| 1. |
- Bergenholm, David, 1987, et al.
(författare)
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Modulation of saturation and chain length of fatty acids in Saccharomyces cerevisiae for production of cocoa butter-like lipids
- 2018
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 115:4, s. 932-942
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Tidskriftsartikel (refereegranskat)abstract
- Chain length and degree of saturation plays an important role for the characteristics of various products derived from fatty acids, such as fuels, cosmetics, and food dditives. The seeds of Theobroma cacao are the source of cocoa butter, a natural lipid of high interest for the food and cosmetics industry. Cocoa butter is rich in saturated fatty acids that are stored in the form of triacylglycerides (TAGs). One of the major TAG species of cocoa butter, consisting of two stearic acid molecules and one oleic acid molecule (stearic acid-oleic acid-stearic acid, sn-SOS), is particularly rare in nature as the saturated fatty acid stearic acid is typically found only in low abundance. Demand for cocoa butter is increasing, yet T. cacao can only be cultivated in some parts of the tropics. Alternative means of production of cocoa butter lipids (CBLs) are, therefore, sought after. Yeasts also store fatty acids in the form of TAGs, but these are typically not rich in saturated fatty acids. To make yeast an attractive host for microbial production of CBLs, its fatty acid composition needs to be optimized. We engineered Saccharomyces cerevisiae yeast strains toward a modified fatty acid synthesis. Analysis of the fatty acid profile of the modified strains showed that the fatty acid content as well as the titers of saturated fatty acids and the titers of TAGs were increased. The relative content of potential CBLs in the TAG pool reached up to 22% in our engineered strains, which is a 5.8-fold increase over the wild-type. SOS content reached a level of 9.8% in our engineered strains, which is a 48-fold increase over the wild type.
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| 2. |
- David, Florian, 1981, et al.
(författare)
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A Perspective on Synthetic Biology in Drug Discovery and Development - Current Impact and Future Opportunities
- 2021
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Ingår i: SLAS Discovery. - : Elsevier BV. - 2472-5552 .- 2472-5560. ; 26:5, s. 581-603
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Forskningsöversikt (refereegranskat)abstract
- The global impact of synthetic biology has been accelerating, because of the plummeting cost of DNA synthesis, advances in genetic engineering, growing understanding of genome organization, and explosion in data science. However, much of the discipline’s application in the pharmaceutical industry remains enigmatic. In this review, we highlight recent examples of the impact of synthetic biology on target validation, assay development, hit finding, lead optimization, and chemical synthesis, through to the development of cellular therapeutics. We also highlight the availability of tools and technologies driving the discipline. Synthetic biology is certainly impacting all stages of drug discovery and development, and the recognition of the discipline’s contribution can further enhance the opportunities for the drug discovery and development value chain.
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| 3. |
- Ferreira, Raphael, 1990, et al.
(författare)
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Metabolic engineering of Saccharomyces cerevisiae for overproduction of triacylglycerols
- 2018
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Ingår i: Metabolic Engineering Communications. - : Elsevier BV. - 2214-0301. ; 6, s. 22-27
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Tidskriftsartikel (refereegranskat)abstract
- Triacylglycerols (TAGs) are valuable versatile compounds that can be used as metabolites for nutrition and health, as well as feedstocks for biofuel production. Although Saccharomyces cerevisiae is the favored microbial cell factory for industrial production of biochemicals, it does not produce large amounts of lipids and TAGs comprise only ~1% of its cell dry weight. Here, we engineered S. cerevisiae to reorient its metabolism for overproduction of TAGs, by regulating lipid droplet associated-proteins involved in TAG synthesis and hydrolysis. We implemented a push-and-pull strategy by overexpressing genes encoding a deregulated acetyl-CoA carboxylase, ACC1 S659A/S1157A (ACC1**), as well as the last two steps of TAG formation: phosphatidic phosphatase (PAH1) and diacylglycerol acyltransferase (DGA1), ultimately leading to 129 mg∙gCDW −1 of TAGs. Disruption of TAG lipase genes TGL3, TGL4, TGL5 and sterol acyltransferase gene ARE1 increased the TAG content to 218 mg∙gCDW −1 . Further disruption of the beta-oxidation by deletion of POX1, as well as glycerol-3-phosphate utilization through deletion of GUT2, did not affect TAGs levels. Finally, disruption of the peroxisomal fatty acyl-CoA transporter PXA1 led to accumulation of 254 mg∙gCDW −1 . The TAG levels achieved here are the highest titer reported in S. cerevisiae, reaching 27.4% of the maximum theoretical yield in minimal medium with 2% glucose. This work shows the potential of using an industrially established and robust yeast species for high level lipid production.
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| 4. |
- Gossing, Michael, 1982, et al.
(författare)
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Impact of forced fatty acid synthesis on metabolism and physiology of Saccharomyces cerevisiae
- 2018
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 18:8
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Tidskriftsartikel (refereegranskat)abstract
- Nutrient sensing and signaling controls the cellular response to extracellular nutrients and intracellular metabolites. Nutrient-dependent regulation of metabolism ensures balanced energy production and expenditure. We show that disturbing energy balance by forcing fatty acid synthesis has profound impact on metabolism and physiology of the yeast cell. In addition to an expected increase in storage lipids, we observed increased β-oxidation and reduced amino acid biosynthesis, indicating increased activity of nutrient-sensitive kinase Snf1p. We also observed increased sensitivity to rapamycin as well as decreased ribosome biogenesis and translation, indicating reduced activity of nutrient-sensitive kinase target of rapamycin complex 1. Additionally, we detected increased levels of oxidative stress and lower levels of amino acids. This study provides detailed insight into cellular resource redistribution in response to forced fatty acid synthesis and enables optimized engineering of microbial lipid production.
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| 5. |
- Otto, Maximilian, 1991, et al.
(författare)
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Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based Applications, Genomic Integrations and Combinatorial Libraries
- 2021
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 10:12, s. 3461-3474
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Tidskriftsartikel (refereegranskat)abstract
- Standardisation of genetic parts has become a topic of increasing interest over the last decades. The promise of simplifying molecular cloning procedures, while at the same time making them more predictable and reproducible has led to the design of several biological standards, one of which is modular cloning (MoClo). The Yeast MoClo toolkit provides a large library of characterised genetic parts combined with a comprehensive and flexible assembly strategy. Here we aimed to (1) simplify the adoption of the standard by providing a simple design tool for including new parts in the MoClo library, (2) characterise the toolkit further by demonstrating the impact of a BglII site in promoter parts on protein expression, and (3) expand the toolkit to enable efficient construction of gRNA arrays, marker-less integration cassettes and combinatorial libraries. These additions make the toolkit more applicable for common engineering tasks and will further promote its adoption in the yeast biological engineering community.
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| 6. |
- Pfleger, B. F., et al.
(författare)
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Metabolic engineering strategies for microbial synthesis of oleochemicals
- 2015
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 29, s. 1-11
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Microbial synthesis of oleochemicals has advanced significantly in the last decade. Microbes have been engineered to convert renewable substrates to a wide range of molecules that are ordinarily made from plant oils. This approach is attractive because it can reduce a motivation for converting tropical rainforest into farmland while simultaneously enabling access to molecules that are currently expensive to produce from oil crops. In the last decade, enzymes responsible for producing oleochemicals in nature have been identified, strategies to circumvent native regulation have been developed, and high yielding strains have been designed, built, and successfully demonstrated. This review will describe the metabolic pathways that lead to the diverse molecular features found in natural oleochemicals, highlight successful metabolic engineering strategies, and comment on areas where future work could further advance the field.
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| 7. |
- Wei, Yongjun, 1986, et al.
(författare)
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Expression of cocoa genes in Saccharomyces cerevisiae improves cocoa butter production
- 2018
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Cocoa butter (CB) extracted from cocoa beans (Theobroma cacao) is the main raw material for chocolate production, but CB supply is insufficient due to the increased chocolate demand and limited CB production. CB is mainly composed of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0-C18:1-C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0-C18:1-C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18:0). In general, Saccharomyces cerevisiae produces TAGs as storage lipids, which consist of C16 and C18 fatty acids. However, cocoa butter-like lipids (CBL, which are composed of POP, POS and SOS) are not among the major TAG forms in yeast. TAG biosynthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT), and it is essential to modulate the yeast TAG biosynthetic pathway for higher CBL production. Results: We cloned seven GPAT genes and three LPAT genes from cocoa cDNA, in order to screen for CBL biosynthetic gene candidates. By expressing these cloned cocoa genes and two synthesized cocoa DGAT genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some of the strains. In the best producer, the potential CBL content was eightfold higher than the control strain, suggesting the cocoa genes expressed in this strain were functional and might be responsible for CBL biosynthesis. Moreover, the potential CBL content increased 134-fold over the control Y29-TcD1 (IMX581 sct1 ale1 lro1 dga1 with TcDGAT1 expression) in strain Y29-441 (IMX581 sct1 ale1 lro1 dga1 with TcGPAT4, TcLPAT4 and TcDGAT1 expression) further suggesting cocoa GPAT and LPAT genes functioned in yeast. Conclusions: We demonstrated that cocoa TAG biosynthetic genes functioned in S. cerevisiae and identified cocoa genes that may be involved in CBL production. Moreover, we found that expression of some cocoa CBL biosynthetic genes improved potential CBL production in S. cerevisiae, showing that metabolic engineering of yeast for cocoa butter production can be realized by manipulating the key enzymes GPAT, LPAT and DGAT in the TAG biosynthetic pathway.
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| 8. |
- Wei, Yongjun, 1986, et al.
(författare)
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Increasing cocoa butter-like lipid production of Saccharomyces cerevisiae by expression of selected cocoa genes
- 2017
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Cocoa butter (CB) extracted from cocoa beans mainly consists of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0–C18:1–C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0–C18:1–C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0–C18:1–C18:0), but CB supply is limited. Therefore, CB-like lipids (CBL, which are composed of POP, POS and SOS) are in great demand. Saccharomyces cerevisiae produces TAGs as storage lipids, which are also mainly composed of C16 and C18 fatty acids. However, POP, POS and SOS are not among the major TAG forms in yeast. TAG synthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT). In order to produce CBL in S. cerevisiae, we selected six cocoa genes encoding GPAT, LPAT and DGAT potentially responsible for CB biosynthesis from the cocoa genome using a phylogenetic analysis approach. By expressing the selected cocoa genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some S. cerevisiae strains. The relative CBL content in three yeast strains harboring cocoa genes increased 190, 230 and 196% over the control strain, respectively; especially, the potential SOS content of the three yeast strains increased 254, 476 and 354% over the control strain. Moreover, one of the three yeast strains had a 2.25-fold increased TAG content and 6.7-fold higher level of CBL compared with the control strain. In summary, CBL production by S. cerevisiae were increased through expressing selected cocoa genes potentially involved in CB biosynthesis. © 2017, The Author(s).
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