Engineering the cbh1 Promoter of Trichoderma reesei for Enhanced Protein Production by Replacing the Binding Sites of a Transcription Repressor ACE1 to Those of the Activators

2020 ◽  
Vol 68 (5) ◽  
pp. 1337-1346 ◽  
Author(s):  
Xianhua Sun ◽  
Xuhuan Zhang ◽  
Huoqing Huang ◽  
Yuan Wang ◽  
Tao Tu ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Binding Sites ◽  
Protein Production

scholarly journals Comparative analysis of early divergent land plants and construction of DNA tools for hyper-expression in Marchantia chloroplasts

2020 ◽  
Author(s):  
Eftychios Frangedakis ◽  
Fernando Guzman-Chavez ◽  
Marius Rebmann ◽  
Kasey Markel ◽  
Ying Yu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Protein Production ◽  
Marchantia Polymorpha ◽  
Pentatricopeptide Repeat ◽  
Test Bed ◽  
High Ploidy ◽  
Post Transcriptional Regulation ◽  
Very High ◽  
Rapid Transformation

ABSTRACTChloroplast genes are present at high ploidy in plants, and capable of driving very high levels of gene expression if mRNA production and stability are properly regulated. Marchantia polymorpha is a simple model plant that allows rapid transformation studies, however post-transcriptional regulation in plastids is poorly characterized in this liverwort. We have mapped patterns of transcription in Marchantia chloroplasts. Furthermore, we have obtained and compared sequences from 51 early-divergent plant species, and identified putative sites for pentatricopeptide repeat protein binding that are thought to play important roles in mRNA stabilisation. Candidate binding sites were tested for their ability to confer high levels of reporter gene expression in Marchantia chloroplasts, and levels of protein production and effects on growth were measured in homoplasmic transformed plants. We have produced novel DNA tools for protein hyper-expression in a facile plant system that is a test-bed for chloroplast engineering.

scholarly journals Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Daniel P. Kiesenhofer ◽  
Robert L. Mach ◽  
Astrid R. Mach-Aigner
Keyword(s):  
Trichoderma Reesei ◽  
Transcription Start Site ◽  
Binding Sites ◽  
Inverted Repeat ◽  
Promoter Activity ◽  
Promoter Strength ◽  
Start Site ◽  
Transcription Start ◽  
Content Type ◽  
Sheer Number

ABSTRACTTrichoderma reeseican produce up to 100 g/liter of extracellular proteins. The major and industrially relevant products are cellobiohydrolase I (CBHI) and the hemicellulase XYNI. The genes encoding both enzymes are transcriptionally activated by the regulatory protein Xyr1. The first 850 nucleotides of thecbh1promoter contain 14 Xyr1-binding sites (XBS), and 8 XBS are present in thexyn1promoter. Some of these XBS are arranged in tandem and others as inverted repeats. One suchciselement, an inverted repeat, plays a crucial role in the inducibility of thexyn1promoter. We investigated the impact of the properties of suchciselements by shuffling them by insertion, exchange, deletion, and rearrangement ofciselements in both thecbh1andxyn1promoter. A promoter-reporter assay using theAspergillus nigergoxAgene allowed us to measure changes in the promoter strength and inducibility. Most strikingly, we found that an inverted repeat of XBS causes an important increase incbh1promoter strength and allows induction by xylan or wheat straw. Furthermore, evidence is provided that the distances ofciselements to the transcription start site have important influence on promoter activity. Our results suggest that the arrangement and distances ofciselements have large impacts on the strength of thecbh1promoter, whereas the sheer number of XBS has only secondary importance. Ultimately, the biotechnologically importantcbh1promoter can be improved byciselement rearrangement.IMPORTANCEIn the present study, we demonstrate that the arrangement ofciselements has a major impact on promoter strength and inducibility. We discovered an influence on promoter activity by the distances ofciselements to the transcription start site. Furthermore, we found that the configuration ofciselements has a greater effect on promoter strength than does the sheer number of transactivator binding sites present in the promoter. Altogether, the arrangement ofciselements is an important factor that should not be overlooked when enhancement of gene expression is desired.

The effect of specific growth rate on protein synthesis and secretion in the filamentous fungus Trichoderma reesei

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 135-143 ◽  
Author(s):  
Tiina M. Pakula ◽  
Katri Salonen ◽  
Jaana Uusitalo ◽  
Merja Penttilä
Keyword(s):  
Growth Rate ◽  
Protein Synthesis ◽  
Specific Growth Rate ◽  
Trichoderma Reesei ◽  
Growth Rates ◽  
Protein Production ◽  
Specific Growth ◽  
Synthesis Rate ◽  
Specific Rate ◽  
Specific Growth Rates

Trichoderma reesei was cultivated in chemostat cultures on lactose-containing medium. The cultures were characterized for growth, consumption of the carbon source and protein production. Secreted proteins were produced most efficiently at low specific growth rates, 0·022–0·033 h−1, the highest specific rate of total protein production being 4·1 mg g−1 h−1 at the specific growth rate 0·031 h−1. At low specific growth rates, up to 29 % of the proteins produced were extracellular, in comparison to only 6–8 % at high specific growth rates, 0·045–0·066 h−1. To analyse protein synthesis and secretion in more detail, metabolic labelling of proteins was applied to analyse production of the major secreted protein, cellobiohydrolase I (CBHI, Cel7A). Intracellular and extracellular labelled CBHI was quantified and analysed for pI isoforms in two-dimensional gels, and the synthesis and secretion rates of the molecule were determined. Both the specific rates of CBHI synthesis and secretion were highest at low specific growth rates, the optimum being at 0·031 h−1. However, at low specific growth rates the secretion rate/synthesis rate ratio was significantly lower than that at high specific growth rates, indicating that at low growth rates the capacity of cells to transport the protein becomes limiting. In accordance with the high level of protein production and limitation in the secretory capacity, the transcript levels of the unfolded protein response (UPR) target genes pdi1 and bip1 as well as the gene encoding the UPR transcription factor hac1 were induced.

Identification of specific binding sites for XYR1, a transcriptional activator of cellulolytic and xylanolytic genes in Trichoderma reesei

2009 ◽  
Vol 46 (8) ◽  
pp. 564-574 ◽  
Author(s):  
Takanori Furukawa ◽  
Yosuke Shida ◽  
Naoki Kitagami ◽  
Kazuki Mori ◽  
Masashi Kato ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Binding Sites ◽  
Specific Binding ◽  
Transcriptional Activator ◽  
Specific Binding Sites

scholarly journals Differences in Vaccine and SARS-CoV-2 Replication Derived mRNA: Implications for Cell Biology and Future Disease

2021 ◽  
Author(s):  
Kevin McKernan ◽  
Anthony M. Kyriakopoulos ◽  
Peter McCullough
Keyword(s):  
Binding Sites ◽  
Cell Biology ◽  
Protein Production ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Codon Optimization ◽  
Synonymous Codon ◽  
Gc Content ◽  
Rna Sequences ◽  
G Quadruplex

Codon optimization describes the process used to increase protein production by use of alternative but synonymous codon changes. In SARS-CoV-2 mRNA vaccines codon optimizations can result in differential secondary conformations that inevitably affect a protein’s function with significant consequences to the cell. Importantly, when codon optimization increases the GC content of synthetic mRNAs, there can be an inevitable enrichment of G-quartets which potentially form G-quadruplex structures. The emerging G-quadruplexes are favorable binding sites of RNA binding proteins like helicases that inevitably affect epigenetic reprogramming of the cell by altering transcription, translation and replication. In this study, we performed a RNAfold analysis to investigate alterations in secondary structures of mRNAs in SARS-CoV-2 vaccines due to codon optimization. We show a significant increase in the GC content of mRNAs in vaccines as compared to native SARS-CoV-2 RNA sequences encoding the spike protein. As the GC enrichment leads to more G-quadruplex structure formations, these may contribute to potential pathological processes initiated by SARS-CoV-2 molecular vaccination.

scholarly journals Novel genetic tools that enable highly pure protein production in Trichoderma reesei

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Anssi Rantasalo ◽  
Marika Vitikainen ◽  
Toni Paasikallio ◽  
Jussi Jäntti ◽  
Christopher P. Landowski ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Protein Production ◽  
Genetic Tools ◽  
Pure Protein

Influence of sorbitol on protein production and glycosylation and cell wall formation in Trichoderma reesei

2010 ◽  
Vol 114 (10) ◽  
pp. 855-862 ◽  
Author(s):  
Wioletta Górka-Nieć ◽  
Urszula Perlińska-Lenart ◽  
Patrycja Zembek ◽  
Grażyna Palamarczyk ◽  
Joanna S. Kruszewska
Keyword(s):  
Cell Wall ◽  
Trichoderma Reesei ◽  
Protein Production ◽  
Cell Wall Formation ◽  
Wall Formation

scholarly journals Modification of transcriptional factor ACE3 enhances protein production in Trichoderma reesei in the absence of cellulase gene inducer

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Yun Luo ◽  
Mari Valkonen ◽  
Raymond E. Jackson ◽  
Jonathan M. Palmer ◽  
Aditya Bhalla ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Protein Production ◽  
Transcriptional Factor ◽  
Cellulase Gene
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