| 1. |
- Zhan, Chunjun, 1986, et al.
(author)
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Reprogramming methanol utilization pathways to convert Saccharomyces cerevisiae to a synthetic methylotroph
- 2023
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In: Nature Catalysis. - 2520-1158. ; 6:5, s. 435-450
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Journal article (peer-reviewed)abstract
- Methanol, an organic one-carbon (C1) compound, represents an attractive alternative carbon source for microbial fermentation. Despite considerable advancements in methanol utilization by prokaryotes such as Escherichia coli, engineering eukaryotic model organisms such as Saccharomyces cerevisiae into synthetic methylotrophs remains challenging. Here, an engineered module circuit strategy combined with adaptive laboratory evolution was applied to engineer S. cerevisiae to use methanol as the sole carbon source. We revealed that the evolved glyoxylate-based serine pathway plays an important role in methanol-dependent growth by promoting formaldehyde assimilation. Further, we determined that the isoprenoid biosynthetic pathway was upregulated, resulting in an increased concentration of squalene and ergosterol in our evolved strain. These changes could potentially alleviate cell membrane damage in the presence of methanol. This work sets the stage for expanding the potential of exploiting S. cerevisiae as a potential organic one-carbon platform for biochemical or biofuel production. [Figure not available: see fulltext.].
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| 2. |
- Chen, Xin, 1980, et al.
(author)
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Dataset for suppressors of amyloid-beta toxicity and their functions in recombinant protein production in yeast
- 2022
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In: Data in Brief. - : Elsevier BV. - 2352-3409. ; 42
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Journal article (peer-reviewed)abstract
- The production of recombinant proteins at high levels often induces stress-related phenotypes by protein misfolding or aggregation. These are similar to those of the yeast Alzheimer's disease (AD) model in which amyloid-beta peptides (A beta 42) were accumulated [1,2] . We have previously identified suppressors of A beta 42 cytotoxicity via the genome-wide synthetic genetic array (SGA) [3] and here we use them as metabolic engineering targets to evaluate their potentiality on recombinant protein production in yeast Saccharomyces cerevisiae. In order to investigate the mechanisms linking the genetic modifications to the improved recombinant protein production, we perform systems biology approaches (transcriptomics and proteomics) on the resulting strain and intermediate strains. The RNAseq data are preprocessed by the nf-core/RNAseq pipeline and analyzed using the Platform for Integrative Analysis of Omics (PIANO) package [4] . The quantitative proteome is analyzed on an Orbitrap Fusion Lumos mass spectrometer interfaced with an Easy-nLC1200 liquid chromatography (LC) system. LC-MS data files are processed by Proteome Discoverer version 2.4 with Mascot 2.5.1 as a database search engine. The original data presented in this work can be found in the research paper titled "Suppressors of Amyloid-beta Toxicity Improve Recombinant Protein Produc-tion in yeast by Reducing Oxidative Stress and Tuning Cellu-lar Metabolism", by Chen et al. [5] . (C) 2022 The Author(s). Published by Elsevier Inc.
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| 3. |
- Chen, Xin, 1980, et al.
(author)
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Suppressors of amyloid-β toxicity improve recombinant protein production in yeast by reducing oxidative stress and tuning cellular metabolism
- 2022
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In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 72, s. 311-324
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Journal article (peer-reviewed)abstract
- High-level production of recombinant proteins in industrial microorganisms is often limited by the formation of misfolded proteins or protein aggregates, which consequently induce cellular stress responses. We hypothesized that in a yeast Alzheimer's disease (AD) model overexpression of amyloid-β peptides (Aβ42), one of the main peptides relevant for AD pathologies, induces similar phenotypes of cellular stress. Using this humanized AD model, we previously identified suppressors of Aβ42 cytotoxicity. Here we hypothesize that these suppressors could be used as metabolic engineering targets to alleviate cellular stress and improve recombinant protein production in the yeast Saccharomyces cerevisiae. Forty-six candidate genes were individually deleted and twenty were individually overexpressed. The positive targets that increased recombinant α-amylase production were further combined leading to an 18.7-fold increased recombinant protein production. These target genes are involved in multiple cellular networks including RNA processing, transcription, ER-mitochondrial complex, and protein unfolding. By using transcriptomics and proteomics analyses, combined with reverse metabolic engineering, we showed that reduced oxidative stress, increased membrane lipid biosynthesis and repressed arginine and sulfur amino acid biosynthesis are significant pathways for increased recombinant protein production. Our findings provide new insights towards developing synthetic yeast cell factories for biosynthesis of valuable proteins.
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| 4. |
- Li, Feiran, 1993, et al.
(author)
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Improving recombinant protein production by yeast through genome-scale modeling using proteome constraints
- 2022
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 13:1
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Journal article (peer-reviewed)abstract
- Eukaryotic cells are used as cell factories to produce and secrete multitudes of recombinant pharmaceutical proteins, including several of the current top-selling drugs. Due to the essential role and complexity of the secretory pathway, improvement for recombinant protein production through metabolic engineering has traditionally been relatively ad-hoc; and a more systematic approach is required to generate novel design principles. Here, we present the proteome-constrained genome-scale protein secretory model of yeast Saccharomyces cerevisiae (pcSecYeast), which enables us to simulate and explain phenotypes caused by limited secretory capacity. We further apply the pcSecYeast model to predict overexpression targets for the production of several recombinant proteins. We experimentally validate many of the predicted targets for alpha-amylase production to demonstrate pcSecYeast application as a computational tool in guiding yeast engineering and improving recombinant protein production. Due to the complexity of the protein secretory pathway, strategy suitable for the production of a certain recombination protein cannot be generalized. Here, the authors construct a proteome-constrained genome-scale protein secretory model for yeast and show its application in the production of different misfolded or recombinant proteins.
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| 5. |
- Li, Xiaowei, 1986, et al.
(author)
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Metabolic network remodelling enhances yeast’s fitness on xylose using aerobic glycolysis
- 2021
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In: Nature Catalysis. - : Springer Science and Business Media LLC. - 2520-1158. ; 4:9, s. 783-796
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Journal article (peer-reviewed)abstract
- The reprogramming of metabolism in response to switching the carbon source from glucose to non-preferred carbon sources is well-studied for yeast. However, understanding how metabolic networks respond to utilize a non-natural carbon source such as xylose is limited due to the incomplete knowledge of cellular response mechanisms. Here we applied a combination of metabolic engineering, systems biology and adaptive laboratory evolution to gain insights into how yeast can perform a global rewiring of cellular processes to efficiently accompany metabolic transitions. Through metabolic engineering, we substantially enhanced the cell growth on xylose after the growth on glucose. Transcriptome analysis of the engineered strains demonstrated that multiple pathways were involved in the cellular reprogramming. Through genome resequencing of the evolved strains and reverse engineering, we further identified that SWI/SNF chromatin remodelling, osmotic response and aldehyde reductase were responsible for the improved growth. Combined, our analysis showed that glycerol-3-phosphate and xylitol serve as two key metabolites that affect cellular adaptation to growth on xylose. [Figure not available: see fulltext.].
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| 6. |
- Wang, Yanyan, 1989, et al.
(author)
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CRISPR/Cas9-mediated point mutations improve alpha-amylase secretion in Saccharomyces cerevisiae
- 2022
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In: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 22:1
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Journal article (peer-reviewed)abstract
- The rapid expansion of the application of pharmaceutical proteins and industrial enzymes requires robust microbial workhorses for high protein production. The budding yeast Saccharomyces cerevisiae is an attractive cell factory due to its ability to perform eukaryotic post-translational modifications and to secrete proteins. Many strategies have been used to engineer yeast platform strains for higher protein secretion capacity. Herein, we investigated a line of strains that have previously been selected after UV random mutagenesis for improved alpha-amylase secretion. A total of 42 amino acid altering point mutations identified in this strain line were reintroduced into the parental strain AAC to study their individual effects on protein secretion. These point mutations included missense mutations (amino acid substitution), nonsense mutations (stop codon generation), and frameshift mutations. For comparison, single gene deletions for the corresponding target genes were also performed in this study. A total of 11 point mutations and seven gene deletions were found to effectively improve alpha-amylase secretion. These targets were involved in several bioprocesses, including cellular stresses, protein degradation, transportation, mRNA processing and export, DNA replication, and repair, which indicates that the improved protein secretion capacity in the evolved strains is the result of the interaction of multiple intracellular processes. Our findings will contribute to the construction of novel cell factories for recombinant protein secretion. Systematic characterization of point mutations from evolved strains using CRISPR/Cas9 technology revealed a set of gene alterations that improved recombinant protein secretion in Saccharomyces cerevisiae.
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| 7. |
- Wang, Yanyan, 1989
(author)
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Engineering yeast for improved recombinant protein production
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Recombinant proteins are broadly used from basic research to therapeutic development and include industrial enzymes and pharmaceutical proteins. The increasing demand for improved production and enhanced quality of recombinant proteins requires robust biotech-based strategies to overcome the limitations of protein extraction from natural sources. A variety of cell factories are therefore established for the large-scale production of recombinant proteins of interest. In comparison to other expression systems, the budding yeast Saccharomyces cerevisiae is an attractive production platform due to its high tolerance to harsh fermentation conditions, and importantly its capability to perform eukaryotic post-translational modifications and to secrete the biologically active product to the extracellular medium. Thus, many strategies have been applied to engineer this organism for increasing its recombinant protein secretory capacity and productivity. The major aim of this thesis work was to study and develop efficient yeast platforms for the production of different heterologous proteins for medical or industrial use through diverse engineering strategies. The first part of this work explored in depth a line of previously evolved yeast strains with improved protein secretory capacity. The universal applicability of the evolved strains was evaluated to produce different antibody fragments, but it was concluded that this secretion platform was not suitable for all types of pharmaceutical proteins tested. Furthermore, by re-introducing all 42 protein-sequence-altering mutations identified in the evolved strains into the parental strain using the CRISPR/Cas9 technology, 14 targets were shown to be beneficial for protein production and 11 out of these 14 beneficial targets were newly identified to be related to recombinant protein production. The second part of this work focused on investigating novel targets related to the cellular stress response and the protein secretory process to rationally optimize S. cerevisiae . Furthermore, screening for suppressors of amyloid-β cytotoxicity in a yeast Alzheimer’s disease model revealed a number of gene targets that reduced oxidative stress and improved production of recombinant proteins. Additionally, a proteome-constrained genome-scale protein secretory model of S. cerevisiae (pcSecYeast) was constructed to simulate the secretion of various recombinant proteins and predict system-level engineering targets for increasing protein production. In summary, the work presented in this thesis provides different efficient strategies to develop yeast platforms for the high-level production of valuable industrial or pharmaceutical proteins, and also provides general guidelines for designing other cell platforms for efficient protein production. Integrated application of various engineering approaches will make meaningful advancements in the field of recombinant protein production in the future.
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| 8. |
- Wang, Yanyan, 1989, et al.
(author)
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Expression of antibody fragments in Saccharomyces cerevisiae strains evolved for enhanced protein secretion
- 2021
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 20:1
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Journal article (peer-reviewed)abstract
- Monoclonal antibodies, antibody fragments and fusion proteins derived thereof have revolutionized the practice of medicine. Major challenges faced by the biopharmaceutical industry are however high production costs, long processing times and low productivities associated with their production in mammalian cell lines. The yeast Saccharomyces cerevisiae, a well-characterized eukaryotic cell factory possessing the capacity of posttranslational modifications, has been industrially exploited as a secretion host for production of a range of products, including pharmaceuticals. However, due to the incompatible surface glycosylation, few antibody molecules have been functionally expressed in S. cerevisiae. Here, three non-glycosylated antibody fragments from human and the Camelidae family were chosen for expression in a S. cerevisiae strain (HA) previously evolved for high α-amylase secretion. These included the Fab fragment Ranibizumab (Ran), the scFv peptide Pexelizumab (Pex), and a nanobody consisting of a single V-type domain (Nan). Both secretion and biological activities of the antibody fragments were confirmed. In addition, the secretion level of each protein was compared in the wild type (LA) and two evolved strains (HA and MA) with different secretory capacities. We found that the secretion of Ran and Nan was positively correlated with the strains’ secretory capacity, while Pex was most efficiently secreted in the parental strain. To investigate the mechanisms for different secretion abilities in these selected yeast strains for the different antibody fragments, RNA-seq analysis was performed. The results showed that several bioprocesses were significantly enriched for differentially expressed genes when comparing the enriched terms between HA.Nan vs. LA.Nan and HA.Pex vs. LA.Pex, including amino acid metabolism, protein synthesis, cell cycle and others, which indicates that there are unique physiological needs for each antibody fragment secretion.
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