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Identification of two integration sites in favor of transgene expression in Trichoderma reesei

BACKGROUND: The ascomycete fungus Trichoderma reesei was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of T. reesei can g...

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Autores principales: Qin, Lina, Jiang, Xianzhang, Dong, Zhiyang, Huang, Jianzhong, Chen, Xiuzhen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5956788/
https://www.ncbi.nlm.nih.gov/pubmed/29796083
http://dx.doi.org/10.1186/s13068-018-1139-3
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author Qin, Lina
Jiang, Xianzhang
Dong, Zhiyang
Huang, Jianzhong
Chen, Xiuzhen
author_facet Qin, Lina
Jiang, Xianzhang
Dong, Zhiyang
Huang, Jianzhong
Chen, Xiuzhen
author_sort Qin, Lina
collection PubMed
description BACKGROUND: The ascomycete fungus Trichoderma reesei was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of T. reesei can generate series of strains that express different levels of the indicated transgene. The insertion site of the GOI plays an important role in the ultimate production of the targeted proteins. However, so far no systematic studies have been made to identify transgene integration loci for optimal expression of the GOI in T. reesei. Currently, only the locus of exocellobiohydrolases I encoding gene (cbh1) is widely used as a promising integration site to lead to high expression level of the GOI. No additional sites associated with efficient gene expression have been characterized. RESULTS: To search for gene integration sites that benefit for the secreted expression of GOI, the food-and-mouth disease virus 2A protein was applied for co-expression of an Aspergillus niger lipA gene and Discosoma sp. DsRed1 gene in T. reesei, by random integration of the expression cassette into the genome. We demonstrated that the fluorescent intensity of RFP (red fluorescent protein) inside of the cell was well correlated with the secreted lipase yields, based on which, we successfully developed a high-throughput screening method to screen strains with relatively higher secreted expression of the GOI (in this study, lipase). The copy number and the insertion sites of the transgene were investigated among the selected highly expressed strains. Eventually, in addition to cbh1 gene locus, two other genome insertion loci that efficiently facilitate gene expression in T. reesei were identified. CONCLUSIONS: We have successfully developed a high-throughput screening method to screen strains with optimal expression of the indicated secreted proteins in T. reesei. Moreover, we identified two optimal genome loci for transgene expression, which could provide new approach to modulate gene expression levels while retaining the indicated promoter and culture conditions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13068-018-1139-3) contains supplementary material, which is available to authorized users.
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spelling pubmed-59567882018-05-24 Identification of two integration sites in favor of transgene expression in Trichoderma reesei Qin, Lina Jiang, Xianzhang Dong, Zhiyang Huang, Jianzhong Chen, Xiuzhen Biotechnol Biofuels Research BACKGROUND: The ascomycete fungus Trichoderma reesei was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of T. reesei can generate series of strains that express different levels of the indicated transgene. The insertion site of the GOI plays an important role in the ultimate production of the targeted proteins. However, so far no systematic studies have been made to identify transgene integration loci for optimal expression of the GOI in T. reesei. Currently, only the locus of exocellobiohydrolases I encoding gene (cbh1) is widely used as a promising integration site to lead to high expression level of the GOI. No additional sites associated with efficient gene expression have been characterized. RESULTS: To search for gene integration sites that benefit for the secreted expression of GOI, the food-and-mouth disease virus 2A protein was applied for co-expression of an Aspergillus niger lipA gene and Discosoma sp. DsRed1 gene in T. reesei, by random integration of the expression cassette into the genome. We demonstrated that the fluorescent intensity of RFP (red fluorescent protein) inside of the cell was well correlated with the secreted lipase yields, based on which, we successfully developed a high-throughput screening method to screen strains with relatively higher secreted expression of the GOI (in this study, lipase). The copy number and the insertion sites of the transgene were investigated among the selected highly expressed strains. Eventually, in addition to cbh1 gene locus, two other genome insertion loci that efficiently facilitate gene expression in T. reesei were identified. CONCLUSIONS: We have successfully developed a high-throughput screening method to screen strains with optimal expression of the indicated secreted proteins in T. reesei. Moreover, we identified two optimal genome loci for transgene expression, which could provide new approach to modulate gene expression levels while retaining the indicated promoter and culture conditions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13068-018-1139-3) contains supplementary material, which is available to authorized users. BioMed Central 2018-05-17 /pmc/articles/PMC5956788/ /pubmed/29796083 http://dx.doi.org/10.1186/s13068-018-1139-3 Text en © The Author(s) 2018 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
Qin, Lina
Jiang, Xianzhang
Dong, Zhiyang
Huang, Jianzhong
Chen, Xiuzhen
Identification of two integration sites in favor of transgene expression in Trichoderma reesei
title Identification of two integration sites in favor of transgene expression in Trichoderma reesei
title_full Identification of two integration sites in favor of transgene expression in Trichoderma reesei
title_fullStr Identification of two integration sites in favor of transgene expression in Trichoderma reesei
title_full_unstemmed Identification of two integration sites in favor of transgene expression in Trichoderma reesei
title_short Identification of two integration sites in favor of transgene expression in Trichoderma reesei
title_sort identification of two integration sites in favor of transgene expression in trichoderma reesei
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5956788/
https://www.ncbi.nlm.nih.gov/pubmed/29796083
http://dx.doi.org/10.1186/s13068-018-1139-3
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