Production and characterization of in planta transiently produced polygalacturanase from Aspergillus nigerand its fusions with hydrophobin or ELP tags

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.

In‐solution antibody harvesting with a plant‐produced hydrophobin–Protein A fusion

“…The HFBI fusion has previously been reported to enhance the accumulation of some fusion proteins in plants (Gutiérrez et al ., ; Jacquet et al ., ; Joensuu et al ., ). However, this effect has not been consistent and several studies have shown no improvement in yields (Pereira et al ., ; Phan et al ., ). This is the first report on improved product accumulation in BY‐2 cells using a HFB tag.…”

“…HFB fusion proteins have been produced in filamentous fungi (Linder et al, 2004;Mustalahti et al, 2013), insect cell cultures (Lahtinen et al, 2008), plants (Guti errez et al, 2013;Jacquet et al, 2014;Joensuu et al, 2010;Pereira et al, 2014;Phan et al, 2014;Saberianfar et al, 2015) and in plant cell cultures (Reuter et al, 2014). Whereas production of HFB fusion proteins has been challenging in some other hosts, plants have shown to be an especially suitable production platform.…”

Co-expression with the Type 3 Secretion Chaperone CesT from Enterohemorrhagic E. coli Increases Accumulation of Recombinant Tir in Plant Chloroplasts

“…Stable plastome transformation of the male-sterile, low-alkaloid N. tabacum cultivar 81V9 (Menassa et al, 2001) was done using standard microparticle bombardment of leaf tissue followed by three consecutive rounds of regeneration on selective medium (500 μg/mL spectinomycin), as previously described (Kolotilin et al, 2013). Transient transformation of N. benthamiana was done using standard agro-infiltration (Pereira et al, 2014). For expression of one or two genes of interest, each culture of Agrobacterium tumefaciens EHA-105 carrying a different construct of interest was diluted to a final optical density at 600 nm (OD 600 ) of 0.45 in Gamborg’s solution (3.2 g/L Gamborg’s B5 salts with vitamins, 20 g/L sucrose, 10 mM MES, pH 5.6, 200 μM acetosyringone), and A. tumefaciens carrying a construct for expression of the p19 suppressor of gene silencing (Silhavy et al, 2002) was suspended at a final OD 600 of 0.10.…”

A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates

“…We chose to target the protein to the ER because it has consistently been shown to provide a superior environment for folding and storing recombinant proteins in plant cells, while cytosolic expression results in little to no protein accumulation in the majority of the cases [11, 16, 18], reviewed in [19]. To determine if PFA would accumulate in plants, the PFA protein was produced in tobacco leaves via Agrobacterium -mediated transient expression.…”