代谢工程改造大肠杆菌高效合成烟酰胺核糖

Metabolic engineering of Escherichia coli for efficient production of nicotinamide riboside

摘要:

烟酰胺核糖(nicotinamide riboside, NR)是烟酰胺腺嘌呤二核苷酸(NAD?)的重要前体分子，因其具有抗衰老活性及多种药理活性而受到广泛关注。为了开发一种高效、可持续且环境友好的合成方法，本研究基于模块化代谢工程策略，构建了一株以烟酰胺为底物高效合成NR的工程菌株。首先，通过敲除rihA等6个基因，阻断了NR及其关键中间体的降解途径；其次，引入关键酶BaPrs^L135I和NR转运蛋白MdtL，显著提高了NR的积累与外排能力；随后敲除pncA优化了烟酰胺利用效率；进一步通过解除purR介导的反馈调控并增强磷酸戊糖途径，有效提高前体物质的供给能力。在上述多模块协同优化的基础上引入毒素-抗毒素系统，在无抗生素添加的条件下，工程菌株NK22在摇瓶发酵36 h时NR产量达到3.84 g/L；在5 L补料分批发酵罐中，NR产量达到27.23 g/L。本研究创制得到的发酵过程中无需添加抗生素的新一代NR生产菌株，为高效、可持续的NR及其高值衍生物微生物细胞工厂构建提供了思路与参考。

Abstract:

Nicotinamide riboside (NR), an essential precursor for nicotinamide adenine dinucleotide (NAD⁺), has garnered increasing attention for its anti-aging properties and diverse pharmacological activities. Developing an efficient, sustainable, and environmentally friendly synthesis approach is of great significance for the industrial production of NR. In this study, we developed a modular metabolic engineering strategy to construct an engineered strain capable of synthesizing NR from nicotinamide efficiently. First, six genes, including rihA, were deleted to block the degradation pathways of NR and its key intermediates. Second, the key enzyme BaPrs^L135I and the NR transporter MdtL were introduced, significantly enhancing NR accumulation and extracellular export. Subsequently, pncA was deleted to improve the nicotinamide utilization efficiency. Furthermore, precursor supply was substantially increased by relieving purR-mediated feedback regulation and strengthening the pentose phosphate pathway. Through the coordinated optimization of these modules and introduction of the toxin-antitoxin system, the engineered strain NK22 produced 3.84 g/L NR after 36 h of shake-flask fermentation. Moreover, the strain achieved a final NR titer of 27.23 g/L in a 5 L fed-batch bioreactor under antibiotic-free conditions. This study establishes a next-generation NR-producing strain capable of antibiotic-free fermentation and provides a robust framework for the development of efficient and sustainable microbial cell factories for NR and its high-value derivatives.