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Using Transcriptomics To Improve Butanol Tolerance of Synechocystis sp Strain PCC 6803
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-2430-2682
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0001-8993-048X
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2013 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 79, no 23, 7419-7427 p.Article in journal (Refereed) Published
Abstract [en]

Cyanobacteria are emerging as promising hosts for production of advanced biofuels such as n-butanol and alkanes. However, cyanobacteria suffer from the same product inhibition problems as those that plague other microbial biofuel hosts. High concentrations of butanol severely reduce growth, and even small amounts can negatively affect metabolic processes. An understanding of how cyanobacteria are affected by their biofuel product can enable identification of engineering strategies for improving their tolerance. Here we used transcriptome sequencing (RNA-Seq) to assess the transcriptome response of Synechocystis sp. strain PCC 6803 to two concentrations of exogenous n-butanol. Approximately 80 transcripts were differentially expressed at 40 mg/liter butanol, and 280 transcripts were different at 1 g/liter butanol. Our results suggest a compromised cell membrane, impaired photosynthetic electron transport, and reduced biosynthesis. Accumulation of intracellular reactive oxygen species (ROS) scaled with butanol concentration. Using the physiology and transcriptomics data, we selected several genes for overexpression in an attempt to improve butanol tolerance. We found that overexpression of several proteins, notably, the small heat shock protein HspA, improved tolerance to butanol. Transcriptomics-guided engineering created more solvent-tolerant cyanobacteria strains that could be the foundation for a more productive biofuel host.

Place, publisher, year, edition, pages
2013. Vol. 79, no 23, 7419-7427 p.
Keyword [en]
Advanced biofuels, Metabolic process, Photosynthetic electron transport, Product inhibition, Reactive oxygen species, Small heat shock proteins, Solvent-tolerant, Transcriptome response
National Category
URN: urn:nbn:se:kth:diva-139282DOI: 10.1128/AEM.02694-13ISI: 000327544700035ScopusID: 2-s2.0-84888217221OAI: diva2:684634

QC 20140108

Available from: 2014-01-08 Created: 2014-01-08 Last updated: 2016-04-22Bibliographically approved
In thesis
1. Metabolic engineering strategies to increase n-butanol production from cyanobacteria
Open this publication in new window or tab >>Metabolic engineering strategies to increase n-butanol production from cyanobacteria
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The development of sustainable replacements for fossil fuels has been spurred by concerns over global warming effects. Biofuels are typically produced through fermentation of edible crops, or forest or agricultural residues requiring cost-intensive pretreatment. An alternative is to use photosynthetic cyanobacteria to directly convert CO2 and sunlight into fuel. In this thesis, the cyanobacterium Synechocystis sp. PCC 6803 was genetically engineered to produce the biofuel n­-butanol. Several metabolic engineering strategies were explored with the aim to increase butanol titers and tolerance.

In papers I-II, different driving forces for n-butanol production were evaluated. Expression of a phosphoketolase increased acetyl-CoA levels and subsequently butanol titers. Attempts to increase the NADH pool further improved titers to 100 mg/L in four days.

In paper III, enzymes were co-localized onto a scaffold to aid intermediate channeling. The scaffold was tested on a farnesene and polyhydroxybutyrate (PHB) pathway in yeast and in E. coli, respectively, and could be extended to cyanobacteria. Enzyme co-localization increased farnesene titers by 120%. Additionally, fusion of scaffold-recognizing proteins to the enzymes improved farnesene and PHB production by 20% and 300%, respectively, even in the absence of scaffold.

In paper IV, the gene repression technology CRISPRi was implemented in Synechocystis to enable parallel repression of multiple genes. CRISPRi allowed 50-95% repression of four genes simultaneously. The method will be valuable for repression of competing pathways to butanol synthesis.

Butanol becomes toxic at high concentrations, impeding growth and thus limiting titers. In papers V-VI, butanol tolerance was increased by overexpressing a heat shock protein or a stress-related sigma factor.

Taken together, this thesis demonstrates several strategies to improve butanol production from cyanobacteria. The strategies could ultimately be combined to increase titers further.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. 79 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:4
cyanobacteria, metabolic engineering, biofuels, butanol, synthetic scaffold, CRISPRi, solvent tolerance
National Category
Industrial Biotechnology
Research subject
urn:nbn:se:kth:diva-185548 (URN)978-91-7595-927-6 (ISBN)
Public defence
2016-05-27, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Swedish Research Council FormasKnut and Alice Wallenberg FoundationSwedish Foundation for Strategic Research
Available from: 2016-04-22 Created: 2016-04-21 Last updated: 2016-04-28Bibliographically approved

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