1,721,064 research outputs found
Photosynthetic generation of heterologous terpenoids in cyanobacteria
Full text linkThe work aims to convert the secondary slow metabolism of the terpenoid biosynthetic pathway into a primary activity in cyanobacteria and to generate heterologous products using these photosynthetic microorganisms as cell factories. Case study is the production of the 10-carbon monoterpene β-phellandrene (PHL) in Synechocystis sp. PCC 6803 (Synechocystis). Barriers to this objective include the slow catalytic activity of the terpenoid metabolism enzymes that limit rates and yield of product synthesis and accumulation. "Fusion constructs as protein overexpression vectors" were applied in the overexpression of the geranyl diphosphate synthase (GPPS) and β-phellandrene synthase (PHLS) genes, causing accumulation of GPPS up to 4% and PHLS up to 10% of the total cellular protein. Such GPPS and PHLS protein overexpression compensated for their slow catalytic activity and enabled transformant Synechocystis to constitutively generate 24 mg of PHL per g biomass (2.4% PHL:biomass, w-w), a substantial improvement over earlier yields. The work showed that a systematic overexpression, at the protein level, of the terpenoid biosynthetic pathway genes is a promising approach to achieving high yields of prenyl product biosynthesis, on the way to exploiting the cellular terpenoid metabolism for commodity product generation
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Cyanobacterial Production of Biopharmaceutical and Biotherapeutic Proteins
Full text linkEfforts to express human therapeutic proteins in photosynthetic organisms have been described in the literature. Regarding microalgae, most of the research entailed a heterologous transformation of the chloroplast, but transformant cells failed to accumulate the desired recombinant proteins in high quantity. The present work provides methods and DNA construct formulations for over-expressing in photosynthetic cyanobacteria, at the protein level, human-origin bio-pharmaceutical and bio-therapeutic proteins. Proof-of-concept evidence is provided for the design and reduction to practice of "fusion constructs as protein overexpression vectors" for the generation of the bio-therapeutic protein interferon alpha-2 (IFN). IFN is a member of the Type I interferon cytokine family, well-known for its antiviral and anti-proliferative functions. Fusion construct formulations enabled accumulation of IFN up to 12% of total cellular protein in soluble form. In addition, the work reports on the isolation and purification of the fusion IFN protein and preliminary verification of its antiviral activity. Combining the expression and purification protocols developed here, it is possible to produce fairly large quantities of interferon in these photosynthetic microorganisms, generated from sunlight, CO2, and H2O
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Phycocyanin Fusion Constructs for Heterologous Protein Expression Accumulate as Functional Heterohexameric Complexes in Cyanobacteria
Full text linkOverexpression of heterologous proteins from plants, bacteria, and human as fusion constructs in cyanobacteria has been documented in the literature. Typically, the heterologous protein "P" of interest is expressed as a fusion with the abundant CpcB β-subunit of phycocyanin (PC), which was placed in the leader sequence position. The working hypothesis for such overexpressions is that CpcB*P fusion proteins somehow accumulate in a soluble and stable form in the cytosol of the cyanobacteria, retaining the activity of the trailing heterologous "P" protein of interest. The present work revealed a substantially different and previously unobvious picture, comprising the following properties of the above-mentioned CpcB*P fusion constructs: (i) the CpcB*P proteins assemble as functional (α,β*P)3CpcG heterohexameric discs, where α is the CpcA α-subunit of PC, β*P is the CpcB*P fusion protein, the asterisk denotes fusion, and CpcG is the 28.9 kDa PC disc linker polypeptide CpcG1. (ii) The (α,β*P)3CpcG1 complexes covalently bind one open tetrapyrrole bilin co-factor per α-subunit and two bilins per β-subunit. (iii) The (α,β*P)3CpcG1 heterohexameric discs are functionally attached to the Synechocystis allophycocyanin (AP) core cylinders and efficiently transfer excitation energy from the assembled (α,β*P)3CpcG1 heterohexamer to the PSII reaction center, enhancing the rate of photochemical charge separation and electron transfer activity in this photosystem. (iv) In addition to the human interferon α-2 and tetanus toxin fragment C tested in this work, we have shown that enzymes such as the plant-origin isoprene synthase, β-phellandrene synthase, geranyl diphosphate synthase, and geranyl linalool synthase are also overexpressed, while retaining their catalytic activity in the respective fusion construct configuration. (v) Folding models for the (α,β*P)3CpcG1 heterohexameric discs showed the recombinant proteins P to be radially oriented with respect to the (α,β)3 compact disc. Elucidation of the fusion construct configuration and function will pave the way for the rational design of fusion constructs harboring and overexpressing multiple proteins of scientific and commercial interest
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Recombinant Protein Stability in Cyanobacteria
Full text linkThe living cell possesses extraordinary molecular and biochemical mechanisms by which to recognize and efficiently remove foreign, damaged, or denatured proteins. This essential function has been a barrier to the overexpression of recombinant proteins in most expression systems. A notable exception is the overexpression in E. coli of recombinant proteins, most of which, however, end-up as "inclusion bodies", i.e., cytoplasmic aggregates of proteins that are inaccessible to the cell's proteasome. "Fusion constructs as protein overexpression vectors" proved to be unparalleled in their ability to cause substantial accumulation of recombinant proteins from plants, animals, and bacteria, as soluble proteins in unicellular cyanobacteria. Recombinant protein levels in the range of 10-20% of the total cellular protein can be achieved. The present work investigated this unique property in the context of recombinant protein stability in Synechocystis sp. PCC 6803 by developing and applying an in vivo cellular tobacco etch virus cleavage system with the objective of separating the target heterologous proteins from their fusion leader sequences. The work provides new insights about the overexpression, cellular stability, and exploitation of transgenes with commercial interest, highly expressed in a cyanobacterial biofactory. The results support the notion that eukaryotic plant- and animal-origin recombinant proteins are unstable, when free in the cyanobacterial cytosol but stable when in a fusion configuration with a highly expressed cyanobacterial native or heterologous protein. Included in this analysis are recombinant proteins of the plant isoprenoid biosynthetic pathway (isoprene synthase, β-phellandrene synthase, geranyl diphosphate synthase), the human interferon protein, as well as prokaryotic proteins (tetanus toxin fragment C and the antibiotic resistance genes kanamycin and chloramphenicol). The future success of synthetic biology approaches with cyanobacteria and other systems would require overexpression of pathway enzymes to attain product volume, and the work reported in this paper sets the foundation for such recombinant pathway enzyme overexpression
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins
Full text linkMicroalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.Y
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Cyanobacterial Production of Isoprene
Full text linkPhotosynthesis for the generation of isoprene in cyanobacteria was demonstrated with Synechocystis, entailing a process where a single host microorganism acts both as photocatalyst and processor, photosynthesizing and emitting isoprene hydrocarbons. A practical aspect of the commercial exploitation of this process in mass culture is the need to prevent invading microorganisms that might cause a culture to crash, and to provide an alternative to fresh water in scale-up applications. Growth media poised at alkaline pH are desirable in this respect, as high pH might favor the growth of the cyanobacteria, while at the same time discouraging the growth of invading predatory microbes and grazers. In addition, demonstration of salinity tolerance would enable the use of seawater for cyanobacteria cultivations. However, it is not known if Synechocystis growth and the isoprene-producing metabolism can be retained under such theoretically non-physiological conditions. We applied the gaseous/aqueous two-phase photobioreactor system with Synechocystis transformed with the codon-optimized isoprene synthase gene (SkIspS) of Pueraria montana (kudzu). Rates of growth and isoprene production are reported under control, and a combination of alkalinity and salinity conditions. The results showed that alkalinity and salinity do not exert a negative effect on either cell growth or isoprene rate and yield in Synechocystis. The work points to a practical approach in the design of cyanobacterial growth media for applications in commercial scale up and isoprene production. The concept applied in this work entails application of an isoprene synthase transgene from terrestrial plants, heterologously expressed in cyanobacteria, thereby reprogramming carbon flux in the terpenoid biosynthetic pathway toward formation and spontaneous release of this volatile chemical from the cell and liquid culture. However, flux manipulations and carbon-partitioning reactions between isoprene (the product) and native terpenoid biosynthesis for cellular needs are not yet optimized for isoprene yield. The primary reactant for isoprene biosynthesis is dimethylallyl diphosphate (DMAPP), whereas both DMAPP and its isopentenyl diphosphate (IPP) isomer are needed for cellular terpenoid biosynthesis. This aspect of the work addressed the function of an isopentenyl diphosphate (IPP) isomerase in cyanobacteria and its role in carbon partitioning between IPP and DMAPP, both of which serve, in variable ratios, as reactants for the synthesis of different cellular terpenoids. The project was approached upon the heterologous expression in Synechocystis of the isopentenyl diphosphate isomerase gene (FNI) from Streptococcus pneumoniae, a non-photosynthetic bacterium, using isoprene production as a “reporter process” for substrate partitioning between DMAPP and IPP. It is shown that transgenic expression of the FNI gene in Synechocystis resulted in a 250% increase in the isoprene production rate and yield, suggesting that the FNI isomerase shifted the composition of the endogenous DMAPP-IPP steady-state pool toward DMAPP, thereby enhancing rates and yield of isoprene production. Efforts to heterologously produce quantities of isoprene hydrocarbons (C5H8) renewably from CO2 and H2O through the photosynthesis of cyanobacteria face barriers, including low levels of recombinant enzyme accumulation compounded by their slow innate catalytic activity. This aspect of the work sought to alleviate the “expression level” barrier upon placing the isoprene synthase (IspS) enzyme in different fusion configurations with the cpcB protein, the highly expressed β-subunit of phycocyanin. Different cpcB*IspS fusion constructs were made, distinguished by the absence or presence of linker amino acids between the two proteins. Length and composition of linker amino acids was variable with lengths of 7, 10, 16 and 65 amino acids designed to confer optimal activity to the IspS through spatial positioning between the cpcB and IspS. Results showed that fusion constructs with the highly expressed cpcB gene, as the leader sequence, improved transgene expression in the range of 61- to 275-fold over what was measured with the unfused IspS control. However, the specific activity of the IspS enzyme was attenuated in all fusion transformants, possibly because of allosteric effects exerted by the leader cpcB fusion protein. This inhibition varied depending on the nature of the linker amino acids between the cpcB and IspS proteins. In terms of isoprene production, the results further showed a tradeoff between specific activity and transgenic enzyme accumulation. For example, the cpcB*L7*IspS strain showed only about 10% the isoprene synthase specific activity of the unfused cpcB-IspS control but it accumulated 254-fold more IspS enzyme. The latter more than countered the slower specific activity and made the cpcB*L7*IspS transformant the best isoprene-producing strain in this work. Isoprene to biomass yield ratios improved from 0.2 mg g-1 (isoprene to biomass, w:w) in the unfused cpcB-IspS control to 5.4 mg g-1 in the cpcB*L7*IspS strain, a 27-fold improvement.Our final approach unified the lessons previously learned. The combination of an optimized cpcB*IspS fusion construct with the overexpression of the FNI protein resulted in up to a 60-fold increase in isoprene yield, relative to that obtained upon the expression of the IspS gene alone in Synechocystis, reaching a yield of 12.3 mg g-1 (isoprene to biomass, w:w). The latter is the highest verifiable constitutive photosynthetic isoprene production measured with homoplasmic lines, in which the heterologous isoprene synthase gene is encoded by the Synechocystis genomic DNA. Thus, the work constitutes a major step forward in the development of a cyanobacterial engineering platform for isoprene production
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