Optimization of transplastomic production of hemicellulases in tobacco: effects of expression cassette configuration and tobacco cultivar used as production platform on recombinant protein yields

Abstract

Background: Chloroplast transformation in tobacco has been used extensively to produce recombinant proteins and enzymes. Chloroplast expression cassettes can be designed with different configurations of the cis-acting elements that govern foreign gene expression. With the aim to optimize production of recombinant hemicellulases in transplastomic tobacco, we developed a set of cassettes that incorporate elements known to facilitate protein expression in chloroplasts and examined expression and accumulation of a bacterial xylanase XynA. Biomass production is another important factor in achieving sustainable and high-volume production of cellulolytic enzymes. Therefore, we compared productivity of two tobacco cultivars - a low-alkaloid and a high-biomass - as transplastomic expression platforms.

Results: Four different cassettes expressing XynA produced various mutant phenotypes of the transplastomic plants, affected their growth rate and resulted in different accumulation levels of the XynA enzyme. The most productive cassette was identified and used further to express XynA and two additional fungal xylanases, Xyn10A and Xyn11B, in a high-biomass tobacco cultivar. The high biomass cultivar allowed for a 60% increase in XynA production per plant. Accumulation of the fungal enzymes reached more than 10-fold higher levels than the bacterial enzyme, constituting up to 6% of the total soluble protein in the leaf tissue. Use of a well-characterized translational enhancer with the selected expression cassette revealed inconsistent effects on accumulation of the recombinant xylanases. Additionally, differences in the enzymatic activity of crude plant extracts measured in leaves of different age suggest presence of a specific xylanase inhibitor in the green leaf tissue.

Conclusion: Our results demonstrate the pivotal importance of the expression cassette design and appropriate tobacco cultivar for high-level transplastomic production of recombinant proteins.

Figure 1

Chloroplast Expression Cassettes (CECs) used in this study; Phenotype of primary transformants (cv. 81V9); Confirmation of homoplastomy; Confirmation of XynA expression. A. Four Chloroplast Expression Cassettes (CECs, designated CEC1 through -C4) with varying configuration of the cis-acting regulatory elements are shown. The integration of the CECs into the tobacco plastome was designed to occur in the transcriptionally-active spacer region between the trnI and trnA genes. The wild type (WT) plastome trnI - trnA region is shown at the bottom. The expected sizes of Rsr II-digested fragments are indicated. Thick black lines represent hybridization sites for the probe used in Southern blot analyses. IEE = Intercistronic Expression Element with the Shine-Dalgarno sequence from the 5' UTR of bacteriophage T7 gene 10 fused to the 3' end; aadA = gene encoding aminoglycoside 3' adenylyltransferase; TpsbC and TrbcL = 3' UTRs of psbC and rbcL from the white poplar plastome; PpsbA = promoter and 5’ UTR of tobacco psbA gene; mPrrn – mutated chloroplast rrn operon promoter; XynA::T = gene encoding the XynA protein fused at the C-terminal with c-myc and strepII tags. B. Transformation with different CECs produced different phenotypes in primary transformants (T₀). C. Southern blot RFLP analysis and confirmation of homoplastomy of T₀ transformants. Total DNA extracted from 2 independent transformants for each CEC and 1 untransformed plant (WT) was digested with Rsr II and analyzed using Southern blotting. All T₀ transformants displayed a single band of expected size, confirming homoplastomy. D. Immunoblot confirmation of XynA expression in plants transformed with different CECs. Lanes 1 through 4 – extracts from CEC1 through CEC4, respectively. Lane 5 – cv. 81V9 WT as negative control. Each lane contains equal amounts of extracted leaf tissue (equivalent to 4.0 mg/lane). Known amounts (ng) of a c-myc-tagged control protein are indicated above the standard curve lanes.

Figure 2

Synchronized germination and growth of cv. 81V9 T₁ plants – phenotype. A. Phenotypic differences displayed by two-week-old T₁ transplastomic seedlings (CEC1 through CEC4) germinated on selective medium. Black bar = 1 cm. Untransformed (WT) seedlings germinated on selective (+Spec) and non-selective (−Spec) medium were used as a control. Impact of different cassettes on growth rate of the T₁ transplastomic plants at 40 days (B) and at 80 days (C) post-germination is shown in comparison to WT plants.

Figure 3

Analysis of activity of different cassettes introduced into cv. 81V9 T₁ plants. A. Northern blot analysis of xynA and aadA transcript abundance produced from different CECs. Lanes 1 through 4 represent total plant RNA extracted from T₁ transplastomic plants for CEC1- through CEC4-XynA, respectively. Lane 5 represents WT RNA. Equal amounts of RNA were loaded in each lane and confirmed by staining (left panel); the blot hybridized to a xynA-specific probe (middle panel) and aadA-specific probe (right panel) displayed differences in transcript levels and sizes for both genes produced from different CECs. B, C. Immunoblot of XynA (B) and AadA (C) protein product produced from different CECs and assessed in the same leaves sampled for the RNA analyses (A). Each lane represents 0.4 mg of extracted leaf tissue. Lane 5 - WT leaf extract. Known amounts (in ng) of a c-myc-tagged control protein are indicated above the standard curve lanes (B).

Figure 4

Spatial accumulation of XynA in cv. 81V9 T₁ transplastomic plants transformed with different CECs. A. Schematic representation of the sampling procedure to obtain spatial accumulation pattern of XynA expressed from different CECs in mature T₁ transplastomic plants. Samples from ten leaves (top to bottom) were extracted using equal sample weight/buffer volume (w/v) ratio and equal amounts of extracts (each lane represents 4.0 mg of extracted leaf tissue) were analysed by immunoblotting after SDS-PAGE (B). The leaf #3 corresponds to the size of a leaf used for initial expression analyses (indicated with an arrow). Untransformed (WT) plants were used as a negative control. Known amounts (in ng) of a c-myc-tagged control protein are indicated above the standard curve lanes.

Figure 5

Constructs for fungal xyn10A and xyn11B expression; Confirmation of homoplastomy of T₀ plants (cv. I64) and effects of T7G10 translational enhancer on accumulation levels of Xyn10A and Xyn11B. A. CEC4 was used to express fungal xylanases Xyn10A and Xyn11B in high-biomass tobacco cv. I64. The sequences of the xyn10A and xyn11B genes were cloned into the GOI position of pCEC4.The expected Rsr II-generated fragment sizes for Southern blot RFLP analysis are indicated for each construct and for the wild type (WT) plastome. B. Southern blot RFLP analysis of cv. I64 T₀ transplastomic lines transformed with pCEC4-Xyn10A and pCEC4-Xyn11B to confirm homoplastomy, two clones per construct analysed. C. Phenotype of T₀ cv. I64 transplastomic lines is identical to WT plants. D. Immunoblot-assisted accumulation analysis for Xyn10A and Xyn11B expressed from CEC4. Two independent primary transformants per construct were examined (lanes 1 and 2 for each protein). Extractions were performed using equal ratio of sample weight/extraction buffer volume (w/v = 1/5). Each lane contains extract equivalent to 4.0 mg of extracted leaf tissue. Untransformed WT extract was used as negative control. Known amounts (ng) of a c-myc-tagged control protein are indicated above the standard curve lanes. E. CEC5 construct (identical to CEC4, but lacking the T7g10 DB element) was used for expression of native forms of Xyn10A and Xyn11B without the T7g10 N-terminal fusion. F. Homoplastomy confirmation was carried out as described above for T₀ cv. I64 transplastomic lines expressing Xyn10A and Xyn11B from CEC5 (B). G. Phenotype of T₀ cv. I64 transplastomic lines is identical to WT plants. H. Accumulation analysis for Xyn10A and Xyn11B expressed from CEC5 was carried out as described in (D).

Figure 6

Tobacco cv. I64 T₁ transplastomic plant development and spatial expression patterns of different xylanases. A. Phenotypes of T₁ transplastomic cv. I64 plants at 40 days post-germination. A slight developmental delay, which was observed to various extent in the transplastomic lines, was completely compensated during further growth as the plants reached maturity, displaying very similar size and flowering time when compared with WT untransformed plants (B, also Table 2). One-meter ruler is pictured on the left for a size reference. C. Western blot-assisted assessment of the spatial accumulation profiles of XynA, Xyn10A and Xyn11B in mature cv. I64 plants. Lanes 1 through 10 represent extracts from 10 leaves (top to bottom), each lane represents extract equivalent to 2.5 mg leaf tissue for XynA and Xyn10A expressed from CEC4; for Xyn10A expressed from CEC5 and Xyn11B expressed from CEC4 and CEC5 each lane represents extract equivalent to 0.1 mg leaf tissue. Known amounts (in ng) of a c-myc-tagged control protein are indicated above the standard curve lanes.

Figure 7

Determination of enzyme amounts in crude extracts of XynA-, Xyn10A- and Xyn11B-expressing cv. I64 plants measured in mature green leaves (GL) and lower senescing leaves (SL) and a zymogram of the extracts.A. Three repeated extractions from GL and SL of the cv.I64 lines expressing XynA and Xyn10A from CEC4 and Xyn11B from CEC5 were analysed by western blot for quantification of recombinant proteins content. Each lane of XynA extracts from both GL and SL represents 2.5 mg of extracted leaf tissue; for Xyn10A and Xyn11B each lane represents 0.05 mg and 0.25 mg of extracted leaf tissue for GL and SL extracts, respectively. WT – extract of untransformed WT plants used as a negative control. Known amounts (in ng) of a c-myc-tagged control protein are indicated above the standard curve lanes. B. Zymogram of GL and SL extracts (combined of three repeats) for XynA, Xyn10A and Xyn11B, resolved on a 12% SDS-PAGE gel containing 0.1% (w/v) xylan. Each lane contains equivalent of 2.5 mg of extracted leaf tissue. Equal amount of extracts from WT leaves were used as a negative control. Arrows indicate expected sizes of the protein bands detected on western blots.