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.

Keywords: hyperthermophilic cellobiohydrolase; plant cell wall; plant immunity; protein storage vacuole; saccharification; transgenic Nicotiana tabacum.

Figure 1

Transient expression of CBM3GH5 in Nicotiana tabacum. (A) Schematic representation of the vacuolar and apoplast version of CBM3GH5, referred to as CBM3GH5-HA-VAC and CBM3GH5-HA, respectively. Expected molecular weights: 80 kDa. (B) Immuno-decoration analysis of leaf extracts from CBM3GH5-HA-VAC-agroinfiltrated plants, using α-HA as primary antibody. Extraction was performed 2 days post-agro-infiltration (dpi) using a Tween 20-supplemented buffer plus heat (HT-T) or a NaCl-supplemented buffer (NaCl). Agroinfiltration with the empty vector was used as negative control. (C) (Upper panel) α-HA immuno-decoration analysis of leaf extracts from agroinfiltrated leaves. Extraction was performed two and three days post-agro-infiltration (2, 3) using the same buffers described in (B). In total, 70 ng of recombinant CBM3GH5-HA from C. reinhardtii (CR) was used as reference. (Lower panel) Activity of CBM3GH5 in the same extracts, determined by activity assay and expressed as Enzyme milliUnits (nmol reducing ends released from carboxy-methylcellulose (CMC) min⁻¹) per sample.

Figure 2

Stable transformation of CBM3GH5 in Nicotiana tabacum. (A) Comparison of the transformation efficiency of different constructs on Agrobacterium-mediated N. tabacum transformation. Numbers of regenerated plants and activity in T1 transformants are reported. The association between groups (N rooted calli and T1 plants with activity) and the CBM3GH5 version is statistically significant according to Fischer’s exact test (* p value < 0.05). n/a, not available. (B) Activity of CBM3GH5 in leaf extract from 30-day-old T1 CBM3GH5-HA-VAC plants, as determined by activity assay. (C) Activity of CBM3GH5 in leaf extracts from 30-day-old T1 CBM3GH5-HA-VAC#4 using different ratios of Tween 20-supplemented buffer per gram of tissue. (D) Activity of CBM3GH5 in leaf extract from 30-day-old T2 CBM3GH5-HA-VAC#4 plants as determined by activity assay. Enzyme activity is expressed as Enzyme Units (µmol reducing ends released from CMC min⁻¹) per gram dry weight (DW) leaf.

Figure 3

Spatial and temporal accumulation of CBM3GH5-HA-VAC in T3 transgenic plants. (A) Representative picture of 55-day-old CBM3GH5-HA-VAC#4-11, CBM3GH5-HA-VAC#6-7 and wild-type (WT) plants. (B,C) Relative expression (B) and activity (C) of CBM3GH5 in leaves (L5–L10) from 55-day-old CBM3GH5-HA-VAC#4-11 and #6-7 plants. (D) Activity of CBM3GH5 in leaf extracts from CBM3GH5-HA-VAC#4-11 at five different developmental stages (35-d: 35-day-old plant, 55-d: 55-day-old plant, 75-d: 75-day-old plant, flowering plant and seed—i.e., plant upon seed maturation). (E,F) Activity of CBM3GH5 in different tissue extracts from flowering CBM3GH5-HA-VAC#4-11 plants (i.e., L5–L10 leaf, stem, root and leaf rib) plus seeds per gram DW (E) and per gram of total soluble proteins (TSP) (F); data are from two independent experiments with consistent results.

Figure 4

Biochemical characterization of CBM3GH5-HA-VAC purified from mature leaves of CBM3GH5-HA-VAC#4-11 plants. (A) SDS-PAGE analysis of fractions (fx) eluted from anionic exchange chromatography (AEC). Black arrow points to CBM3GH5-HA. Recombinant CBM3GH5-HA from C. reinhardtii (CR) was used as reference. (B) Activity of CBM3GH5-HA-VAC in the same fractions (fx) shown in (A), expressed as relative activity (%). (C) Specific activity of CBM3GH5 from C. reinhardtii (CR) and N. tabacum (NT) towards 1% CMC; specific activity was expressed as Enzyme Units per mg of enzyme (pH 5.5, 75 °C). (D) Relative activity (%) of CBM3GH5 from N. tabacum protein storage vacuole (NT) and C. reinhardtii chloroplast (CR) towards 1% CMC, after 7 h incubation at different temperatures.

Figure 5

CBM3GH5-HA-VAC plants showed an increased temperature-dependent saccharification efficiency. (A) Sugars released upon acid-hydrolysis of leaf (black bar) and stem (grey bar) material from wild-type and transgenic CBM3GH5-HA-VAC plants, as determined by phenol–sulfuric acid assay. (B,C) Saccharification efficiency of leaf (B) and stem (C) material from CBM3GH5-HA-VAC#4-11, CBM3GH5-HA-VAC#6-7 and WT plants after 24 (black bar) and 48 (grey bar) hours of incubation with 1% Celluclast at 55 °C. (D) Saccharification efficiency of leaf material from CBM3GH5HAVAC#4-11, CBM3GH5HAVAC#6-7 and WT plants after 24 (black bar) and 48 (grey bar) hours of incubation with 1% Celluclast at 25 °C. Data are expressed as mean ± SD (N ≥ 3). Asterisks indicate statistically significant difference against control (WT) according to Student’s t test (* p < 0.05; ** p < 0.01).