Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
Vol. 18, No. 3, 2015, pp. 161-168
Bioline Code: ej15028
Full paper language: English
Document type: Research Article
Document available free of charge
Electronic Journal of Biotechnology, Vol. 18, No. 3, 2015, pp. 161-168
© Copyright 2015 - Electronic Journal of Biotechnology
Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast|
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
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
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.
Cell wall; Chloroplast genetic transformation; Hydrolytic enzyme; Nicotiana tabacum
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