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Enhancing Production of Pinene in Escherichia coli by Using a Combination of Tolerance, Evolution, and Modular Co-culture Engineering

α-Pinene is a natural and active monoterpene, which is widely used as a flavoring agent and in fragrances, pharmaceuticals, and biofuels. Although it has been successfully produced by genetically engineered microorganisms, the production level of pinene is much lower than that of hemiterpene (isopre...

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Autores principales: Niu, Fu-Xing, He, Xin, Wu, Ya-Qin, Liu, Jian-Zhong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079208/
https://www.ncbi.nlm.nih.gov/pubmed/30108554
http://dx.doi.org/10.3389/fmicb.2018.01623
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author Niu, Fu-Xing
He, Xin
Wu, Ya-Qin
Liu, Jian-Zhong
author_facet Niu, Fu-Xing
He, Xin
Wu, Ya-Qin
Liu, Jian-Zhong
author_sort Niu, Fu-Xing
collection PubMed
description α-Pinene is a natural and active monoterpene, which is widely used as a flavoring agent and in fragrances, pharmaceuticals, and biofuels. Although it has been successfully produced by genetically engineered microorganisms, the production level of pinene is much lower than that of hemiterpene (isoprene) and sesquiterpenes (farnesene) to date. We first improved pinene tolerance to 2.0% and pinene production by adaptive laboratory evolution after atmospheric and room temperature plasma (ARTP) mutagenesis and overexpression of the efflux pump to obtain the pinene tolerant strain Escherichia coli YZFP, which is resistant to fosmidomycin. Through error-prone PCR and DNA shuffling, we isolated an Abies grandis geranyl pyrophosphate synthase variant that outperformed the wild-type enzyme. To balance the expression of multiple genes, a tunable intergenic region (TIGR) was inserted between A. grandis GPPS(D90G/L175P) and Pinus taeda Pt1(Q457L). In an effort to improve the production, an E. coli-E. coli modular co-culture system was engineered to modularize the heterologous mevalonate (MEV) pathway and the TIGR-mediated gene cluster of A. grandis GPPS(D90G/L175P) and P. taeda Pt1(Q457L). Specifically, the MEV pathway and the TIGR-mediated gene cluster were integrated into the chromosome of the pinene tolerance strain E. coli YZFP and then evolved to a higher gene copy number by chemically induced chromosomal evolution, respectively. The best E. coli-E. coli co-culture system of fermentation was found to improve pinene production by 1.9-fold compared to the mono-culture approach. The E. coli-E. coli modular co-culture system of whole-cell biocatalysis further improved pinene production to 166.5 mg/L.
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spelling pubmed-60792082018-08-14 Enhancing Production of Pinene in Escherichia coli by Using a Combination of Tolerance, Evolution, and Modular Co-culture Engineering Niu, Fu-Xing He, Xin Wu, Ya-Qin Liu, Jian-Zhong Front Microbiol Microbiology α-Pinene is a natural and active monoterpene, which is widely used as a flavoring agent and in fragrances, pharmaceuticals, and biofuels. Although it has been successfully produced by genetically engineered microorganisms, the production level of pinene is much lower than that of hemiterpene (isoprene) and sesquiterpenes (farnesene) to date. We first improved pinene tolerance to 2.0% and pinene production by adaptive laboratory evolution after atmospheric and room temperature plasma (ARTP) mutagenesis and overexpression of the efflux pump to obtain the pinene tolerant strain Escherichia coli YZFP, which is resistant to fosmidomycin. Through error-prone PCR and DNA shuffling, we isolated an Abies grandis geranyl pyrophosphate synthase variant that outperformed the wild-type enzyme. To balance the expression of multiple genes, a tunable intergenic region (TIGR) was inserted between A. grandis GPPS(D90G/L175P) and Pinus taeda Pt1(Q457L). In an effort to improve the production, an E. coli-E. coli modular co-culture system was engineered to modularize the heterologous mevalonate (MEV) pathway and the TIGR-mediated gene cluster of A. grandis GPPS(D90G/L175P) and P. taeda Pt1(Q457L). Specifically, the MEV pathway and the TIGR-mediated gene cluster were integrated into the chromosome of the pinene tolerance strain E. coli YZFP and then evolved to a higher gene copy number by chemically induced chromosomal evolution, respectively. The best E. coli-E. coli co-culture system of fermentation was found to improve pinene production by 1.9-fold compared to the mono-culture approach. The E. coli-E. coli modular co-culture system of whole-cell biocatalysis further improved pinene production to 166.5 mg/L. Frontiers Media S.A. 2018-07-31 /pmc/articles/PMC6079208/ /pubmed/30108554 http://dx.doi.org/10.3389/fmicb.2018.01623 Text en Copyright © 2018 Niu, He, Wu and Liu. http://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 Microbiology
Niu, Fu-Xing
He, Xin
Wu, Ya-Qin
Liu, Jian-Zhong
Enhancing Production of Pinene in Escherichia coli by Using a Combination of Tolerance, Evolution, and Modular Co-culture Engineering
title Enhancing Production of Pinene in Escherichia coli by Using a Combination of Tolerance, Evolution, and Modular Co-culture Engineering
title_full Enhancing Production of Pinene in Escherichia coli by Using a Combination of Tolerance, Evolution, and Modular Co-culture Engineering
title_fullStr Enhancing Production of Pinene in Escherichia coli by Using a Combination of Tolerance, Evolution, and Modular Co-culture Engineering
title_full_unstemmed Enhancing Production of Pinene in Escherichia coli by Using a Combination of Tolerance, Evolution, and Modular Co-culture Engineering
title_short Enhancing Production of Pinene in Escherichia coli by Using a Combination of Tolerance, Evolution, and Modular Co-culture Engineering
title_sort enhancing production of pinene in escherichia coli by using a combination of tolerance, evolution, and modular co-culture engineering
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079208/
https://www.ncbi.nlm.nih.gov/pubmed/30108554
http://dx.doi.org/10.3389/fmicb.2018.01623
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