41 results found
Perin G, Jones PR, 2019, Economic feasibility and long-term sustainability criteria on the path to enable a transition from fossil fuels to biofuels., Current Opinion in Biotechnology, Vol: 57, Pages: 175-182, ISSN: 0958-1669
Currently the production of liquid biofuels relies on plant biomass, which in turn depends on the photosynthetic conversion of light and CO2 into chemical energy. As a consequence, the process is renewable on a far shorter time-scale than its fossil counterpart, thus rendering a potential to reduce the environmental impact of the transportation sector. However, the global economy is not intensively pursuing this route, as current generation biofuel production does not meet two key criteria: (1) economic feasibility and (2) long-term sustainability. Herein, we argue that microalgal systems are valuable alternatives to consider, although it is currently technologically immature and therefore not possible to reach criterion 1, nor evaluate criterion 2. In this review we discuss the major limiting factors for this technology and highlight how further research efforts could be deployed to concretize an industrial reality.
Yunus IS, Wichmann J, Wördenweber R, et al., 2018, Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel, Metabolic Engineering, Vol: 49, Pages: 201-211, ISSN: 1096-7176
Liquid fuels sourced from fossil sources are the dominant energy form for mobile transport today. The consumption of fossil fuels is still increasing, resulting in a continued search for more sustainable methods to renew our supply of liquid fuel. Photosynthetic microorganisms naturally accumulate hydrocarbons that could serve as a replacement for fossil fuel, however productivities remain low. We report successful introduction of five synthetic metabolic pathways in two green cell factories, prokaryotic cyanobacteria and eukaryotic algae. Heterologous thioesterase expression enabled high-yield conversion of native fatty acyl-acyl carrier protein (ACP) into free fatty acids (FFA) in Synechocystis sp. PCC 6803 but not in Chlamydomonas reinhardtii where the polar lipid fraction instead was enhanced. Despite no increase in measurable FFA in Chlamydomonas, genetic recoding and over-production of the native fatty acid photodecarboxylase (FAP) resulted in increased accumulation of 7-heptadecene. Implementation of a carboxylic acid reductase (CAR) and aldehyde deformylating oxygenase (ADO) dependent synthetic pathway in Synechocystis resulted in the accumulation of fatty alcohols and a decrease in the native saturated alkanes. In contrast, the replacement of CAR and ADO with Pseudomonas mendocina UndB (so named as it is responsible for 1-undecene biosynthesis in Pseudomonas) or Chlorella variabilis FAP resulted in high-yield conversion of thioesterase-liberated FFAs into corresponding alkenes and alkanes, respectively. At best, the engineering resulted in an increase in hydrocarbon accumulation of 8- (from 1 to 8.5 mg/g cell dry weight) and 19-fold (from 4 to 77 mg/g cell dry weight) for Chlamydomonas and Synechocystis, respectively. In conclusion, reconstitution of the eukaryotic algae pathway in the prokaryotic cyanobacteria host generated the most effective system, highlighting opportunities for mix-and-match synthetic metabolism. These studies describe functioning synt
Yunus IS, Jones PR, 2018, Photosynthesis-dependent biosynthesis of medium chain-length fatty acids and alcohols, Metabolic Engineering, Vol: 49, Pages: 59-68, ISSN: 1096-7176
Cyanobacteria can directly channel atmospheric CO2 into a wide range of versatile carbon products such as fatty acids and fatty alcohols with applications including fuel, cosmetics, and health products. Works on alcohol production in cyanobacteria have so far focused on either long (C12-C18) or short (C2-C4) chain-length products. In the present work, we report the first synthetic pathway for 1-octanol (C8) biosynthesis in Synechocystis sp. PCC 6803, employing a carboxylic acid reductase and C8-preferring fatty acyl-ACP thioesterase. The first engineered strain produced 1-octanol but exhibited poor productivity and cellular health issues. We therefore proceeded to systematically optimize the strain and cultivation conditions in order to understand what the limiting factors were. The identification of optimal promoters and ribosomal binding sites, in combination with isopropyl myristate solvent overlay, resulted in a combined (C8-OH and C10-OH) titer of more than 100 mg/L (a 25-fold improvement relative to the first engineered strain) and a restoration of cellular health. Additionally, more than 905 mg/L 1-octanol was produced when the strain expressing sfp (phosphopantetheinyl transferase) and car (carboxylic acid reductase) was fed with octanoic acid. A combination of feeding experiments and protein quantification indicated that the supply of octanoic acid from the introduced thioesterase, and possibly also native fatty acid synthesis pathway, were the main bottlenecks of the pathway.
Kreula SM, Kaewphan S, Ginter F, et al., 2018, Finding novel relationships with integrated gene-gene association network analysis of Synechocystis sp. PCC 6803 using species-independent text-mining, PeerJ, Vol: 6, ISSN: 2167-8359
The increasing move towards open access full-text scientific literature enhances our ability to utilize advanced text-mining methods to construct information-rich networks that no human will be able to grasp simply from 'reading the literature'. The utility of text-mining for well-studied species is obvious though the utility for less studied species, or those with no prior track-record at all, is not clear. Here we present a concept for how advanced text-mining can be used to create information-rich networks even for less well studied species and apply it to generate an open-access gene-gene association network resource for Synechocystis sp. PCC 6803, a representative model organism for cyanobacteria and first case-study for the methodology. By merging the text-mining network with networks generated from species-specific experimental data, network integration was used to enhance the accuracy of predicting novel interactions that are biologically relevant. A rule-based algorithm (filter) was constructed in order to automate the search for novel candidate genes with a high degree of likely association to known target genes by (1) ignoring established relationships from the existing literature, as they are already 'known', and (2) demanding multiple independent evidences for every novel and potentially relevant relationship. Using selected case studies, we demonstrate the utility of the network resource and filter to (i) discover novel candidate associations between different genes or proteins in the network, and (ii) rapidly evaluate the potential role of any one particular gene or protein. The full network is provided as an open-source resource.
De Porcellinis AJ, Norgaard H, Brey LMF, et al., 2018, Overexpression of bifunctional fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase leads to enhanced photosynthesis and global reprogramming of carbon metabolism in Synechococcus sp PCC 7002, Metabolic Engineering, Vol: 47, Pages: 170-183, ISSN: 1096-7176
Cyanobacteria fix atmospheric CO2 to biomass and through metabolic engineering can also act as photosynthetic factories for sustainable productions of fuels and chemicals. The Calvin Benson cycle is the primary pathway for CO2 fixation in cyanobacteria, algae and C3 plants. Previous studies have overexpressed the Calvin Benson cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (hereafter BiBPase), in both plants and algae, although their impacts on cyanobacteria have not yet been rigorously studied. Here, we show that overexpression of BiBPase and RuBisCO have distinct impacts on carbon metabolism in the cyanobacterium Synechococcus sp. PCC 7002 through physiological, biochemical, and proteomic analyses. The former enhanced growth, cell size, and photosynthetic O2 evolution, and coordinately upregulated enzymes in the Calvin Benson cycle including RuBisCO and fructose-1,6-bisphosphate aldolase. At the same time it downregulated enzymes in respiratory carbon metabolism (glycolysis and the oxidative pentose phosphate pathway) including glucose-6-phosphate dehydrogenase (G6PDH). The content of glycogen was also significantly reduced while the soluble carbohydrate content increased. These results indicate that overexpression of BiBPase leads to global reprogramming of carbon metabolism in Synechococcus sp. PCC 7002, promoting photosynthetic carbon fixation and carbon partitioning towards non-storage carbohydrates. In contrast, whilst overexpression of RuBisCO had no measurable impact on growth and photosynthetic O2 evolution, it led to coordinated increase in the abundance of proteins involved in pyruvate metabolism and fatty acid biosynthesis. Our results underpin that singular genetic modifications in the Calvin Benson cycle can have far broader cellular impact than previously expected. These features could be exploited to more efficiently direct carbons towards desired
Kreula S, Kaewphan S, Ginter F, et al., 2017, Finding novel relationships with integrated gene-gene association network analysis of Synechocystis sp. PCC 6803 using species-independent text-mining, Publisher: PeerJ Inc.
The increasing move towards open access full-text scientific literature enhances our ability to utilize advanced text-mining methods to construct information-rich networks that no human will be able to grasp simply from 'reading the literature'. The utility of text-mining for well-studied species is obvious though the utility for less studied species, or those with no prior track-record at all, is not clear. Here we present a concept for how advanced text-mining can be used to create information-rich networks even for less well studied species and apply it to generate an open-access gene-gene association network resource for Synechocystis sp. PCC 6803, a representative model organism for cyanobacteria and first case-study for the methodology. By merging the text-mining network with networks generated from species-specific experimental data, network integration was used to enhance the accuracy of predicting novel interactions that are biologically relevant. A rule-based algorithm was constructed in order to automate the search for novel candidate genes with a high degree of likely association to known target genes by (1) ignoring established relationships from the existing literature, as they are already 'known', and (2) demanding multiple independent evidences for every novel and potentially relevant relationship. Using selected case studies, we demonstrate the utility of the network resource and rule-based algorithm to (i) discover novel candidate associations between different genes or proteins in the network, and (ii) rapidly evaluate the potential role of any one particular gene or protein. The full network is provided as an open source resource.
Kamarainen J, Huokko T, Kreula S, et al., 2017, Pyridine nucleotide transhydrogenase PntAB is essential for optimal growth and photosynthetic integrity under low-light mixotrophic conditions in Synechocystis sp PCC 6803, New Phytologist, Vol: 214, Pages: 194-204, ISSN: 1469-8137
Pyridine nucleotide transhydrogenase (PntAB) is an integral membrane protein complex participating in the regulation of NAD(P)+:NAD(P)H redox homeostasis in various prokaryotic and eukaryotic organisms. In the present study we addressed the function and biological role of PntAB in oxygenic photosynthetic cyanobacteria capable of both autotrophic and heterotrophic growth, with support from structural three-dimensional (3D)-modeling. The pntA gene encoding the α subunit of heteromultimeric PntAB in Synechocystis sp. PCC 6803 was inactivated, followed by phenotypic and biophysical characterization of the ΔpntA mutant under autotrophic and mixotrophic conditions. Disruption of pntA resulted in phenotypic growth defects observed under low light intensities in the presence of glucose, whereas under autotrophic conditions the mutant did not differ from the wild-type strain. Biophysical characterization and protein-level analysis of the ΔpntA mutant revealed that the phenotypic defects were accompanied by significant malfunction and damage of the photosynthetic machinery. Our observations link the activity of PntAB in Synechocystis directly to mixotrophic growth, implicating that under these conditions PntAB functions to balance the NADH: NADPH equilibrium specifically in the direction of NADPH. The results also emphasize the importance of NAD(P)+:NAD(P)H redox homeostasis and associated ATP:ADP equilibrium for maintaining the integrity of the photosynthetic apparatus under low-light glycolytic metabolism.
Erb TJ, Jones PR, Bar-Even A, 2017, Synthetic metabolism: metabolic engineering meets enzyme design., Current Opinion in Chemical Biology, Vol: 37, Pages: 56-62, ISSN: 1879-0402
Metabolic engineering aims at modifying the endogenous metabolic network of an organism to harness it for a useful biotechnological task, for example, production of a value-added compound. Several levels of metabolic engineering can be defined and are the topic of this review. Basic 'copy, paste and fine-tuning' approaches are limited to the structure of naturally existing pathways. 'Mix and match' approaches freely recombine the repertoire of existing enzymes to create synthetic metabolic networks that are able to outcompete naturally evolved pathways or redirect flux toward non-natural products. The space of possible metabolic solution can be further increased through approaches including 'new enzyme reactions', which are engineered on the basis of known enzyme mechanisms. Finally, by considering completely 'novel enzyme chemistries' with de novo enzyme design, the limits of nature can be breached to derive the most advanced form of synthetic pathways. We discuss the challenges and promises associated with these different metabolic engineering approaches and illuminate how enzyme engineering is expected to take a prime role in synthetic metabolic engineering for biotechnology, chemical industry and agriculture of the future.
Malatinszky D, Steuer R, Jones PR, 2017, A comprehensively curated genome-scale two-cell model for the cyanobacterium Anabaena sp. PCC 7120, Plant Physiology, Vol: 173, Pages: 509-523, ISSN: 0032-0889
Anabaena sp. PCC 7120 is a nitrogen-fixing filamentous cyanobacterium. Under nitrogen limiting conditions, a fraction of the vegetative cells in each filament terminally differentiate to non-growing heterocysts. Heterocysts are metabolically and structurally specialized to enable O2 -sensitive nitrogen fixation. The functionality of the filament, as an association of vegetative cells and heterocysts, is postulated to depend on metabolic exchange of electrons, carbon and fixed nitrogen. In the present work, we compile and evaluate a comprehensive curated stoichiometric model of this two-cell system, with the objective function based on the growth of the filament under diazotrophic conditions. The predicted growth rate under nitrogen replete and deplete conditions, as well as the effect of external carbon and nitrogen sources, was thereafter verified. Furthermore, the model was utilized to comprehensively evaluate the optimality of putative metabolic exchange reactions between heterocysts and vegetative cells. The model suggested that optimal growth requires at least four exchange metabolites. Several combinations of exchange metabolites resulted in predicted growth rates that are higher than growth rates achieved by only considering exchange of metabolites previously suggested in the literature. The curated model of the metabolic network of Anabaena sp. PCC 7120 enhances our ability to understand the metabolic organization of multi-cellular cyanobacteria and provides a platform for further study and engineering of their metabolism
Zavrel T, Knoop H, Steuer R, et al., 2016, A quantitative evaluation of ethylene production in the recombinant cyanobacterium Synechocystis sp PCC 6803 harboring the ethylene-forming enzyme by membrane inlet mass spectrometry, BIORESOURCE TECHNOLOGY, Vol: 202, Pages: 142-151, ISSN: 0960-8524
Vuorijoki L, Isojarvi J, Kallio P, et al., 2015, Development of a Quantitative SRM-Based Proteomics Method to Study Iron Metabolism of Synechocystis sp. PCC 6803, Journal of Proteome Research, Vol: 15, Pages: 266-279, ISSN: 1535-3907
Kremer F, Jones PR, Blank LM, et al., 2015, A comparison of the microbial production and combustion characteristics of three alcohol biofuels: ethanol, 1-butanol, and 1-octanol, Frontiers in Bioengineering and Biotechnology, Vol: 3, ISSN: 2296-4185
Over the last decade, microbes have been engineered for the manufacture of a variety of biofuels. Saturated linear-chain alcohols have great potential as transport biofuels. Their hydrocarbon backbones, as well as oxygenated content, confer combustive properties that make it suitable for use in internal combustion engines. Herein, we compared the microbial production and combustion characteristics of ethanol, 1-butanol, and 1-octanol. In terms of productivity and efficiency, current microbial platforms favor the production of ethanol. From a combustion standpoint, the most suitable fuel for spark-ignition engines would be ethanol, while for compression-ignition engines it would be 1-octanol. However, any general conclusions drawn at this stage regarding the most superior biofuel would be premature, as there are still many areas that need to be addressed, such as large-scale purification and pipeline compatibility. So far, the difficulties in developing and optimizing microbial platforms for fuel production, particularly for newer fuel candidates, stem from our poor understanding of the myriad biological factors underpinning them. A great deal of attention therefore needs to be given to the fundamental mechanisms that govern biological processes. Additionally, research needs to be undertaken across a wide range of disciplines to overcome issues of sustainability and commercial viability.
Menon N, Pasztor A, Menon BRK, et al., 2015, A microbial platform for renewable propane synthesis based on a fermentative butanol pathway, Biotechnology for Biofuels, Vol: 8, ISSN: 1754-6834
Akhtar MK, Dandapani H, Thiel K, et al., 2014, Microbial production of 1-octanol: a naturally excreted biofuel with diesel-like properties, Metab Eng Commun, Vol: 2, Pages: 1-5, ISSN: 2214-0301
The development of sustainable, bio-based technologies to convert solar energy and carbon dioxide into fuels is a grand challenge. A core part of this challenge is to produce a fuel that is compatible with the existing transportation infrastructure. This task is further compounded by the commercial desire to separate the fuel from the biotechnological host. Based on its fuel characteristics, 1-octanol was identified as an attractive metabolic target with diesel-like properties. We therefore engineered a synthetic pathway specifically for the biosynthesis of 1-octanol in Escherichia coli BL21(DE3) by over-expression of three enzymes (thioesterase, carboxylic acid reductase and aldehyde reductase) and one maturation factor (phosphopantetheinyl transferase). Induction of this pathway in a shake flask resulted in 4.4 mg 1-octanol L−1 h−1 which exceeded the productivity of previously engineered strains. Furthermore, the majority (73%) of the fatty alcohol was localised within the media without the addition of detergent or solvent overlay. The deletion of acrA reduced the production and excretion of 1-octanol by 3-fold relative to the wild-type, suggesting that the AcrAB–TolC complex may be responsible for the majority of product efflux. This study presents 1-octanol as a potential fuel target that can be synthesised and naturally accumulated within the media using engineered microbes.
Kallio P, Pasztor A, Thiel K, et al., 2014, An engineered pathway for the biosynthesis of renewable propane, NATURE COMMUNICATIONS, Vol: 5, ISSN: 2041-1723
Akhtar MK, Jones PR, 2014, Cofactor engineering for enhancing the flux of metabolic pathways., Front Bioeng Biotechnol, Vol: 2, Pages: 30-30, ISSN: 2296-4185
The manufacture of a diverse array of chemicals is now possible with biologically engineered strains, an approach that is greatly facilitated by the emergence of synthetic biology. This is principally achieved through pathway engineering in which enzyme activities are coordinated within a genetically amenable host to generate the product of interest. A great deal of attention is typically given to the quantitative levels of the enzymes with little regard to their overall qualitative states. This highly constrained approach fails to consider other factors that may be necessary for enzyme functionality. In particular, enzymes with physically bound cofactors, otherwise known as holoenzymes, require careful evaluation. Herein, we discuss the importance of cofactors for biocatalytic processes and show with empirical examples why the synthesis and integration of cofactors for the formation of holoenzymes warrant a great deal of attention within the context of pathway engineering.
Jones PR, Pásztor AP, Kallio P, et al., 2014, A synthetic O2-tolerant butanol pathway exploiting native fatty acid biosynthesis in Escherichia coli, Biotechnology and Bioengineering, Vol: 112, Pages: 120-128, ISSN: 1097-0290
Jones PR, 2014, Genetic instability in cyanobacteria – an elephant in the room?, Frontiers in Bioengineering and Biotechnology, Vol: 2, Pages: 1-5
Many research groups are interested in engineering the metabolism of cyanobacteria with the objective to convert solar energy, CO2, and water (perhaps also N2) into commercially valuable products. Toward this objective, many challenges stand in the way before sustainable production can be realized. One of these challenges, potentially, is genetic instability. Although only a handful of reports of this phenomenon are available in the scientific literature, it does appear to be a real issue that so far has not been studied much in cyanobacteria. With this brief perspective, I wish to raise the awareness of this potential issue and hope to inspire future studies on the topic as I believe it will make an important contribution to enabling sustainable large-scale biotechnology in the future using aquatic photobiological microorganisms.
Akhtar MK, Turner NJ, Jones PR, 2013, Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 110, Pages: 87-92, ISSN: 0027-8424
Kaewphan S, Kreula S, Van Landeghem S, et al., 2013, Integrating Large-Scale Text Mining and Co-Expression Networks: Targeting NADP (H) Metabolism in E. coli with Event Extraction, BioTxtM 2012 Third workshop on Building and Evaluating Resources for Biomedical Text Mining, Pages: 8-15
We present an application of EVEX, a literature-scale event extraction resource, in the concrete biological use case of NADP(H)metabolism regulation inEscherichia coli. We make extensive use of the EVEX event generalization based on gene family definitionsin Ensembl Genomes, to extract cross-species candidate regulators. We manually evaluate the resulting network so as to only preservecorrect events and facilitate its integration with microarray-based co-expression data. When analysing the combined network obtainedfrom text mining and co-expression, we identify 41 candidate genes involved in triangular patterns involving both subnetworks. Severalof these candidates are of particular interest, and we discuss their biological relevance further. This study is the first to present areal-world evaluation of the EVEX resource in particular and literature-scale application of the systems emerging from the BioNLPShared Task series in general. We summarize the lessons learned from this use case in order to focus future development of EVEX andsimilar literature-scale resources.
Kamarainen J, Knoop H, Stanford NJ, et al., 2012, Physiological tolerance and stoichiometric potential of cyanobacteria for hydrocarbon fuel production, JOURNAL OF BIOTECHNOLOGY, Vol: 162, Pages: 67-74, ISSN: 0168-1656
Guerrero F, Carbonell V, Cossu M, et al., 2012, Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp PCC 6803, PLOS ONE, Vol: 7, ISSN: 1932-6203
Eser BE, Das D, Han J, et al., 2011, Oxygen-Independent Alkane Formation by Non-Heme Iron-Dependent Cyanobacterial Aldehyde Decarbonylase: Investigation of Kinetics and Requirement for an External Electron Donor, BIOCHEMISTRY, Vol: 50, Pages: 10743-10750, ISSN: 0006-2960
Akhtar MK, Jones PR, 2009, Construction of a synthetic YdbK-dependent pyruvate:H-2 pathway in Escherichia coli BL21(DE3), METABOLIC ENGINEERING, Vol: 11, Pages: 139-147, ISSN: 1096-7176
Jones PR, 2008, Improving fermentative biomass-derived H-2-production by engineering microbial metabolism, 2nd Asian Bio-Hydrogen Symposium, Publisher: PERGAMON-ELSEVIER SCIENCE LTD, Pages: 5122-5130, ISSN: 0360-3199
Veit A, Akhtar MK, Mizutani T, et al., 2008, Constructing and testing the thermodynamic limits of synthetic NAD(P)H:H-2 pathways, MICROBIAL BIOTECHNOLOGY, Vol: 1, Pages: 382-394, ISSN: 1751-7915
Jones PR, Gawel R, Francis IL, et al., 2008, The influence of interactions between major white wine components on the aroma, flavour and texture of model white wine, FOOD QUALITY AND PREFERENCE, Vol: 19, Pages: 596-607, ISSN: 0950-3293
Akhtar MK, Jones PR, 2008, Deletion of iscR stimulates recombinant clostridial Fe-Fe hydrogenase activity and H-2-accumulation in Escherichia coli BL21(DE3), APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, Vol: 78, Pages: 853-862, ISSN: 0175-7598
Akhtar MK, Jones PR, 2008, Engineering of a synthetic hydF-hydE-hydG-hydA operon for biohydrogen production, ANALYTICAL BIOCHEMISTRY, Vol: 373, Pages: 170-172, ISSN: 0003-2697
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