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Bacterial fermentation platform for producing artificial aromatic amines.

Masuo S, Zhou S, Kaneko T, Takaya N - Sci Rep (2016)

Bottom Line: We designed and produced artificial pathways that mimicked the fungal Ehrlich pathway in E. coli and converted 4-aminophenylalanine into 4-aminophenylethanol and 4-aminophenylacetate at 90% molar yields.Combining these conversion systems or fungal phenylalanine decarboxylases, the 4-aminophenylalanine-producing platform fermented glucose to 4-aminophenylethanol, 4-aminophenylacetate, and 4-phenylethylamine.This original bacterial platform for producing artificial aromatic amines highlights their potential as heteroatoms containing bio-based materials that can replace those derived from petroleum.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.

ABSTRACT
Aromatic amines containing an aminobenzene or an aniline moiety comprise versatile natural and artificial compounds including bioactive molecules and resources for advanced materials. However, a bio-production platform has not been implemented. Here we constructed a bacterial platform for para-substituted aminobenzene relatives of aromatic amines via enzymes in an alternate shikimate pathway predicted in a Pseudomonad bacterium. Optimization of the metabolic pathway in Escherichia coli cells converted biomass glucose to 4-aminophenylalanine with high efficiency (4.4 g L(-1) in fed-batch cultivation). We designed and produced artificial pathways that mimicked the fungal Ehrlich pathway in E. coli and converted 4-aminophenylalanine into 4-aminophenylethanol and 4-aminophenylacetate at 90% molar yields. Combining these conversion systems or fungal phenylalanine decarboxylases, the 4-aminophenylalanine-producing platform fermented glucose to 4-aminophenylethanol, 4-aminophenylacetate, and 4-phenylethylamine. This original bacterial platform for producing artificial aromatic amines highlights their potential as heteroatoms containing bio-based materials that can replace those derived from petroleum.

No MeSH data available.


Related in: MedlinePlus

Production of 4APE and 4APAA.(a,b) Bioconversion of 4APhe to 4APE and 4APAA by E. coli BL21 Star (DE3) harboring either pRSF-aro10 (a) or pRSF-aro10ald2 (b). After incubation with 0.1 mM IPTG, cells were washed twice and incubated in reaction buffer containing 3.6 g L−1 4APhe. Error bars indicate standard deviation (n = 3). (c, d) HPLC chromatogram of culture supernatant of NDPG harboring pRSF-aro10 (c) and pRSF-aro10ald2 (d). Cells were shaken in fermentation medium containing 5 g L−1 tryptone, 2.5 g L−1 yeast extract and 10 g L−1 glucose in flasks at 30 °C, induced with 0.1 mM IPTG, and cultured for 36 h.
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f4: Production of 4APE and 4APAA.(a,b) Bioconversion of 4APhe to 4APE and 4APAA by E. coli BL21 Star (DE3) harboring either pRSF-aro10 (a) or pRSF-aro10ald2 (b). After incubation with 0.1 mM IPTG, cells were washed twice and incubated in reaction buffer containing 3.6 g L−1 4APhe. Error bars indicate standard deviation (n = 3). (c, d) HPLC chromatogram of culture supernatant of NDPG harboring pRSF-aro10 (c) and pRSF-aro10ald2 (d). Cells were shaken in fermentation medium containing 5 g L−1 tryptone, 2.5 g L−1 yeast extract and 10 g L−1 glucose in flasks at 30 °C, induced with 0.1 mM IPTG, and cultured for 36 h.

Mentions: The fungal Ehrlich pathway deaminates phenylalanine to phenylpyruvate, which phenylpyruvate decarboxylase (PDC) then converts to phenylacetaldehyde. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) reduce and oxidize phenylacetaldehyde to 2-phenylethanol and to phenylacetic acid, respectively22. Artificial pathways mimicking the Ehrlich pathway were designed to produce 4APE and 4APAA (Fig. 1) via pathway enzymes, although a 4-amino-substituted AA has never been assessed as an Ehrlich pathway intermediate. We constructed recombinant E. coli BL21 Star (DE3) producing ARO10 encoding PDC from the fungus S. cerevisiae22, and examined the bioconversion of 4APhe in resting cells. The reaction consumed 1.8 g L1 (10 mM) of 4APhe to generate 1.2 g L−1 of 4APE with a 90% molar yield (Table 1), implying that PDC efficiently produced 4-aminophenylacetaldehyde, which endogenous E. coli ADH reduced to 4APE. The resting E. coli BL21 Star (DE3) cells producing both ARO10 and ALDH encoding ALD322 converted 1.8 g L−1 of 4APhe to 0.9 g L−1 of 4APAA with a 63% molar yield (Table 1). The strain produced 0.4 g L−1 4APE as a byproduct, indicating that the dehydrogenation of 4-aminophenylacetaldehyde limits 4APAA production. Replacing ALD3 with E. coli padA23 and S. cerevisiae ALD222 in the bacterium expressing ARO10 and ALD3 increased the yields of 4APAA to 85% and 90%, respectively (Table 1). We also combined PDC, Pichia pastoris PpARO1024 or Aspergillus oryzae ppdA25 with ALD3, but they produced less 4APAA than those expressing ARO10 and ALD3 (Table 1). These results indicated that the heterologous expression of ARO10 alone and of the ARO10 and ALD2 set converted 4APhe to 4APE and APAA the most efficiently. Figure 4a,b shows the time-dependent bioconversion of 4APhe (3.6 g L−1) in the optimized systems that produced 2.8 g L−1 4APE and 2.7 g L−1 4APAA.


Bacterial fermentation platform for producing artificial aromatic amines.

Masuo S, Zhou S, Kaneko T, Takaya N - Sci Rep (2016)

Production of 4APE and 4APAA.(a,b) Bioconversion of 4APhe to 4APE and 4APAA by E. coli BL21 Star (DE3) harboring either pRSF-aro10 (a) or pRSF-aro10ald2 (b). After incubation with 0.1 mM IPTG, cells were washed twice and incubated in reaction buffer containing 3.6 g L−1 4APhe. Error bars indicate standard deviation (n = 3). (c, d) HPLC chromatogram of culture supernatant of NDPG harboring pRSF-aro10 (c) and pRSF-aro10ald2 (d). Cells were shaken in fermentation medium containing 5 g L−1 tryptone, 2.5 g L−1 yeast extract and 10 g L−1 glucose in flasks at 30 °C, induced with 0.1 mM IPTG, and cultured for 36 h.
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Related In: Results  -  Collection

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f4: Production of 4APE and 4APAA.(a,b) Bioconversion of 4APhe to 4APE and 4APAA by E. coli BL21 Star (DE3) harboring either pRSF-aro10 (a) or pRSF-aro10ald2 (b). After incubation with 0.1 mM IPTG, cells were washed twice and incubated in reaction buffer containing 3.6 g L−1 4APhe. Error bars indicate standard deviation (n = 3). (c, d) HPLC chromatogram of culture supernatant of NDPG harboring pRSF-aro10 (c) and pRSF-aro10ald2 (d). Cells were shaken in fermentation medium containing 5 g L−1 tryptone, 2.5 g L−1 yeast extract and 10 g L−1 glucose in flasks at 30 °C, induced with 0.1 mM IPTG, and cultured for 36 h.
Mentions: The fungal Ehrlich pathway deaminates phenylalanine to phenylpyruvate, which phenylpyruvate decarboxylase (PDC) then converts to phenylacetaldehyde. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) reduce and oxidize phenylacetaldehyde to 2-phenylethanol and to phenylacetic acid, respectively22. Artificial pathways mimicking the Ehrlich pathway were designed to produce 4APE and 4APAA (Fig. 1) via pathway enzymes, although a 4-amino-substituted AA has never been assessed as an Ehrlich pathway intermediate. We constructed recombinant E. coli BL21 Star (DE3) producing ARO10 encoding PDC from the fungus S. cerevisiae22, and examined the bioconversion of 4APhe in resting cells. The reaction consumed 1.8 g L1 (10 mM) of 4APhe to generate 1.2 g L−1 of 4APE with a 90% molar yield (Table 1), implying that PDC efficiently produced 4-aminophenylacetaldehyde, which endogenous E. coli ADH reduced to 4APE. The resting E. coli BL21 Star (DE3) cells producing both ARO10 and ALDH encoding ALD322 converted 1.8 g L−1 of 4APhe to 0.9 g L−1 of 4APAA with a 63% molar yield (Table 1). The strain produced 0.4 g L−1 4APE as a byproduct, indicating that the dehydrogenation of 4-aminophenylacetaldehyde limits 4APAA production. Replacing ALD3 with E. coli padA23 and S. cerevisiae ALD222 in the bacterium expressing ARO10 and ALD3 increased the yields of 4APAA to 85% and 90%, respectively (Table 1). We also combined PDC, Pichia pastoris PpARO1024 or Aspergillus oryzae ppdA25 with ALD3, but they produced less 4APAA than those expressing ARO10 and ALD3 (Table 1). These results indicated that the heterologous expression of ARO10 alone and of the ARO10 and ALD2 set converted 4APhe to 4APE and APAA the most efficiently. Figure 4a,b shows the time-dependent bioconversion of 4APhe (3.6 g L−1) in the optimized systems that produced 2.8 g L−1 4APE and 2.7 g L−1 4APAA.

Bottom Line: We designed and produced artificial pathways that mimicked the fungal Ehrlich pathway in E. coli and converted 4-aminophenylalanine into 4-aminophenylethanol and 4-aminophenylacetate at 90% molar yields.Combining these conversion systems or fungal phenylalanine decarboxylases, the 4-aminophenylalanine-producing platform fermented glucose to 4-aminophenylethanol, 4-aminophenylacetate, and 4-phenylethylamine.This original bacterial platform for producing artificial aromatic amines highlights their potential as heteroatoms containing bio-based materials that can replace those derived from petroleum.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.

ABSTRACT
Aromatic amines containing an aminobenzene or an aniline moiety comprise versatile natural and artificial compounds including bioactive molecules and resources for advanced materials. However, a bio-production platform has not been implemented. Here we constructed a bacterial platform for para-substituted aminobenzene relatives of aromatic amines via enzymes in an alternate shikimate pathway predicted in a Pseudomonad bacterium. Optimization of the metabolic pathway in Escherichia coli cells converted biomass glucose to 4-aminophenylalanine with high efficiency (4.4 g L(-1) in fed-batch cultivation). We designed and produced artificial pathways that mimicked the fungal Ehrlich pathway in E. coli and converted 4-aminophenylalanine into 4-aminophenylethanol and 4-aminophenylacetate at 90% molar yields. Combining these conversion systems or fungal phenylalanine decarboxylases, the 4-aminophenylalanine-producing platform fermented glucose to 4-aminophenylethanol, 4-aminophenylacetate, and 4-phenylethylamine. This original bacterial platform for producing artificial aromatic amines highlights their potential as heteroatoms containing bio-based materials that can replace those derived from petroleum.

No MeSH data available.


Related in: MedlinePlus