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Increased Carbon Fixation for Chemical Production in Cyanobacteria
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Uppsala University.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The combustion of fossil fuels has created many environmental problems, the major one, the greenhouse effect. Thus, we need solutions in order to replace fossil fuels and recycle the CO2 in the atmosphere. Renewable energies have created attention the last decades but electricity is the main energy form obtained. Photosynthetic organisms (including cyanobacteria) can be used as cell factories since they can convert solar energy to chemical energy. In addition, the requisites to grow them are few; light water, CO2 and inorganic nutrients. Cyanobacteria have been genetically engineered in order to produce numerous chemicals and fuels of human interest in direct processes. However, the amount of product obtained is still low. Increased carbon fixation in cyanobacteria results in higher production of carbon-based substances. This thesis focuses on the effects of overexpressing the native phosphoenolpyruvate carboxylase (PEPc) in the model cyanobacterium Synechocystis PCC 6803. PEPc is an essential enzyme and provides oxaloacetate, an intermediate of the tricarboxylic acid cycle (TCA cycle). The TCA cycle is involved in connecting the carbon and nitrogen metabolism in cyanobacteria. The strains were further engineered to produce ethylene and succinate, two examples of interests for the chemical and fuel industry. Strains with additional PEPc produced significantly more ethylene and succinate. Moreover, an in vitro characterization of PEPc from the cyanobacterium Synechococcus PCC 7002 was performed. The focus was on oligomerization state, kinetics and the structure of the carboxylase. This thesis demonstrates that increasing carbon fixation and discovering the bottlenecks in chemical production can lead to higher yields and gives us hope that cyanobacteria can be commercialized.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1848
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:uu:diva-392234ISBN: 978-91-513-0736-7 (print)OAI: oai:DiVA.org:uu-392234DiVA, id: diva2:1347571
Public defence
2019-10-18, Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-09-25 Created: 2019-09-01 Last updated: 2019-10-15
List of papers
1. Mechanisms of carbon fixation and engineering for increased carbon fixation in cyanobacteria
Open this publication in new window or tab >>Mechanisms of carbon fixation and engineering for increased carbon fixation in cyanobacteria
2015 (English)In: ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS, ISSN 2211-9264, Vol. 11, p. 263-270Article, review/survey (Refereed) Published
Abstract [en]

Cyanobacteria, gram-negative prokaryotic microorganisms, perform oxygenic photosynthesis with a photosynthetic machinery similar to higher plants which includes ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) as the main CO2-fixing enzyme. Currently, there is a growing interest to use cyanobacteria as photosynthetic microbial cell factories for the direct production of solar fuels or other compounds of human interest. However, rates and efficiencies to produce e.g. biofuels are still very low. The amount of available fixed carbon for the synthesis of desired product(s) may be one of the limiting steps. This contribution reviews CO2-fixation in cyanobacteria with focus on CO2-concentrating mechanisms, RuBisCO, phosphoenolpyruvate carboxylase and other carboxylases, engineering approaches for increased carbon fixation, and finally the synthetic malonyl-CoA-oxaloacetate-glyoxylate pathways.

Keywords
Carbon fixation, Cyanobacteria, Genetic engineering, MOG pathway, PEPc, RuBisCO, Synechocystis PCC 6803
National Category
Other Biological Topics Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-267607 (URN)10.1016/j.algal.2015.07.002 (DOI)000363046900033 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2015-11-25 Created: 2015-11-25 Last updated: 2019-09-01Bibliographically approved
2. Enhanced growth at low light intensity in the cyanobacterium Synechocystis PCC 6803 by overexpressing phosphoenolpyruvate carboxylase
Open this publication in new window or tab >>Enhanced growth at low light intensity in the cyanobacterium Synechocystis PCC 6803 by overexpressing phosphoenolpyruvate carboxylase
2016 (English)In: ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS, ISSN 2211-9264, Vol. 16, p. 275-281Article in journal (Refereed) Published
Abstract [en]

Synechocystis PCC 6803 strains overexpressing pepc, gene encoding the carbon fixing enzyme phosphoenolpyruvate carboxylase (PEPc), were constructed and characterized for growth, PEPc protein content and in vitro PEPc activities. Synechocystis strains WT + Km(r) - one (native) copy of pepc (control), WT + 2xPEPc - native copy of pepc and two additional native copies of pepc (in total three copies of pepc), and WT + PPM - native copies of ppsa (encoding phosphoenolpyruvate synthase), pepc and mdh (encoding malate dehydrogenase) and one additional copy of each gene (in total two copies each of ppsa, pepc and mdh) were analyzed for growth under normal and low light intensities, and in darkness (no growth). No significant differences in the growth rates were observed when the cells were grown under normal light intensity. However, growth under low light intensity (3 mu mol photons.m(-2).sec(-1)) resulted in increased growth rate, in particular in the strain with 3 copies of pepc. SDS-PAGE/Western immunoblots using antibodies directed against PEPc demonstrated an increased level of PEPc protein with increasing number of copies of pepc. This was followed by increased levels of in vitro PEPc activities. A less efficient ribulose 1,5-bisphosphate carboxylase/oxygenase in combination with reduced levels of NADPH and ATP under low light condition may make the relatively more efficient carbon fixing enzyme PEPc the limiting step for growth under this condition.

Keywords
Genetic engineering, Enhanced growth, pepc, Phosphoenolpyruvate carboxylase, Synechocystis
National Category
Bio Materials
Identifiers
urn:nbn:se:uu:diva-297764 (URN)10.1016/j.algal.2016.03.027 (DOI)000375610000032 ()
Funder
Knut and Alice Wallenberg Foundation, 2011.0067Swedish Energy Agency
Available from: 2016-06-28 Created: 2016-06-28 Last updated: 2019-09-01Bibliographically approved
3. Phosphoenolpyruvate carboxylase in Synechococcus PCC 7002: Oligomerization, structure, and characteristics
Open this publication in new window or tab >>Phosphoenolpyruvate carboxylase in Synechococcus PCC 7002: Oligomerization, structure, and characteristics
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-392227 (URN)
Available from: 2019-09-01 Created: 2019-09-01 Last updated: 2019-09-01
4. Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803
Open this publication in new window or tab >>Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-392232 (URN)
Available from: 2019-09-01 Created: 2019-09-01 Last updated: 2019-09-01
5. Increased succinate production by expressing a glyoxylate shunt in the engineered Synechocystis PCC 6803
Open this publication in new window or tab >>Increased succinate production by expressing a glyoxylate shunt in the engineered Synechocystis PCC 6803
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-392233 (URN)
Available from: 2019-09-01 Created: 2019-09-01 Last updated: 2019-09-01
6. Engineering Cyanobacteria for Biofuel Production
Open this publication in new window or tab >>Engineering Cyanobacteria for Biofuel Production
Show others...
2017 (English)In: Modern Topics in the Phototrophic Prokaryotes: Environmental and Applied Aspects / [ed] Hallenbeck, Patrick, USA: Springer, 2017, p. 351-393Chapter in book (Refereed)
Place, publisher, year, edition, pages
USA: Springer, 2017
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-338078 (URN)978-3-319-46259-2 (ISBN)978-3-319-46261-5 (ISBN)
Available from: 2018-01-07 Created: 2018-01-07 Last updated: 2019-09-01Bibliographically approved

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