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

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.

Transgenic tobacco plants with improved cyanobacterial Rubisco expression but no extra assembly factors grow at near wild‐type rates if provided with elevated CO2

“…The chloroplast transformation technology used in the present work has the capacity to introduce multiple transgenes and appears ideal for the expression of β–carboxysomes or other CCMs in higher‐plant chloroplasts to improve photosynthesis (Lu et al ., ). The discovery of the inter‐cistronic expression element (IEE) has greatly facilitated stacking of multiple transgenes in synthetic operons for reliable expression of downstream genes in the chloroplast genome (Zhou et al ., ; Kolotilin et al ., ; Lu et al ., ). Although processing at the IEE sites was incomplete in our transformants, it is not surprising that the genes located downstream were still efficiently translated, as proteins from unprocessed multigene transcripts are often successfully produced from tobacco chloroplast transformants (Quesada‐Vargas et al ., ; Whitney et al ., ).…”

Increased bioethanol production from commercial tobacco cultivars overexpressing thioredoxin f grown under field conditions

“…The chloroplast is an ideal subcellular compartment for this purpose with very high levels of foreign protein accumulation. Exoand endoglucanases, β-glucosidases (Gray et al 2009;Ziegelhoffer et al 2009;Verma et al 2010;Gray et al 2011;Petersen and Bock 2011), endo-β-mannanase (Agrawal et al 2011), hemicellulases (Kolotilin et al 2013) and additional enzymes (pectate lyase, cutinase, swollenin, xylanase, acetyl xylan esterase, and lipase) (Verma et al 2010) have been successfully expressed in transplastomic tobacco plants in their active forms. In some cases, plant crude-extract enzyme cocktails showed a higher activity level and yielded more glucose in contrast to commercial cocktails (Verma et al 2010), demonstrating the great potential associated to plant-derived enzyme production.…”

Co-expression with the Type 3 Secretion Chaperone CesT from Enterohemorrhagic E. coli Increases Accumulation of Recombinant Tir in Plant Chloroplasts

“…All coding regions were sequenced to confirm identity and proper insertion of genes. Stable plastome transformation of the male-sterile, low-alkaloid N. tabacum cultivar 81V9 (Menassa et al, 2001) was done using standard microparticle bombardment of leaf tissue followed by three consecutive rounds of regeneration on selective medium (500 μg/mL spectinomycin), as previously described (Kolotilin et al, 2013). Transient transformation of N. benthamiana was done using standard agro-infiltration (Pereira et al, 2014).…”