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Engineering Cyanobacteria for Biofuel Production
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)
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)
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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. p. 351-393
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-338078ISBN: 978-3-319-46259-2 (print)ISBN: 978-3-319-46261-5 (electronic)OAI: oai:DiVA.org:uu-338078DiVA, id: diva2:1171380
Available from: 2018-01-07 Created: 2018-01-07 Last updated: 2018-09-09Bibliographically 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)
Opponent
Supervisors
Available from: 2018-02-01 Created: 2018-01-07 Last updated: 2018-03-07
2. Metabolic Engineering of Synechocystis PCC 6803 for Butanol Production
Open this publication in new window or tab >>Metabolic Engineering of Synechocystis PCC 6803 for Butanol Production
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There is an urgent demand for renewable alternatives to fossil fuels since the extraction and utilization cause a series of environmental problems in the world. Thus, the utilization of solar energy has attracted much attention in the last decades since there is excess amount of light on Earth. Photosynthetic microorganisms, such as cyanobacteria, can be a good biological chassis to convert solar energy directly to chemical energy. It has been demonstrated that cyanobacteria can produce various compounds which can be used asfourth-generation biofuels. This thesis focuses on the photo-autotrophic production of two biofuel compounds, isobutanol and 1-butanol, in the unicellular cyanobacterial strain Synechocystis PCC 6803. In the studies of isobutanol production, the endogenous alcohol dehydrogenase of Synechocystis encoded by slr1192 showed impressive activity in isobutanol formation. In addition, a-ketoisovalerate decarboxylase (Kivd) was identified as the only heterologous enzyme needed to be introduced for isobutanol production in Synechocystis. Kivd was further recognized as a bottleneck in the isobutanol production pathway. Therefore, Kivd was engineered via rational design to shift the preferential activity towards the production of isobutanol instead of the by-product 3-methyl-1-butanol. The best strain pEEK2-ST expressing KivdS286T showed dramatically increased productivity, and the activity of Kivd was successfully shifted further towards isobutanol production. A cumulative isobutanol titer of 911 mg L-1 was observed from this strain after 46 days growth under 50 μmol photons m−2 s−1 with pH adjusted to between 7 and 8. A maximum production rate of nearly 44 mg L-1d-1was reached between days 4 and 6. Similar metabolic engineering strategies were employed to generate 1-butanol producing Synechocystis strains and then to stepwise enhance the production. By selecting the best enzymes and promotors, 836 mg L-1 in-flask 1-butanol was produced. By optimizing the cultivation condition, an in-flask titer of 2.1 g L-1 and a maximal cumulative titer of 4.7 g L-1 were observed in the long-term cultivation. This thesis demonstrates different metabolic engineering strategies for producing valuable compounds in Synechocystis, exemplified with butanol, and how to enhance production systematically. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1721
Keywords
Synechocystis PCC 6803, biofuel, isobutanol, 1-butanol, metabolic engineering, protein engineering
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-360031 (URN)978-91-513-0441-0 (ISBN)
Public defence
2018-10-26, Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2018-10-05 Created: 2018-09-09 Last updated: 2018-10-16

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Miao, RuiWegelius, AdamDurall de la Fuente, ClaudiaLiang, FeiyanKhanna, NamitaLindblad, Peter

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