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Sammendrag

Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost‐effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large‐scale protein production, and extensive host‐specific post‐translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum‐based transient expression technology, and this recombinant enzyme (TrCel7Arec) was compared with the native fungal enzyme (TrCel7Anat) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N‐terminal glutamate of TrCel7Arec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7Arec was vulnerable to proteolytic digestion during protein production due to the absence of O‐mannosylation in the plant host as compared with the native protein. In general, the purified full‐length TrCel7Arec had 25% lower catalytic activity than TrCel7Anat and impaired substrate‐binding properties, which can be attributed to larger N‐glycans and lack of O‐glycans in TrCel7Arec. All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases.

Sammendrag

1. Neodiprion sertifer nucleopolyhedrovirus (NeseNPV) is widely used as a viral bio-insecticide against larvae of the European pine sawfly N. sertifer (Geoff.) (Hymenoptera: Diprionidae), which is one of the most harmful defoliators of pines in Northern Europe. A major obstacle to studying this pathogenic virus in nature is the difficulty of confirming and quantifying the presence of NeseNPV. 2. In the present study, we developed real-time polymerase chain reaction (PCR) primers, based on the caspid gene 39 sequence, for the specific and quantitative detection of NeseNPV. The quantitative real-time PCR (qPCR) assay can detect virus from any substrate tested, including different insect life stages (egg, larval, adult), pine foliage, and litter or ground vegetation. The reproducible detection limit for the real-time assay is 0.013 pg of viral DNA (0.013 × 10−12 g), corresponding to 136 viral genomes or approximately one to seven virus occlusion bodies per sample. 3. qPCR is a specific, quantitative, sensitive, reliable and flexible procedure, and is a good supplement to conventional microscopy- or bioassay-based methods for detection of the virus. We have used qPCR to quantify the level of NeseNPV in samples collected in the field after aerial application of the virus, and demonstrated significantly higher virus levels in sawfly larvae from sprayed areas compared with unsprayed control areas 4 weeks after spraying. 4. This qPCR assay can be used to determine important aspects of the biology of NeseNPV (e.g. virus levels in different insect life stages and in their microhabitats on pine foliage and in forest litter).