| 1. |
- Liu, Zihe, 1984, et al.
(författare)
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Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering
- 2014
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Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 1098-5336 .- 0099-2240. ; 80:17, s. 5542-5550
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Tidskriftsartikel (refereegranskat)abstract
- The increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in the VTA1 gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.
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| 2. |
- Hou, Jin, 1982, et al.
(författare)
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Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae
- 2013
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:8, s. 3559-3568
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high level over-expression of HSF1-R206S increased heterologous alpha-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously.
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| 3. |
- Hou, Jin, 1982, et al.
(författare)
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Management of the endoplasmic reticulum stress by activation of the heat shock response in yeast
- 2014
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 14:3, s. 481-494
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Tidskriftsartikel (refereegranskat)abstract
- In yeast Saccharomyces cerevisiae, accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates the unfolded protein response (UPR), which is mediated by Hac1p. The heat shock response (HSR) mediated by Hsf1p, mainly regulates cytosolic processes and protects the cell from stresses. Here, we find that a constitutive activation of the HSR could increase ER stress resistance in both wild-type and UPR-deficient cells. Activation of HSR decreased UPR activation in the WT (as shown by the decreased HAC1 mRNA splicing). We analyzed the genome-wide transcriptional response in order to propose regulatory mechanisms that govern the interplay between UPR and HSR and followed up for the hypotheses by experiments in vivo and in vitro. Interestingly, we found that the regulation of ER stress response via HSR is (1) only partially dependent on over-expression of Kar2p (ER resident chaperone induced by ER stress); (2) does not involve the increase in protein turnover via the proteasome activity; (3) is related to the oxidative stress response. From the transcription data, we also propose that HSR enhances ER stress resistance mainly through facilitation of protein folding and secretion. We also find that HSR coordinates multiple stress-response pathways, including the repression of the overall transcription and translation.
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| 4. |
- Liu, Zihe, 1984, et al.
(författare)
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Anaerobic alpha-Amylase Production and Secretion with Fumarate as the Final Electron Acceptor in Saccharomyces cerevisiae
- 2013
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Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 79:9, s. 2962-2967
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we focus on production of heterologous alpha-amylase in the yeast Saccharomyces cerevisiae under anaerobic conditions. We compare the metabolic fluxes and transcriptional regulation under aerobic and anaerobic conditions, with the objective of identifying the final electron acceptor for protein folding under anaerobic conditions. We find that yeast produces more amylase under anaerobic conditions than under aerobic conditions, and we propose a model for electron transfer under anaerobic conditions. According to our model, during protein folding the electrons from the endoplasmic reticulum are transferred to fumarate as the final electron acceptor. This model is supported by findings that the addition of fumarate under anaerobic (but not aerobic) conditions improves cell growth, specifically in the alpha-amylase-producing strain, in which it is not used as a carbon source. Our results provide a model for the molecular mechanism of anaerobic protein secretion using fumarate as the final electron acceptor, which may allow for further engineering of yeast for improved protein secretion under anaerobic growth conditions.
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| 5. |
- Feizi, Amir, 1980, et al.
(författare)
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Genome-Scale Modeling of the Protein Secretory Machinery in Yeast
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 8:5
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Tidskriftsartikel (refereegranskat)abstract
- The protein secretory machinery in Eukarya is involved in post-translational modification (PTMs) and sorting of the secretory and many transmembrane proteins. While the secretory machinery has been well-studied using classic reductionist approaches, a holistic view of its complex nature is lacking. Here, we present the first genome-scale model for the yeast secretory machinery which captures the knowledge generated through more than 50 years of research. The model is based on the concept of a Protein Specific Information Matrix (PSIM: characterized by seven PTMs features). An algorithm was developed which mimics secretory machinery and assigns each secretory protein to a particular secretory class that determines the set of PTMs and transport steps specific to each protein. Protein abundances were integrated with the model in order to gain system level estimation of the metabolic demands associated with the processing of each specific protein as well as a quantitative estimation of the activity of each component of the secretory machinery.
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| 6. |
- Kristiansson, Erik, 1978, et al.
(författare)
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A novel method for cross-species gene expression analysis
- 2013
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Ingår i: BMC Bioinformatics. - : Springer Science and Business Media LLC. - 1471-2105. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background Analysis of gene expression from different species is a powerful way to identify evolutionarily conserved transcriptional responses. However, due to evolutionary events such as gene duplication, there is no one-to-one correspondence between genes from different species which makes comparison of their expression profiles complex. Results In this paper we describe a new method for cross-species meta-analysis of gene expression. The method takes the homology structure between compared species into account and can therefore compare expression data from genes with any number of orthologs and paralogs. A simulation study shows that the proposed method results in a substantial increase in statistical power compared to previously suggested procedures. As a proof of concept, we analyzed microarray data from heat stress experiments performed in eight species and identified several well-known evolutionarily conserved transcriptional responses. The method was also applied to gene expression profiles from five studies of estrogen exposed fish and both known and potentially novel responses were identified. Conclusions The method described in this paper will further increase the potential and reliability of meta-analysis of gene expression profiles from evolutionarily distant species. The method has been implemented in R and is freely available at http://bioinformatics.math.chalmers.se/Xspecies/ webcite.
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| 7. |
- Liu, Lifang, 1979, et al.
(författare)
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Genome-scale analysis of the high-efficient protein secretion system of Aspergillus oryzae
- 2014
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Ingår i: BMC Systems Biology. - : Springer Science and Business Media LLC. - 1752-0509. ; 8:73
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Tidskriftsartikel (refereegranskat)abstract
- Background: The koji mold, Aspergillus oryzae is widely used for the production of industrial enzymes due to its particularly high protein secretion capacity and ability to perform post-translational modifications. However, systemic analysis of its secretion system is lacking, generally due to the poorly annotated proteome. Results: Here we defined a functional protein secretory component list of A. oryzae using a previously reported secretory model of S. cerevisiae as scaffold. Additional secretory components were obtained by blast search with the functional components reported in other closely related fungal species such as Aspergillus nidulans and Aspergillus niger. To evaluate the defined component list, we performed transcriptome analysis on three a-amylase over-producing strains with varying levels of secretion capacities. Specifically, secretory components involved in the ER-associated processes (including components involved in the regulation of transport between ER and Golgi) were significantly up-regulated, with many of them never been identified for A. oryzae before. Furthermore, we defined a complete list of the putative A. oryzae secretome and monitored how it was affected by overproducing amylase. Conclusion: In combination with the transcriptome data, the most complete secretory component list and the putative secretome, we improved the systemic understanding of the secretory machinery of A. oryzae in response to high levels of protein secretion. The roles of many newly predicted secretory components were experimentally validated and the enriched component list provides a better platform for driving more mechanistic studies of the protein secretory pathway in this industrially important fungus.
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| 8. |
- Österlund, Tobias, 1984, et al.
(författare)
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Fifteen years of large scale metabolic modeling of yeast: Developments and impacts
- 2012
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Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 30:5, s. 979-988
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Forskningsöversikt (refereegranskat)abstract
- Since the first large-scale reconstruction of the Saccharomyces cerevisiae metabolic network 15 years ago the development of yeast metabolic models has progressed rapidly, resulting in no less than nine different yeast genome-scale metabolic models. Here we review the historical development of large-scale mathematical modeling of yeast metabolism and the growing scope and impact of applications of these models in four different areas: as guide for metabolic engineering and strain improvement, as a tool for biological interpretation and discovery, applications of novel computational framework and for evolutionary studies.
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| 9. |
- Österlund, Tobias, 1984, et al.
(författare)
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Mapping condition-dependent regulation of metabolism in yeast through genome-scale modeling
- 2013
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Ingår i: BMC Systems Biology. - : Springer Science and Business Media LLC. - 1752-0509. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Background:The genome-scale metabolic model of Saccharomyces cerevisiae, first presented in 2003, was the first genome-scale network reconstruction for a eukaryotic organism. Since then continuous efforts have been made in order to improve and expand the yeast metabolic network.Results:Here we present iTO977, a comprehensive genome-scale metabolic model that contains more reactions, metabolites and genes than previous models. The model was constructed based on two earlier reconstructions, namely iIN800 and the consensus network, and then improved and expanded using gap-filling methods and by introducing new reactions and pathways based on studies of the literature and databases. The model was shown to perform well both for growth simulations in different media and gene essentiality analysis for single and double knock-outs. Further, the model was used as a scaffold for integrating transcriptomics, and flux data from four different conditions in order to identify transcriptionally controlled reactions, i.e. reactions that change both in flux and transcription between the compared conditions.Conclusion:We present a new yeast model that represents a comprehensive up-to-date collection of knowledge on yeast metabolism. The model was used for simulating the yeast metabolism under four different growth conditions and experimental data from these four conditions was integrated to the model. The model together with experimental data is a useful tool to identify condition-dependent changes of metabolism between different environmental conditions.
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| 10. |
- Österlund, Tobias, 1984
(författare)
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Reconstruction of Biological Networks for Integrative Analysis
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biological systems can be very complex and consist of several thousand components that interact with each other in the cell. One of the goals of systems biology is to study biological systems from a systemic viewpoint in order to get an increased understanding of the behavior of the cell. Biological network reconstructions are important tools in systems biology in order to model the behavior of different biological systems. The biological networks can also be used as a scaffold for integrative analysis where high-throughput data from different conditions or different strains are integrated into the biological network to reduce the dimension of the data and to group the response between conditions or strains into biological pathways or key metabolites etc. The biological interpretation and discovery using integrative analysis can be facilitated by constructing more comprehensive and diverse biological networks.In this thesis I expanded current biological network reconstructions for the yeast Saccharomyces cereveisae in three steps and used them as a scaffold for biological interpretation and discovery. First I constructed an up-to-date yeast genome-scale metabolic model. The model is a comprehensive description of yeast metabolism and contains more genes, reactions and metabolites than previous models. The model performs well in simulating the metabolism under different conditions. Second, I studied the transcriptional regulatory network of yeast in terms of topology and structure of the network and compared it to transcriptional regulation in E. coli, human and mouse. I also used high-throughput data from many different conditions to study the condition-dependent response of the yeast transcriptional regulatory network. Third, I was involved in reconstruction of models of the protein secretion machinery in S. cerevisiae and for the high protein producer Aspergillus oryzae, describing protein folding, post-translational modifications and protein transport etc. High-throughput data from several different strains producing α-amylase were integrated into the models in order to get an insight in the mechanisms and bottlenecks of protein secretion in these organisms.The biological networks presented here were also used for data integration and the results and interpretation of the cellular behavior under different conditions can give us a deeper understanding and insight in for example condition-specific transcriptional regulation and protein production.
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