Abstract
BackgroundPectinases play an important role in plant cell wall deconstruction and have potential in diverse industries such as food, wine, animal feed, textile, paper, fuel, and others. The demand for such enzymes is increasing exponentially, as are the efforts to improve their production and to implement their use in several industrial processes. The goal of this study was to examine the potential of producing polygalacturonase I from Aspergillus niger in plants and to investigate the effects of subcellular compartmentalization and protein fusions on its accumulation and activity.ResultsPolygalacturonase I from Aspergillus niger (AnPGI) was transiently produced in Nicotiana benthamiana by targeting it to five different cellular compartments: apoplast, endoplasmic reticulum (ER), vacuole, chloroplast and cytosol. Accumulation levels of 2.5%, 3.0%, and 1.9% of total soluble protein (TSP) were observed in the apoplast, ER, and vacuole, respectively, and specific activity was significantly higher in vacuole-targeted AnPGI compared to the same enzyme targeted to the ER or apoplast. No accumulation was found for AnPGI when targeted to the chloroplast or cytosol. Analysis of AnPGI fused with elastin-like polypeptide (ELP) revealed a significant increase in the protein accumulation level, especially when targeted to the vacuole where the protein doubles its accumulation to 3.6% of TSP, while the hydrophobin (HFBI) fusion impaired AnPGI accumulation and both tags impaired activity, albeit to different extents. The recombinant protein showed activity against polygalacturonic acid with optimum conditions at pH 5.0 and temperature from 30 to 50°C, depending on its fusion. In vivo analysis of reducing sugar content revealed a higher release of reducing sugars in plant tissue expressing recombinant AnPGI compared to wild type N. benthamiana leaves.ConclusionOur results demonstrate that subcellular compartmentalization of enzymes has an impact on both the target protein accumulation and its activity, especially in the case of proteins that undergo post-translational modifications, and should be taken into consideration when protein production strategies are designed. Using plants to produce heterologous enzymes for the degradation of a key component of the plant cell wall could reduce the cost of biomass pretreatment for the production of cellulosic biofuels.
Highlights
Pectinases play an important role in plant cell wall deconstruction and have potential in diverse industries such as food, wine, animal feed, textile, paper, fuel, and others
Using a series of constructs targeting the polygalacturonase I from Aspergillus niger (AnPGI) to different subcellular compartments, we report here the effects of subcellular targeting on the accumulation and activity of Aspergillus niger polygalacturonase I (AnPGI) in leaves of N. benthamiana
Analysis of heterologous polygalacturonase I transiently expressed in N. benthamiana Several factors need to be taken into consideration for achieving high levels of recombinant protein accumulation in plants, including high transcript levels, correct post translational modifications, and protein turnover
Summary
Pectinases play an important role in plant cell wall deconstruction and have potential in diverse industries such as food, wine, animal feed, textile, paper, fuel, and others. The demand for such enzymes is increasing exponentially, as are the efforts to improve their production and to implement their use in several industrial processes. The goal of pretreatment is to break down the physical structure of the cell wall and make the crystalline structure of cellulose more accessible to cellulases This pretreatment process may account for up to 30% of the cost of biofuel production [1]. Enzymatic pretreatment represents a more promising and environmentally friendly technology
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