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Construction of T7-Like Expression System in Pseudomonas putida KT2440 to Enhance the Heterologous Expression Level

Pseudomonas putida KT2440 has become an attractive chassis for heterologous expression with the development of effective genetic manipulation tools. Improving the level of transcriptional regulation is particularly important for extending the potential of P. putida KT2440 in heterologous expression....

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Autores principales: Liang, Tianxin, Sun, Jun, Ju, Shuyun, Su, Shenyi, Yang, Lirong, Wu, Jianping
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8322953/
https://www.ncbi.nlm.nih.gov/pubmed/34336782
http://dx.doi.org/10.3389/fchem.2021.664967
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author Liang, Tianxin
Sun, Jun
Ju, Shuyun
Su, Shenyi
Yang, Lirong
Wu, Jianping
author_facet Liang, Tianxin
Sun, Jun
Ju, Shuyun
Su, Shenyi
Yang, Lirong
Wu, Jianping
author_sort Liang, Tianxin
collection PubMed
description Pseudomonas putida KT2440 has become an attractive chassis for heterologous expression with the development of effective genetic manipulation tools. Improving the level of transcriptional regulation is particularly important for extending the potential of P. putida KT2440 in heterologous expression. Although many strategies have been applied to enhance the heterologous expression level in P. putida KT2440, it was still at a relatively low level. Herein we constructed a T7-like expression system in P. putida KT2440, mimicking the pET expression system in Escherichia coli, which consisted of T7-like RNA polymerase (MmP1) integrated strain and the corresponding expression vector for the heterologous expression enhancement. With the optimization of the insertion site and the copy number of RNA polymerase (RNAP), the relative fluorescence intensity (RFI) of the super-folder green fluorescent protein (sfGFP) was improved by 1.4-fold in MmP1 RNAP integrated strain. The induction point and IPTG concentration were also optimized. This strategy was extended to the gene-reduced strain EM42 and the expression of sfGFP was improved by 2.1-fold. The optimal RNAP integration site was also used for introducing T7 RNAP in P. putida KT2440 and the expression level was enhanced, indicating the generality of the integration site for the T7 expression system. Compared to other inducible expression systems in KT2440, the heterologous expression level of the Mmp1 system and T7 system were more than 2.5 times higher. Furthermore, the 3.6-fold enhanced expression level of a difficult-to-express nicotinate dehydrogenase from Comamonas testosteroni JA1 verified the efficiency of the T7-like expression system in P. putida KT2440. Taken together, we constructed and optimized the T7-like and T7 expression system in P. putida, thus providing a set of applicable chassis and corresponding plasmids to improve recombinant expression level, expecting to be used for difficult-to-express proteins.
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spelling pubmed-83229532021-07-31 Construction of T7-Like Expression System in Pseudomonas putida KT2440 to Enhance the Heterologous Expression Level Liang, Tianxin Sun, Jun Ju, Shuyun Su, Shenyi Yang, Lirong Wu, Jianping Front Chem Chemistry Pseudomonas putida KT2440 has become an attractive chassis for heterologous expression with the development of effective genetic manipulation tools. Improving the level of transcriptional regulation is particularly important for extending the potential of P. putida KT2440 in heterologous expression. Although many strategies have been applied to enhance the heterologous expression level in P. putida KT2440, it was still at a relatively low level. Herein we constructed a T7-like expression system in P. putida KT2440, mimicking the pET expression system in Escherichia coli, which consisted of T7-like RNA polymerase (MmP1) integrated strain and the corresponding expression vector for the heterologous expression enhancement. With the optimization of the insertion site and the copy number of RNA polymerase (RNAP), the relative fluorescence intensity (RFI) of the super-folder green fluorescent protein (sfGFP) was improved by 1.4-fold in MmP1 RNAP integrated strain. The induction point and IPTG concentration were also optimized. This strategy was extended to the gene-reduced strain EM42 and the expression of sfGFP was improved by 2.1-fold. The optimal RNAP integration site was also used for introducing T7 RNAP in P. putida KT2440 and the expression level was enhanced, indicating the generality of the integration site for the T7 expression system. Compared to other inducible expression systems in KT2440, the heterologous expression level of the Mmp1 system and T7 system were more than 2.5 times higher. Furthermore, the 3.6-fold enhanced expression level of a difficult-to-express nicotinate dehydrogenase from Comamonas testosteroni JA1 verified the efficiency of the T7-like expression system in P. putida KT2440. Taken together, we constructed and optimized the T7-like and T7 expression system in P. putida, thus providing a set of applicable chassis and corresponding plasmids to improve recombinant expression level, expecting to be used for difficult-to-express proteins. Frontiers Media S.A. 2021-07-16 /pmc/articles/PMC8322953/ /pubmed/34336782 http://dx.doi.org/10.3389/fchem.2021.664967 Text en Copyright © 2021 Liang, Sun, Ju, Su, Yang and Wu. 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 Chemistry
Liang, Tianxin
Sun, Jun
Ju, Shuyun
Su, Shenyi
Yang, Lirong
Wu, Jianping
Construction of T7-Like Expression System in Pseudomonas putida KT2440 to Enhance the Heterologous Expression Level
title Construction of T7-Like Expression System in Pseudomonas putida KT2440 to Enhance the Heterologous Expression Level
title_full Construction of T7-Like Expression System in Pseudomonas putida KT2440 to Enhance the Heterologous Expression Level
title_fullStr Construction of T7-Like Expression System in Pseudomonas putida KT2440 to Enhance the Heterologous Expression Level
title_full_unstemmed Construction of T7-Like Expression System in Pseudomonas putida KT2440 to Enhance the Heterologous Expression Level
title_short Construction of T7-Like Expression System in Pseudomonas putida KT2440 to Enhance the Heterologous Expression Level
title_sort construction of t7-like expression system in pseudomonas putida kt2440 to enhance the heterologous expression level
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8322953/
https://www.ncbi.nlm.nih.gov/pubmed/34336782
http://dx.doi.org/10.3389/fchem.2021.664967
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