Blocking endocytotic mechanisms to improve heterologous protein titers in <i>Saccharomyces cerevisiae</i>

Rodríguez-Limas, William A.; Tannenbaum, Victoria; Tyo, Keith E.J.

doi:10.1002/bit.25360

Cited by 17 publications

(11 citation statements)

References 57 publications

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“…For example, compared with other categories, the most enriched (3.7-fold) GO process category was "Endocytosis." This suggests that genes coding for endocytosis are potential targets for rational design for increasing protein secretion, which is consistent with a recent study reporting improvement of protein production through the blocking of endocytosis (15). Similarly, the GO process categories "organelle fusion," "vesicle organization," and "membrane fusion," all related to protein trafficking, show enrichment (more than twofold) among mutated genes.…”

Section: Resultssupporting

confidence: 73%

Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

Huang

Bai

Sjöström

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

161

143

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There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used highthroughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed wholegenome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.protein secretion | yeast cell factories | droplet microfluidics | random mutagenesis | systems biology T he production of recombinant proteins by cell factories, including biopharmaceutical proteins and industrial enzymes, is a growing multibillion-dollar industry (1) and demands continuous improvement of the chosen cell factories. The improvements involve optimization of transcription and translation, but also of protein posttranslational modifications, folding, and trafficking. One of the remaining challenges is the rational engineering design for the optimization of the protein secretory capacity, which involves a complex secretory network (2). The complexity of the protein secretory machinery and lack of a complete understanding of its underlying mechanisms has limited the utility of rational metabolic engineering for the improvement of recombinant protein production (3). Designing an efficient cell platform for protein secretion may often require overcoming limitations at different levels, e.g., translation, protein folding, and protein trafficking, and the modification of a single metabolic engineering target may therefore be insufficient (4). Although adaptive laboratory evolution has proven a useful strategy to acquire desired phenotypes with accumulation of beneficial mutations under selective pressure (5), this approach can be more cumbersome when trying to select clones with improved protein secretion.Alternatively, a cell library can be constructed by introduci...

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Section: Resultssupporting

confidence: 73%

Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

Huang

Bai

Sjöström

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

161

143

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“…In prokaryotes, the tagged protein is stable, until degradation is induced by expression of a protease or adaptor protein, resulting in rapid decrease in protein concentration [ 17 – 19 ]. Several demonstrations of targeted, exogenous protein degradation have been applied recently for metabolic engineering [ 20 , 21 ], fundamental study of essential proteins [ 22 ] , enhancing recombinant protein production [ 23 ] , and protein-based circuits [ 24 ]. Most of these studies target essential proteins that otherwise could not be genetically knocked out.…”

Section: Introductionmentioning

confidence: 99%

N-Terminal-Based Targeted, Inducible Protein Degradation in Escherichia coli

et al. 2016

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Dynamically altering protein concentration is a central activity in synthetic biology. While many tools are available to modulate protein concentration by altering protein synthesis rate, methods for decreasing protein concentration by inactivation or degradation rate are just being realized. Altering protein synthesis rates can quickly increase the concentration of a protein but not decrease, as residual protein will remain for a while. Inducible, targeted protein degradation is an attractive option and some tools have been introduced for higher organisms and bacteria. Current bacterial tools rely on C-terminal fusions, so we have developed an N-terminal fusion (Ntag) strategy to increase the possible proteins that can be targeted. We demonstrate Ntag dependent degradation of mCherry and beta-galactosidase and reconfigure the Ntag system to perform dynamic, exogenously inducible degradation of a targeted protein and complement protein depletion by traditional synthesis repression. Model driven analysis that focused on rates, rather than concentrations, was critical to understanding and engineering the system. We expect this tool and our model to enable inducible protein degradation use particularly in metabolic engineering, biological study of essential proteins, and protein circuits.

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“…Although previous studies suggested various strategies for improving protein production at different levels including transcription, translation, posttranslational processes, etc. (12)(13)(14)(15)(16)(17), they rarely focused on processes associated with intracellular retention of the protein in the secretory pathway.…”

mentioning

confidence: 99%

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

Huang

Wang

Qin

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Blocking endocytotic mechanisms to improve heterologous protein titers in Saccharomyces cerevisiae

Cited by 17 publications

References 57 publications

Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

N-Terminal-Based Targeted, Inducible Protein Degradation in Escherichia coli

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

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