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Enhancing surfactin production by using systematic CRISPRi repression to screen amino acid biosynthesis genes in Bacillus subtilis
BACKGROUND: Surfactin is a cyclic lipopeptide that is of great industrial use owing to its extraordinary surfactant power and antimicrobial, antiviral, and antitumor activities. Surfactin is synthesized by a condensation reaction in microbes, which uses fatty acids and four kinds of amino acids (l-g...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533722/ https://www.ncbi.nlm.nih.gov/pubmed/31122258 http://dx.doi.org/10.1186/s12934-019-1139-4 |
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author | Wang, Congya Cao, Yingxiu Wang, Yongping Sun, Liming Song, Hao |
author_facet | Wang, Congya Cao, Yingxiu Wang, Yongping Sun, Liming Song, Hao |
author_sort | Wang, Congya |
collection | PubMed |
description | BACKGROUND: Surfactin is a cyclic lipopeptide that is of great industrial use owing to its extraordinary surfactant power and antimicrobial, antiviral, and antitumor activities. Surfactin is synthesized by a condensation reaction in microbes, which uses fatty acids and four kinds of amino acids (l-glutamate, l-aspartate, l-leucine and l-valine) as precursors. Surfactin biosynthesis could be improved by increasing the supply of fatty acids; however, the effect of the regulation of amino acid metabolism on surfactin production was not yet clear. RESULTS: In this study, we aimed to improve surfactin production in B. subtilis by repressing the genes on the branch metabolic pathways of amino acid biosynthesis using CRISPRi technology. First, 20 genes were inhibited individually, resulting in 2.5- to 627-fold decreases in transcriptional level as determined by RT-qPCR. Among the 20 recombinant strains, 16 strains obtained higher surfactin titres than that produced by the parent BS168NU-Sd strain (the surfactin production of BS168NU-Sd with only dCas9 but no sgRNA expression was 0.17 g/L). In particular, the strains in which the yrpC, racE or murC genes were inhibited individually produced 0.54, 0.41, or 0.42 g/L surfactin, respectively. All three genes are related to the metabolism of l-glutamate, whose acylation is the first step in the surfactin condensation reaction. Furthermore, these three genes were repressed in combination, and the strain with co-inhibition of yrpC and racE produced 0.75 g/L surfactin, which was 4.69-fold higher than that of the parent strain. In addition, the inhibition of bkdAA and bkdAB, which are related to the metabolism of l-leucine and l-valine, not only improved surfactin production but also increased the proportion of the C(14) isoform. CONCLUSIONS: This study, to the best of our knowledge for the first time, systematically probed the regulatory effect of increasing the supply of amino acids on surfactin production. It provided an effective strategy and a new perspective for systematic studies on surfactin and other amino acid-derived chemicals. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-019-1139-4) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6533722 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-65337222019-05-28 Enhancing surfactin production by using systematic CRISPRi repression to screen amino acid biosynthesis genes in Bacillus subtilis Wang, Congya Cao, Yingxiu Wang, Yongping Sun, Liming Song, Hao Microb Cell Fact Research BACKGROUND: Surfactin is a cyclic lipopeptide that is of great industrial use owing to its extraordinary surfactant power and antimicrobial, antiviral, and antitumor activities. Surfactin is synthesized by a condensation reaction in microbes, which uses fatty acids and four kinds of amino acids (l-glutamate, l-aspartate, l-leucine and l-valine) as precursors. Surfactin biosynthesis could be improved by increasing the supply of fatty acids; however, the effect of the regulation of amino acid metabolism on surfactin production was not yet clear. RESULTS: In this study, we aimed to improve surfactin production in B. subtilis by repressing the genes on the branch metabolic pathways of amino acid biosynthesis using CRISPRi technology. First, 20 genes were inhibited individually, resulting in 2.5- to 627-fold decreases in transcriptional level as determined by RT-qPCR. Among the 20 recombinant strains, 16 strains obtained higher surfactin titres than that produced by the parent BS168NU-Sd strain (the surfactin production of BS168NU-Sd with only dCas9 but no sgRNA expression was 0.17 g/L). In particular, the strains in which the yrpC, racE or murC genes were inhibited individually produced 0.54, 0.41, or 0.42 g/L surfactin, respectively. All three genes are related to the metabolism of l-glutamate, whose acylation is the first step in the surfactin condensation reaction. Furthermore, these three genes were repressed in combination, and the strain with co-inhibition of yrpC and racE produced 0.75 g/L surfactin, which was 4.69-fold higher than that of the parent strain. In addition, the inhibition of bkdAA and bkdAB, which are related to the metabolism of l-leucine and l-valine, not only improved surfactin production but also increased the proportion of the C(14) isoform. CONCLUSIONS: This study, to the best of our knowledge for the first time, systematically probed the regulatory effect of increasing the supply of amino acids on surfactin production. It provided an effective strategy and a new perspective for systematic studies on surfactin and other amino acid-derived chemicals. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-019-1139-4) contains supplementary material, which is available to authorized users. BioMed Central 2019-05-23 /pmc/articles/PMC6533722/ /pubmed/31122258 http://dx.doi.org/10.1186/s12934-019-1139-4 Text en © The Author(s) 2019 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 Wang, Congya Cao, Yingxiu Wang, Yongping Sun, Liming Song, Hao Enhancing surfactin production by using systematic CRISPRi repression to screen amino acid biosynthesis genes in Bacillus subtilis |
title | Enhancing surfactin production by using systematic CRISPRi repression to screen amino acid biosynthesis genes in Bacillus subtilis |
title_full | Enhancing surfactin production by using systematic CRISPRi repression to screen amino acid biosynthesis genes in Bacillus subtilis |
title_fullStr | Enhancing surfactin production by using systematic CRISPRi repression to screen amino acid biosynthesis genes in Bacillus subtilis |
title_full_unstemmed | Enhancing surfactin production by using systematic CRISPRi repression to screen amino acid biosynthesis genes in Bacillus subtilis |
title_short | Enhancing surfactin production by using systematic CRISPRi repression to screen amino acid biosynthesis genes in Bacillus subtilis |
title_sort | enhancing surfactin production by using systematic crispri repression to screen amino acid biosynthesis genes in bacillus subtilis |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533722/ https://www.ncbi.nlm.nih.gov/pubmed/31122258 http://dx.doi.org/10.1186/s12934-019-1139-4 |
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