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Enhancement of photosynthetic isobutanol production in engineered cells of Synechocystis PCC 6803
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström.
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.
2018 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 11, article id 267Article in journal (Refereed) Published
Abstract [en]

Background: Cyanobacteria, oxygenic photoautotrophic prokaryotes, can be engineered to produce various valuable chemicals from solar energy and CO2 in direct processes. The concept of photosynthetic production of isobutanol, a promising chemical and drop-in biofuel, has so far been demonstrated for Synechocystis PCC 6803 and Synechococcus elongatus PCC 7942. In Synechocystis PCC 6803, a heterologous expression of alpha-ketoisovalerate decarboxylase (Kivd) from Lactococcus lactis resulted in an isobutanol and 3-methyl-1-butanol producing strain. Kivd was identified as a bottleneck in the metabolic pathway and its activity was further improved by reducing the size of its substrate-binding pocket with a single replacement of serine-286 to threonine (Kivd(S286T)). However, isobutanol production still remained low. Results: In the present study, we report on how cultivation conditions significantly affect the isobutanol production in Synechocystis PCC 6803. A HCl-titrated culture grown under medium light (50 mu mol photons m(-2) s(-1)) showed the highest isobutanol production with an in-flask titer of 194 mg l(-1) after 10 days and 435 mg l(-1) at day 40. This corresponds to a cumulative isobutanol production of 911 mg l(-1), with a maximal production rate of 43.6 mg l(-1) day(-1) observed between days 4 and 6. Additional metabolic bottlenecks in the isobutanol biosynthesis pathway were further addressed. The expression level of Kivd(S286T) was significantly affected when co-expressed with another gene downstream in a single operon and in a convergent oriented operon. Moreover, the expression of the ADH encoded by codon-optimized slr1192 and co-expression of IlvC and IlvD were identified as potential approaches to further enhance isobutanol production in Synechocystis PCC 6803. Conclusion: The present study demonstrates the importance of a suitable cultivation condition to enhance isobutanol production in Synechocystis PCC 6803. Chemostat should be used to further increase both the total titer as well as the rate of production. Furthermore, identified bottleneck, Kivd, should be expressed at the highest level to further enhance isobutanol production.

Place, publisher, year, edition, pages
BioMed Central, 2018. Vol. 11, article id 267
Keywords [en]
Synechocystis, Cumulative titer, Cultivation condition, Metabolic bottleneck, Co-expression
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-359862DOI: 10.1186/s13068-018-1268-8ISI: 000445749100004PubMedID: 30275907OAI: oai:DiVA.org:uu-359862DiVA, id: diva2:1245995
Funder
EU, Horizon 2020, 640720NordForsk, 82845Available from: 2018-09-06 Created: 2018-09-06 Last updated: 2018-10-18Bibliographically 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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