BACKGROUND 2-Acetamidophenol (AAP) is an aromatic compound with the potential for antifungal, anti-inflammatory, antitumor, anti-platelet, and anti-arthritic activities. Due to the biosynthesis of AAP is not yet fully understood, AAP is mainly produced by chemical synthesis. Currently, metabolic engineering of natural microbial pathway to produce valuable aromatic compound has remarkable advantages and exhibits attractive potential. Thus, it is of paramount importance to develop a dominant strain to produce AAP by elucidating the AAP biosynthesis pathway. RESULT In this study, the active aromatic compound AAP was first purified and identified in gene phzB disruption strain HT66ΔphzB, which was derived from Pseudomonas chlororaphis HT66. The titer of AAP in the strain HT66ΔphzB was 236.89 mg/L. Then, the genes involved in AAP biosynthesis were determined. Through the deletion of genes phzF, Nat and trpE, AAP was confirmed to have the same biosynthesis route as phenazine-1-carboxylic (PCA). Moreover, a new arylamine N-acetyltransferases (NATs) was identified and proved to be the key enzyme required for generating AAP by in vitro assay. P. chlororaphis P3, a chemical mutagenesis mutant strain of HT66, has been demonstrated to have a robust ability to produce antimicrobial phenazines. Therefore, genetic engineering, precursor addition, and culture optimization strategies were used to enhance AAP production in P. chlororaphis P3. The inactivation of phzB in P3 increased AAP production by 92.4%. Disrupting the phenazine negative regulatory genes lon and rsmE and blocking the competitive pathway gene pykA in P3 increased AAP production 2.08-fold, which also confirmed that AAP has the same biosynthesis route as PCA. Furthermore, adding 2-amidophenol to the KB medium increased AAP production by 64.6%, which suggested that 2-amidophenol is the precursor of AAP. Finally, by adding 5 mM 2-amidophenol and 2 mM Fe3+ to the KB medium, the production of AAP reached 1209.58 mg/L in the engineered strain P3ΔphzBΔlonΔpykAΔrsmE using a shaking-flask culture. This is the highest microbial-based AAP production achieved to date. CONCLUSION In conclusion, this study clarified the biosynthesis process of AAP in Pseudomonas and provided a promising host for industrial-scale biosynthesis of AAP from renewable resources.

中文翻译：

---

鉴定新的芳基胺N-乙酰基转移酶并增强假单胞菌HT66中2-乙酰氨基苯酚的生产。

背景技术2-乙酰胺基苯酚（AAP）是具有抗真菌，抗炎，抗肿瘤，抗血小板和抗关节炎活性的潜力的芳香族化合物。由于尚未完全了解AAP的生物合成，因此AAP主要通过化学合成来生产。当前，天然微生物途径的代谢工程以产生有价值的芳族化合物具有显着的优势并且展现出诱人的潜力。因此，通过阐明AAP的生物合成途径，开发出能产生AAP的显性菌株至关重要。结果在本研究中，活性芳香族化合物AAP首次在phzB破坏株HT66ΔphzB中得到了纯化和鉴定，该菌株来源于绿假单胞菌HT66。HT66ΔphzB菌株中AAP的效价为236.89 mg / L。然后，确定了参与AAP生物合成的基因。通过删除phzF，Nat和trpE基因，证实AAP具有与吩嗪-1-羧酸（PCA）相同的生物合成途径。此外，鉴定了一种新的芳基胺N-乙酰基转移酶（NATs），并证明它是通过体外测定生成AAP所需的关键酶。业已证明HT66的化学诱变突变株P. chlororaphis P3具有强大的抗微生物吩嗪生产能力。因此，基因工程，前体添加和培养优化策略被用来增强P. chlororaphis P3中的AAP生产。P3中phzB的失活使AAP产生增加了92.4％。破坏吩嗪阴性调节基因lon和rsmE并阻断P3中的竞争途径基因pykA使AAP产量增加2.08倍，这也证实了AAP具有与PCA相同的生物合成途径。此外，在KB培养基中添加2-酰胺基苯酚可使AAP产量增加64.6％，这表明2-酰胺基苯酚是AAP的前体。最后，通过向烧瓶培养基中加入5 mM 2-酰胺基酚和2 mM Fe3 +，在工程菌株P3ΔphzBΔlonΔpykAΔrsmE中使用摇瓶培养法生产的AAP达到1209.58 mg / L。这是迄今为止实现的最高的基于微生物的AAP生产。结论总之，本研究阐明了假单胞菌中AAP的生物合成过程，并为工业规模的可再生资源AAP生物合成提供了有希望的宿主。这表明2-酰胺基苯酚是AAP的前体。最后，通过向烧瓶培养基中加入5 mM 2-酰胺基酚和2 mM Fe3 +，在工程菌株P3ΔphzBΔlonΔpykAΔrsmE中使用摇瓶培养法生产的AAP达到1209.58 mg / L。这是迄今为止实现的最高的基于微生物的AAP生产。结论总之，本研究阐明了假单胞菌中AAP的生物合成过程，并为工业规模的可再生资源AAP的生物合成提供了有希望的宿主。这表明2-酰胺基苯酚是AAP的前体。最后，通过向烧瓶培养基中加入5 mM 2-酰胺基酚和2 mM Fe3 +，在工程菌株P3ΔphzBΔlonΔpykAΔrsmE中使用摇瓶培养法生产的AAP达到1209.58 mg / L。这是迄今为止实现的最高的基于微生物的AAP生产。结论总之，本研究阐明了假单胞菌中AAP的生物合成过程，并为工业规模的可再生资源AAP的生物合成提供了有希望的宿主。