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Evaluation of Baltic Sea metagenome-derived aldehyde deformylating oxygenases in Synechocystis sp. strain PCC 6803
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.ORCID iD: 0000-0001-8317-1654
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-249933OAI: oai:DiVA.org:kth-249933DiVA, id: diva2:1306302
Note

QC 20190521

Available from: 2019-04-23 Created: 2019-04-23 Last updated: 2019-05-21Bibliographically approved
In thesis
1. Synthetic biology approaches for improving production of fatty acid-derived compounds in cyanobacteria
Open this publication in new window or tab >>Synthetic biology approaches for improving production of fatty acid-derived compounds in cyanobacteria
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The environmental consequences associated with the use of fossil-sourced fuels and chemicals have brought with it a realization that future development must move in a more sustainable direction. Currently available biofuels or renewably produced chemical, such as bioethanol or biodiesel, are produced from microbial fermentation of sugar-rich crops or by chemical conversion of natural oils or fats. However, these strategies are not sustainable in the long run as fuel and chemical production competes with food supply and arable land usage. Instead of relying on photosynthetic feedstocks that require further conversion, one can engineer photosynthetic cyanobacteria to produce a product of interest directly from CO2 and sunlight. The first part of this thesis aimed to develop new synthetic biology tools for the model cyanobacteria Synechocystis sp. PCC 6803. The second part of the thesis focused on evaluating the regulation of fatty acid synthesis in cyanobacteria, and the production of fatty acid-derived chemicals in Synechocystis.

In paper I, fusion of small affinity proteins (Affibodies) to the major type IV pili protein was shown to mediate successful surface display of the affibody. This surface display strategy was further shown to allow inter-species binding between Synechocystis and Escherichia coli or Staphylococcus carnosus displaying complementary polymerizing affibodies.

In paper II, a CRISPR-interference tool was successfully implemented in Synechocystis for inducible gene repression. Further, its multiplexing ability was proven by simultaneous repression of up to four aldehyde reductase/dehydrogenase genes. In paper III, this established CRISPRi tool was used to target and repress native pathways competing with heterologous fatty alcohol production in Synechocystis. Repressing the gene encoding the PlsX phosphate acyltransferase allowed re-direction of carbon-flux from membrane lipids to fatty alcohol production, with a final best strain producing 10.4 mg g-1 DCW octadecanol and hexadecanol.

In paper IV, the transcriptional response towards perturbations within the fatty acid synthesis pathway was evaluated for the two model cyanobacteria Synechocystis and Synechococcus elongatus PCC 7942. Preliminary results indicate that blocking fatty acid synthesis initiation/elongation causes a transcriptional response of the involved pathway genes only in S. elongatus PCC 7942, indicating differential transcriptional responses in these two strains.

In paper V, metagenomically sourced aldehyde deformylating oxygenase (Ado) variants were evaluated for their alka(e)ne synthesizing ability. Several of these novel Ado enzymes outperformed the generally well-performing Ado from S. elongatus when relating alka(e)ne production to the soluble enzyme amount.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2019. p. 63
Series
TRITA-CBH-FOU ; 2019:16
Keywords
cyanobacteria, metabolic engineering, surface display, CRISPRi, fatty alcohols, fatty acid synthesis, aldehyde deformylating oxygenase
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-249935 (URN)978-91-7873-178-7 (ISBN)
Public defence
2019-05-17, Air & Fire auditorium, Science for Life Laboratory, Tomtebodavägen 23A, Solna, 10:00 (English)
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Note

QC 20190423

Available from: 2019-04-23 Created: 2019-04-23 Last updated: 2019-05-16Bibliographically approved

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