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Production of Squalene in Synechocystis sp. PCC 6803
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
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2014 (English)In: PLoS ONE, Vol. 9, no 3, e90270- p.Article in journal (Refereed) Published
Abstract [en]

In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode squalene hopene cyclase (Shc), an enzyme converting squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce squalene using cyanobacteria. The inactivation led to accumulation of squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750−1 L−1. We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a squalene hopene cyclase, and the second identification of squalene synthase, in cyanobacteria.

Place, publisher, year, edition, pages
2014. Vol. 9, no 3, e90270- p.
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-220620DOI: 10.1371/journal.pone.0090270ISI: 000332851300017OAI: oai:DiVA.org:uu-220620DiVA: diva2:705848
Available from: 2014-03-18 Created: 2014-03-18 Last updated: 2016-11-23Bibliographically approved
In thesis
1. Metabolic Engineering of Synechocystis sp. PCC 6803 for Terpenoid Production
Open this publication in new window or tab >>Metabolic Engineering of Synechocystis sp. PCC 6803 for Terpenoid Production
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the Paris Agreement from 2015, nations agreed to limit the effects of global warming to well below 2°C. To be able to reach those goals, cheap, abundant and carbon neutral energy alternatives needs to be developed. The microorganisms that several billion years ago oxygenated the atmosphere; cyanobacteria, might hold the key for creating those energy technologies. Due to their capacity for photosynthesis, metabolic engineering of cyanobacteria can reroute the carbon dioxide they fix from the atmosphere into valuable products, thereby converting them into solar powered cell factories.

Of the many products bacteria can be engineered to make, the production of terpenoids has gained increasing attention for their attractive properties as fuels, pharmaceuticals, fragrances and food additives. In this thesis, I detail the work I have done on engineering the unicellular cyanobacterium Synechocystis sp. PCC 6803 for terpenoid production. By deleting an enzyme that converts squalene into hopanoids, we could create a strain that accumulates squalene, a molecule with uses as a fuel or chemical feedstock. In another study, we integrated two terpene synthases from the traditional medical plant Coleus forskohlii, into the genome of Synechocystis. Expression of those genes led to the formation of manoyl oxide, a precursor to the pharmaceutically active compound forskolin. Production of manoyl oxide in Synechocystis was further enhanced by engineering in two additional genes from C. forskohlii that boosted the flux to the product. To learn how to increase the production of squalene, manoyl oxide or any other terpenoid, we conducted a detailed investigation of each step in the MEP biosynthesis pathway, which creates the two common building blocks for all terpenoids. Each enzymatic step in the pathway was overexpressed, and increased flux was assayed by using isoprene as a reporter and several potential targets for overexpression were identified. The final part of this thesis details the characterization of native, inducible promoters and ribosomal binding sites in Synechocystis

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 63 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1455
Keyword
Metabolic engineering, Cyanobacteria, Synechocystis, Terpenoids, Genetic tools
National Category
Biochemistry and Molecular Biology Microbiology
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-308099 (URN)978-91-554-9761-3 (ISBN)
Public defence
2017-01-13, Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2016-12-20 Created: 2016-11-23 Last updated: 2016-12-28

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Englund, EliasPattanaik, BagmiUbhayasekera, Sarojini J.K.A.Stensjö, KarinBergquist, JonasLindberg, Pia

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