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Production of p-amino-l-phenylalanine (l-PAPA) from glycerol by metabolic grafting of Escherichia coli
BACKGROUND: The non-proteinogenic aromatic amino acid, p-amino-l-phenylalanine (l-PAPA) is a high-value product with a broad field of applications. In nature, l-PAPA occurs as an intermediate of the chloramphenicol biosynthesis pathway in Streptomyces venezuelae. Here we demonstrate that the model o...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148955/ https://www.ncbi.nlm.nih.gov/pubmed/30241531 http://dx.doi.org/10.1186/s12934-018-0996-6 |
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author | Mohammadi Nargesi, Behrouz Trachtmann, Natalie Sprenger, Georg A. Youn, Jung-Won |
author_facet | Mohammadi Nargesi, Behrouz Trachtmann, Natalie Sprenger, Georg A. Youn, Jung-Won |
author_sort | Mohammadi Nargesi, Behrouz |
collection | PubMed |
description | BACKGROUND: The non-proteinogenic aromatic amino acid, p-amino-l-phenylalanine (l-PAPA) is a high-value product with a broad field of applications. In nature, l-PAPA occurs as an intermediate of the chloramphenicol biosynthesis pathway in Streptomyces venezuelae. Here we demonstrate that the model organism Escherichia coli can be transformed with metabolic grafting approaches to result in an improved l-PAPA producing strain. RESULTS: Escherichia coli K-12 cells were genetically engineered for the production of l-PAPA from glycerol as main carbon source. To do so, genes for a 4-amino-4-deoxychorismate synthase (pabAB from Corynebacterium glutamicum), and genes encoding a 4-amino-4-deoxychorismate mutase and a 4-amino-4-deoxyprephenate dehydrogenase (papB and papC, both from Streptomyces venezuelae) were cloned and expressed in E. coli W3110 (lab strain LJ110). In shake flask cultures with minimal medium this led to the formation of ca. 43 ± 2 mg l(−1) of l-PAPA from 5 g l(−1) glycerol. By expression of additional chromosomal copies of the tktA and glpX genes, and of plasmid-borne aroFBL genes in a tyrR deletion strain, an improved l-PAPA producer was obtained which gave a titer of 5.47 ± 0.4 g l(−1) l-PAPA from 33.3 g l(−1) glycerol (0.16 g l-PAPA/g of glycerol) in fed-batch cultivation (shake flasks). Finally, in a fed-batch fermenter cultivation, a titer of 16.7 g l(−1) l-PAPA was obtained which is the highest so far reported value for this non-proteinogenic amino acid. CONCLUSION: Here we show that E. coli is a suitable chassis strain for l-PAPA production. Modifying the flux to the product and improved supply of precursor, by additional gene copies of glpX, tkt and aroFBL together with the deletion of the tyrR gene, increased the yield and titer. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-018-0996-6) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6148955 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-61489552018-09-24 Production of p-amino-l-phenylalanine (l-PAPA) from glycerol by metabolic grafting of Escherichia coli Mohammadi Nargesi, Behrouz Trachtmann, Natalie Sprenger, Georg A. Youn, Jung-Won Microb Cell Fact Research BACKGROUND: The non-proteinogenic aromatic amino acid, p-amino-l-phenylalanine (l-PAPA) is a high-value product with a broad field of applications. In nature, l-PAPA occurs as an intermediate of the chloramphenicol biosynthesis pathway in Streptomyces venezuelae. Here we demonstrate that the model organism Escherichia coli can be transformed with metabolic grafting approaches to result in an improved l-PAPA producing strain. RESULTS: Escherichia coli K-12 cells were genetically engineered for the production of l-PAPA from glycerol as main carbon source. To do so, genes for a 4-amino-4-deoxychorismate synthase (pabAB from Corynebacterium glutamicum), and genes encoding a 4-amino-4-deoxychorismate mutase and a 4-amino-4-deoxyprephenate dehydrogenase (papB and papC, both from Streptomyces venezuelae) were cloned and expressed in E. coli W3110 (lab strain LJ110). In shake flask cultures with minimal medium this led to the formation of ca. 43 ± 2 mg l(−1) of l-PAPA from 5 g l(−1) glycerol. By expression of additional chromosomal copies of the tktA and glpX genes, and of plasmid-borne aroFBL genes in a tyrR deletion strain, an improved l-PAPA producer was obtained which gave a titer of 5.47 ± 0.4 g l(−1) l-PAPA from 33.3 g l(−1) glycerol (0.16 g l-PAPA/g of glycerol) in fed-batch cultivation (shake flasks). Finally, in a fed-batch fermenter cultivation, a titer of 16.7 g l(−1) l-PAPA was obtained which is the highest so far reported value for this non-proteinogenic amino acid. CONCLUSION: Here we show that E. coli is a suitable chassis strain for l-PAPA production. Modifying the flux to the product and improved supply of precursor, by additional gene copies of glpX, tkt and aroFBL together with the deletion of the tyrR gene, increased the yield and titer. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-018-0996-6) contains supplementary material, which is available to authorized users. BioMed Central 2018-09-21 /pmc/articles/PMC6148955/ /pubmed/30241531 http://dx.doi.org/10.1186/s12934-018-0996-6 Text en © The Author(s) 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Mohammadi Nargesi, Behrouz Trachtmann, Natalie Sprenger, Georg A. Youn, Jung-Won Production of p-amino-l-phenylalanine (l-PAPA) from glycerol by metabolic grafting of Escherichia coli |
title | Production of p-amino-l-phenylalanine (l-PAPA) from glycerol by metabolic grafting of Escherichia coli |
title_full | Production of p-amino-l-phenylalanine (l-PAPA) from glycerol by metabolic grafting of Escherichia coli |
title_fullStr | Production of p-amino-l-phenylalanine (l-PAPA) from glycerol by metabolic grafting of Escherichia coli |
title_full_unstemmed | Production of p-amino-l-phenylalanine (l-PAPA) from glycerol by metabolic grafting of Escherichia coli |
title_short | Production of p-amino-l-phenylalanine (l-PAPA) from glycerol by metabolic grafting of Escherichia coli |
title_sort | production of p-amino-l-phenylalanine (l-papa) from glycerol by metabolic grafting of escherichia coli |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148955/ https://www.ncbi.nlm.nih.gov/pubmed/30241531 http://dx.doi.org/10.1186/s12934-018-0996-6 |
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