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Metabolic engineering strategies to increase n-butanol production from cyanobacteria
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-2430-2682
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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:4
Keyword [en]
cyanobacteria, metabolic engineering, biofuels, butanol, synthetic scaffold, CRISPRi, solvent tolerance
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-185548ISBN: 978-91-7595-927-6 (print)OAI: oai:DiVA.org:kth-185548DiVA: diva2:922039
Public defence
2016-05-27, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Funder
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
List of papers
1. Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production
Open this publication in new window or tab >>Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production
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2015 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, 167Article in journal (Refereed) Published
Abstract [en]

Background: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. Results: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. Conclusion: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.

Place, publisher, year, edition, pages
BioMed Central, 2015
Keyword
Biofuel, Butanol, Cyanobacteria, Metabolic engineering, Phosphoketolase, Starvation
National Category
Other Industrial Biotechnology Microbiology
Identifiers
urn:nbn:se:kth:diva-176965 (URN)10.1186/s12934-015-0355-9 (DOI)000362875500001 ()26474754 (PubMedID)2-s2.0-84944474444 (Scopus ID)
Note

QC 20151118

Available from: 2015-11-18 Created: 2015-11-13 Last updated: 2017-12-01Bibliographically approved
2. Complementary effects of ATP, acetyl-CoA and NADH driving forces increase butanol production in Synechocystis sp. PCC 6803
Open this publication in new window or tab >>Complementary effects of ATP, acetyl-CoA and NADH driving forces increase butanol production in Synechocystis sp. PCC 6803
(English)Manuscript (preprint) (Other academic)
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-185546 (URN)
Note

OS 2016

Available from: 2016-04-21 Created: 2016-04-21 Last updated: 2016-04-25Bibliographically approved
3. Affibody scaffolds improve sesquiterpene production in Saccharomyces cerevisiae
Open this publication in new window or tab >>Affibody scaffolds improve sesquiterpene production in Saccharomyces cerevisiae
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-185547 (URN)
Note

QC 20160422

Available from: 2016-04-21 Created: 2016-04-21 Last updated: 2016-04-22Bibliographically approved
4. 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, ISSN 2186-2311, E-ISSN 2161-5063, Vol. 5, no 3, 207-212 p.Article 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
Keyword
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: 2017-11-30Bibliographically approved
5. Using Transcriptomics To Improve Butanol Tolerance of Synechocystis sp Strain PCC 6803
Open this publication in new window or tab >>Using Transcriptomics To Improve Butanol Tolerance of Synechocystis sp Strain PCC 6803
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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.

Keyword
Advanced biofuels, Metabolic process, Photosynthetic electron transport, Product inhibition, Reactive oxygen species, Small heat shock proteins, Solvent-tolerant, Transcriptome response
National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-139282 (URN)10.1128/AEM.02694-13 (DOI)000327544700035 ()2-s2.0-84888217221 (Scopus ID)
Funder
Formas
Note

QC 20140108

Available from: 2014-01-08 Created: 2014-01-08 Last updated: 2017-12-06Bibliographically approved
6. Overexpression of sigma factor SigB improves temperature and butanol tolerance of Synechocystis sp PCC6803
Open this publication in new window or tab >>Overexpression of sigma factor SigB improves temperature and butanol tolerance of Synechocystis sp PCC6803
2014 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 182, no 1, 54-60 p.Article in journal (Refereed) Published
Abstract [en]

Among phenotypes of interest for an industrial cyanobacteria host are improved tolerance to temperature, salt, and solvent stress. Cellular responses to many stresses are controlled by the network of sensory receptors and downstream regulatory proteins. We applied transcription factor engineering to Synechocystis and tested mutant strains for tolerance to temperature and the biofuel 1-butanol. Histidine kinases (Hik), response regulators (Rre), and an RNA polymerase sigma factor (SigB) were overexpressed or deleted. Overexpression of SigB increased both temperature and butanol tolerance and lowered the intracellular concentration of reactive oxygen species. This report demonstrates that alteration of regulatory proteins in a cyanobacterium can be a useful tool to improve stress tolerance.

Keyword
Cyanobacteria, Biofuels, Tolerance, Reactive oxygen species, Transcription factors
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-148606 (URN)10.1016/j.jbiotec.2014.04.017 (DOI)000338812100007 ()2-s2.0-84901023888 (Scopus ID)
Funder
FormasKnut and Alice Wallenberg Foundation
Note

QC 20140812

Available from: 2014-08-12 Created: 2014-08-11 Last updated: 2017-12-05Bibliographically approved

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