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Protein engineering of α-ketoisovalerate decarboxylase for improved isobutanol production in Synechocystis PCC 6803
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: 0000-0001-7731-3396
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.ORCID iD: 0000-0001-7256-0275
2018 (English)In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 47, p. 42-48Article in journal (Refereed) Published
Abstract [en]

Protein engineering is a powerful tool to modify e.g. protein stability, activity and substrate selectivity. Heterologous expression of the enzyme α-ketoisovalerate decarboxylase (Kivd) in the unicellular cyanobacterium Synechocystis PCC 6803 results in cells producing isobutanol and 3-methyl-1-butanol, with Kivd identified as a potential bottleneck. In the present study, we used protein engineering of Kivd to improve isobutanol production in Synechocystis PCC 6803. Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. Single replacement, either Val461 to isoleucine or Ser286 to threonine, increased the Kivd activity significantly, both in vivo and in vitro resulting in increased overall production while isobutanol production was increased more than 3-methyl-1-butanol production. Moreover, among all the engineered strains examined, the strain with the combined modification V461I/S286T showed the highest (2.4 times) improvement of isobutanol-to-3M1B molar ratio, which was due to a decrease of the activity towards 3M1B production. Protein engineering of Kivd resulted in both enhanced total catalytic activity and preferential shift towards isobutanol production in Synechocystis PCC 6803.

Place, publisher, year, edition, pages
2018. Vol. 47, p. 42-48
Keywords [en]
Cyanobacteria, Isobutanol, alpha-ketoisovalerate decarboxylase, Site mutagenesis, Substrate pocket, Enzyme activity
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-357581DOI: 10.1016/j.ymben.2018.02.014ISI: 000433423600005PubMedID: 29501927OAI: oai:DiVA.org:uu-357581DiVA, id: diva2:1239793
Funder
Knut and Alice Wallenberg Foundation, 2011.0067Swedish Energy Agency, 44728-1NordForsk, 82845EU, Horizon 2020, 640720Available from: 2018-08-17 Created: 2018-08-17 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)
Opponent
Supervisors
Available from: 2018-10-05 Created: 2018-09-09 Last updated: 2018-10-16

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