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Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae

BACKGROUND: Saccharomyces cerevisiae (baker’s yeast) has great potential as a whole-cell biocatalyst for multistep synthesis of various organic molecules. To date, however, few examples exist in the literature of the successful biosynthetic production of chemical compounds, in yeast, that do not exi...

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Autores principales: Weber, Nora, Hatsch, Anaëlle, Labagnere, Ludivine, Heider, Harald
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364695/
https://www.ncbi.nlm.nih.gov/pubmed/28335772
http://dx.doi.org/10.1186/s12934-017-0667-z
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author Weber, Nora
Hatsch, Anaëlle
Labagnere, Ludivine
Heider, Harald
author_facet Weber, Nora
Hatsch, Anaëlle
Labagnere, Ludivine
Heider, Harald
author_sort Weber, Nora
collection PubMed
description BACKGROUND: Saccharomyces cerevisiae (baker’s yeast) has great potential as a whole-cell biocatalyst for multistep synthesis of various organic molecules. To date, however, few examples exist in the literature of the successful biosynthetic production of chemical compounds, in yeast, that do not exist in nature. Considering that more than 30% of all drugs on the market are purely chemical compounds, often produced by harsh synthetic chemistry or with very low yields, novel and environmentally sound production routes are highly desirable. Here, we explore the biosynthetic production of enantiomeric precursors of the anti-tuberculosis and anti-epilepsy drugs ethambutol, brivaracetam, and levetiracetam. To this end, we have generated heterologous biosynthetic pathways leading to the production of (S)-2-aminobutyric acid (ABA) and (S)-2-aminobutanol in baker’s yeast. RESULTS: We first designed a two-step heterologous pathway, starting with the endogenous amino acid l-threonine and leading to the production of enantiopure (S)-2-aminobutyric acid. The combination of Bacillus subtilis threonine deaminase and a mutated Escherichia coli glutamate dehydrogenase resulted in the intracellular accumulation of 0.40 mg/L of (S)-2-aminobutyric acid. The combination of a threonine deaminase from Solanum lycopersicum (tomato) with two copies of mutated glutamate dehydrogenase from E. coli resulted in the accumulation of comparable amounts of (S)-2-aminobutyric acid. Additional l-threonine feeding elevated (S)-2-aminobutyric acid production to more than 1.70 mg/L. Removing feedback inhibition of aspartate kinase HOM3, an enzyme involved in threonine biosynthesis in yeast, elevated (S)-2-aminobutyric acid biosynthesis to above 0.49 mg/L in cultures not receiving additional l-threonine. We ultimately extended the pathway from (S)-2-aminobutyric acid to (S)-2-aminobutanol by introducing two reductases and a phosphopantetheinyl transferase. The engineered strains produced up to 1.10 mg/L (S)-2-aminobutanol. CONCLUSIONS: Our results demonstrate the biosynthesis of (S)-2-aminobutyric acid and (S)-2-aminobutanol in yeast. To our knowledge this is the first time that the purely synthetic compound (S)-2-aminobutanol has been produced in vivo. This work paves the way to greener and more sustainable production of chemical entities hitherto inaccessible to synthetic biology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-017-0667-z) contains supplementary material, which is available to authorized users.
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spelling pubmed-53646952017-03-24 Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae Weber, Nora Hatsch, Anaëlle Labagnere, Ludivine Heider, Harald Microb Cell Fact Research BACKGROUND: Saccharomyces cerevisiae (baker’s yeast) has great potential as a whole-cell biocatalyst for multistep synthesis of various organic molecules. To date, however, few examples exist in the literature of the successful biosynthetic production of chemical compounds, in yeast, that do not exist in nature. Considering that more than 30% of all drugs on the market are purely chemical compounds, often produced by harsh synthetic chemistry or with very low yields, novel and environmentally sound production routes are highly desirable. Here, we explore the biosynthetic production of enantiomeric precursors of the anti-tuberculosis and anti-epilepsy drugs ethambutol, brivaracetam, and levetiracetam. To this end, we have generated heterologous biosynthetic pathways leading to the production of (S)-2-aminobutyric acid (ABA) and (S)-2-aminobutanol in baker’s yeast. RESULTS: We first designed a two-step heterologous pathway, starting with the endogenous amino acid l-threonine and leading to the production of enantiopure (S)-2-aminobutyric acid. The combination of Bacillus subtilis threonine deaminase and a mutated Escherichia coli glutamate dehydrogenase resulted in the intracellular accumulation of 0.40 mg/L of (S)-2-aminobutyric acid. The combination of a threonine deaminase from Solanum lycopersicum (tomato) with two copies of mutated glutamate dehydrogenase from E. coli resulted in the accumulation of comparable amounts of (S)-2-aminobutyric acid. Additional l-threonine feeding elevated (S)-2-aminobutyric acid production to more than 1.70 mg/L. Removing feedback inhibition of aspartate kinase HOM3, an enzyme involved in threonine biosynthesis in yeast, elevated (S)-2-aminobutyric acid biosynthesis to above 0.49 mg/L in cultures not receiving additional l-threonine. We ultimately extended the pathway from (S)-2-aminobutyric acid to (S)-2-aminobutanol by introducing two reductases and a phosphopantetheinyl transferase. The engineered strains produced up to 1.10 mg/L (S)-2-aminobutanol. CONCLUSIONS: Our results demonstrate the biosynthesis of (S)-2-aminobutyric acid and (S)-2-aminobutanol in yeast. To our knowledge this is the first time that the purely synthetic compound (S)-2-aminobutanol has been produced in vivo. This work paves the way to greener and more sustainable production of chemical entities hitherto inaccessible to synthetic biology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-017-0667-z) contains supplementary material, which is available to authorized users. BioMed Central 2017-03-23 /pmc/articles/PMC5364695/ /pubmed/28335772 http://dx.doi.org/10.1186/s12934-017-0667-z Text en © The Author(s) 2017 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
Weber, Nora
Hatsch, Anaëlle
Labagnere, Ludivine
Heider, Harald
Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae
title Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae
title_full Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae
title_fullStr Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae
title_full_unstemmed Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae
title_short Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae
title_sort production of (s)-2-aminobutyric acid and (s)-2-aminobutanol in saccharomyces cerevisiae
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364695/
https://www.ncbi.nlm.nih.gov/pubmed/28335772
http://dx.doi.org/10.1186/s12934-017-0667-z
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