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Synechocystis PCC 6803 overexpressing RuBisCO grow faster with increased photosynthesis
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. (Microbial chemistry)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. (Microbial Chemistry)
2017 (English)In: Metabolic Engineering Communications, ISSN 2214-0301, Vol. 4, p. 29-36Article in journal (Refereed) Published
Abstract [en]

The ribulose-1,5-bisphosphate (RuBP) oxygenation reaction catalyzed by Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is competing with carboxylation, being negative for both energy and carbon balances in photoautotrophic organisms. This makes RuBisCO one of the bottlenecks for oxygenic photosynthesis and carbon fixation. In this study, RuBisCO was overexpressed in the unicellular cyanobacterium Synechocystis PCC 6803. Relative RuBisCO levels in the engineered strains FL50 and FL52 increased 2.1 times and 1.4 times, respectively, and both strains showed increased growth, photosynthesis and in vitro RuBisCO activity. The oxygen evolution rate increased by 54% and 42% on per chlorophyll basis, while the in vitro RuBisCO activity increased by 52% and 8.6%, respectively. The overexpressed RuBisCO were tagged with a FLAG tag, in strain FL50 on the N terminus of the large subunit while in strain FL52 on the C terminus of the small subunit. The presence of a FLAG tag enhanced transcription of the genes encoding RuBisCO, and, with high possibility, also enhanced the initiation of translation or stability of the enzyme. However, when using a streptavidin-binding tag II (strep-tag II), we did not observe a similar effect. Tagged RuBisCO offers an opportunity for further studying RuBisCO expression and stability. Increased levels of RuBisCO can further improve photosynthesis and growth in the cyanobacterium Synechocystis PCC 6803 under certain growth conditions.

Place, publisher, year, edition, pages
2017. Vol. 4, p. 29-36
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-338075DOI: 10.1016/j.meteno.2017.02.002PubMedID: 29468130OAI: oai:DiVA.org:uu-338075DiVA, id: diva2:1171376
Available from: 2018-01-07 Created: 2018-01-07 Last updated: 2018-04-04Bibliographically approved
In thesis
1. Engineering cyanobacteria for increased growth and productivity
Open this publication in new window or tab >>Engineering cyanobacteria for increased growth and productivity
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Increasing the photosynthetic efficiency is one of the strategies to increase the crop yields to meet the requirement of 50% more food by 2050. Due to the similarity on photosynthesis between crops and cyanobacteria, cyanobacteria are ideal alternatives to study photosynthesis since cyanobacteria are prokaryotes, easier to engineer and have shorter life cycle. On the other hand, cyanobacteria are promising cell factories for food additives, biofuels, and other products. To get the desired products from cyanobacteria directly will consume atmospheric CO2 and avoid additional releasing of CO2 from the usage of fossil resources.

In this thesis, four CBB cycle enzymes were overexpressed individually in the model cyanobacterium Synechocystis PCC 6803. To get ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) overexpressed, two methods were used. One was to introduce another copy of the carboxysome protein CcmM gene into the cells since CcmM is essential for packing RuBisCO into the carboxysome. Another way was to tag the RuBisCO gene either on the N terminus of the large subunit or on the C terminus of the small subunit by FLAG. Even though the RuBisCO level increased, the specific RuBisCO activity did not change. Fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), aldolase (FBA) and transketolase (TK) were overexpressed by introducing a second copy of corresponding gene. The engineered strains with increased levels of RuBisCO, FBP/SBPase, and FBA grew faster, had higher maximum net oxygen evolution rate and accumulated more biomass when cultivated under 100µmol photons m-2 s-1 light intensity. The strain carrying more TK showed a chlorotic phenotype but still accumulated more biomass under the same light condition. Four strains with one of the CBB cycle enzymes overexpressed were selected to investigate the effects on ethanol production. Increased ethanol production and ethanol to total biomass rate were observed in the CBB cycle engineered strains. The best strain produced almost 50% ethanol out of the total biomass.

This work shows that overexpressing selected enzymes of the CBB cycle in cyanobacteria resulted in enhanced total biomass accumulation and increased compound (exampled as ethanol) production under certain growth conditions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 63
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1616
Keywords
Cyanobacteria, CBB cycle, growth, biomass, photosynthesis, ethanol
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-338081 (URN)978-91-513-0201-0 (ISBN)
Public defence
2018-02-23, Häggsalen, Ang/10132, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 09:15 (English)
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Supervisors
Available from: 2018-02-01 Created: 2018-01-07 Last updated: 2018-03-07

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