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Metabolic Engineering of Synechocystis PCC 6803 for Butanol Production
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]

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 [en]
Synechocystis PCC 6803, biofuel, isobutanol, 1-butanol, metabolic engineering, protein engineering
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-360031ISBN: 978-91-513-0441-0 (print)OAI: oai:DiVA.org:uu-360031DiVA, id: diva2:1246651
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
List of papers
1. Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide
Open this publication in new window or tab >>Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide
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2015 (English)In: ACS Synthetic Biology, E-ISSN 2161-5063, Vol. 4, no 12, p. 1270-1278Article in journal (Refereed) Published
Abstract [en]

Forskolin is a high value diterpenoid with a broad range of pharmaceutical applications, naturally found in root bark of the plant Coleus forskohlii. Because of its complex molecular structure, chemical synthesis of forskolin is not commercially attractive. Hence, the labor and resource intensive extraction and purification from C. forskohlii plants remains the current source of the compound. We have engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO), paving the way for light driven biotechnological production of this high value compound. In the course of this work, a new series of integrative vectors for use in Synechocystis was developed and used to create stable lines expressing chromosomally integrated CfTPS2 and CfTPS3, the enzymes responsible for the formation of 13R-MO in C. forskohlii. The engineered strains yielded production titers of up to 0.24 mg g(-1) DCW 13R-MO. To increase the yield, 13R-MO producing strains were further engineered by introduction of selected enzymes from C. forskohlii, improving the titer to 0.45 mg g(-1) DCW. This work forms a basis for further development of production of complex plant diterpenoids in cyanobacteria.

Keywords
Synechocystis; manoyl oxide; forskolin; diterpenoid; MEP-pathway; genetic tools
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-259966 (URN)10.1021/acssynbio.5b00070 (DOI)000366884700002 ()26133196 (PubMedID)
Funder
Swedish Energy AgencyKnut and Alice Wallenberg FoundationEU, European Research Council, ERC-2012-ADG_20120314EU, European Research Council, 323034
Available from: 2015-08-13 Created: 2015-08-13 Last updated: 2018-09-09Bibliographically approved
2. Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases
Open this publication in new window or tab >>Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases
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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.

Keywords
Cyanobacteria, Alcohol dehydrogenase, α-ketoisovalerate decarboxylase, Synechocystis PCC 6803, Isobutanol production
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-359893 (URN)10.1016/j.meteno.2017.07.003 (DOI)29188183 (PubMedID)
Available from: 2018-09-06 Created: 2018-09-06 Last updated: 2018-09-09Bibliographically approved
3. Protein engineering of α-ketoisovalerate decarboxylase for improved isobutanol production in Synechocystis PCC 6803
Open this publication in new window or tab >>Protein engineering of α-ketoisovalerate decarboxylase for improved isobutanol production in Synechocystis PCC 6803
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.

Keywords
Cyanobacteria, Isobutanol, alpha-ketoisovalerate decarboxylase, Site mutagenesis, Substrate pocket, Enzyme activity
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-357581 (URN)10.1016/j.ymben.2018.02.014 (DOI)000433423600005 ()29501927 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, 2011.0067Swedish Energy Agency, 44728-1NordForsk, 82845EU, Horizon 2020, 640720
Available from: 2018-08-17 Created: 2018-08-17 Last updated: 2018-09-09Bibliographically approved
4. Enhancement of photosynthetic isobutanol production in engineered cells of Synechocystis PCC 6803
Open this publication in new window or tab >>Enhancement of photosynthetic isobutanol production in engineered cells of Synechocystis PCC 6803
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
Keywords
Synechocystis, Cumulative titer, Cultivation condition, Metabolic bottleneck, Co-expression
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-359862 (URN)10.1186/s13068-018-1268-8 (DOI)000445749100004 ()30275907 (PubMedID)
Funder
EU, Horizon 2020, 640720NordForsk, 82845
Available from: 2018-09-06 Created: 2018-09-06 Last updated: 2018-10-18Bibliographically approved
5. Metabolic engineering of Synechocystis PCC 6803 for photosynthetic 1-butanol production
Open this publication in new window or tab >>Metabolic engineering of Synechocystis PCC 6803 for photosynthetic 1-butanol production
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-359891 (URN)
Available from: 2018-09-06 Created: 2018-09-06 Last updated: 2018-09-17
6. Engineering Cyanobacteria for Biofuel Production
Open this publication in new window or tab >>Engineering Cyanobacteria for Biofuel Production
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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
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: 2019-09-01Bibliographically approved

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