into the cell, pairing the NMN+ accumulating strain with the NMN(H)-cycling system may enable the first example of an in vivo noncanonical redox cofactor system which does not require the supplementation of cofactor into the reaction system.

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This item is under embargo until December 15, 2022.