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Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-2430-2682
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
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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. Vol. 14, 167
Keyword [en]
Biofuel, Butanol, Cyanobacteria, Metabolic engineering, Phosphoketolase, Starvation
National Category
Other Industrial Biotechnology Microbiology
URN: urn:nbn:se:kth:diva-176965DOI: 10.1186/s12934-015-0355-9ISI: 000362875500001PubMedID: 26474754ScopusID: 2-s2.0-84944474444OAI: diva2:872391

QC 20151118

Available from: 2015-11-18 Created: 2015-11-13 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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