| 1. |
- Albertsen, L., et al.
(författare)
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Diversion of Flux toward Sesquiterpene Production in Saccharomyces cerevisiae by Fusion of Host and Heterologous Enzymes
- 2011
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Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 77:3, s. 1033-1040
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Tidskriftsartikel (refereegranskat)abstract
- The ability to transfer metabolic pathways from the natural producer organisms to the well-characterized cell factory Saccharomyces cerevisiae is well documented. However, as many secondary metabolites are produced by collaborating enzymes assembled in complexes, metabolite production in yeast may be limited by the inability of the heterologous enzymes to collaborate with the native yeast enzymes. This may cause loss of intermediates by diffusion or degradation or due to conversion of the intermediate through competitive pathways. To bypass this problem, we have pursued a strategy in which key enzymes in the pathway are expressed as a physical fusion. As a model system, we have constructed several fusion protein variants in which farnesyl diphosphate synthase (FPPS) of yeast has been coupled to patchoulol synthase (PTS) of plant origin (Pogostemon cablin). Expression of the fusion proteins in S. cerevisiae increased the production of patchoulol, the main sesquiterpene produced by PTS, up to 2-fold. Moreover, we have demonstrated that the fusion strategy can be used in combination with traditional metabolic engineering to further increase the production of patchoulol. This simple test case of synthetic biology demonstrates that engineering the spatial organization of metabolic enzymes around a branch point has great potential for diverting flux toward a desired product.
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| 2. |
- Asadollahi, M. A., et al.
(författare)
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Enhancement of Farnesyl Diphosphate Pool as Direct Precursor of Sesquiterpenes Through Metabolic Engineering of the Mevalonate Pathway in Saccharomyces cerevisiae
- 2010
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 106:1, s. 86-96
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Tidskriftsartikel (refereegranskat)abstract
- The mevalonate pathway in the yeast Saccharomyces cerevisiae was deregulated in order to enhance the intracellular pool of farnesyl diphosphate (FPP), the direct precursor for the biosynthesis of sesquiterpenes. Overexpression of the catalytic domain of HMG1, both from the genome and plasmid, resulted in higher production of cubebol, a plant originating sesquiterpene, and increased squalene accumulation. Down-regulation of ERG9 by replacing its native promoter with the regulatable MET3 promoter, enhanced cubebol titers but simultaneous overexpression of tHMG1 and repression of ERG9 did not further improve cubebol production. Furtheremore, the concentrations of squalene and ergosterol were measured in the engineered strains. Unexpectedly, significant accumulation of squalene and restoring the ergosterol biosynthesis were observed in the ERG9 repressed strains transformed with the plasmids harboring cubebol synthase gene. This could be explained by a toxicity effect of cubebol, possibly resulting in higher transcription levels for the genes under control of MET3 promoter, which could lead to accumulation of squalene and ergosterol. Biotechnol. Bioeng. 2010;106: 86-96. (C) 2010 Wiley Periodicals, Inc.
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| 3. |
- Asadollahi, M. A., et al.
(författare)
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Enhancing sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic engineering
- 2009
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 11:6, s. 328-334
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Tidskriftsartikel (refereegranskat)abstract
- A genome-scale metabolic model was used to identify new target genes for enhanced biosynthesis of sesquiterpenes in the yeast Saccharomyces cerevisiae. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using Opt Gene as the modeling framework and minimization of metabolic adjustments (MOMA) as objective function. Deletion of NADPH-dependent glutamate dehydrogenase encoded by GDH1 was identified as the best target gene for the improvement of sesquiterpene biosynthesis in yeast. Deletion of this gene enhances the available NADPH in the cytosol for other NADPH requiring enzymes, including HMG-CoA reductase. However, since disruption of GDH1 impairs the ammonia utilization, simultaneous over-expression of the NADH-dependent glutamate dehydrogenase en coded by GDH2 was also considered in this study. Deletion of GDH1 led to an approximately 85% increase in the final cubebol titer. However, deletion of this gene also caused a significant decrease in the maximum specific growth rate. Over-expression of GDH2 did not show a further effect on the final cubebol titer but this alteration significantly improved the growth rate compared to the GDH1 deleted strain. (C) 2009 Elsevier Inc. All rights reserved.
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| 4. |
- Borodina, I., et al.
(författare)
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Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via beta-alanine
- 2015
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 27, s. 57-64
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Tidskriftsartikel (refereegranskat)abstract
- Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharolnyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the beta-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of beta-alanine and its subsequent conversion into 3HP using a novel beta-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7 +/- 0.3 g L-1 with a 0.14 +/- 0.0 C-mol C-mol(-1) yield on glucose in 80 h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production.
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| 5. |
- Jensen, N. B., et al.
(författare)
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EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae
- 2014
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 14:2, s. 238-248
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Tidskriftsartikel (refereegranskat)abstract
- Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log(10) mean +/- 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out.
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| 6. |
- Kildegaard, K. R., et al.
(författare)
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Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway
- 2016
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: In the future, oil-and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs. Results: Here we describe the metabolic engineering of baker's yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella enterica SEacsL641P. Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 +/- 0.4 g L-1 3HP with a yield of 13 % C-mol C-mol(-1) glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by C-13 metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed. Conclusions: In this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents a good platform for further optimization of 3HP production and hence an important step towards potential commercial bio-based production of 3HP.
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| 7. |
- Maury, J., et al.
(författare)
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EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae
- 2016
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 11:3
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Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR) of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP) reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the beta-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP). The best 3HP producing clone, with 5.45 g.L-1 of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.
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| 8. |
- Maury, J., et al.
(författare)
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Metabolic engineering of isoprenoid production: Reconstruction of multistep heterologous pathways in tractable hosts
- 2013
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Ingår i: Isoprenoid Synthesis in Plants and Microorganisms: New Concepts and Experimental Approaches. - New York, NY : Springer New York. - 9781461440635 ; , s. 73-89
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Isoprenoids represent a wide group of chemically active compounds that can find a wide range of applications as flavors, perfumes, vitamins, nutraceuticals, and pharmaceuticals. Many isoprenoids are naturally produced in very low quantities by plants, which make their use in broader perspectives difficult. Microbial production of plant-originating isoprenoids quickly appeared as an alternative of choice. Metabolic engineering methods were applied and proved successful to improve the supply of precursors to derive toward isoprenoid compounds of interest. Combinations between metabolic engineering and the flourishing field of synthetic biology have also been observed with researchers attempting to reconstruct and optimize complex biosynthetic pathways in well-characterized and tractable microbial hosts. In this chapter, we review recent metabolic engineering studies for isoprenoid production in yeast and try to show, through key examples, that the fields of synthetic biology and metabolic engineering can go hand in hand to establish microbial cell factories for isoprenoid production.
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| 9. |
- Maury, J., et al.
(författare)
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Reconstruction of a bacterial isoprenoid biosynthetic pathway in Saccharomyces cerevisiae
- 2008
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 582:29, s. 4032-4038
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Tidskriftsartikel (refereegranskat)abstract
- A eukaryotic mevalonate pathway transferred and expressed in Escherichia coli, and a mammalian hydrocortisone biosynthetic pathway rebuilt in Saccharomyces cerevisiae are examples showing that transferring metabolic pathways from one organism to another can have a powerful impact on cell properties. In this study, we reconstructed the E. coli isoprenoid biosynthetic pathway in S. cerevisiae. Genes encoding the seven enzymatic steps of the pathway were cloned and expressed in S. cerevisiae. mRNA from the seven genes was detected, and the pathway was shown able to sustain growth of yeast in conditions of inhibition of its constitutive isoprenoid biosynthetic pathway. © 2008 Federation of European Biochemical Societies.
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| 10. |
- Otero, José Manuel, 1979, et al.
(författare)
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Whole genome sequencing of Saccharomyces cerevisiae: from genotype to phenotype for improved metabolic engineering applications
- 2010
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 11:1, s. 723-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The need for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering.Results: In this work we performed whole genome high-throughput sequencing and annotation can be used to identify single nucleotide polymorphisms (SNPs) between Saccharomyces cerevisiae strains S288c and CEN.PK113-7D. The yeast strain S288c was the first eukaryote sequenced, serving as the reference genome for the Saccharomyces Genome Database, while CEN.PK113-7D is a preferred laboratory strain for industrial biotechnology research. A total of 13,787 high-quality SNPs were detected between both strains (reference strain: S288c). Considering only metabolic genes (782 of 5,596 annotated genes), a total of 219 metabolism specific SNPs are distributed across 158 metabolic genes, with 85 of the SNPs being nonsynonymous (e. g., encoding amino acid modifications). Amongst metabolic SNPs detected, there was pathway enrichment in the galactose uptake pathway (GAL1, GAL10) and ergosterol biosynthetic pathway (ERG8, ERG9). Physiological characterization confirmed a strong deficiency in galactose uptake and metabolism in S288c compared to CEN.PK113-7D, and similarly, ergosterol content in CEN.PK113-7D was significantly higher in both glucose and galactose supplemented cultivations compared to S288c. Furthermore, DNA microarray profiling of S288c and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that genotype to phenotype correlations are manifested post-transcriptionally or post-translationally either through protein concentration and/or function.Conclusions: With an intensifying need for microbial cell factories that produce a wide array of target compounds, whole genome high-throughput sequencing and annotation for SNP detection can aid in better reducing and defining the metabolic landscape. This work demonstrates direct correlations between genotype and phenotype that provides clear and high-probability of success metabolic engineering targets. The genome sequence, annotation, and a SNP viewer of CEN.PK113-7D are deposited at http://www.sysbio.se/cenpk.
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