Optimization of Acidothermus cellulolyticus Endoglucanase (E1) Production in Transgenic Tobacco Plants by Transcriptional, Post-transcription and Post-translational Modification

Abstract

An attempt was made to obtain a high-level production of intact Acidothermus cellulolyticus endoglucanase (E1) in transgenic tobacco plants. The E1 expression was examined under the control of the constitutive and strong Mac promoter or light-inducible tomato Rubisco small sub-unit (RbcS-3C) promoter with its original or Alfalfa Mosaic Virus (AMV) RNA4 5'-untranslated leader (UTL) and targeted to different sub-cellular compartments via transit peptides. The transit peptides included native E1, endoplasmic reticulum, vacuole, apoplast, and chloroplast. E1 expression and its stability in transgenic plants were determined via E1 activity, protein immunoblotting, and RNA gel-blotting analyses. Effects of sub-cellular compartments on E1 production and its stability were determined in transgenic tobacco plants carrying one of six transgene expression vectors, where the E1 was under the control of Mac promoter, mannopine synthase transcription terminator, and one of the five transit peptides. Transgenic tobacco plants with an apoplastic transit peptide had the highest average E1 activity and protein accumulation, which was about 0.25% of total leaf soluble proteins estimated via E1 specific activity and protein gel blots. Intercellular fluid analyses confirmed that E1 signal peptide functioned properly in tobacco cells to secret E1 protein into the apoplast. By replacing RbcS-3C UTL with AMV RNA4 UTL E1 production was enhanced more than twofold, while it was less effective than the mannopine synthase UTL. It was observed that RbcS-3C promoter was more favorable for E1 expression in transgenic plants than the Mac promoter. E1 activity in dried tobacco seeds stored one year at room temperature was 45% higher than that observed immediately after harvesting, suggesting that E1 protein can be stored at room temperature for a long period. E1 stability in different sub-cellular compartments and the optimal combination of promoter, 5'-UTL, and sub-cellular compartmentation for heterologous protein production in transgenic plants are discussed.

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

“…The various factors that affect the expression of a particular heterologous gene and subsequent production of its protein in plant cells include: (i) transcriptional determinants such as transgene copy number, site of integration of the T-DNA in the chromosome, and promoter activity [50]; (ii) posttranscriptional factors including, 5 0 -untranslated leader sequence, 3 0 -polyadenylation, and mRNA stability [51,52]; and (iii) translational/post-translational factors such as protein stability, modification, and trafficking [52,53]. Promoters and enhancers (cis-acting regulatory elements) play a crucial role in regulating spatial and temporal patterns of transgene expression at particular stages or in response to particular conditions [50][51][52][53][54][55].…”

“…Promoters and enhancers (cis-acting regulatory elements) play a crucial role in regulating spatial and temporal patterns of transgene expression at particular stages or in response to particular conditions [50][51][52][53][54][55]. Several promoters and other cis-acting elements including signal peptides of plant, plant pathogen, or bacterial origin used in heterologous expression vectors have been described (Table S1 in the supplementary material online).…”

“…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].…”

“…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].…”

“…This enzyme was also expressed at a higher level (2.6% TSP) when targeted to the apoplast using plant-based signal peptides [57]. Expression of A. cellulolyticus E1 and E1cd proteins in the tobacco leaf apoplastic space was superior to either the chloroplast or the cytosol [41,51]. Several other thermophilic enzyme genes have been successfully and functionally expressed in plant apoplasts (Table S1 in the supplementary material online).…”