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  • 1.
    Anfelt, Josefine
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Metabolic engineering strategies to increase n-butanol production from cyanobacteria2016Doctoral 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.

  • 2.
    Anfelt, Josefine
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hallström, Björn
    Nielsen, Jens
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hudson, Elton Paul
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Using Transcriptomics To Improve Butanol Tolerance of Synechocystis sp Strain PCC 68032013In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 79, no 23, p. 7419-7427Article in journal (Refereed)
    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.

  • 3.
    Anfelt, Josefine
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Kaczmarzyk, Danuta
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Shabestary, Kiyan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Renberg, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Nielsen, Jens
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Tech Univ Denmark.
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production2015In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, article id 167Article in journal (Refereed)
    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.

  • 4.
    Anfelt, Josefine
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Shabestary, Kiyan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Complementary effects of ATP, acetyl-CoA and NADH driving forces increase butanol production in Synechocystis sp. PCC 6803Manuscript (preprint) (Other academic)
  • 5.
    Kaczmarzyk, Danuta
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Anfelt, Josefine
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Särnegrim, Amanda
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hudson, Paul
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Overexpression of sigma factor SigB improves temperature and butanol tolerance of Synechocystis sp PCC68032014In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 182, no 1, p. 54-60Article in journal (Refereed)
    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.

  • 6.
    Konrad, Anna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Anfelt, Josefin
    KTH, School of Biotechnology (BIO), Proteomics.
    Andersson, Sandra
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Optimization of an antibody labeling strategy through the use of photoactivable synthetic Z domainsManuscript (preprint) (Other academic)
  • 7.
    Shabestary, Kiyan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Anfelt, Josefin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ljungqvist, Emil
    KTH.
    Jahn, Michael
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Yao, Lun
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton P.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Targeted Repression of Essential Genes To Arrest Growth and Increase Carbon Partitioning and Biofuel Titers in Cyanobacteria2018In: ACS Synthetic Biology, E-ISSN 2161-5063, Vol. 7, no 7, article id diva2:1239079Article in journal (Refereed)
    Abstract [en]

    Photoautotrophic production of fuels and chemicals by cyanobacteria typically gives lower volumetric productivities and titers than heterotrophic production. Cyanobacteria cultures become light limited above an optimal cell density, so that this substrate is not supplied to all cells sufficiently. Here, we investigate genetic strategies for a two-phase cultivation, where biofuel-producing Synechocystis cultures are limited to an optimal cell density through inducible CRISPR interference (CRISPRi) repression of cell growth. Fixed CO2 is diverted to ethanol or n-butanol. Among the most successful strategies was partial repression of citrate synthase gltA. Strong repression (>90%) of gitA at low culture densities increased carbon partitioning to n-butanol 5-fold relative to a nonrepression strain, but sacrificed volumetric productivity due to severe growth restriction. CO2 fixation continued for at least 3 days after growth was arrested. By targeting sgRNAs to different regions of the gitA gene, we could modulate GItA expression and carbon partitioning between growth and product to increase both specific and volumetric productivity. These growth arrest strategies can be useful for improving performance of other photoautotrophic processes.

  • 8. Tippmann, S.
    et al.
    Anfelt, Josefin
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    David, F.
    Rand, J. M.
    Siewers, V.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Denmark.
    Nielsen, J.
    Hudson, E. Paul
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Affibody Scaffolds Improve Sesquiterpene Production in Saccharomyces cerevisiae2017In: ACS Photonics, E-ISSN 2330-4022, Vol. 6, no 1, p. 19-28Article in journal (Refereed)
    Abstract [en]

    Enzyme fusions have been widely used as a tool in metabolic engineering to increase pathway efficiency by reducing substrate loss and accumulation of toxic intermediates. Alternatively, enzymes can be colocalized through attachment to a synthetic scaffold via noncovalent interactions. Here we describe the use of affibodies for enzyme tagging and scaffolding. The scaffolding is based on the recognition of affibodies to their anti-idiotypic partners in vivo, and was first employed for colocalization of farnesyl diphosphate synthase and farnesene synthase in S. cerevisiae. Different parameters were modulated to improve the system, and the enzyme:scaffold ratio was most critical for its functionality. Ultimately, the yield of farnesene on glucose YSFar could be improved by 135% in fed-batch cultivations using a 2-site affibody scaffold. The scaffolding strategy was then extended to a three-enzyme polyhydroxybutyrate (PHB) pathway, heterologously expressed in E. coli. Within a narrow range of enzyme and scaffold induction, the affibody tagging and scaffolding increased PHB production 7-fold. This work demonstrates how the versatile affibody can be used for metabolic engineering purposes.

  • 9. Tippmann, Stefan
    et al.
    Anfelt, Josefine
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    David, Florian
    Rand, Jacqueline M.
    Siewers, Verena
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Tech Univ Denmark.
    Nielsen, Jens
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Affibody scaffolds improve sesquiterpene production in Saccharomyces cerevisiaeManuscript (preprint) (Other academic)
  • 10.
    Yao, Lun
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Cengic, Ivana
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Anfelt, Josefine
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Multiple Gene Repression in Cyanobacteria Using CRISPRi2016In: ACS Photonics, E-ISSN 2330-4022, Vol. 5, no 3, p. 207-212Article in journal (Refereed)
    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.

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