Phonbuppha, J. | Search bioRxiv

bioRxiv2023

A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase levels of a pool of sugar-phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells; demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase, GDH) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology. TeaserUse of sugar and sugar reductase to increase sugar phosphates for enhancing in situ synthesis of cofactors upon cellular demand for synthetic biology.

synthetic biology↗

bioRxiv2021

High Sensitivity and Low-Cost Flavin luciferase (FLUX)-based Reporter Gene for Mammalian Cell Expression

Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most common, and thus most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase for Mammalian Cell Expression (FLUX) by engineering luciferase from Vibrio campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells. We found that the FLUX reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUX/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We demonstrated that FLUX can be used for testing inhibitors of the NF-{kappa}B signaling pathway, validating FLUX applications for various assays in the future.

biochemistry↗