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

Abstract

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.

Reducing biomass recalcitrance by heterologous expression of a bacterial peroxidase in tobacco (Nicotiana benthamiana)

“…Transgenic expression of other classes of fungal peroxidases such as the P . chrysosporium manganese peroxidase isozyme H3 (MnP-2) in tobacco chloroplast 70 , MnP from Coriolus versicolor 71 and the secretory Trametes . versicolor LiP 72 also in tobacco, and T .…”

“…Although plant heterologous expression of ligninolytic enzymes such as LiP and MnP have been reported previously, those prior studies were aimed at studying the feasibility of recombinant enzyme production 70 , 71 , 99 and phytoremediation of toxic chemicals from the environment 71 – 73 . Accumulation of lignin degrading enzymes such as LiP, MnP, DyPs and laccases with the purpose of improving saccharification efficiency has never been reported.…”

Biotechnological Applications of Plastid Foreign Gene Expression

“…To supply the necessity of efficient enzymes in the industry, the microorganisms are the first potential source of enzymes to be used in genetic engineering [26,38] and currently, from them it has been possible to obtain endoglucanases, xylanases, cellobiohydrolases, exoglucanases, glycosyl hydrolases, glucuronoyl esterases, ferulic acid esterase and acetylesterases [19]. However, the enzymatic activity obtained from microorganism's expression is limited by the enzymatic capacity inherent to own system and the protein overexpression is only possible with the change of expression system; to supply the high quantities of proteins required by industry, currently, the chloroplast genetic engineering has been used to express several hydrolytic enzymes genes such as cellulases (bgl1C, cel6B, cel9A, xeg74, celA, celB, bgl1, Cel6, Cel7, EndoV, CelKI, Cel3, TF6A, Pga2, Vlp2 peroxidase genes), pectinases (PelA genes), ligninases (MnP-2 genes) and xylanases (xyn, xynA genes) [14,[39][40][41][42][43].…”

“…On the other hand, there are reports where transgenic plants are morphologically and agronomically indistinguishable from the untransformed plants. These open new avenues for large scale production of several other industrially useful cellulolytic enzymes through chloroplast expression [39,48,49].…”

Strategies for the production of cell wall‐deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production

“…Targeting expression to locations other than the cytosol and apoplast can also help prevent phenotypic defects. Constitutive expression of manganese peroxidase in vegetative tissues can cause severe negative phenotypic impacts including cell death and lesions (Clough et al ., ); however, this was not observed when the enzyme was targeted to the chloroplasts (Espinoza‐Sánchez et al ., ), or produced in seeds (Clough et al ., ).…”