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Metabolic Engineering of Synechocystis sp. PCC 6803 for Terpenoid Production
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
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. , p. 63
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1455
Keyword [en]
Metabolic engineering, Cyanobacteria, Synechocystis, Terpenoids, Genetic tools
National Category
Biochemistry and Molecular Biology Microbiology
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-308099ISBN: 978-91-554-9761-3 (print)OAI: oai:DiVA.org:uu-308099DiVA, id: diva2:1049208
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
List of papers
1. Production of Squalene in Synechocystis sp. PCC 6803
Open this publication in new window or tab >>Production of Squalene in Synechocystis sp. PCC 6803
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2014 (English)In: PLoS ONE, Vol. 9, no 3, p. e90270-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.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-220620 (URN)10.1371/journal.pone.0090270 (DOI)000332851300017 ()
Available from: 2014-03-18 Created: 2014-03-18 Last updated: 2016-11-23Bibliographically approved
2. Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide
Open this publication in new window or tab >>Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide
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2015 (English)In: ACS Synthetic Biology, ISSN 0065-0897, E-ISSN 2161-5063, Vol. 4, no 12, p. 1270-1278Article in journal (Refereed) Published
Abstract [en]

Forskolin is a high value diterpenoid with a broad range of pharmaceutical applications, naturally found in root bark of the plant Coleus forskohlii. Because of its complex molecular structure, chemical synthesis of forskolin is not commercially attractive. Hence, the labor and resource intensive extraction and purification from C. forskohlii plants remains the current source of the compound. We have engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO), paving the way for light driven biotechnological production of this high value compound. In the course of this work, a new series of integrative vectors for use in Synechocystis was developed and used to create stable lines expressing chromosomally integrated CfTPS2 and CfTPS3, the enzymes responsible for the formation of 13R-MO in C. forskohlii. The engineered strains yielded production titers of up to 0.24 mg g(-1) DCW 13R-MO. To increase the yield, 13R-MO producing strains were further engineered by introduction of selected enzymes from C. forskohlii, improving the titer to 0.45 mg g(-1) DCW. This work forms a basis for further development of production of complex plant diterpenoids in cyanobacteria.

Keyword
Synechocystis; manoyl oxide; forskolin; diterpenoid; MEP-pathway; genetic tools
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-259966 (URN)10.1021/acssynbio.5b00070 (DOI)000366884700002 ()26133196 (PubMedID)
Funder
Swedish Energy AgencyKnut and Alice Wallenberg FoundationEU, European Research Council, ERC-2012-ADG_20120314EU, European Research Council, 323034
Available from: 2015-08-13 Created: 2015-08-13 Last updated: 2017-12-04Bibliographically approved
3. Effect of expression of MEP pathway enzymes on production of isoprene in Escherichia coli and Synechocystis PCC 6803
Open this publication in new window or tab >>Effect of expression of MEP pathway enzymes on production of isoprene in Escherichia coli and Synechocystis PCC 6803
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-308077 (URN)
Available from: 2016-11-23 Created: 2016-11-23 Last updated: 2016-11-23
4. Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803
Open this publication in new window or tab >>Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803
2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 36640Article in journal (Refereed) Published
Abstract [en]

For effective metabolic engineering, a toolbox of genetic components that enables predictable control of gene expression is needed. Here we present a systematic study of promoters and ribosome binding sites in the unicellular cyanobacterium Synechocystis sp. PCC 6803. A set of metal ion inducible promoters from Synechocystis were compared to commonly used constitutive promoters, by measuring fluorescence of a reporter protein in a standardized setting to allow for accurate comparisons of promoter activity. The most versatile and useful promoter was found to be PnrsB, which from a relatively silent expression could be induced almost 40-fold, nearly up to the activity of the strong psbA2 promoter. By varying the concentrations of the two metal ion inducers Ni(2+) and Co(2+), expression from the promoter was highly tunable, results that were reproduced with PnrsB driving ethanol production. The activities of several ribosomal binding sites were also measured, and tested in parallel in Synechocystis and Escherichia coli. The results of the study add useful information to the Synechocystis genetic toolbox for biotechnological applications.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-308074 (URN)10.1038/srep36640 (DOI)000388069200002 ()27857166 (PubMedID)
Funder
Swedish Energy Agency, 38334-1
Available from: 2016-11-23 Created: 2016-11-23 Last updated: 2018-01-07Bibliographically approved

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