scholarly journals N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Hongting Tang ◽  
Shenghuan Wang ◽  
Jiajing Wang ◽  
Meihui Song ◽  
Mengyang Xu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Secretory Pathway ◽  
Cell Wall Integrity

scholarly journals Different Routes of Protein Folding Contribute to Improved Protein Production in Saccharomyces cerevisiae

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Qi Qi ◽  
Feiran Li ◽  
Rosemary Yu ◽  
Martin K. M. Engqvist ◽  
Verena Siewers ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Folding ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Strain Engineering ◽  
Folding Pathway ◽  
Original Strain ◽  
Precision Control ◽  
Folding Process

ABSTRACT Protein folding is often considered the flux controlling process in protein synthesis and secretion. Here, two previously isolated Saccharomyces cerevisiae strains with increased α-amylase productivity were analyzed in chemostat cultures at different dilution rates using multi-omics data. Based on the analysis, we identified different routes of the protein folding pathway to improve protein production. In the first strain, the increased abundance of proteins working on the folding process, coordinated with upregulated glycogen metabolism and trehalose metabolism, helped increase α-amylase productivity 1.95-fold compared to the level in the original strain in chemostat culture at a dilution rate of 0.2/h. The second strain further strengthened the folding precision to improve protein production. More precise folding helps the cell improve protein production efficiency and reduce the expenditure of energy on the handling of misfolded proteins. As calculated using an enzyme-constrained genome-scale metabolic model, the second strain had an increased productivity of 2.36-fold with lower energy expenditure than that of the original under the same condition. Further study revealed that the regulation of N-glycans played an important role in the folding precision control and that overexpression of the glucosidase Cwh41p can significantly improve protein production, especially for the strains with improved folding capacity but lower folding precision. Our findings elucidated in detail the mechanisms in two strains having improved protein productivity and thereby provided novel insights for industrial recombinant protein production as well as demonstrating how multi-omics analysis can be used for identification of novel strain-engineering targets. IMPORTANCE Protein folding plays an important role in protein maturation and secretion. In recombinant protein production, many studies have focused on the folding pathway to improve productivity. Here, we identified two different routes for improving protein production by yeast. We found that improving folding precision is a better strategy. Dysfunction of this process is also associated with several aberrant protein-associated human diseases. Here, our findings about the role of glucosidase Cwh41p in the precision control system and the characterization of the strain with a more precise folding process could contribute to the development of novel therapeutic strategies.

scholarly journals Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production

2018 ◽  
Vol 115 (47) ◽  
pp. E11025-E11032 ◽  
Author(s):  
Mingtao Huang ◽  
Guokun Wang ◽  
Jiufu Qin ◽  
Dina Petranovic ◽  
Jens Nielsen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Recombinant Protein ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Pathway ◽  
Synergistic Effects ◽  
Production Capacity ◽  
Market Demand ◽  
Protein Retention ◽  
Cell Factories

Baker’s yeast Saccharomyces cerevisiae is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. Here we identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications on the endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, we enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.

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.

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