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  • 1.
    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.

  • 2.
    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.

  • 3.
    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)
  • 4.
    Björk, Sara
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Shabestary, Kiyan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Yao, Lun
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ljungqvist, Emil
    Jönsson, Håkan
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton P.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Droplet microfluidic screening of a Synechocystis sp. CRISPRi library based on L-lactate productionManuscript (preprint) (Other academic)
  • 5.
    Cengic, Ivana
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Asplund-Samuelsson, Johannes
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Hudson, Elton P.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Indications of differential transcriptional regulation of fatty acid synthesis in the model cyanobacteria Synechocystis sp. Strain PCC 6803 and Synechococcus elongatus PCC 7942Manuscript (preprint) (Other academic)
  • 6.
    Cengic, Ivana
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kaczmarzyk, Danuta
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Yao, Lun
    KTH.
    Hudson, Paul
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    CRISPRi for metabolic engineering and fatty alcohol production in cyanobacteria2016In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 33, p. S37-S37Article in journal (Refereed)
  • 7.
    Cengic, Ivana
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton P.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Surface Display of Small Affinity Proteins on Synechocystis sp Strain PCC 6803 Mediated by Fusion to the Major Type IV Pilin PilA12018In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 200, no 16, article id e00270-18Article in journal (Refereed)
    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.

  • 8.
    Englund, Elias
    et al.
    KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab. Department of Chemistry – Ångström, Uppsala University, Box 523, SE-751 20 Uppsala, Sweden.
    Shabestary, Kiyan
    KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton P.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    Lindberg, P.
    Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound2018In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 49, p. 164-177Article in journal (Refereed)
    Abstract [en]

    Of the two natural metabolic pathways for making terpenoids, biotechnological utilization of the mevalonate (MVA) pathway has enabled commercial production of valuable compounds, while the more recently discovered but stoichiometrically more efficient methylerythritol phosphate (MEP) pathway is underdeveloped. We conducted a study on the overexpression of each enzyme in the MEP pathway in the unicellular cyanobacterium Synechocystis sp. PCC 6803, to identify potential targets for increasing flux towards terpenoid production, using isoprene as a reporter molecule. Results showed that the enzymes Ipi, Dxs and IspD had the biggest impact on isoprene production. By combining and creating operons out of those genes, isoprene production was increased 2-fold compared to the base strain. A genome-scale model was used to identify targets upstream of the MEP pathway that could redirect flux towards terpenoids. A total of ten reactions from the Calvin-Benson-Bassham cycle, lower glycolysis and co-factor synthesis pathways were probed for their effect on isoprene synthesis by co-expressing them with the MEP enzymes, resulting in a 60% increase in production from the best strain. Lastly, we studied two isoprene synthases with the highest reported catalytic rates. Only by expressing them together with Dxs and Ipi could we get stable strains that produced 2.8 mg/g isoprene per dry cell weight, a 40-fold improvement compared to the initial strain. 

  • 9.
    Forsström, Bjorn
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Axnäs, Barbara Bislawska
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Stengele, Klaus-Peter
    Buehler, Jochen
    Albert, Thomas J.
    Richmond, Todd A.
    Hu, Francis Jingxin
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton Paul
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays2014In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, no 6, p. 1585-1597Article in journal (Refereed)
    Abstract [en]

    Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on-and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.

  • 10.
    Hammar, Petter
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Angermayr, S. Andreas
    Sjöström, Staffan L.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    van der Meer, Josefin
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hellingwerf, Klaas J.
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Jönsson, Håkan N.
    Novo Nordisk Foundation Center for Biosustainability.
    Single-cell screening of photosynthetic growth and lactate production by cyanobacteria2015In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 8, article id 193Article in journal (Refereed)
    Abstract [en]

    Background: Photosynthetic cyanobacteria are attractive for a range of biotechnological applications including biofuel production. However, due to slow growth, screening of mutant libraries using microtiter plates is not feasible. Results: We present a method for high-throughput, single-cell analysis and sorting of genetically engineered l-lactate-producing strains of Synechocystis sp. PCC6803. A microfluidic device is used to encapsulate single cells in picoliter droplets, assay the droplets for L-lactate production, and sort strains with high productivity. We demonstrate the separation of low- and high-producing reference strains, as well as enrichment of a more productive L-lactate-synthesizing population after UV-induced mutagenesis. The droplet platform also revealed population heterogeneity in photosynthetic growth and lactate production, as well as the presence of metabolically stalled cells. Conclusions: The workflow will facilitate metabolic engineering and directed evolution studies and will be useful in studies of cyanobacteria biochemistry and physiology.

  • 11.
    Hammar, Petter
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sjöström, Staffan L.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Angermayr, Andreas
    Hellingwerf, Klaas J.
    Hudson, Paul
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan N.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Single-cell screening of secreted lactate production in cyanobacteriaManuscript (preprint) (Other academic)
  • 12.
    Hudson, Elton P.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Nikoshkov, Andrej
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 5, p. e37429-Article in journal (Refereed)
    Abstract [en]

    In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.

  • 13.
    Hudson, Elton P.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes2012In: Scientific Reports, ISSN 2045-2322, Vol. 2, p. 706-Article in journal (Refereed)
    Abstract [en]

    As antibody-based diagnosis and therapy grow at an increased pace, there is a need for methods which rapidly and accurately determine antibody-antigen interactions. Here, we report a method for the multiplex determination of antibody epitopes using bacterial cell-surface display. A protein-fragment library with 107 cell clones, covering 60 clinically-relevant protein targets, was created and characterized with massively parallel sequencing. Using this multi-target fragment library we determined simultaneously epitopes of commercial monoclonal and polyclonal antibodies targeting PSMA, EGFR, and VEGF. Off-target binding was observed for one of the antibodies, which demonstrates the method's ability to reveal cross-reactivity. We exemplify the detection of structural epitopes by mapping the therapeutic antibody Avastin. Based on our findings we suggest this method to be suitable for mapping linear and structural epitopes of monoclonal and polyclonal antibodies in a multiplex fashion and could find applicability in serum profiling as well as other protein-protein interaction studies.

  • 14.
    Jahn, Michael
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab. K.
    Vialas, Vital
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Karlsen, Jan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Maddalo, Gianluca
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Edfors, Fredrik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsström, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käll, Lukas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton P.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Growth of Cyanobacteria Is Constrained by the Abundance of Light and Carbon Assimilation Proteins2018In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 25, no 2, p. 478-+Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria must balance separate demands for energy generation, carbon assimilation, and biomass synthesis. We used shotgun proteomics to investigate proteome allocation strategies in the model cyanobacterium Synechocystis sp. PCC 6803 as it adapted to light and inorganic carbon (C-i) limitation. When partitioning the proteome into seven functional sectors, we find that sector sizes change linearly with growth rate. The sector encompassing ribosomes is significantly smaller than in E. coli, which may explain the lower maximum growth rate in Synechocystis. Limitation of light dramatically affects multiple proteome sectors, whereas the effect of C-i limitation is weak. Carbon assimilation proteins respond more strongly to changes in light intensity than to C-i. A coarse-grained cell economy model generally explains proteome trends. However, deviations from model predictions suggest that the large proteome sectors for carbon and light assimilation are not optimally utilized under some growth conditions and may constrain the proteome space available to ribosomes.

  • 15.
    Janasch, Markus
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Asplund-Samuelsson, Johannes
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Steuer, R.
    Hudson, Elton P.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kinetic modeling of the Calvin cycle identifies flux control and stable metabolomes in Synechocystis carbon fixation2019In: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 70, no 3, p. 973-983Article in journal (Refereed)
    Abstract [en]

    Biological fixation of atmospheric CO2 via the Calvin-Benson-Bassham cycle has massive ecological impact and offers potential for industrial exploitation, either by improving carbon fixation in plants and autotrophic bacteria, or by installation into new hosts. A kinetic model of the Calvin-Benson-Bassham cycle embedded in the central carbon metabolism of the cyanobacterium Synechocystis sp. PCC 6803 was developed to investigate its stability and underlying control mechanisms. To reduce the uncertainty associated with a single parameter set, random sampling of the steady-state metabolite concentrations and the enzyme kinetic parameters was employed, resulting in millions of parameterized models which were analyzed for flux control and stability against perturbation. Our results show that the Calvin cycle had an overall high intrinsic stability, but a high concentration of ribulose 1,5-bisphosphate was associated with unstable states. Low substrate saturation and high product saturation of enzymes involved in highly interconnected reactions correlated with increased network stability. Flux control, that is the effect that a change in one reaction rate has on the other reactions in the network, was distributed and mostly exerted by energy supply (ATP), but also by cofactor supply (NADPH). Sedoheptulose 1,7-bisphosphatase/fructose 1,6-bisphosphatase, fructose-bisphosphate aldolase, and transketolase had a weak but positive effect on overall network flux, in agreement with published observations. The identified flux control and relationships between metabolite concentrations and system stability can guide metabolic engineering. The kinetic model structure and parameterizing framework can be expanded for analysis of metabolic systems beyond the Calvin cycle.

  • 16.
    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.

  • 17.
    Kaczmarzyk, Danuta
    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.
    Yao, Lun
    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.
    Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX2018In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 45, p. 59-66Article in journal (Refereed)
    Abstract [en]

    Fatty alcohol production in Synechocystis sp. PCC 6803 was achieved through heterologous expression of the fatty acyl-CoA/ACP reductase Maqu2220 from the bacteria Marinobacter aquaeolei VT8 and the fatty acyl-ACP reductase DPW from the rice Oryza sativa. These platform strains became models for testing multiplex CRISPR-interference (CRISPRi) metabolic engineering strategies to both improve fatty alcohol production and to study membrane homeostasis. CRISPRi allowed partial repression of up to six genes simultaneously, each encoding enzymes of acyl-ACP-consuming pathways. We identified the essential phosphate acyltransferase enzyme PlsX (slr1510) as a key node in C18 fatty acyl-ACP consumption, repression of slr1510 increased octadecanol productivity threefold over the base strain and gave the highest specific titers reported for this host, 10.3 mg g−1 DCW. PlsX catalyzes the first committed step of phosphatidic acid synthesis, and has not been characterized in Synechocystis previously. We found that accumulation of fatty alcohols impaired growth, altered the membrane composition, and caused a build-up of reactive oxygen species.

  • 18.
    Kaczmarzyk, Danuta
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Georg-August-University, Germany.
    Hudson, Elton P.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Fulda, Martin
    Arabidopsis acyl-acyl carrier protein synthetase AAE15 with medium chain fatty acid specificity is functional in cyanobacteria2016In: AMB Express, ISSN 2191-0855, E-ISSN 2191-0855, Vol. 6, no 1, p. 1-9, article id 7Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria are potential hosts for the biosynthesis of oleochemical compounds. The metabolic precursors for such compounds are fatty acids and their derivatives, which require chemical activation to become substrates in further conversion steps. We characterized the acyl activating enzyme AAE15 of Arabidopsis encoded by At4g14070, which is a homologue of a cyanobacterial acyl-ACP synthetase (AAS). We expressed AAE15 in insect cells and demonstrated its AAS activity with medium chain fatty acid (C10-C14) substrates in vitro. Furthermore, we used AAE15 to complement a Synechocystis aas deletion mutant and showed that the new strain preferentially incorporates supplied medium chain fatty acids into internal lipid molecules. Based on this data we propose that AAE15 can be utilized in metabolic engineering strategies for cyanobacteria that aim to produce compounds based on medium chain fatty acids.

  • 19.
    Karlsen, Jan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Asplund-Samuelsson, Johannes
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Thomas, Quentin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). KTH, Centres, Science for Life Laboratory, SciLifeLab. Univ Copenhagen, Copenhagen Plant Sci Ctr, Dept Plant & Environm Sci, Frederiksberg, Denmark..
    Jahn, Michael
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton Paul
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ribosome Profiling of Synechocystis Reveals Altered Ribosome Allocation at Carbon Starvation2018In: MSYSTEMS, ISSN 2379-5077, Vol. 3, no 5, article id e00126-18Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria experience both rapid and periodic fluctuations in light and inorganic carbon (C-i) and have evolved regulatory mechanisms to respond to these, including extensive posttranscriptional gene regulation. We report the first genome-wide ribosome profiling data set for cyanobacteria, where ribosome occupancy on mRNA is quantified with codon-level precision. We measured the transcriptome and translatome of Synechocystis during autotrophic growth before (high carbon [HC] condition) and 24 h after removing CO2 from the feedgas (low carbon [LC] condition). Ribosome occupancy patterns in the 5' untranslated region suggest that ribosomes can assemble there and slide to the Shine-Dalgarno site, where they pause. At LC, total translation was reduced by 80% and ribosome pausing was increased at stop and start codons and in untranslated regions, which may be a sequestration mechanism to inactivate ribosomes in response to rapid C-i depletion. Several stress response genes, such as thioredoxin M (sll1057), a putative endonuclease (slr0915), protease HtrA (slr1204), and heat shock protein HspA (sll1514) showed marked increases in translational efficiency at LC, indicating translational control in response to Ci depletion. Ribosome pause scores within open reading frames were mostly constant, though several ribosomal proteins had significantly altered pause score distributions at LC, which might indicate translational regulation of ribosome biosynthesis in response to Ci depletion. We show that ribosome profiling is a powerful tool to decipher dynamic gene regulation strategies in cyanobacteria. IMPORTANCE Ribosome profiling accesses the translational step of gene expression via deep sequencing of ribosome-protected mRNA footprints. Pairing of ribosome profiling and transcriptomics data provides a translational efficiency for each gene. Here, the translatome and transcriptome of the model cyanobacterium Synechocystis were compared under carbon-replete and carbon starvation conditions. The latter may be experienced when cyanobacteria are cultivated in poorly mixed bioreactors or engineered to be product-secreting cell factories. A small fraction of genes (<200), including stress response genes, showed changes in translational efficiency during carbon starvation, indicating condition-dependent translation-level regulation. We observed ribosome occupancy in untranslated regions, possibly due to an alternative translation initiation mechanism in Synechocystis. The higher proportion of ribosomes residing in untranslated regions during carbon starvation may be a mechanism to quickly inactivate superfluous ribosomes. This work provides the first ribosome profiling data for cyanobacteria and reveals new regulation strategies for coping with nutrient limitation.

  • 20.
    Lundqvist, Magnus
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Edfors, Fredrik
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Sivertsson, Åsa
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hallström, Björn M
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tegel, Hanna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Holmberg, Anders
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Solid-phase cloning for high-throughput assembly of single and multiple DNA parts2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 7, article id e49Article in journal (Refereed)
    Abstract [en]

    We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.

  • 21.
    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.

  • 22.
    Shabestary, Kiyan
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Computational metabolic engineering strategies for growth-coupled biofuel production by Synechocystis2016In: Metabolic Engineering Communications, ISSN 2214-0301, Vol. 3, p. 216-226Article in journal (Refereed)
    Abstract [en]

    Chemical and fuel production by photosynthetic cyanobacteria is a promising technology but to date has not reached competitive rates and titers. Genome-scale metabolic modeling can reveal limitations in cyanobacteria metabolism and guide genetic engineering strategies to increase chemical production. Here, we used constraint-based modeling and optimization algorithms on a genome-scale model of Synechocystis PCC6803 to find ways to improve productivity of fermentative, fatty-acid, and terpene-derived fuels. OptGene and MOMA were used to find heuristics for knockout strategies that could increase biofuel productivity. OptKnock was used to find a set of knockouts that led to coupling between biofuel and growth. Our results show that high productivity of fermentation or reversed beta-oxidation derived alcohols such as 1-butanol requires elimination of NADH sinks, while terpenes and fatty-acid based fuels require creating imbalances in intracellular ATP and NADPH production and consumption. The FBA-predicted productivities of these fuels are at least 10-fold higher than those reported so far in the literature. We also discuss the physiological and practical feasibility of implementing these knockouts. This work gives insight into how cyanobacteria could be engineered to reach competitive biofuel productivities.

  • 23. 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)
  • 24. Xue, Chuang
    et al.
    Liu, Min
    Guo, Xinwen
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Chen, Lijie
    Bai, Fengwu
    Liu, Fangfang
    Yang, Shang-Tian
    Bridging chemical- and bio-catalysis: high-value liquid transportation fuel production from renewable agricultural residues2017In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 19, no 3, p. 660-669Article in journal (Refereed)
    Abstract [en]

    Catalytic conversion of lignocellulosic biomass to high-value transportation petrol, jet and diesel fuels is of great importance to develop versatile renewable energy and boost the rural economy, thus it is receiving worldwide attention. However, the state-of-the-art processes still suffer from a low efficiency in yield and productivity, which hinders the progress of its commercialization. Thus, it is essential to explore the consolidated catalytic reaction and refinery process. Here we report an advanced approach/process that could continuously produce long-chain (C-5-C-15) ketones from lignocellulosic biomass in a cascade mode by integrating several bio- and chemical catalysis reactions/processes, which exhibits a high efficiency and stable conversion rate. This technology consolidated the acetone-butanol-ethanol (ABE) fermentation, palladium catalyzed alkylation and in situ product recovery, allowing the catalytic reaction to proceed consistently and smoothly. The remarkable conversion properties are mainly attributed to the excellent compatible linkage of chemical catalysis with biosynthesis and process design to stabilize the Pd catalytic reaction. This validated strategy for the rational process design and integration of chemical-and bio-catalysis offers great demonstration in producing high-value transportation biofuels derived from agricultural residues.

  • 25.
    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.

1 - 25 of 25
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