| 1. |
- Fletcher, Eugene, 1986, et al.
(författare)
-
Industrial systems biology and its impact on synthetic biology of yeast cell factories
- 2016
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 113:6, s. 1164-1170
-
Forskningsöversikt (refereegranskat)abstract
- Engineering industrial cell factories to effectively yield a desired product while dealing with industrially relevant stresses is usually the most challenging step in the development of industrial production of chemicals using microbial fermentation processes. Using synthetic biology tools, microbial cell factories such as Saccharomyces cerevisiae can be engineered to express synthetic pathways for the production of fuels, biopharmaceuticals, fragrances, and food flavors. However, directing fluxes through these synthetic pathways towards the desired product can be demanding due to complex regulation or poor gene expression. Systems biology, which applies computational tools and mathematical modeling to understand complex biological networks, can be used to guide synthetic biology design. Here, we present our perspective on how systems biology can impact synthetic biology towards the goal of developing improved yeast cell factories. (C) 2015 Wiley Periodicals, Inc.
|
|
| 2. |
- Hu, Yating, 1991, et al.
(författare)
-
Metabolic engineering of Saccharomyces cerevisiae for production of germacrene A, a precursor of beta-elemene
- 2017
-
Ingår i: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 44:7, s. 1065-1072
-
Tidskriftsartikel (refereegranskat)abstract
- Beta-elemene, a sesquiterpene and the major component of the medicinal herb Curcuma wenyujin, has antitumor activity against various types of cancer and could potentially serve as a potent antineoplastic drug. However, its current mode of production through extraction from plants has been inefficient and suffers from limited natural resources. Here, we engineered a yeast cell factory for the sustainable production of germacrene A, which can be transformed to beta-elemene by a one-step chemical reaction in vitro. Two heterologous germacrene A synthases (GASs) converting farnesyl pyrophosphate (FPP) to germacrene A were evaluated in yeast for their ability to produce germacrene A. Thereafter, several metabolic engineering strategies were used to improve the production level. Overexpression of truncated 3-hydroxyl-3-methylglutaryl-CoA reductase and fusion of FPP synthase with GAS, led to a sixfold increase in germacrene A production in shake-flask culture. Finally, 190.7 mg/l of germacrene A was achieved. The results reported in this study represent the highest titer of germacrene A reported to date. These results provide a basis for creating an efficient route for further industrial application re-placing the traditional extraction of beta-elemene from plant sources.
|
|
| 3. |
- Krivoruchko, Anastasia, 1984, et al.
(författare)
-
Activation of the carbohydrate response element binding protein (ChRESP) in response to anoxia in the turtle Trachemys scripta elegans
- 2014
-
Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 1872-8006 .- 0304-4165. ; 1840:10, s. 3000-3005
-
Tidskriftsartikel (refereegranskat)abstract
- Background: ChREBP (carbohydrate response element binding protein) is a glucose-responsive transcription factor that is known to be an important regulator of glycolytic and lipogenic genes in response to glucose. We hypothesized that activation of ChREBP could be relevant to anoxia survival by the anoxia-tolerant turtle, Trachemys scripta elegans. Methods: Expression of ChREBP in response to 5 and 20 h of anoxia was examined using RT-PCR and Western immunoblotting. In addition, subcellular localization and DNA-binding activity of ChREBP protein were assessed and transcript levels of liver pyruvate kinase (LPK), a downstream gene under ChREBP control were quantified using RT-PCR. Results: ChREBP was anoxia-responsive in kidney and liver, with transcript levels increasing by 1.2-1.8 fold in response to anoxia and protein levels increasing by 1.8-1.9 fold. Enhanced nuclear presence under anoxia was also observed in both tissues by 22-2.8 fold. A 4.2 fold increase in DNA binding activity of ChREBP was also observed in liver in response to 5 h of anoxia. In addition, transcript levels of LPK increased by 2.1 fold in response to 5 h of anoxia in the liver. Conclusions: The results suggest that activation of ChREBP in response to anoxia might be a crucial factor for anoxia survival in turtle liver by contributing to elevated glycolytic flux in the initial phases of oxygen limitation. General significance: This study provides the first demonstration of activation of ChREBP in response to anoxia in a natural model of anoxia tolerance, further improving our understanding of the molecular nature of anoxia tolerance.
|
|
| 4. |
- Krivoruchko, Anastasia, 1984, et al.
(författare)
-
Activation of the unfolded protein response during anoxia exposure in the turtle Trachemys scripta elegans
- 2013
-
Ingår i: Molecular and Cellular Biochemistry. - : Springer Science and Business Media LLC. - 0300-8177 .- 1573-4919. ; 374:1-2, s. 91-103
-
Tidskriftsartikel (refereegranskat)abstract
- Red-eared slider turtles, Trachemys scripta elegans, can survive for several weeks without oxygen when submerged in cold water. We hypothesized that anaerobiosis is aided by adaptive up-regulation of the unfolded protein response (UPR), a stress-responsive pathway that is activated by accumulation of unfolded proteins in the endoplasmic reticulum (ER) and functions to restore ER homeostasis. RT-PCR, western immunoblotting and DNA-binding assays were used to quantify the responses and/or activation status of UPR-responsive genes and proteins in turtle tissues after animal exposure to 5 or 20 h of anoxic submergence at 4 C. The phosphorylation state of protein kinase-like ER kinase (PERK) (a UPR-regulated kinase) and eukaryotic initiation factor 2 (eIF2α) increased by 1.43-2.50 fold in response to anoxia in turtle heart, kidney, and liver. Activation of the PERK-regulated transcription factor, activating transcription factor 4 (ATF4), during anoxia was documented by elevated atf4 transcripts and total ATF4 protein (1.60-2.43 fold), increased nuclear ATF4 content, and increased DNA-binding activity (1.44-2.32 fold). ATF3 and GADD34 (downstream targets of ATF4) also increased by 1.38-3.32 fold in heart and liver under anoxia, and atf3 transcripts were also elevated in heart. Two characteristic chaperones of the UPR, GRP78, and GRP94, also responded positively to anoxia with strong increases in both the transcript and protein levels. The data demonstrate that the UPR is activated in turtle heart, kidney, and liver in response to anoxia, suggesting that this pathway mediates an integrated stress response to protect tissues during oxygen deprivation. © 2012 Springer Science+Business Media New York.
|
|
| 5. |
- Krivoruchko, Anastasia, 1984, et al.
(författare)
-
Anoxia-responsive regulation of the FoxO transcription factors in freshwater turtles, Trachemys scripta elegans
- 2013
-
Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 1872-8006 .- 0304-4165. ; 1830:11, s. 4990-4998
-
Tidskriftsartikel (refereegranskat)abstract
- Background The forkhead class O (FoxO) transcription factors are important regulators of multiple aspects of cellular metabolism. We hypothesized that activation of these transcription factors could play crucial roles in low oxygen survival in the anoxia-tolerant turtle, Trachemys scripta elegans. Methods Two FoxOs, FoxO1 and FoxO3, were examined in turtle tissues in response to 5 and 20 h of anoxic submergence using techniques of RT-PCR, western immunoblotting and DNA-binding assays to assess activation. Transcript levels of FoxO-responsive genes were also quantified using RT-PCR. Results FoxO1 was anoxia-responsive in the liver, with increases in transcript levels, protein levels, nuclear levels and DNA-binding of 1.7-4.8 fold in response to anoxia. Levels of phosphorylated FoxO1 also decreased to 57% of control values in response to 5 h of anoxia, indicating activation. FoxO3 was activated in the heart, kidney and liver in response to anoxia, with nuclear levels increasing by 1.5-3.7 fold and DNA-binding activity increasing by 1.3-2.9 fold. Transcript levels of two FoxO-target genes, p27kip1 and catalase, also rose by 2.4-2.5 fold in the turtle liver under anoxia. Conclusions The results suggest that the FoxO transcription factors are activated in response to anoxia in T. scripta elegans, potentially contributing to the regulation of stress resistance and metabolic depression. General significance This study provides the first demonstration of activation of FoxOs in a natural model for vertebrate anoxia tolerance, further improving understanding of how tissues can survive without oxygen. © 2013 Elsevier B.V.
|
|
| 6. |
- Krivoruchko, Anastasia, 1984, et al.
(författare)
-
Improving biobutanol production in engineered Saccharomyces cerevisiae by manipulation of acetyl-CoA metabolism
- 2013
-
Ingår i: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 40:9, s. 1051-1056
-
Tidskriftsartikel (refereegranskat)abstract
- Recently, butanols (1-butanol, 2-butanol and iso-butanol) have generated attention as alternative gasoline additives. Butanols have several properties favorable in comparison to ethanol, and strong interest therefore exists in the reconstruction of the 1-butanol pathway in commonly used industrial microorganisms. In the present study, the biosynthetic pathway for 1-butanol production was reconstructed in the yeast Saccharomyces cerevisiae. In addition to introducing heterologous enzymes for butanol production, we engineered yeast to have increased flux toward cytosolic acetyl-CoA, the precursor metabolite for 1-butanol biosynthesis. This was done through introduction of a plasmid-containing genes for alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA synthetase (ACS), and acetyl-CoA acetyltransferase (ERG10), as well as the use of strains containing deletions in the malate synthase (MLS1) or citrate synthase (CIT2) genes. Our results show a trend to increased butanol production in strains engineered for increased cytosolic acetyl-CoA levels, with the best-producing strains having maximal butanol titers of 16.3 mg/l. This represents a 6.5-fold improvement in butanol titers compared to previous values reported for yeast and demonstrates the importance of an improved cytosolic acetyl-CoA supply for heterologous butanol production by this organism.
|
|
| 7. |
- Krivoruchko, Anastasia, 1984, et al.
(författare)
-
Microbial acetyl-CoA metabolism and metabolic engineering
- 2015
-
Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 28, s. 28-42
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Recent concerns over the sustainability of petrochemical-based processes for production of desired chemicals have fueled research into alternative modes of production. Metabolic engineering of microbial cell factories such as Saccharomyces cerevisiae and Escherichia coli offers a sustainable and flexible alternative for the production of various molecules. Acetyl-CoA is a key molecule in microbial central carbon metabolism and is involved in a variety of cellular processes. In addition, it functions as a precursor for many molecules of biotechnological relevance. Therefore, much interest exists in engineering the metabolism around the acetyl-CoA pools in cells in order to increase product titers. Here we provide an overview of the acetyl-CoA metabolism in eukaryotic and prokaryotic microbes (with a focus on S. cerevisiae and E. coli), with an emphasis on reactions involved in the production and consumption of acetyl-CoA. In addition, we review various strategies that have been used to increase acetyl-CoA production in these microbes.
|
|
| 8. |
- Krivoruchko, Anastasia, 1984, et al.
(författare)
-
Production of natural products through metabolic engineering of Saccharomyces cerevisiae
- 2015
-
Ingår i: Current Opinion in Biotechnology. - : Elsevier BV. - 0958-1669 .- 1879-0429. ; 35, s. 7-15
-
Forskningsöversikt (refereegranskat)abstract
- Many high-value metabolites are produced in nature by organisms that are not ideal for large-scale production. Therefore, interest exists in expressing the biosynthetic pathways of these compounds in organisms that are more suitable for industrial production. Recent years have seen developments in both the discovery of various biosynthetic pathways, as well as development of metabolic engineering tools that allow reconstruction of complex pathways in microorganisms. In the present review we discuss recent advances in reconstruction of the biosynthetic pathways of various high-value products in the yeast Saccharomyces cerevisiae, a commonly used industrial microorganism. Key achievements in the production of different isoprenoids, aromatics and polyketides are presented and the metabolic engineering strategies underlying these accomplishments are discussed.
|
|
| 9. |
- Liu, Yi, 1986, et al.
(författare)
-
Engineering yeast phospholipid metabolism for de novo oleoylethanolamide production
- 2020
-
Ingår i: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 16:2, s. 197-205
-
Tidskriftsartikel (refereegranskat)abstract
- Phospholipids, the most abundant membrane lipid components, are crucial in maintaining membrane structures and homeostasis for biofunctions. As a structurally diverse and tightly regulated system involved in multiple organelles, phospholipid metabolism is complicated to manipulate. Thus, repurposing phospholipids for lipid-derived chemical production remains unexplored. Herein, we develop a Saccharomyces cerevisiae platform for de novo production of oleoylethanolamide, a phospholipid derivative with promising pharmacological applications in ameliorating lipid dysfunction and neurobehavioral symptoms. Through deregulation of phospholipid metabolism, screening of biosynthetic enzymes, engineering of subcellular trafficking and process optimization, we could produce oleoylethanolamide at a titer of 8,115.7 µg l−1 and a yield on glucose of 405.8 µg g−1. Our work provides a proof-of-concept study for systemically repurposing phospholipid metabolism for conversion towards value-added biological chemicals, and this multi-faceted framework may shed light on tailoring phospholipid metabolism in other microbial hosts.
|
|
| 10. |
- Qin, Jiufu, 1985, et al.
(författare)
-
Engineering yeast metabolism for the discovery and production of polyamines and polyamine analogues
- 2021
-
Ingår i: Nature Catalysis. - : Springer Science and Business Media LLC. - 2520-1158. ; 4:6, s. 498-509
-
Tidskriftsartikel (refereegranskat)abstract
- Structurally complex and diverse polyamines and polyamine analogues are potential therapeutics and agrochemicals that can address grand societal challenges, for example, healthy ageing and sustainable food production. However, their structural complexity and low abundance in nature hampers either bulk chemical synthesis or extraction from natural resources. Here we reprogrammed the metabolism of baker’s yeast Saccharomyces cerevisiae and recruited nature’s diverse reservoir of biochemical tools to enable a complete biosynthesis of multiple polyamines and polyamine analogues. Specifically, we adopted a systematic engineering strategy to enable gram-per-litre-scale titres of spermidine, a central metabolite in polyamine metabolism. To demonstrate the potential of our polyamine platform, various polyamine synthases and ATP-dependent amide-bond-forming systems were introduced for the biosynthesis of natural and unnatural polyamine analogues. The yeast platform serves as a resource to accelerate the discovery and production of polyamines and polyamine analogues, and thereby unlocks this chemical space for further pharmacological and insecticidal studies. [Figure not available: see fulltext.]
|
|
