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Synthetic biology approaches for improving production of fatty acid-derived compounds in cyanobacteria
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
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 [en]
cyanobacteria, metabolic engineering, surface display, CRISPRi, fatty alcohols, fatty acid synthesis, aldehyde deformylating oxygenase
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-249935ISBN: 978-91-7873-178-7 (print)OAI: oai:DiVA.org:kth-249935DiVA, id: diva2:1306324
Public defence
2019-05-17, Air & Fire auditorium, Science for Life Laboratory, Tomtebodavägen 23A, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20190423

Available from: 2019-04-23 Created: 2019-04-23 Last updated: 2019-05-16Bibliographically approved
List of papers
1. Surface Display of Small Affinity Proteins on Synechocystis sp Strain PCC 6803 Mediated by Fusion to the Major Type IV Pilin PilA1
Open this publication in new window or tab >>Surface Display of Small Affinity Proteins on Synechocystis sp Strain PCC 6803 Mediated by Fusion to the Major Type IV Pilin PilA1
2018 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 200, no 16, article id e00270-18Article in journal (Refereed) Published
Abstract [en]

Functional surface display of small affinity proteins, namely, affibodies (6.5 kDa), was evaluated for the model cyanobacterium Synechocystis sp. strain PCC 6803 through anchoring to native surface structures. These structures included confirmed or putative subunits of the type IV pili, the S-layer protein, and the heterologous Escherichia coli autotransporter antigen 43 system. The most stable display system was determined to be through C-terminal fusion to PilA1, the major type IV pilus subunit in Synechocystis, in a strain unable to retract these pili (Delta pilT1). Type IV pilus synthesis was upheld, albeit reduced, when fusion proteins were incorporated. However, pilus-mediated functions, such as motility and transformational competency, were negatively affected. Display of affibodies on Synechocystis and the complementary anti-idiotypic affibodies on E. coli or Staphylococcus carnosus was able to mediate interspecies cell-cell binding by affibody complex formation. The same strategy, however, was not able to drive cell-cell binding and aggregation of Synechocystis-only mixtures. Successful affibody tagging of the putative minor pilin PilA4 showed that it locates to the type IV pili in Synechocystis and that its extracellular availability depends on PilA1. In addition, affibody tagging of the S-layer protein indicated that the domains responsible for the anchoring and secretion of this protein are located at the N and C termini, respectively. This study can serve as a basis for future surface display of proteins on Synechocystis for biotechnological applications. IMPORTANCE Cyanobacteria are gaining interest for their potential as autotrophic cell factories. Development of efficient surface display strategies could improve their suitability for large-scale applications by providing options for designed microbial consortia, cell immobilization, and biomass harvesting. Here, surface display of small affinity proteins was realized by fusing them to the major subunit of the native type IV pili in Synechocystis sp. strain PCC 6803. The display of complementary affinity proteins allowed specific cell-cell binding between Synechocystis and Escherichia coli or Staphylococcus carnosus. Additionally, successful tagging of the putative pilin PilA4 helped determine its localization to the type IV pili. Analogous tagging of the S-layer protein shed light on the regions involved in its secretion and surface anchoring.

Place, publisher, year, edition, pages
American Society for Microbiology, 2018
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-232872 (URN)10.1128/JB.00270-18 (DOI)000439777600014 ()29844032 (PubMedID)2-s2.0-85050469301 (Scopus ID)
Note

QC 20180810

Available from: 2018-08-10 Created: 2018-08-10 Last updated: 2019-04-23Bibliographically approved
2. Multiple Gene Repression in Cyanobacteria Using CRISPRi
Open this publication in new window or tab >>Multiple Gene Repression in Cyanobacteria Using CRISPRi
2016 (English)In: ACS Photonics, E-ISSN 2330-4022, Vol. 5, no 3, p. 207-212Article in journal (Refereed) Published
Abstract [en]

We describe the application of clustered regularly interspaced short palindromic repeats interference (CRISPRi) for gene repression in the model cyanobacterium Synechcocystis sp. PCC 6803. The nuclease-deficient Cas9 from the type-II. CRISPR/Cas of Streptrococcus pyogenes was used to repress green fluorescent protein (GFP) to negligible levels. CRISPRi was also used to repress formation of carbon storage compounds polyhydroxybutryate (PHB) and glycogen during nitrogen starvation. As an example of the potential of CRISPRi for basic and applied cyanobacteria research, we simultaneously knocked down 4 putative aldehyde reductases and dehydrogenases at 50-95% repression. This work also demonstrates that tightly repressed promoters allow for inducible and reversible CRISPRi in cyanobacteria.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016
Keywords
Strain Pcc 6803, Sequence-Specific Control, Synechocystis Sp Pcc6803, Escherichia-Coli, Carbon-Dioxide, Light, Expression, Dehydrogenase, Tolerance, Aldehyde
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-185366 (URN)10.1021/acssynbio.5b00264 (DOI)000372672500003 ()26689101 (PubMedID)2-s2.0-84961794371 (Scopus ID)
Funder
Swedish Research Council Formas, 213-2011-1655Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Foundation for Strategic Research , RBP14-0013
Note

QC 20160418

Available from: 2016-04-18 Created: 2016-04-18 Last updated: 2019-04-23Bibliographically approved
3. Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX
Open this publication in new window or tab >>Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX
2018 (English)In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 45, p. 59-66Article in journal (Refereed) Published
Abstract [en]

Fatty alcohol production in Synechocystis sp. PCC 6803 was achieved through heterologous expression of the fatty acyl-CoA/ACP reductase Maqu2220 from the bacteria Marinobacter aquaeolei VT8 and the fatty acyl-ACP reductase DPW from the rice Oryza sativa. These platform strains became models for testing multiplex CRISPR-interference (CRISPRi) metabolic engineering strategies to both improve fatty alcohol production and to study membrane homeostasis. CRISPRi allowed partial repression of up to six genes simultaneously, each encoding enzymes of acyl-ACP-consuming pathways. We identified the essential phosphate acyltransferase enzyme PlsX (slr1510) as a key node in C18 fatty acyl-ACP consumption, repression of slr1510 increased octadecanol productivity threefold over the base strain and gave the highest specific titers reported for this host, 10.3 mg g−1 DCW. PlsX catalyzes the first committed step of phosphatidic acid synthesis, and has not been characterized in Synechocystis previously. We found that accumulation of fatty alcohols impaired growth, altered the membrane composition, and caused a build-up of reactive oxygen species.

Place, publisher, year, edition, pages
Academic Press, 2018
Keywords
Acyl-ACP, Acyltransferase, CRISPRi, Cyanobacteria, Fatty alcohols, Membranes
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-220198 (URN)10.1016/j.ymben.2017.11.014 (DOI)000424292100007 ()2-s2.0-85036651462 (Scopus ID)
Funder
Swedish Foundation for Strategic Research , RBP14-0013Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20171218

Available from: 2017-12-18 Created: 2017-12-18 Last updated: 2019-04-23Bibliographically approved
4. Indications of differential transcriptional regulation of fatty acid synthesis in the model cyanobacteria Synechocystis sp. Strain PCC 6803 and Synechococcus elongatus PCC 7942
Open this publication in new window or tab >>Indications of differential transcriptional regulation of fatty acid synthesis in the model cyanobacteria Synechocystis sp. Strain PCC 6803 and Synechococcus elongatus PCC 7942
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-249932 (URN)
Note

QC 20190521

Available from: 2019-04-23 Created: 2019-04-23 Last updated: 2019-05-21Bibliographically approved
5. Evaluation of Baltic Sea metagenome-derived aldehyde deformylating oxygenases in Synechocystis sp. strain PCC 6803
Open this publication in new window or tab >>Evaluation of Baltic Sea metagenome-derived aldehyde deformylating oxygenases in Synechocystis sp. strain PCC 6803
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-249933 (URN)
Note

QC 20190521

Available from: 2019-04-23 Created: 2019-04-23 Last updated: 2019-05-21Bibliographically approved

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