Abstract
BackgroundChloroplast 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.ResultsFour 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.ConclusionOur results demonstrate the pivotal importance of the expression cassette design and appropriate tobacco cultivar for high-level transplastomic production of recombinant proteins.
Highlights
Chloroplast transformation in tobacco has been used extensively to produce recombinant proteins and enzymes
In the large single copy (LSC) region a silent intergenic spacer between trnfM and trnG genes has been extensively utilized [6,11,16,21,22]. Cassettes integrated into these plastome loci were reported to produce abundant yields of recombinant proteins, some reaching a massive accumulation of 70% of the total soluble protein (TSP) in the plant leaf tissue and overburdening the protein synthesis machinery in the plastid [23,24,25]
We hypothesized that testing levels of accumulation of the foreign proteins produced from different cassettes would determine an ideal configuration, capable of expressing other recombinant proteins at high levels
Summary
Chloroplast transformation in tobacco has been used extensively to produce recombinant proteins and enzymes. 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. In the large single copy (LSC) region a silent intergenic spacer between trnfM and trnG genes has been extensively utilized [6,11,16,21,22] Cassettes integrated into these plastome loci were reported to produce abundant yields of recombinant proteins, some reaching a massive accumulation of 70% of the total soluble protein (TSP) in the plant leaf tissue and overburdening the protein synthesis machinery in the plastid [23,24,25]. Factors such as mRNA stability, mRNA-rRNA interactions, appropriate codon usage, efficient processing of polycistronic transcripts, the N-terminal amino acid residue and sequences downstream the initial methionine of the nascent polypeptide chain, as well as protein secondary structure - all exert tight control over recombinant protein production and accumulation in chloroplasts [26,27,28,29,30]
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