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. 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. 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.

中文翻译：

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大肠杆菌的代谢工程，用于优化烟酰胺单核苷酸（一种非经典氧化还原辅因子）的生物合成。


非经典氧化还原辅助因子正在成为无细胞生物合成中的重要工具，以提高经济可行性、实现精确控制并扩大可利用的化学范围。然而，这些非规范氧化还原辅因子需要进行生物合成，以实现与可再生生物制造过程的完全整合。在这项工作中，我们改造了大肠杆菌细胞来生物合成非经典辅因子烟酰胺单核苷酸（NMN+），该物质已被有效地用于无细胞生物合成。首先，我们开发了一个基于生长的筛选平台，以确定大肠杆菌中有效的 NMN+ 生物合成途径。其次，我们探索了各种途径组合和宿主基因破坏，以在最佳菌株中实现约 1.5 mM NMN+ 的细胞内水平，比细胞基础水平增加 130 倍，该菌株具有以前未表征的来自 Ralstonia 的烟酰胺磷酸核糖基转移酶 (NadV)青枯菌。最后，我们揭示了 NMN+ 积累影响大肠杆菌细胞适应性的机制，这为未来旨在提高这种非经典氧化还原辅因子产量的工作提供了线索。这些结果进一步加深了对 NMN+ 有效生产和整合到大肠杆菌中的理解。这可能使得 NMN+ 定向生物催化的实施无需外源辅因子供应。