| 1. |
- 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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| 2. |
- Ferreira, Raphael, 1990, et al.
(författare)
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Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion
- 2018
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:6, s. 1262-1267
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Tidskriftsartikel (refereegranskat)abstract
- Bio-based production of fatty acids and fatty acid-derived products can enable sustainable substitution of petroleum-derived fuels and chemicals. However, developing new microbial cell factories for producing high levels of fatty acids requires extensive engineering of lipid metabolism, a complex and tightly regulated metabolic network. Here we generated a Saccharomyces cerevisiae platform strain with a simplified lipid metabolism network with high-level production of free fatty acids (FFAs) due to redirected fatty acid metabolism and reduced feedback regulation. Deletion of the main fatty acid activation genes (the first step in β-oxidation), main storage lipid formation genes, and phosphatidate phosphatase genes resulted in a constrained lipid metabolic network in which fatty acid flux was directed to a large extent toward phospholipids. This resulted in simultaneous increases of phospholipids by up to 2.8- fold and of FFAs by up to 40-fold compared with wild-type levels. Further deletion of phospholipase genes PLB1 and PLB2 resulted in a 46% decrease in FFA levels and 105% increase in phospholipid levels, suggesting that phospholipid hydrolysis plays an important role in FFA production when phospholipid levels are increased. The multiple deletion mutant generated allowed for a study of fatty acid dynamics in lipid metabolism and represents a platform strain with interesting properties that provide insight into the future development of lipid-related cell factories.
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| 3. |
- Teixeira, Paulo, 1990, et al.
(författare)
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Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae
- 2017
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 16:1, s. 45-
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Tidskriftsartikel (refereegranskat)abstract
- Background: In vivo production of fatty acid-derived chemicals in Saccharomyces cerevisiae requires strategies to increase the intracellular supply of either acyl-CoA or free fatty acids (FFAs), since their cytosolic concentrations are quite low in a natural state for this organism. Deletion of the fatty acyl-CoA synthetase genes FAA1 and FAA4 is an effective and straightforward way to disable re-activation of fatty acids and drastically increase FFA levels. However, this strategy causes FFA over-accumulation and consequential release to the extracellular medium, which results in a significant loss of precursors that compromises the process yield. In the present study, we aimed for dynamic expression of the fatty acyl-CoA synthetase gene FAA1 to regulate FFA and acyl-CoA pools in order to improve fatty alcohol production yields. Results: We analyzed the metabolite dynamics of a faa1 Delta faa4 Delta strain constitutively expressing a carboxylic acid reductase from Mycobacterium marinum (MmCAR) and an endogenous alcohol dehydrogenase (Adh5) for in vivo production of fatty alcohols from FFAs. We observed production of fatty acids and fatty alcohols with different rates leading to high levels of FFAs not being converted to the final product. To address the issue, we expressed the MmCAR + Adh5 pathway together with a fatty acyl-CoA reductase from Marinobacter aquaeolei to enable fatty alcohol production simultaneously from FFA and acyl-CoA, respectively. Then, we expressed FAA1 under the control of different promoters in order to balance FFA and acyl-CoA interconversion rates and to achieve optimal levels for conversion to fatty alcohols. Expressing FAA1 under control of the HXT1 promoter led to an increased accumulation of fatty alcohols per OD600 up to 41% while FFA levels were decreased by 63% compared with the control strain. Conclusions: Fine-tuning and dynamic regulation of key metabolic steps can be used to improve cell factories when the rates of downstream reactions are limiting. This avoids loss of precursors to the extracellular medium or to competing reactions, hereby potentially improving the process yield. The study also provides knowledge of a key point of fatty acid regulation and homeostasis, which can be used for future design of cells factories for fatty acid-derived chemicals.
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| 4. |
- Blitzblau, Hannah G., et al.
(författare)
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Production of 10-methyl branched fatty acids in yeast
- 2021
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Despite the environmental value of biobased lubricants, they account for less than 2% of global lubricant use due to poor thermo-oxidative stability arising from the presence of unsaturated double bonds. Methyl branched fatty acids (BFAs), particularly those with branching near the acyl-chain mid-point, are a high-performance alternative to existing vegetable oils because of their low melting temperature and full saturation. Results: We cloned and characterized two pathways to produce 10-methyl BFAs isolated from actinomycetes and γ-proteobacteria. In the two-step bfa pathway of actinomycetes, BfaB methylates Δ9 unsaturated fatty acids to form 10-methylene BFAs, and subsequently, BfaA reduces the double bond to produce a fully saturated 10-methyl branched fatty acid. A BfaA-B fusion enzyme increased the conversion efficiency of 10-methyl BFAs. The ten-methyl palmitate production (tmp) pathway of γ-proteobacteria produces a 10-methylene intermediate, but the TmpA putative reductase was not active in E. coli or yeast. Comparison of BfaB and TmpB activities revealed a range of substrate specificities from C14-C20 fatty acids unsaturated at the Δ9, Δ10 or Δ11 position. We demonstrated efficient production of 10-methylene and 10-methyl BFAs in S. cerevisiae by secretion of free fatty acids and in Y. lipolytica as triacylglycerides, which accumulated to levels more than 35% of total cellular fatty acids. Conclusions: We report here the characterization of a set of enzymes that can produce position-specific methylene and methyl branched fatty acids. Yeast expression of bfa enzymes can provide a platform for the large-scale production of branched fatty acids suitable for industrial and consumer applications.
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| 5. |
- Costa, J., et al.
(författare)
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Intragenic Recombination Has a Critical Role on the Evolution of Legionella pneumophila Virulence-Related Effector sidJ
- 2014
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 9:10
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Tidskriftsartikel (refereegranskat)abstract
- SidJ is a Dot/Icm effector involved in the trafficking or retention of ER-derived vesicles to Legionella pneumophila vacuoles whose mutation causes an observable growth defect, both in macrophage and amoeba hosts. Given the crucial role of this effector in L. pneumophila virulence we investigated the mechanisms shaping its molecular evolution. The alignment of SidJ sequences revealed several alleles with amino acid variations that may influence the protein properties. The identification of HGT events and the detection of balancing selection operating on sidJ evolution emerge as a clear result. Evidence suggests that intragenic recombination is an important strategy in the evolutionary adaptive process playing an active role on sidJ genetic plasticity. This pattern of evolution is in accordance with the life style of L. pneumophila as a broad host-range pathogen by preventing host-specialization and contributing to the resilience of the species.
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| 6. |
- Dabirian, Yasaman, 1992, et al.
(författare)
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FadR-Based Biosensor-Assisted Screening for Genes Enhancing Fatty Acyl-CoA Pools in Saccharomyces cerevisiae
- 2019
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 8:8, s. 1788-1800
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Tidskriftsartikel (refereegranskat)abstract
- Fatty acid-derived compounds have a range of industrial applications, from chemical building blocks to biofuels. Due to the highly dynamic nature of fatty acid metabolism, it is difficult to identify genes modulating fatty acyl-CoA levels using a rational approach. Metabolite biosensors can be used to screen genes from large-scale libraries in vivo in a high throughput manner. Here, a fatty acyl-CoA sensor based on the transcription factor FadR from Escherichia coli was established in Saccharomyces cerevisiae and combined with a gene overexpression library to screen for genes increasing the fatty acyl-CoA pool. Fluorescence-activated cell sorting, followed by data analysis, identified genes enhancing acyl-CoA levels. From these, overexpression of RTC3, GGA2, and LPP1 resulted in about 80% increased fatty alcohol levels. Changes in fatty acid saturation and chain length distribution could also be observed. These results indicate that the use of this acyl-CoA biosensor combined with a gene overexpression library allows for identification of gene targets improving production of fatty acids and derived products.
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| 7. |
- Leite, Melina de Souza, et al.
(författare)
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Major axes of variation in tree demography across global forests
- 2024
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Ingår i: Ecography. - 0906-7590 .- 1600-0587.
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Tidskriftsartikel (refereegranskat)abstract
- The future trajectory of global forests is closely intertwined with tree demography, and a major fundamental goal in ecology is to understand the key mechanisms governing spatio-temporal patterns in tree population dynamics. While previous research has made substantial progress in identifying the mechanisms individually, their relative importance among forests remains unclear mainly due to practical limitations. One approach to overcome these limitations is to group mechanisms according to their shared effects on the variability of tree vital rates and quantify patterns therein. We developed a conceptual and statistical framework (variance partitioning of Bayesian multilevel models) that attributes the variability in tree growth, mortality, and recruitment to variation in species, space, and time, and their interactions – categories we refer to as organising principles (OPs). We applied the framework to data from 21 forest plots covering more than 2.9 million trees of approximately 6500 species. We found that differences among species, the species OP, proved a major source of variability in tree vital rates, explaining 28–33% of demographic variance alone, and 14–17% in interaction with space, totalling 40–43%. Our results support the hypothesis that the range of vital rates is similar across global forests. However, the average variability among species declined with species richness, indicating that diverse forests featured smaller interspecific differences in vital rates. Moreover, decomposing the variance in vital rates into the proposed OPs showed the importance of unexplained variability, which includes individual variation, in tree demography. A focus on how demographic variance is organized in forests can facilitate the construction of more targeted models with clearer expectations of which covariates might drive a vital rate. This study therefore highlights the most promising avenues for future research, both in terms of understanding the relative contributions of groups of mechanisms to forest demography and diversity, and for improving projections of forest ecosystems.
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| 8. |
- Otto, Maximilian, 1991, et al.
(författare)
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Integration of a multi-step heterologous pathway in Saccharomyces cerevisiae for the production of abscisic acid
- 2019
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: The sesquiterpenoid abscisic acid (ABA) is mostly known for regulating developmental processes and abiotic stress responses in higher plants. Recent studies show that ABA also exhibits a variety of pharmacological activities. Affordable and sustainable production will be required to utilize the compound in agriculture and as a potential pharmaceutical. Saccharomyces cerevisiae is an established workhorse for the biotechnological production of chemicals. In this study, we constructed and characterised an ABA-producing S. cerevisiae strain using the ABA biosynthetic pathway from Botrytis cinerea. Results: Expression of the B. cinerea genes bcaba1, bcaba2, bcaba3 and bcaba4 was sufficient to establish ABA production in the heterologous host. We characterised the ABA-producing strain further by monitoring ABA production over time and, since the pathway contains two cytochrome P450 enzymes, by investigating the effects of overexpressing the native S. cerevisiae or the B. cinerea cytochrome P450 reductase. Both, overexpression of the native or heterologous cytochrome P450 reductase, led to increased ABA titres. We were able to show that ABA production was not affected by precursor or NADPH supply, which suggested that the heterologous enzymes were limiting the flux towards the product. The B. cinerea cytochrome P450 monooxygenases BcABA1 and BcABA2 were identified as pathway bottlenecks and balancing the expression levels of the pathway enzymes resulted in 4.1-fold increased ABA titres while reducing by-product formation. Conclusion: This work represents the first step towards a heterologous ABA cell factory for the commercially relevant sesquiterpenoid. © 2019 The Author(s).
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| 9. |
- Teixeira, Paulo, 1990, et al.
(författare)
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Engineering lipid droplet assembly mechanisms for improved triacylglycerol accumulation in Saccharomyces cerevisiae
- 2018
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 18:6
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Tidskriftsartikel (refereegranskat)abstract
- Production of triacylglycerols (TAGs) through microbial fermentation is an emerging alternative to plant and animal-derived sources. The yeast Saccharomyces cerevisiae is a preferred organism for industrial use but has natively a very poor capacity of TAG production and storage. Here, we engineered S. cerevisiae for accumulation of high TAG levels through the use of structural and physiological factors that influence assembly and biogenesis of lipid droplets. First, human and fungal perilipin genes were expressed, increasing TAG content by up to 36% when expressing the human perilipin gene PLIN3. Secondly, expression of the FIT2 homologue YFT2 resulted in a 26% increase in TAG content. Lastly, the genes ERD1 and PMR1 were deleted in order to induce an endoplasmic reticulum stress response and stimulate lipid droplet formation, increasing TAG content by 72% for Δerd1. These new approaches were implemented in previously engineered strains that carry high flux of fatty acid biosynthesis and conversion of acyl-CoA into TAGs, resulting in improvements of up to 138% over those high-producing strains without any substantial growth effects or abnormal cell morphology. We find that these approaches not only represent a significant improvement of S. cerevisiae for TAG production, but also highlight the importance of lipid droplet dynamics for high lipid accumulation in yeast.
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| 10. |
- Teixeira, Paulo, 1990
(författare)
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Engineering Lipid Metabolism for Production of Oleochemicals in Saccharomyces cerevisiae
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oleochemicals are chemicals usually derived from plant oils or animal fat. Large use of plant oil derivatives as replacements for petroleum-derived chemicals brings sustainability issues from extensive cultivation of oil plants in restricted regions. This project studied and developed the baker’s yeast Saccharomyces cerevisiae as a platform for sustainable production of oleochemical precursors. The first part of this work studied the dynamics of free fatty acids (FFAs) production. First, an alternative fatty acid synthesis system based on the reverse β-oxidation pathway was evaluated for its in vivo function but concluding that it was not an efficient route for fatty acid synthesis. The subsequent studies were based on high level production of FFA and secretion to the extracellular medium through removal of acyl-CoA synthase activity by deleting the FAA1-4 genes. This phenotype was coupled to a pathway that converts FFA to fatty alcohols, which allowed the observation that while FFA are more efficiently converted to fatty alcohols during growth on glucose, the production of FFA is highly increased during growth on ethanol. Fine-tuning of FAA1 expression resulted in improved production of fatty alcohols without FFA secretion in this strain. Following up, the pathways leading to FFA formation in a Δfaa1 Δfaa4 background were studied through construction of a strain with a constrained lipid metabolism network. It was observed that upon removing storage lipid formation, phospholipid synthesis had a strong correlation with FFA production and FFA formation was mostly derived from phospholipid hydrolysis. On the second part of this work, S. cerevisiae was engineered for the highest TAG production levels reported so far. This relied on overexpressing genes involved in malonyl-CoA supply and TAG synthesis from acyl-CoA, and removing genes involved in TAG hydrolysis, β-oxidation and glycerol-3-phosphate usage. On a second approach, TAG accumulation properties were further improved in these strains through enhancing lipid droplet assembly processes. This was achieved through expression of perilipins and FIT proteins and through stimulation of ER stress mechanisms. In conclusion, lipid metabolism is an important part of cell homeostasis and engineering this system requires overcoming its tight regulation networks and mastering the processes involved in the physical structural organization of the system. Here this was highlighted using both knowledge-driven studies and engineering approaches, leading to important advancements in the field.
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| 11. |
- Zhiwei, Zhu, 1985, et al.
(författare)
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Multidimensional engineering of Saccharomyces cerevisiae for efficient synthesis of medium-chain fatty acids
- 2020
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Ingår i: Nature Catalysis. - : Springer Science and Business Media LLC. - 2520-1158. ; 3:1, s. 64-74
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Tidskriftsartikel (refereegranskat)abstract
- Medium-chain fatty acids (MCFAs; C6–C12) are valuable molecules used for biofuel and oleochemical production; however, it is challenging to synthesize these fatty acids efficiently using microbial biocatalysts due to the cellular toxicity of MCFAs. In this study, both the endogenous fatty acid synthase (FAS) and an orthogonal bacterial type I FAS were engineered for MCFA production in the yeast Saccharomyces cerevisiae. To improve cellular tolerance to toxic MCFAs, we performed directed evolution of the membrane transporter Tpo1 and strain adaptive laboratory evolution, which elevated the MCFA production by 1.3 ± 0.3- and 1.7 ± 0.2-fold, respectively. We therefore further engineered the highly resistant strain to augment the metabolic flux towards MCFAs. This multidimensional engineering of the yeast at the single protein/enzyme level, the pathway level and the cellular level, combined with an optimized cultivation process, resulted in the production of >1 g l−1 extracellular MCFAs—a more than 250-fold improvement over the original strain.
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