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bioRxiv · 10.1101/2020.05.11.089011

Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor

Black, W. B.·Aspacio, D.·Bever, D.·King, E.·Zhang, L.·Li, H.
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Abstract

Background Noncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes.Results In this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN+), which has been efficiently used in cell-free biosynthesis. 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.Conclusion These results further the understanding of effective production and integration of NMN+ into E. coli. This may enable the implementation of NMN+-directed biocatalysis without the need for exogenous cofactor supply.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsNAD+nicotinamide adenine dinucleotideNADP+nicotinamide adenine dinucleotide phosphateP2NA+3-carbomoyl-1-phenethylpyridin-1-ium chlorideNMN+nicotinamide mononucleotideNadVnicotinamide phosphoribosyltransferasesNadE*nicotinamide mononucleotide synthasePncCnicotinamide mononucleotideNaMN+nicotinic acid mononucleotideNRnicotinamide ribosidePnuCnicotinamide riboside transporterPnuC*mutant nicotinamide riboside transporterNrk1nicotinamide riboside kinase from Saccharomyces cerevisiaeNadRnicotinamide riboside kinase (Salmonella enterica)NAnicotinamideLC-MSliquid chromatography-mass spectrometryNaADnicotinic acid adenine dinucleotidePCRpolymerase chain reactionIPTGisopropyl-β-D-thiogalactopyranosider.p.m.rotations per minuteView Full Text

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Black, W. B., Aspacio, D., Bever, D., King, E., Zhang, L., Li, H.. 2020-05-13. Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor. https://doi.org/10.1101/2020.05.11.089011

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