Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • 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
Engineering cyanobacteria for increased growth and productivity
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. (Microbial chemistry)
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 [en]
Cyanobacteria, CBB cycle, growth, biomass, photosynthesis, ethanol
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-338081ISBN: 978-91-513-0201-0 (print)OAI: oai:DiVA.org:uu-338081DiVA, id: diva2:1171383
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
List of papers
1. Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803
Open this publication in new window or tab >>Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803
2016 (English)In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 38, p. 56-64Article in journal (Refereed) Published
Abstract [en]

Synechocystis PCC 6803 is a model unicellular cyanobacterium used in e.g. photosynthesis and CO2 assimilation research. In the present study we examined the effects of overexpressing Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), sedoheptulose 1,7-biphosphatase (SBPase), fructose-bisphosphate aldolase (FBA) and transketolase (TK), confirmed carbon flux control enzymes of the Calvin-Bassham-Benson (CBB) cycle in higher plants, in Synechocystis PCC 6803. Overexpressing RuBisCO, SBPase and FBA resulted in increased in vivo oxygen evolution (maximal 115%), growth rate and biomass accumulation (maximal 52%) under 100μmolphotonsm(-2)s(-1) light condition. Cells overexpressing TK showed a chlorotic phenotype but increased biomass by approximately 42% under 100μmolphotonsm(-2)s(-1) light condition. Under 15μmolphotonsm(-2)s(-1) light condition, cells overexpressing TK showed enhanced in vivo oxygen evolution. This study demonstrates increased growth and biomass accumulation when overexpressing selected enzymes of the CBB cycle. RuBisCO, SBPase, FBA and TK are identified as four potential targets to improve growth and subsequently also yield of valuable products from Synechocystis PCC 6803.

Keywords
RuBisCO, CcmM, SBPase, FBA, TK, Synechocystis PCC 6803
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-306885 (URN)10.1016/j.ymben.2016.06.005 (DOI)000387984600007 ()27328433 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, 2011.0067Swedish Energy Agency, 11674-5
Available from: 2016-11-04 Created: 2016-11-04 Last updated: 2018-01-07Bibliographically approved
2. Synechocystis PCC 6803 overexpressing RuBisCO grow faster with increased photosynthesis
Open this publication in new window or tab >>Synechocystis PCC 6803 overexpressing RuBisCO grow faster with increased photosynthesis
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-338075 (URN)10.1016/j.meteno.2017.02.002 (DOI)29468130 (PubMedID)
Available from: 2018-01-07 Created: 2018-01-07 Last updated: 2018-04-04Bibliographically approved
3. 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
4. Engineered cyanobacteria with enhanced growth show increased ethanol production and higher biofuel to biomass ratio
Open this publication in new window or tab >>Engineered cyanobacteria with enhanced growth show increased ethanol production and higher biofuel to biomass ratio
(English)In: Article in journal (Refereed) Submitted
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-338077 (URN)
Available from: 2018-01-07 Created: 2018-01-07 Last updated: 2018-01-14
5. 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: 2018-09-09Bibliographically approved

Open Access in DiVA

fulltext(2911 kB)154 downloads
File information
File name FULLTEXT01.pdfFile size 2911 kBChecksum SHA-512
53ca4b13ca1dffe7c4ca1190453f26bee34a27963c7ec69304b573c23dabad3def7c5c2607d81af00f90c94df55f603e88a9df4d4d9659d569dc889e94c13dec
Type fulltextMimetype application/pdf
Buy this publication >>

Search in DiVA

By author/editor
Liang, Feiyan
By organisation
Molecular Biomimetics
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 154 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 776 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • 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