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Combinatorial pathway enzyme engineering and host engineering overcomes pyruvate overflow and enhances overproduction of N-acetylglucosamine in Bacillus subtilis

BACKGROUND: Glucosamine-6-phosphate N-acetyltransferase (GNA1) is the key enzyme that causes overproduction of N-acetylglucosamine in Bacillus subtilis. Previously, we increased GlcNAc production by promoting the expression of GNA1 from Caenorhabditis elegans (CeGNA1) in an engineered B. subtilis st...

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Autores principales: Ma, Wenlong, Liu, Yanfeng, Lv, Xueqin, Li, Jianghua, Du, Guocheng, Liu, Long
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318901/
https://www.ncbi.nlm.nih.gov/pubmed/30609921
http://dx.doi.org/10.1186/s12934-018-1049-x
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author Ma, Wenlong
Liu, Yanfeng
Lv, Xueqin
Li, Jianghua
Du, Guocheng
Liu, Long
author_facet Ma, Wenlong
Liu, Yanfeng
Lv, Xueqin
Li, Jianghua
Du, Guocheng
Liu, Long
author_sort Ma, Wenlong
collection PubMed
description BACKGROUND: Glucosamine-6-phosphate N-acetyltransferase (GNA1) is the key enzyme that causes overproduction of N-acetylglucosamine in Bacillus subtilis. Previously, we increased GlcNAc production by promoting the expression of GNA1 from Caenorhabditis elegans (CeGNA1) in an engineered B. subtilis strain BSGN12. In this strain overflow metabolism to by-products acetoin and acetate had been blocked by mutations, however pyruvate accumulated as an overflow metabolite. Although overexpression of CeGNA1 drove carbon flux from pyruvate to the GlcNAc synthesis pathway and decreased pyruvate accumulation, the residual pyruvate reduced the intracellular pH, resulting in inhibited CeGNA1 activity and limited GlcNAc production. RESULTS: In this study, we attempted to further overcome pyruvate overflow by enzyme engineering and host engineering for enhanced GlcNAc production. To this end, the key enzyme CeGNA1 was evolved through error-prone PCR under pyruvate stress to enhance its catalytic activity. Then, the urease from Bacillus paralicheniformis was expressed intracellularly to neutralize the intracellular pH, making it more robust in growth and more efficient in GlcNAc production. It was found that the activity of mutant CeGNA1 increased by 11.5% at pH 6.5–7.5, with the catalytic efficiency increasing by 27.5% to 1.25 s(−1) µM(−1). Modulated expression of urease increased the intracellular pH from 6.0 to 6.8. The final engineered strain BSGN13 overcame pyruvate overflow, produced 25.6 g/L GlcNAc with a yield of 0.43 g GlcNAc/g glucose in a shake flask fermentation and produced 82.5 g/L GlcNAc with a yield of 0.39 g GlcNAc/g glucose by fed-batch fermentation, which was 1.7- and 1.2-times, respectively, of the yield achieved previously. CONCLUSIONS: This study highlights a strategy that combines pathway enzyme engineering and host engineering to resolve overflow metabolism in B. subtilis for the overproduction of GlcNAc. By means of modulated expression of urease reduced pyruvate burden, conferred bacterial survival fitness, and enhanced GlcNAc production, all of which improved our understanding of co-regulation of cell growth and metabolism to construct more efficient B. subtilis cell factories. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-018-1049-x) contains supplementary material, which is available to authorized users.
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spelling pubmed-63189012019-01-08 Combinatorial pathway enzyme engineering and host engineering overcomes pyruvate overflow and enhances overproduction of N-acetylglucosamine in Bacillus subtilis Ma, Wenlong Liu, Yanfeng Lv, Xueqin Li, Jianghua Du, Guocheng Liu, Long Microb Cell Fact Research BACKGROUND: Glucosamine-6-phosphate N-acetyltransferase (GNA1) is the key enzyme that causes overproduction of N-acetylglucosamine in Bacillus subtilis. Previously, we increased GlcNAc production by promoting the expression of GNA1 from Caenorhabditis elegans (CeGNA1) in an engineered B. subtilis strain BSGN12. In this strain overflow metabolism to by-products acetoin and acetate had been blocked by mutations, however pyruvate accumulated as an overflow metabolite. Although overexpression of CeGNA1 drove carbon flux from pyruvate to the GlcNAc synthesis pathway and decreased pyruvate accumulation, the residual pyruvate reduced the intracellular pH, resulting in inhibited CeGNA1 activity and limited GlcNAc production. RESULTS: In this study, we attempted to further overcome pyruvate overflow by enzyme engineering and host engineering for enhanced GlcNAc production. To this end, the key enzyme CeGNA1 was evolved through error-prone PCR under pyruvate stress to enhance its catalytic activity. Then, the urease from Bacillus paralicheniformis was expressed intracellularly to neutralize the intracellular pH, making it more robust in growth and more efficient in GlcNAc production. It was found that the activity of mutant CeGNA1 increased by 11.5% at pH 6.5–7.5, with the catalytic efficiency increasing by 27.5% to 1.25 s(−1) µM(−1). Modulated expression of urease increased the intracellular pH from 6.0 to 6.8. The final engineered strain BSGN13 overcame pyruvate overflow, produced 25.6 g/L GlcNAc with a yield of 0.43 g GlcNAc/g glucose in a shake flask fermentation and produced 82.5 g/L GlcNAc with a yield of 0.39 g GlcNAc/g glucose by fed-batch fermentation, which was 1.7- and 1.2-times, respectively, of the yield achieved previously. CONCLUSIONS: This study highlights a strategy that combines pathway enzyme engineering and host engineering to resolve overflow metabolism in B. subtilis for the overproduction of GlcNAc. By means of modulated expression of urease reduced pyruvate burden, conferred bacterial survival fitness, and enhanced GlcNAc production, all of which improved our understanding of co-regulation of cell growth and metabolism to construct more efficient B. subtilis cell factories. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-018-1049-x) contains supplementary material, which is available to authorized users. BioMed Central 2019-01-04 /pmc/articles/PMC6318901/ /pubmed/30609921 http://dx.doi.org/10.1186/s12934-018-1049-x 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
Ma, Wenlong
Liu, Yanfeng
Lv, Xueqin
Li, Jianghua
Du, Guocheng
Liu, Long
Combinatorial pathway enzyme engineering and host engineering overcomes pyruvate overflow and enhances overproduction of N-acetylglucosamine in Bacillus subtilis
title Combinatorial pathway enzyme engineering and host engineering overcomes pyruvate overflow and enhances overproduction of N-acetylglucosamine in Bacillus subtilis
title_full Combinatorial pathway enzyme engineering and host engineering overcomes pyruvate overflow and enhances overproduction of N-acetylglucosamine in Bacillus subtilis
title_fullStr Combinatorial pathway enzyme engineering and host engineering overcomes pyruvate overflow and enhances overproduction of N-acetylglucosamine in Bacillus subtilis
title_full_unstemmed Combinatorial pathway enzyme engineering and host engineering overcomes pyruvate overflow and enhances overproduction of N-acetylglucosamine in Bacillus subtilis
title_short Combinatorial pathway enzyme engineering and host engineering overcomes pyruvate overflow and enhances overproduction of N-acetylglucosamine in Bacillus subtilis
title_sort combinatorial pathway enzyme engineering and host engineering overcomes pyruvate overflow and enhances overproduction of n-acetylglucosamine in bacillus subtilis
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318901/
https://www.ncbi.nlm.nih.gov/pubmed/30609921
http://dx.doi.org/10.1186/s12934-018-1049-x
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