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Construction of an Antibiotic-Free Vector and its Application in the Metabolic Engineering of Escherichia Coli for Polyhydroxybutyrate Production
An antibiotic- and inducer-free culture condition was proposed for polyhydroxybutyrate (PHB) production in recombinant Escherichia coli. First, antibiotic-free vectors were constructed by installing the plasmid maintenance system, alp7, hok/sok, and the hok/sok and alp7 combination into the pUC19 ve...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9204107/ https://www.ncbi.nlm.nih.gov/pubmed/35721860 http://dx.doi.org/10.3389/fbioe.2022.837944 |
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author | Liao, Ying-Cheng Saengsawang, Boonyawee Chen, Jun-Wei Zhuo, Xiao-Zhen Li, Si-Yu |
author_facet | Liao, Ying-Cheng Saengsawang, Boonyawee Chen, Jun-Wei Zhuo, Xiao-Zhen Li, Si-Yu |
author_sort | Liao, Ying-Cheng |
collection | PubMed |
description | An antibiotic- and inducer-free culture condition was proposed for polyhydroxybutyrate (PHB) production in recombinant Escherichia coli. First, antibiotic-free vectors were constructed by installing the plasmid maintenance system, alp7, hok/sok, and the hok/sok and alp7 combination into the pUC19 vector. The plasmid stability test showed that pVEC02, the pUC19 vector containing the hok/sok system, was the most effective in achieving antibiotic-free cultivation in the E. coli B strain but not in the K strain. Second, the putative phaCAB operon derived from Caldimonas manganoxidans was inserted into pVEC02 to yield pPHB01 for PHB production in E. coli BL21 (DE3). The putative phaCAB operon was first shown function properly for PHB production and thus, inducer-free conditions were achieved. However, the maintenance of pPHB01 in E. coli requires antibiotics supplementation. Finally, an efficient E. coli ρ factor-independent terminator, thrLABC (ECK120033737), was inserted between the phaCAB operon and the hok/sok system to avoid possible transcriptional carry-over. The newly constructed plasmid pPHB01-1 facilitates an antibiotic- and inducer-free culture condition and induces the production of PHB with a concentration of 3.0 on0.2 g/L, yield of 0.26 /L0.07 g/g-glucose, and content of 44 /g3%. The PHB production using E. coli BL21 (DE3)/pPHB01-1 has been shown to last 84 and 96 h in the liquid and solid cultures. |
format | Online Article Text |
id | pubmed-9204107 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-92041072022-06-18 Construction of an Antibiotic-Free Vector and its Application in the Metabolic Engineering of Escherichia Coli for Polyhydroxybutyrate Production Liao, Ying-Cheng Saengsawang, Boonyawee Chen, Jun-Wei Zhuo, Xiao-Zhen Li, Si-Yu Front Bioeng Biotechnol Bioengineering and Biotechnology An antibiotic- and inducer-free culture condition was proposed for polyhydroxybutyrate (PHB) production in recombinant Escherichia coli. First, antibiotic-free vectors were constructed by installing the plasmid maintenance system, alp7, hok/sok, and the hok/sok and alp7 combination into the pUC19 vector. The plasmid stability test showed that pVEC02, the pUC19 vector containing the hok/sok system, was the most effective in achieving antibiotic-free cultivation in the E. coli B strain but not in the K strain. Second, the putative phaCAB operon derived from Caldimonas manganoxidans was inserted into pVEC02 to yield pPHB01 for PHB production in E. coli BL21 (DE3). The putative phaCAB operon was first shown function properly for PHB production and thus, inducer-free conditions were achieved. However, the maintenance of pPHB01 in E. coli requires antibiotics supplementation. Finally, an efficient E. coli ρ factor-independent terminator, thrLABC (ECK120033737), was inserted between the phaCAB operon and the hok/sok system to avoid possible transcriptional carry-over. The newly constructed plasmid pPHB01-1 facilitates an antibiotic- and inducer-free culture condition and induces the production of PHB with a concentration of 3.0 on0.2 g/L, yield of 0.26 /L0.07 g/g-glucose, and content of 44 /g3%. The PHB production using E. coli BL21 (DE3)/pPHB01-1 has been shown to last 84 and 96 h in the liquid and solid cultures. Frontiers Media S.A. 2022-06-03 /pmc/articles/PMC9204107/ /pubmed/35721860 http://dx.doi.org/10.3389/fbioe.2022.837944 Text en Copyright © 2022 Liao, Saengsawang, Chen, Zhuo and Li. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Bioengineering and Biotechnology Liao, Ying-Cheng Saengsawang, Boonyawee Chen, Jun-Wei Zhuo, Xiao-Zhen Li, Si-Yu Construction of an Antibiotic-Free Vector and its Application in the Metabolic Engineering of Escherichia Coli for Polyhydroxybutyrate Production |
title | Construction of an Antibiotic-Free Vector and its Application in the Metabolic Engineering of Escherichia Coli for Polyhydroxybutyrate Production |
title_full | Construction of an Antibiotic-Free Vector and its Application in the Metabolic Engineering of Escherichia Coli for Polyhydroxybutyrate Production |
title_fullStr | Construction of an Antibiotic-Free Vector and its Application in the Metabolic Engineering of Escherichia Coli for Polyhydroxybutyrate Production |
title_full_unstemmed | Construction of an Antibiotic-Free Vector and its Application in the Metabolic Engineering of Escherichia Coli for Polyhydroxybutyrate Production |
title_short | Construction of an Antibiotic-Free Vector and its Application in the Metabolic Engineering of Escherichia Coli for Polyhydroxybutyrate Production |
title_sort | construction of an antibiotic-free vector and its application in the metabolic engineering of escherichia coli for polyhydroxybutyrate production |
topic | Bioengineering and Biotechnology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9204107/ https://www.ncbi.nlm.nih.gov/pubmed/35721860 http://dx.doi.org/10.3389/fbioe.2022.837944 |
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