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Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases
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.
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.ORCID iD: 0000-0002-6413-1443
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2017 (English)In: Metabolic Engineering Communications, ISSN 2214-0301, Vol. 5, p. 45-53Article in journal (Refereed) Published
Abstract [en]

Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. In this study, we examined the capacity of engineered strains of Synechocystis PCC 6803 containing the α-ketoisovalerate decarboxylase from Lactococcus lactis and different heterologous and endogenous alcohol dehydrogenases (ADH) for isobutanol production. A strain expressing an introduced kivdwithout any additional copy of ADH produced 3 mg L−1 OD750−1 isobutanol in 6 days. After the cultures were supplemented with external addition of isobutyraldehyde, the substrate for ADH, 60.8 mg L−1 isobutanol was produced after 24 h when OD750 was 0.8. The in vivo activities of four different ADHs, two heterologous and two putative endogenous in Synechocystis, were examined and the Synechocystis endogenous ADH encoded by slr1192 showed the highest efficiency for isobutanol production. Furthermore, the strain overexpressing the isobutanol pathway on a self-replicating vector with the strong Ptrc promoter showed significantly higher gene expression and isobutanol production compared to the corresponding strains expressing the same operon introduced on the genome. Hence, this study demonstrates that Synechocystis endogenous AHDs have a high capacity for isobutanol production, and identifies kivd encoded α-ketoisovalerate decarboxylase as one of the likely bottlenecks for further isobutanol production.

Place, publisher, year, edition, pages
2017. Vol. 5, p. 45-53
Keywords [en]
Cyanobacteria, Alcohol dehydrogenase, α-ketoisovalerate decarboxylase, Synechocystis PCC 6803, Isobutanol production
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-359893DOI: 10.1016/j.meteno.2017.07.003PubMedID: 29188183OAI: oai:DiVA.org:uu-359893DiVA, id: diva2:1246027
Available from: 2018-09-06 Created: 2018-09-06 Last updated: 2018-09-09Bibliographically approved
In thesis
1. 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)
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Available from: 2018-10-05 Created: 2018-09-09 Last updated: 2018-10-16

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Miao, RuiLiu, XufengEnglund, EliasLindberg, PiaLindblad, Peter

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