| 1. |
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| 2. |
- Anfelt, Josefine, et al.
(författare)
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Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production
- 2015
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Ingår i: Microbial Cell Factories. - : BioMed Central. - 1475-2859. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. Results: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. Conclusion: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.
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| 3. |
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| 4. |
- Englund, Elias, et al.
(författare)
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Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound
- 2018
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Ingår i: Metabolic engineering. - : Academic Press Inc.. - 1096-7176 .- 1096-7184. ; 49, s. 164-177
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Tidskriftsartikel (refereegranskat)abstract
- Of the two natural metabolic pathways for making terpenoids, biotechnological utilization of the mevalonate (MVA) pathway has enabled commercial production of valuable compounds, while the more recently discovered but stoichiometrically more efficient methylerythritol phosphate (MEP) pathway is underdeveloped. We conducted a study on the overexpression of each enzyme in the MEP pathway in the unicellular cyanobacterium Synechocystis sp. PCC 6803, to identify potential targets for increasing flux towards terpenoid production, using isoprene as a reporter molecule. Results showed that the enzymes Ipi, Dxs and IspD had the biggest impact on isoprene production. By combining and creating operons out of those genes, isoprene production was increased 2-fold compared to the base strain. A genome-scale model was used to identify targets upstream of the MEP pathway that could redirect flux towards terpenoids. A total of ten reactions from the Calvin-Benson-Bassham cycle, lower glycolysis and co-factor synthesis pathways were probed for their effect on isoprene synthesis by co-expressing them with the MEP enzymes, resulting in a 60% increase in production from the best strain. Lastly, we studied two isoprene synthases with the highest reported catalytic rates. Only by expressing them together with Dxs and Ipi could we get stable strains that produced 2.8 mg/g isoprene per dry cell weight, a 40-fold improvement compared to the initial strain.
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| 5. |
- Miao, Rui, et al.
(författare)
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CRISPR interference screens reveal growth–robustness tradeoffs in Synechocystis sp. PCC 6803 across growth conditions
- 2023
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Ingår i: The Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 35:11, s. 3937-3956
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Tidskriftsartikel (refereegranskat)abstract
- Barcoded mutant libraries are a powerful tool for elucidating gene function in microbes, particularly when screened in multiple growth conditions. Here, we screened a pooled CRISPR interference library of the model cyanobacterium Synechocystis sp. PCC 6803 in 11 bioreactor-controlled conditions, spanning multiple light regimes and carbon sources. This gene repression library contained 21,705 individual mutants with high redundancy over all open reading frames and noncoding RNAs. Comparison of the derived gene fitness scores revealed multiple instances of gene repression being beneficial in 1 condition while generally detrimental in others, particularly for genes within light harvesting and conversion, such as antennae components at high light and PSII subunits during photoheterotrophy. Suboptimal regulation of such genes likely represents a tradeoff of reduced growth speed for enhanced robustness to perturbation. The extensive data set assigns condition-specific importance to many previously unannotated genes and suggests additional functions for central metabolic enzymes. Phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, and the small protein CP12 were critical for mixotrophy and photoheterotrophy, which implicates the ternary complex as important for redirecting metabolic flux in these conditions in addition to inactivation of the Calvin cycle in the dark. To predict the potency of sgRNA sequences, we applied machine learning on sgRNA sequences and gene repression data, which showed the importance of C enrichment and T depletion proximal to the PAM site. Fitness data for all genes in all conditions are compiled in an interactive web application.
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| 6. |
- Nilsson, Astrid, et al.
(författare)
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Environmental impacts and limitations of third-generation biobutanol : Life cycle assessment of n-butanol produced by genetically engineered cyanobacteria
- 2020
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Ingår i: Journal of Industrial Ecology. - : WILEY. - 1088-1980 .- 1530-9290. ; 24:1
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Tidskriftsartikel (refereegranskat)abstract
- Photosynthetic cyanobacteria have attracted interest as production organisms for third-generation biofuels, where sunlight and CO2 are used by microbes directly to synthesize fuel molecules. A particularly suitable biofuel is n-butanol, and there have been several laboratory reports of genetically engineered photosynthetic cyanobacteria capable of synthesizing and secreting n-butanol. This work evaluates the environmental impacts and cumulative energy demand (CED) of cyanobacteria-produced n-butanol through a cradle-to-grave consequential life cycle assessment (LCA). A hypothetical production plant in northern Sweden (area 1 ha, producing 5-85 m(3) n-butanol per year) was considered, and a range of cultivation formats and cellular productivity scenarios assessed. Depending on the scenario, greenhouse gas emissions (GHGe) ranged from 16.9 to 58.6 gCO(2)eq/MJ(BuOH) and the CED from 3.8 to 13 MJ/MJ(BuOH). Only with the assumption of a nearby paper mill to supply waste sources for heat and CO2 was the sustainability requirement of at least 60% GHGe savings compared to fossil fuels reached, though placement in northern Sweden reduced energy needed for reactor cooling. A high CED in all scenarios shows that significant metabolic engineering is necessary, such as a carbon partitioning of >90% to n-butanol, as well as improved light utilization, to begin to displace fossil fuels or even first- and second-generation bioethanol.
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| 7. |
- Shabestary, Kiyan, et al.
(författare)
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Computational metabolic engineering strategies for growth-coupled biofuel production by Synechocystis
- 2016
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Ingår i: Metabolic Engineering Communications. - : Elsevier. - 2214-0301. ; 3, s. 216-226
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Tidskriftsartikel (refereegranskat)abstract
- Chemical and fuel production by photosynthetic cyanobacteria is a promising technology but to date has not reached competitive rates and titers. Genome-scale metabolic modeling can reveal limitations in cyanobacteria metabolism and guide genetic engineering strategies to increase chemical production. Here, we used constraint-based modeling and optimization algorithms on a genome-scale model of Synechocystis PCC6803 to find ways to improve productivity of fermentative, fatty-acid, and terpene-derived fuels. OptGene and MOMA were used to find heuristics for knockout strategies that could increase biofuel productivity. OptKnock was used to find a set of knockouts that led to coupling between biofuel and growth. Our results show that high productivity of fermentation or reversed beta-oxidation derived alcohols such as 1-butanol requires elimination of NADH sinks, while terpenes and fatty-acid based fuels require creating imbalances in intracellular ATP and NADPH production and consumption. The FBA-predicted productivities of these fuels are at least 10-fold higher than those reported so far in the literature. We also discuss the physiological and practical feasibility of implementing these knockouts. This work gives insight into how cyanobacteria could be engineered to reach competitive biofuel productivities.
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| 8. |
- Shabestary, Kiyan, et al.
(författare)
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Cycling between growth and production phases increases cyanobacteria bioproduction of lactate
- 2021
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 68, s. 131-141
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Tidskriftsartikel (refereegranskat)abstract
- Decoupling growth from product synthesis is a promising strategy to increase carbon partitioning and maximize productivity in cell factories. However, reduction in both substrate uptake rate and metabolic activity in the production phase are an underlying problem for upscaling. Here, we used CRISPR interference to repress growth in lactate-producing Synechocystis sp. PCC 6803. Carbon partitioning to lactate in the production phase exceeded 90%, but CO2 uptake was severely reduced compared to uptake during the growth phase. We characterized strains during the onset of growth arrest using transcriptomics and proteomics. Multiple genes involved in ATP homeostasis were regulated once growth was inhibited, which suggests an alteration of energy charge that may lead to reduced substrate uptake. In order to overcome the reduced metabolic activity and take advantage of increased carbon partitioning, we tested a novel production strategy that involved alternating growth arrest and recovery by periodic addition of an inducer molecule to activate CRISPRi. Using this strategy, we maintained lactate biosynthesis in Synechocystis for 30 days in a constant light turbidostat cultivation. Cumulative lactate titers were also increased by 100% compared to a constant growth-arrest regime, and reached 1 g/L. Further, the cultivation produced lactate for 30 days, compared to 20 days for the non-growth arrest cultivation. Periodic growth arrest could be applicable for other products, and in cyanobacteria, could be linked to internal circadian rhythms that persist in constant light.
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| 9. |
- Shabestary, Kiyan, 1991-
(författare)
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Improving cyanobacteria productivity: From theory to assay
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bio-based production of biochemicals and biofuels holds great promises for the transition towards a more sustainable society. With increasing levels of carbon dioxide (CO2) in the atmosphere, cyanobacteria stand apart as natural catalysts directly converting CO2 and light to product. However, current product productivities and titers do not meet the standard set by the petroleum-based industry. In particular, the solar-to-product efficiency needs to be drastically improved to make the process economically more interesting. As proof of concept, this thesis puts an emphasis on identifying metabolic limitations towards increased solar-to-product efficiency using model-guided formulation of strategies and genome-wide screening, followed by novel practical implementations. It follows previous works identifying the intracellular ATP/NADPH ratio as an important variable to balance photosynthesis, carbon fixation, product synthesis and biomass formation to ensure more performant metabolic engineering designs of photoautotrophs. In Paper I, we identified in silico growth-coupled metabolic designs linking product formation to growth to increase productivity and stability of the engineered strain. In Paper II, we found computationally and experimentally that carbon rerouting gave the best results to increase product formation. In Paper III, we used the CRISPRi system to further maximize carbon rerouting to product synthesis in growth-arrest strategies. Finally, in Paper IV, we conduct a genome-wide screening using a CRISPRi library and identified key targets to improve product synthesis, product tolerance and growth. We also demonstrate experimentally some of the strategies found in Paper I. This thesis suggests that growth-arrest production is a promising avenue to maximize the solar-to-product efficiency and asserts that systems biology tools will be needed to identify and tackle the remaining strain instability associated with those designs.
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| 10. |
- Shabestary, Kiyan, et al.
(författare)
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Targeted Repression of Essential Genes To Arrest Growth and Increase Carbon Partitioning and Biofuel Titers in Cyanobacteria
- 2018
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 7:7
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Tidskriftsartikel (refereegranskat)abstract
- Photoautotrophic production of fuels and chemicals by cyanobacteria typically gives lower volumetric productivities and titers than heterotrophic production. Cyanobacteria cultures become light limited above an optimal cell density, so that this substrate is not supplied to all cells sufficiently. Here, we investigate genetic strategies for a two-phase cultivation, where biofuel-producing Synechocystis cultures are limited to an optimal cell density through inducible CRISPR interference (CRISPRi) repression of cell growth. Fixed CO2 is diverted to ethanol or n-butanol. Among the most successful strategies was partial repression of citrate synthase gltA. Strong repression (>90%) of gitA at low culture densities increased carbon partitioning to n-butanol 5-fold relative to a nonrepression strain, but sacrificed volumetric productivity due to severe growth restriction. CO2 fixation continued for at least 3 days after growth was arrested. By targeting sgRNAs to different regions of the gitA gene, we could modulate GItA expression and carbon partitioning between growth and product to increase both specific and volumetric productivity. These growth arrest strategies can be useful for improving performance of other photoautotrophic processes.
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