An efficient downstream box fusion allows high-level accumulation of active bacterial beta-glucosidase in tobacco chloroplasts

Abstract

Production of enzymes for lignocellulose hydrolysis in planta has been proposed as a lower-cost alternative to microbial production, with plastid transformation as a preferred method due to high foreign protein yields. An important regulator of chloroplast protein production is the downstream box (DB) region, located immediately downstream of the start codon. Protein accumulation can vary over several orders of magnitude by altering the DB region. Experiments in bacteria have suggested that these differences in protein accumulation may result from changes in translation efficiency, though the precise mechanism of DB function is not known. In this study, three DB regions were fused to the bglC ORF encoding a β-glucosidase from the thermophilic bacterium Thermobifida fusca and inserted into the tobacco (Nicotiana tabacum) plastid genome. More than a two order of magnitude of difference in BglC protein accumulation was observed, dependent on the identity of the DB fusion. Differential transcript accumulation explained some the observed differences in protein accumulation, but in addition, less 3' degradation of bglC transcripts was observed in transgenic plants that accumulated the most BglC enzyme. Chloroplast-produced BglC was active against both pure cellobiose and against tobacco lignocellulose. These experiments demonstrate the potential utility of transplastomic plants as a vehicle for heterologous β-glucosidase production for the cellulosic ethanol industry.

Genetic Engineering for Microalgae Strain Improvement in Relation to Biocrude Production Systems

“…Genetic fusion of endogenous regions to an exogenous protein of interest may represent another effective strategy for high-level transgene expression (Gray et al 2011;Kasai et al 2003). A disadvantage of the fusion protein approach may be reduced industrial or clinical values or increased cost of purifying the protein of interest from its fusion partner.…”

Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion

“…The cauliflower mosaic virus (CaMV) 35S promoter is extensively used in plant expression vectors (reviewed in [31,44]). Multiple copies of enhancer sequences from highly active promoters can be stacked to boost transgene expression [32][33][34]50,51].…”

“…Additionally, 3 0 -untranslated sequences downstream of the stop codon playing a critical role in processing have been included along with signal sequences targeting and stabilizing the message for polyadenylation (Table S1 in the supplementary material online). Polyadenylation strongly enhances the heterologous expression of microbial genes in plants [33,34,[40][41][42]. Sequences that are located immediately around the translational start site, termed Kozak sequences [56], can also be modified to fit the consensus initiation sequence of plants in order to achieve higher rates of translation.…”

“…N-terminal or C-terminal transit peptides generally possess the necessary information for the correct targeting of proteins to subcellular organelles. Subcellular targeting of heterologous proteins generally yields higher accumulation levels in transgenic plants than expression in the cytosol [32][33][34][35][36][37][38][39][40][41][42]47,51]. When expressed in subcellular compartments, heterologous proteins typically display correct folding and activity, appropriate glycosylation, reduced degradation, and increased stability, as compared to expression and accumulation in the cytosol [8,44].…”

“…Development of hyperthermophilic fermentation microbes would confer an additional advantage when using the in planta approach. Plants are increasingly being investigated as potential 'green bioreactors' for the cost-effective and large-scale production of biologically active recombinant enzymes [31][32][33][34][35][36], but the production of cellulolytic enzymes using transgenic plants has mostly been restricted to mesophilic and thermotolerant enzymes [37][38][39][40][41][42][43][44][45]. To the best of our knowledge, the effects of such expression on the efficiency of biomass conversion have been reported in few instances [15].…”