Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast

Background The high capacity of chloroplast genome response to integrate and express transgenes at high levels makes this technology a good option to produce proteins of interest. This report presents the stable expression of Pectin lyase (PelA gene) and the first stable expression of manganese pero...

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Autores principales:	Espinoza-Sánchez,Edward Alexander, Álvarez-Hernández,Marianela Hazel, Torres-Castillo,Jorge Ariel, Rascón-Cruz,Quintín, Gutiérrez-Díez,Adriana, Zavala-García,Francisco, Sinagawa-García,Sugey Ramona
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2015
Materias:	Cell wall Chloroplast genetic transformation Hydrolytic enzyme Nicotiana tabacum
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000300004
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spelling	oai:scielo:S0717-345820150003000042015-06-26Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplastEspinoza-Sánchez,Edward AlexanderÁlvarez-Hernández,Marianela HazelTorres-Castillo,Jorge ArielRascón-Cruz,QuintínGutiérrez-Díez,AdrianaZavala-García,FranciscoSinagawa-García,Sugey Ramona Cell wall Chloroplast genetic transformation Hydrolytic enzyme Nicotiana tabacum Background The high capacity of chloroplast genome response to integrate and express transgenes at high levels makes this technology a good option to produce proteins of interest. This report presents the stable expression of Pectin lyase (PelA gene) and the first stable expression of manganese peroxidase (MnP-2 gene) from the chloroplast genome. Results pES4 and pES5 vectors were derived from pPV111A plasmid and contain the PelA and MnP-2 synthetic genes, respectively. Both genes are flanked by a synthetic rrn16S promoter and the 3'UTR from rbcL gene. Efficient gene integration into both inverted repeats of the intergenic region between rrn16S and 3'rps'12 was confirmed by Southern blot. Stable processing and expression of the RNA were confirmed by Northern blot analysis. Enzymatic activity was evaluated to detect expression and functionality of both enzymes. In general, mature plants showed more activity than young transplastomic plants. Compared to wild type plants, transplastomic events expressing pectin lyase exhibited enzymatic activity above 58.5% of total soluble protein at neutral pH and 60°C. In contrast, MnP-2 showed high activity at pH 6 with optimum temperature at 65°C. Neither transplastomic plant exhibited an abnormal phenotype. Conclusion This study demonstrated that hydrolytic genes PelA and MnP-2 could be integrated and expressed correctly from the chloroplast genome of tobacco plants. A whole plant, having ~ 470 g of biomass could feasibly yield 66,676.25 units of pectin or 21,715.46 units of manganese peroxidase. Also, this study provides new information about methods and strategies for the expression of enzymes with industrial value.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.18 n.3 20152015-05-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000300004en10.1016/j.ejbt.2015.03.002
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	Cell wall Chloroplast genetic transformation Hydrolytic enzyme Nicotiana tabacum
spellingShingle	Cell wall Chloroplast genetic transformation Hydrolytic enzyme Nicotiana tabacum Espinoza-Sánchez,Edward Alexander Álvarez-Hernández,Marianela Hazel Torres-Castillo,Jorge Ariel Rascón-Cruz,Quintín Gutiérrez-Díez,Adriana Zavala-García,Francisco Sinagawa-García,Sugey Ramona Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast
description	Background The high capacity of chloroplast genome response to integrate and express transgenes at high levels makes this technology a good option to produce proteins of interest. This report presents the stable expression of Pectin lyase (PelA gene) and the first stable expression of manganese peroxidase (MnP-2 gene) from the chloroplast genome. Results pES4 and pES5 vectors were derived from pPV111A plasmid and contain the PelA and MnP-2 synthetic genes, respectively. Both genes are flanked by a synthetic rrn16S promoter and the 3'UTR from rbcL gene. Efficient gene integration into both inverted repeats of the intergenic region between rrn16S and 3'rps'12 was confirmed by Southern blot. Stable processing and expression of the RNA were confirmed by Northern blot analysis. Enzymatic activity was evaluated to detect expression and functionality of both enzymes. In general, mature plants showed more activity than young transplastomic plants. Compared to wild type plants, transplastomic events expressing pectin lyase exhibited enzymatic activity above 58.5% of total soluble protein at neutral pH and 60°C. In contrast, MnP-2 showed high activity at pH 6 with optimum temperature at 65°C. Neither transplastomic plant exhibited an abnormal phenotype. Conclusion This study demonstrated that hydrolytic genes PelA and MnP-2 could be integrated and expressed correctly from the chloroplast genome of tobacco plants. A whole plant, having ~ 470 g of biomass could feasibly yield 66,676.25 units of pectin or 21,715.46 units of manganese peroxidase. Also, this study provides new information about methods and strategies for the expression of enzymes with industrial value.
author	Espinoza-Sánchez,Edward Alexander Álvarez-Hernández,Marianela Hazel Torres-Castillo,Jorge Ariel Rascón-Cruz,Quintín Gutiérrez-Díez,Adriana Zavala-García,Francisco Sinagawa-García,Sugey Ramona
author_facet	Espinoza-Sánchez,Edward Alexander Álvarez-Hernández,Marianela Hazel Torres-Castillo,Jorge Ariel Rascón-Cruz,Quintín Gutiérrez-Díez,Adriana Zavala-García,Francisco Sinagawa-García,Sugey Ramona
author_sort	Espinoza-Sánchez,Edward Alexander
title	Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast
title_short	Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast
title_full	Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast
title_fullStr	Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast
title_full_unstemmed	Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast
title_sort	stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2015
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000300004
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Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast

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