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

Manuel Benedetti,Roberto Bassi,Zeno Guardini,Valeria Vecchi,Luca Dall'Osto

doi:10.3390/plants9121799

Manuel Benedetti, Roberto Bassi + Show 3 more

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https://doi.org/10.3390/plants9121799

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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.

Highlights

In recent years, the request for sustainable fuels has promoted research toward the use of plant biomass, an abundant source of renewable energy, for production of biofuels
The protein CBM3GH5 was capped with the sequence encoding the signal peptide of PGIP2 from Phaseolus vulgaris (MTQFNIPVTMSSSLSIILVILVSLRTALSE-) for targeting the enzyme to the apoplast [6,14,29] since the entry in the secretory pathway is required for an efficient vacuole sorting [26,30]; the CBM3GH5-encoding sequence devoid of the C-terminal Pro-Peptide was used to redirect the enzyme to the apoplast (Figure 1A)
Plant cell wall structures can be successfully altered by using focused transgenic-based strategies for increasing the saccharification efficiency of plant biomass: alterations in cell wall structures may be achieved by either modifying the level of endogenous enzymes involved in the metabolism of plant cell wall polysaccharides or accumulating recombinant Cell Wall Degrading Enzymes (CWDEs) into the apoplast

Summary

Introduction

The request for sustainable fuels has promoted research toward the use of plant biomass, an abundant source of renewable energy, for production of biofuels. Biological conversion into fermentable sugars requires production of large amounts of microbial Cell Wall Degrading Enzymes (CWDEs), whose expression in planta might provide a low-cost production platform together with a direct contact with their natural substrates—i.e., plant cell wall polysaccharides. The in muro targeting of CWDEs does enhance the hydrolysis of cell wall polysaccharides into fermentable sugars [1] and yet often leads to undesired side effects [2]. CWDEs are produced by phytopathogens to dismantle the cell wall integrity, supporting the infection process [3] and providing carbon sources for the microbe [4]. Plants evolved a system of plant pattern-recognition receptors (PPRRs) to promptly perceive CWDEs secreted by pathogens—e.g., by sensing the products of their activity (Damage Associated Molecular Patterns, DAMPs) [5,6,7] or by recognizing specific epitopes in CWDEs [8,9] such as Microbe Associated Molecular Patterns (MAMPs) [10].

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