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Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols.

ABSTRACT: The predominant bacterial pathway for nitrobenzene (NB) degradation uses an NB nitroreductase and hydroxylaminobenzene (HAB) mutase to form the ring-fission substrate ortho-aminophenol. We tested the hypothesis that constructed strains might accumulate the aminophenols from nitroacetophenones and other nitroaromatic compounds. We constructed a recombinant plasmid carrying NB nitroreductase (nbzA) and HAB mutase A (habA) genes, both from Pseudomonas pseudoalcaligenes JS45, and expressed the enzymes in Escherichia coli JS995. IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cells of strain JS995 rapidly and stoichiometrically converted NB to 2-aminophenol, 2-nitroacetophenone (2NAP) to 2-amino-3-hydroxyacetophenone (2AHAP), and 3-nitroacetophenone (3NAP) to 3-amino-2-hydroxyacetophenone (3AHAP). We constructed another recombinant plasmid containing the nitroreductase gene (nfs1) from Enterobacter cloacae and habA from strain JS45 and expressed the enzymes in E. coli JS996. Strain JS996 converted NB to 2-aminophenol, 2-nitrotoluene to 2-amino-3-methylphenol, 3-nitrotoluene to 2-amino-4-methylphenol, 4-nitrobiphenyl ether to 4-amino-5-phenoxyphenol, and 1-nitronaphthalene to 2-amino-1-naphthol. In larger-scale biotransformations catalyzed by strain JS995, 75% of the 2NAP transformed was converted to 2AHAP, whereas 3AHAP was produced stoichiometrically from 3NAP. The final yields of the aminophenols after extraction and recovery were >64%. The biocatalytic synthesis of ortho-aminophenols from nitroacetophenones suggests that strain JS995 may be useful in the biocatalytic production of a variety of substituted ortho-aminophenols from the corresponding nitroaromatic compounds.

SUBMITTER: Kadiyala V

PROVIDER: S-EPMC262294 | biostudies-literature | 2003 Nov

REPOSITORIES: biostudies-literature

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Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols.

Kadiyala Venkateswarlu V Nadeau Lloyd J LJ Spain Jim C JC

Applied and environmental microbiology 20031101 11

The predominant bacterial pathway for nitrobenzene (NB) degradation uses an NB nitroreductase and hydroxylaminobenzene (HAB) mutase to form the ring-fission substrate ortho-aminophenol. We tested the hypothesis that constructed strains might accumulate the aminophenols from nitroacetophenones and other nitroaromatic compounds. We constructed a recombinant plasmid carrying NB nitroreductase (nbzA) and HAB mutase A (habA) genes, both from Pseudomonas pseudoalcaligenes JS45, and expressed the enzym ...[more]

PMID: 14602609

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Project description:BACKGROUND: The trisaccharide 2'-fucosyllactose (2'-FL) is one of the most abundant oligosaccharides found in human milk. Due to its prebiotic and anti-infective properties, 2'-FL is discussed as nutritional additive for infant formula. Besides chemical synthesis and extraction from human milk, 2'-FL can be produced enzymatically in vitro and in vivo. The most promising approach for a large-scale formation of 2'-FL is the whole cell biosynthesis in Escherichia coli by intracellular synthesis of GDP-L-fucose and subsequent fucosylation of lactose with an appropriate ?1,2-fucosyltransferase. Even though whole cell approaches have been demonstrated for the synthesis of 2'-FL, further improvements of the engineered E. coli host are required to increase product yields. Furthermore, an antibiotic-free method of whole cell synthesis of 2'-FL is desirable to simplify product purification and to avoid traces of antibiotics in a product with nutritional purpose. RESULTS: Here we report the construction of the first selection marker-free E. coli strain that produces 2'-FL from lactose and glycerol. To construct this strain, recombinant genes of the de novo synthesis pathway for GDP-L-fucose as well as the gene for the H. pylori fucosyltransferase futC were integrated into the chromosome of E. coli JM109 by using the ?-Red recombineering technique. Strains carrying additional copies of the futC gene and/or the gene fkp (from Bacteroides fragilis) for an additional salvage pathway for GDP-L-fucose production were used and shown to further improve production of 2'-FL in shake flask experiments. An increase of the intracellular GDP-L-fucose concentration by expression of fkp gene as well as an additional copy of the futC gene lead to an enhanced formation of 2'-FL. Using an improved production strain, feasibility of large scale 2'-FL production was demonstrated in an antibiotic-free fed-batch fermentation (13 l) with a final 2'-FL concentration of 20.28 ± 0.83 g l(-1) and a space-time-yield of 0.57 g l(-1) h(-1). CONCLUSIONS: By chromosomal integration of recombinant genes, altering the copy number of these genes and analysis of 2'-FL and intracellular GDP-L-fucose levels, we were able to construct and improve the first selection marker-free E. coli strain which is capable to produce 2'-FL without the use of expression plasmids. Analysis of intracellular GDP-L-fucose levels identified the de novo synthesis pathway of GDP-L-fucose as one bottleneck in 2'-FL production. In antibiotic-free fed-batch fermentation with an improved strain, scale-up of 2'-FL could be demonstrated.

Dataset Information

Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols.

Publications

Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols.

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