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Surface Display of Small Affinity Proteins on Synechocystis sp Strain PCC 6803 Mediated by Fusion to the Major Type IV Pilin PilA1
KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-8317-1654
KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-8993-048X
KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0003-1899-7649
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. Vol. 200, no 16, article id e00270-18
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-232872DOI: 10.1128/JB.00270-18ISI: 000439777600014PubMedID: 29844032Scopus ID: 2-s2.0-85050469301OAI: oai:DiVA.org:kth-232872DiVA, id: diva2:1237883
Note

QC 20180810

Available from: 2018-08-10 Created: 2018-08-10 Last updated: 2019-04-23Bibliographically 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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Supervisors
Note

QC 20190423

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

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Cengic, IvanaUhlén, MathiasHudson, Elton P.

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