uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide
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.
Show others and affiliations
2015 (English)In: ACS Synthetic Biology, ISSN 0065-0897, E-ISSN 2161-5063, Vol. 4, no 12, 1270-1278 p.Article 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.

Place, publisher, year, edition, pages
2015. Vol. 4, no 12, 1270-1278 p.
Keyword [en]
Synechocystis; manoyl oxide; forskolin; diterpenoid; MEP-pathway; genetic tools
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-259966DOI: 10.1021/acssynbio.5b00070ISI: 000366884700002PubMedID: 26133196OAI: oai:DiVA.org:uu-259966DiVA: diva2:845940
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
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

Open Access in DiVA

No full text

Other links

Publisher's full textPubMedFulltext

Authority records BETA

Englund, EliasMiao, RuiLindberg, Pia

Search in DiVA

By author/editor
Englund, EliasMiao, RuiLindberg, Pia
By organisation
Molecular Biomimetics
In the same journal
ACS Synthetic Biology
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 945 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf