scholarly journals Genome scale modeling of the protein secretory pathway reveals novel targets for improved recombinant protein production in yeast

2021 ◽  
Author(s):  
Feiran Li ◽  
Yu Chen ◽  
Qi Qi ◽  
Yanyan Wang ◽  
Le Yuan ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Secretory Pathway ◽  
Protein Translation ◽  
Cellular Growth ◽  
Secretory Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genome Scale

Eukaryal cells are used for the production of many recombinant pharmaceutical proteins, including several of the current top-selling products. The protein secretory pathway in eukaryal cells is complex and involves many different processes such as post-translational modifications, translocation, and folding. Furthermore, recombinant protein production competes with native secretory proteins for the limited energy and proteome resources allocated to the protein secretory pathway. Due to the complexity of this pathway, improvement through metabolic engineering has traditionally been relatively ad-hoc; and considering the industrial importance of this pathway, there is a need for more systematic approaches for novel design principles. Here, we present the first proteome-constrained genome-scale protein secretory model of a eukaryal cell, namely for the yeast Saccharomyces cerevisiae (pcSecYeast). The model contains all key processes of this pathway, i.e., protein translation, modification, and degradation coupled with metabolism. The model can capture delicate phenotypic changes such as the switch in the use of specific glucose transporters in response to changing extracellular glucose concentration. Furthermore, the model can also simulate the effects of protein misfolding on cellular growth, suggesting that retro-translocation of misfolded proteins contributes to protein retention in the Endoplasmic reticulum (ER). We used pcSecYeast to simulate various recombinant proteins production and identified overexpression targets for different recombinant proteins overproduction. We experimentally validated many of the predicted targets for α-amylase production in this study, and the results show that the secretory pathways have more limited capacity than metabolism in terms of protein secretion.

Genome-scale analysis of library sorting (GALibSo): Isolation of secretion enhancing factors for recombinant protein production inPichia pastoris

2010 ◽  
Vol 105 (3) ◽  
pp. 543-555 ◽  
Author(s):  
Gerhard Stadlmayr ◽  
Katrin Benakovitsch ◽  
Brigitte Gasser ◽  
Diethard Mattanovich ◽  
Michael Sauer
Keyword(s):  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Scale Analysis ◽  
Genome Scale

scholarly journals Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

2021 ◽  
Vol 12 ◽  
Author(s):  
Gema Lozano Terol ◽  
Julia Gallego-Jara ◽  
Rosa Alba Sola Martínez ◽  
Adrián Martínez Vivancos ◽  
Manuel Cánovas Díaz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Copy Number ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
E Coli

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

scholarly journals CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain

2021 ◽  
Vol 12 ◽  
Author(s):  
Michael Panting ◽  
Inger Baeksted Holme ◽  
Jón Már Björnsson ◽  
Yingxin Zhong ◽  
Henrik Brinch-Pedersen
Keyword(s):  
Recombinant Protein ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Recombinant Proteins ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Protein Accumulation ◽  
Unfolded Protein ◽  
Protein Response ◽  
Positive Effect

The use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of unfolded proteins. The unfolded protein response (UPR) is activated to alleviate the ER-stress. With the aim to increase the yield of human epidermal growth factor (EGF) and mouse leukemia inhibitory factor (mLIF) in barley, we selected genes reported to have increased expression during ER-induced stress. The selected genes were calreticulin (CRT), protein disulfide isomerase (PDI), isopentenyl diphosphate isomerase (IPI), glutathione-s-transferase (GST), HSP70, HSP26, and HSP16.9. These were knocked out using CRISPR/Cas9 or overexpressed by conventional transgenesis. The generated homozygous barley lines were crossed with barley plants expressing EGF or mLIF and the offspring plants analyzed for EGF and mLIF protein accumulation in the mature grain. All manipulated genes had an impact on the expression of UPR genes when plantlets were subjected to tunicamycin (TN). The PDI knockout plant showed decreased protein body formation, with protein evenly distributed in the cells of the endosperm. The two genes, GST and IPI, were found to have a positive effect on recombinant protein production. mLIF expression was increased in a F2 homozygous GST knockout mutant background as compared to a F2 GST wild-type offspring. The overexpression of IPI in a F1 cross showed a significant increase in EGF expression. We demonstrate that manipulation of UPR related genes can have a positive effect on recombinant protein accumulation.

Recent Advances in Recombinant Protein Production by Bacillus subtilis

2020 ◽  
Vol 11 (1) ◽  
pp. 295-318 ◽  
Author(s):  
Kang Zhang ◽  
Lingqia Su ◽  
Jing Wu
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Genetic Manipulation ◽  
Microbial Cell ◽  
Secretory Proteins ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Recent Advances

Bacillus subtilis has become a widely used microbial cell factory for the production of recombinant proteins, especially those associated with foods and food processing. Recent advances in genetic manipulation and proteomic analysis have been used to greatly improve protein production in B. subtilis. This review begins with a discussion of genome-editing technologies and application of the CRISPR–Cas9 system to B. subtilis. A summary of the characteristics of crucial legacy strains is followed by suggestions regarding the choice of origin strain for genetic manipulation. Finally, the review analyzes the genes and operons of B. subtilis that are important for the production of secretory proteins and provides suggestions and examples of how they can be altered to improve protein production. This review is intended to promote the engineering of this valuable microbial cell factory for better recombinant protein production.

scholarly journals Analysis of 11,430 recombinant protein production experiments reveals that protein yield is tunable by synonymous codon changes of translation initiation sites

2021 ◽  
Vol 17 (10) ◽  
pp. e1009461
Author(s):  
Bikash K. Bhandari ◽  
Chun Shen Lim ◽  
Daniela M. Remus ◽  
Augustine Chen ◽  
Craig van Dolleweerd ◽  
...  
Keyword(s):  
Cell Growth ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Translation Initiation ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Synonymous Codon ◽  
Recombinant Protein Expression ◽  
Host Cells ◽  
Over Expression

Recombinant protein production is a key process in generating proteins of interest in the pharmaceutical industry and biomedical research. However, about 50% of recombinant proteins fail to be expressed in a variety of host cells. Here we show that the accessibility of translation initiation sites modelled using the mRNA base-unpairing across the Boltzmann’s ensemble significantly outperforms alternative features. This approach accurately predicts the successes or failures of expression experiments, which utilised Escherichia coli cells to express 11,430 recombinant proteins from over 189 diverse species. On this basis, we develop TIsigner that uses simulated annealing to modify up to the first nine codons of mRNAs with synonymous substitutions. We show that accessibility captures the key propensity beyond the target region (initiation sites in this case), as a modest number of synonymous changes is sufficient to tune the recombinant protein expression levels. We build a stochastic simulation model and show that higher accessibility leads to higher protein production and slower cell growth, supporting the idea of protein cost, where cell growth is constrained by protein circuits during overexpression.

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