Abstract
Background2-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.ResultIn 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.ConclusionIn 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.
Highlights
Aromatic compounds are versatile chemicals used in chemicals, foods, pharmaceuticals, materials, and etc. [1,2,3]
Most of them are derived from benzene, toluene, xylene and manufactured from petroleum [2, 4, 5]. 2-Acetamidophenol (AAP), known as N-(2-hydroxyphenyl)-acetamide or O-acetaminophenol, is an aromatic compound derived from salicylic acid [6]
Inactivation of gene phzB in P. chlororaphis HT66 Our previous study reported the function of gene phzA in the biosynthesis of phenazine-1,6-dicarboxylic acid in P. chlororaphis HT66 [32]
Summary
Aromatic compounds are versatile chemicals used in chemicals, foods, pharmaceuticals, materials, and etc. [1,2,3]. 2-Acetamidophenol (AAP), known as N-(2-hydroxyphenyl)-acetamide or O-acetaminophenol, is an aromatic compound derived from salicylic acid [6]. Owing to its potential antifungal, anti-inflammatory, antitumor, anti-platelet, and anti-arthritic activities [7,8,9,10], and because it is less toxic than aspirin, AAP has been widely used in the pharmaceutical industry, therapeutic applications and synthetic chemistry [11,12,13]. Since the biosynthesis of AAP is not yet fully understood, AAP is mainly produced by chemical synthesis [8]. Metabolic engineering of natural microbial pathway to produce valuable aromatic compounds has remarkable advantages and exhibits attractive potential recently [1,2,3,4,5, 14]. It is of importance to develop a dominant strain to produce AAP by elucidating the AAP biosynthesis pathway
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