Abstract
Aromatic amines are an important class of chemicals which are used as building blocks for the synthesis of polymers and pharmaceuticals. In this study we establish a de novo pathway for the biosynthesis of the aromatic amines para-amino-phenylethanol (PAPE) and para-amino-phenylacetic acid (4-APA) in Escherichia coli. We combined a synthetic para-amino-l-phenylalanine pathway with the fungal Ehrlich pathway. Therefore, we overexpressed the heterologous genes encoding 4-amino-4-deoxychorismate synthase (pabAB from Corynebacterium glutamicum), 4-amino-4-deoxychorismate mutase and 4-amino-4-deoxyprephenate dehydrogenase (papB and papC from Streptomyces venezuelae) and ThDP-dependent keto-acid decarboxylase (aro10 from Saccharomyces cerevisiae) in E. coli. The resulting para-amino-phenylacetaldehyde either was reduced to PAPE or oxidized to 4-APA. The wild type strain E. coli LJ110 with a plasmid carrying these four genes produced (in shake flask cultures) 11 ± 1.5 mg l−1 of PAPE from glucose (4.5 g l−1). By the additional cloning and expression of feaB (phenylacetaldehyde dehydrogenase from E. coli) 36 ± 5 mg l−1 of 4-APA were obtained from 4.5 g l−1 glucose. Competing reactions, such as the genes for aminotransferases (aspC and tyrB) or for biosynthesis of L-phenylalanine and L-tyrosine (pheA, tyrA) and for the regulator TyrR were removed. Additionally, the E. coli genes aroFBL were cloned and expressed from a second plasmid. The best producer strains of E. coli showed improved formation of PAPE and 4-APA, respectively. Plasmid-borne expression of an aldehyde reductase (yahK from E. coli) gave best values for PAPE production, whereas feaB-overexpression led to best values for 4-APA. In fed-batch cultivation, the best producer strains achieved 2.5 ± 0.15 g l−1 of PAPE from glucose (11% C mol mol-1 glucose) and 3.4 ± 0.3 g l−1 of 4-APA (17% C mol mol−1 glucose), respectively which are the highest values for recombinant strains reported so far.
Highlights
Aromatic amines (AA) are an important class of chemicals of which several are already used and characterized for different industrial applications
From this observation we reasoned that PAPE and 4-APA are compounds to be produced with recombinant E. coli strains without extreme toxicity to the producer cells
Takaya and coworkers have studied the microbial biosynthesis of PAPE and 4-APA with recombinant E. coli strains (Masuo et al, 2016)
Summary
Aromatic amines (AA) are an important class of chemicals of which several are already used and characterized for different industrial applications These chemicals can be used for the manufacturing of dyes, pesticides, drugs, plastics, semi-conductive or conductive polymers, and other industrial products (Lawrence, 2004; Vogt and Gerulis, 2005; Sousa et al, 2013; Arora, 2015; Masuo et al, 2016; Tateyama et al, 2016; Tsuge et al, 2016; Kawasaki et al, 2018). The AA para-amino-phenylethanol (PAPE) (see Figure 1), an analog of 2-phenylethanol which has a rose-like odor (Etschmann et al, 2002), is an important building block for chemical syntheses. A classical synthesis of aromatic amines is performed by nitroreduction of aromatic compounds in the presence of acidic compounds and a reducing agent, such as solid metals (Vogt and Gerulis, 2005; Schul’tsev, 2011; Froidevaux et al, 2016)
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