Dataset Information

Expression of a Hyperthermophilic Cellobiohydrolase in Transgenic Nicotiana tabacum by Protein Storage Vacuole Targeting.

ABSTRACT: Plant expression of microbial Cell Wall Degrading Enzymes (CWDEs) is a valuable strategy to produce industrial enzymes at affordable cost. Unfortunately, the constitutive expression of CWDEs may affect plant fitness to variable extents, including developmental alterations, sterility and even lethality. In order to explore novel strategies for expressing CWDEs in crops, the cellobiohydrolase CBM3GH5, from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus, was constitutively expressed in N. tabacum by targeting the enzyme both to the apoplast and to the protein storage vacuole. The apoplast targeting failed to isolate plants expressing the recombinant enzyme despite a large number of transformants being screened. On the opposite side, the targeting of the cellobiohydrolase to the protein storage vacuole led to several transgenic lines expressing CBM3GH5, with an enzyme yield of up to 0.08 mg g DW^-1 (1.67 Units g DW^-1) in the mature leaf tissue. The analysis of CBM3GH5 activity revealed that the enzyme accumulated in different plant organs in a developmental-dependent manner, with the highest abundance in mature leaves and roots, followed by seeds, stems and leaf ribs. Notably, both leaves and stems from transgenic plants were characterized by an improved temperature-dependent saccharification profile.

SUBMITTER: Benedetti M

PROVIDER: S-EPMC7767180 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Publications

Expression of a Hyperthermophilic Cellobiohydrolase in Transgenic <i>Nicotiana tabacum</i> by Protein Storage Vacuole Targeting.

Benedetti Manuel M Vecchi Valeria V Guardini Zeno Z Dall'Osto Luca L Bassi Roberto R

Plants (Basel, Switzerland) 20201218 12

Plant expression of microbial Cell Wall Degrading Enzymes (CWDEs) is a valuable strategy to produce industrial enzymes at affordable cost. Unfortunately, the constitutive expression of CWDEs may affect plant fitness to variable extents, including developmental alterations, sterility and even lethality. In order to explore novel strategies for expressing CWDEs in crops, the cellobiohydrolase CBM3GH5, from the hyperthermophilic bacterium <i>Caldicellulosiruptor saccharolyticus</i>, was constitutiv ...[more]

PMID: 33353085

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Project description:Plant molecular farming (PMF) is an important growing prospective approach in plant biotechnology; it includes production of recombinant pharmaceutical and industrial proteins in large quantities from engineered plants. Elastin is a major protein component of tissues that require elasticity, it helps keep skin smooth as it stretches to allow normal. Elastin is used as a raw material for the cosmetic industry. In this work, we aimed to use plant as a bioreactor for the expression and production of the full human tropoelastin protein. Agrobacterium- mediated transient expression system into Nicotiana tabacum using syringe agroinfiltration was used to provide fast and convenient way to produce recombinant proteins with greater expression overall the plant leaf. This study aimed to establish an efficient and rapid system for transiently expression and production of human recombinant tropoelastin protein in transgenic N. tabacum plants. Modified elastin (ELN) gene was biosynthesized and cloned into pCambia1390 vector to be used into N. tabacum agroinfilteration. Optimization of codon usage for the human tropoelastin gene, without changing the primary structure of the protein was carried out to ensure high expression in tobacco plants. The obtained data proved that the 5(th) day post-infiltration is the optimum interval to obtain the maximum production of our recombinant protein. Southern blot analysis was able to detect 2175 bp fragment length representing the ELN orf (open reding frame). On the other hand, ELN -expression within plant's tissue was visualized by RT-PCR during the period 3-10 days post agroinfiltration. At the protein level, western and ELISA confirmed the expression of recombinant tropoelastin protein. Western blot analysis detected the tropoelastin protein as parent band at ?70 kDa from freshly extracted protein, while two degraded bands of ?55 and ?45 kDa, representing a pattern of tropoelastin were appeared with frozen samples. This study showed that biosynthetic ELN gene was successfully expressed into N. tabacum leaves using agroinfiltration technique.

Dataset Information

Expression of a Hyperthermophilic Cellobiohydrolase in Transgenic Nicotiana tabacum by Protein Storage Vacuole Targeting.

Publications

Expression of a Hyperthermophilic Cellobiohydrolase in Transgenic <i>Nicotiana tabacum</i> by Protein Storage Vacuole Targeting.

Similar Datasets

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