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Shabestary, Kiyan
Publications (4 of 4) Show all publications
Englund, E., Shabestary, K., Hudson, E. P. & Lindberg, P. (2018). Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound. Metabolic engineering, 49, 164-177
Open this publication in new window or tab >>Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound
2018 (English)In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 49, p. 164-177Article in journal (Refereed) Published
Abstract [en]

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. 

Place, publisher, year, edition, pages
Academic Press Inc., 2018
Keywords
Carbon flux, Cyanobacteria, Isoprene, MEP pathway, Metabolic engineering, Metabolic modeling, Enzymes, Lipids, Metabolism, Strain, Carbon fluxes, Commercial productions, Cyanobacterium synechocystis, MEP pathways, Reporter molecules, Synechocystis pcc 6803, bacterial enzyme, enzyme Dxs, enzyme Ipi, enzyme IspD, isoprene synthase, methylerythritol 4 phosphate, phosphate, synthetase, terpene derivative, terpenoid, unclassified drug, Agrobacterium tumefaciens, Article, bacterial genome, bacterial growth, bacterial strain, biotechnological production, Botryococcus braunii, Coleus, Coleus forskohlii, controlled study, Deinococcus radiodurans, enzyme activity, Escherichia coli, gene expression level, gene overexpression, gene targeting, glycolysis, heterologous expression, molecular cloning, nonhuman, operon, priority journal, protein analysis, protein expression level, Synechocystis sp. PCC 6803, upregulation
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-236716 (URN)10.1016/j.ymben.2018.07.004 (DOI)000447634700016 ()2-s2.0-85051682232 (Scopus ID)
Funder
Swedish Energy Agency, 38334-1Swedish Foundation for Strategic Research , RBP-14-0013
Note

Export Date: 22 October 2018; Article; CODEN: MEENF; Correspondence Address: Lindberg, P.; Department of Chemistry – Ångström, Uppsala University, Box 523, Sweden; email: pia.lindberg@kemi.uu.se; QC 20181106

Available from: 2018-10-23 Created: 2018-10-23 Last updated: 2018-11-06Bibliographically approved
Shabestary, K., Anfelt, J., Ljungqvist, E., Jahn, M., Yao, L. & Hudson, E. P. (2018). Targeted Repression of Essential Genes To Arrest Growth and Increase Carbon Partitioning and Biofuel Titers in Cyanobacteria [Letter to the editor]. ACS Synthetic Biology, 7(7), Article ID diva2:1239079.
Open this publication in new window or tab >>Targeted Repression of Essential Genes To Arrest Growth and Increase Carbon Partitioning and Biofuel Titers in Cyanobacteria
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2018 (English)In: ACS Synthetic Biology, E-ISSN 2161-5063, Vol. 7, no 7, article id diva2:1239079Article in journal, Letter (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
Keywords
cyanobacteria, CRISPRi, bioproduction
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-235174 (URN)10.1021/acssynbio.8b00056 (DOI)
Funder
EU, Horizon 2020, 760994
Note

QC 20180920

Available from: 2018-09-17 Created: 2018-09-17 Last updated: 2018-09-20Bibliographically approved
Shabestary, K. & Hudson, E. P. (2016). Computational metabolic engineering strategies for growth-coupled biofuel production by Synechocystis. Metabolic Engineering Communications, 3, 216-226
Open this publication in new window or tab >>Computational metabolic engineering strategies for growth-coupled biofuel production by Synechocystis
2016 (English)In: Metabolic Engineering Communications, ISSN 2214-0301, Vol. 3, p. 216-226Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2016
Keywords
Biofuel, Cyanobacteria, Flux balance analysis, Modeling, MOMA, OptFlux, OptKnock, butanol, reduced nicotinamide adenine dinucleotide phosphate, algorithm, Article, biofuel production, biomass production, cyanobacterium, fatty acid oxidation, fermentation, gene mutation, metabolic engineering, nonhuman, priority journal, Synechocystis
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-195191 (URN)10.1016/j.meteno.2016.07.003 (DOI)2-s2.0-84979497796 (Scopus ID)
Funder
Swedish Foundation for Strategic Research , RBP14–0013
Note

QC 20161202

Available from: 2016-12-02 Created: 2016-11-02 Last updated: 2016-12-02Bibliographically approved
Björk, S., Shabestary, K., Yao, L., Ljungqvist, E., Jönsson, H. & Hudson, E. P.Droplet microfluidic screening of a Synechocystis sp. CRISPRi library based on L-lactate production.
Open this publication in new window or tab >>Droplet microfluidic screening of a Synechocystis sp. CRISPRi library based on L-lactate production
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(English)Manuscript (preprint) (Other academic)
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-259487 (URN)
Note

QC 20191011

Available from: 2019-09-16 Created: 2019-09-16 Last updated: 2019-10-11Bibliographically approved
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